Responses of Plants to UV-B Radiation

Cover Photo: Daniela Lud, Institute of Ecology, Yerseke, The Netherlands

ADVANCES IN VEGETATION SCIENCE

Volume 18
Responses of Plants
to UV-B Radiation

Edited by:

JELTE ROZEMA
Department o,f'Systems Ecology,
Vrije Universiteit, Amsterdam, The Netherlands

YTANNTS MANETAS
Department of Biology, School of Science,
University of Patras, Greece

LARS-OLOF BJORN
Department of' Plant Physiology,
Lund University, Sweden

Reprinted from Plant Ecologv, Volume 154, Nos. 1-2, pp. 1-274

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A c.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-90-481-5353-4 ISBN 978-94-017-2892-8 (eBook)

DOI 10.1007/978-94-017-2892-8

Printed on acid~free paper

AII Rights Reserved

© 200 l Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2001
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system. without written permission [rom the copyright owner.
Table of Contents

Preface I J. Rozema, Y. Manetas & L.O. Bjorn VII

Section 1 - General
Is provitamin D a UV-B receptor in plants~
L.O. Bjorn & T. Wang 1-8
(Poly)phenolic compounds in pollen and spores of Antarctic plants as indicators of solar UV-B:
a new proxy for the construction of past solar UV- B?
J. Rozema. A.J. Noordijk. R.A. Broekman. A. van Beetn. B. B. Meijkamp. N. VJ. de Bakker, 1. WM. van
de Staaij. M. Stroetenga. S.J.P. Bohncke, M. Konert, S. Kars, H. Peat, R.I.L. Smith & P. Convey 9-26
The direct effects of UV-B radiation on Betula puhescens litter decomposing at four
European field sites
S.A. Moody, N.D. Paul, L.O. Bjorn, T.V Callaghan, J.A. Lee, Y. Manetas, 1. Rozema, D. Gwynn-Jones,
U. Johanson, A. Kvparissis & A.M. C. Oudejans 27-36

Section 2 - Terrestrial Plants and Terrestrial Ecosystems

The reduction of aboveground Calamagrostis epigeios mass and tiller number by enhanced UV-B in a
dune-grassland ecosystem
A.M. C. Oudejans, A. Nijssen, J.S. Huls & J. Rozema 37-48
The influence of enhanced UV-B radiation on the spring geophyte Pulmonaria o.fficinalis
A. GabersCik, M. Novak, T. Trost, Z. Maze}. M. Germ & L.O. Bjorn 49-56
The growth, flower properties and demography of Anthemis arvensis exposed to enhanced UV-B
radiation
Y. Petropoulou, 0. Georgiou, G.K. Psaras & Y. Manetas 57-64

Section 3 - Arctic and Antarctic Plants and Ecosystems

Short-term impacts of enhanced UV-B radiation on photo-assimilate allocation and metabolism:
a possible interpretation for time-dependent inhibition of growth
D. Gwynn-Jones 65-73
Field research on the effects of UV- B filters on terrestrial Antarctic vegetation
A.H.L. Huiskes, D. Lud & T.C. W Moerdijk-Poortvliet 75-86
The effects of altered levels of UV-B radiation on an Antarctic grass and lichen
D. Lud, A.H.L. Huiskes, T.C. W Moerdijk & 1. Rozema 87-99
Consequences of depletion of stratospheric ozone for terrestrial Antarctic ecosystems: the response of
Deschampsia antarctica to enhanced UV-B radiation in a controlled environment
J. Rozema, R. Broekman, D. Lud. A.H.J. Huiskes, T. Muerdijk. N. de Bakker, B. B. Meijkamp &
A. van Beem 101-115
Section 4- Interactions of UV-B Radiation with Other Factors of Terrestrial Environments
Reduction of ambient UV-B radiation does not affect growth but may change the flowering pattern of
Rosmarinus officina lis L.
G. Grammatikopou/os, P Drilias, A. Kyparissis, Y. Petropoulou & Y. Manetas 117-122
UV-B and PAR in single and mixed canopies grown under different UV-B exclusions in the field
G. Deckmyn, E. Cayenberghs & R. Ceulemans 123-133
The response of Viciafaba to enhanced UV-B radiation under low and ncar ambient PAR levels
B.B. Meijkamp, G. Doodeman & J. Rozema 135-146
Growth under UV-B radiation increases tolerance to high-light stress in pea and bean plants
E.M. Bolink, I. van Schalkwijk, F. Posthumus & P.R. van Hasselt 147-156
Nutrient availability influences UV-B sensitivity of Plantago lanceolata
M. Tosserams, J. Smet, E. Magendans & J. Rozema 157-168
Increased solar UV-8 radiation may reduce infection by arbuscular mycorrhizal fungi (AMF) in dune
grassland plants: evidence from five years of field exposure
J. van de Staaij, J. Rozema, A. van Beem & R. Aerts 169-177
Combined effects of enhanced UV-B radiation and additional nutrients on growth of two
Mediterranean plant species
E. Levizou & Y. Manetas 179-186
Effects of UV-B radiation and additional irrigation on the Mediterranean evergreen sclerophyll
Ceratonia siliqua L. under field conditions
A. Kyparissis, P Drilias, Y. Petropoulou, G. Grammatikopou/os & Y. Manetas 187-193
Combined effects of C0 2 concentration and enhanced UV-B radiation on faba bean
M. Tosserams, A. Visser, M. Groen, G. Kalis, E. Magendans & J. Rozema 195-210
Enhanced UV-B radiation, artificial wounding and leaf chemical defensive potential in
Phlomisfruticosa L.
E. Levizou & Y. Manetas 211-217

Section 5- Aquatic Plants and Aquatic Ecosystems

Responses of aquatic algae and cyanobacteria to solar UV-B
R.P Sinha, M. Klisch, A. Groniger & D.-P. Hiider 219-236
Effects of UV-8 radiation on a charophycean alga, Chara aspera
N. V..l. de Bakker, A.P van Beem, .!. WM. van de Staaij,.!. Rozema & R. Aerts 237-246
Differential sensitivity to natural ultraviolet radiation among phytoplankton species in Arctic lakes
(Spitsbergen, Norway)
E. van Donk, B.A. Faafeng, H.J. de Lange & D.O. Hessen 247-259
The photoprotective role of humus-DOC for Selenastrum and Daphnia
D.O. He;;sen & Pl. FtErr;Jvig 261-273

Subject Index 275

Species Index 277
 Plan/ Eculor;y 154: vii, 200 l.
Vll

Preface

September 22, 1999 a workshop UV-8 and Plants was held as part of the Eurcco 99 VTIIth European Ecological
Congress, Halkidiki, Greece. The workshop was initiated by Yiannis Manetas, member of the local organizing
committee. The support of the organizers of Eureco for this workshop is gratefully acknowledged. There were
about 100 participants in the workshop and 25 papers and posters were presented. Kluwer Academic Publishers
approved the request of the Subject Editor for Physiological Ecology of the international journal Plant Ecology, to
have the papers published in a special issue entitled "UV-B and Plants". Most papers relate to research of UV-B
effects on plants by European scientists. All papers have been peer-reviewed and we are indebted to all reviewers
involved. Pa1t of the research presented has been funded by the European Commission under the programme
Environment and Climate, which is gratefully acknowledged. Also we acknowledge the financial assistance by the
Eugenides Foundation (Athens, Greece).

The UV-B research presented covers plants from terrestrial and aquatic environments and also studies from the
Arctic and Antarctic are included.

During the spring of 2000 a marked ozone hole developed in the stratosphere over the Arctic. It has now been made
likely that the global warming as a result of the greenhouse effect is linked with cooling of the stratosphere, thus
stimulating the destruction of ozone.

Emission of CFC's has been banned and recovery of ozone has been predicted, but as yet no signs of restoration
have occurred. By the coupling of the greenhouse effect and the stratospheric ozone depletion, the recovery of the
ozone layer may be markedly delayed.

In addition to studies of the atmosphere and stratosphere, research into the impact of enhanced UV-B radiation is
essential in assessing the occurrence and prevention of UV-B damage, as well the analysis of adaptions to UV-B
radiation of terrestrial and aquatic organisms. It is the wish of the editors that the special issue UV-B and Plants
will contribute in this respect.

Jette Rozema
Yiannis Manetas
Lars Olof Bjorn
Guest Editors
 Section 1: General

Mammals need vitamin 0 for several processes. Because of low levels of ultraviolet-B radiation in the Arctic. vitamin 0
could be in limiting supply for terrestrial mammals there, such as these reindeer. But what could be the role of vitamin 0
in plams, algae, and lichens. and how could it be affected hy the UV-B level') (Photograph by Lars-Oiof Bjorn)
 Plant EcologY 154: 3-8, 200 I.
© 2001 KlttWer Academic Puhlishers.
Is provitamin D a UV-B receptor in plants?
Lars Olof Bjorn & Ting Wang
Department of Plant Physiology, Lund University, Box 117, SE-221 00 Lund, Sweden
Key words: Dehydrocholesterol, Ergosterol, Photoreceptor, Provitamin D, Ultraviolet, UV-B, Vitamin D
Abstract
An hypothesis is presented that provitamin D (dehydrocholesterol and/or ergosterol) can act as a UV-B receptor in
plants and algae. We also propose that the proportions between provitamins D, prcvitamins D, and vitamins D (D2
and D 3 ), after calibration, can be used to evaluate UV-B exposure of phytoplankton and terrestrial vegetation.
Introduction
There is an enormous literature on the medical aspects
of vitamin D (see Feldman et al. 1997; Holick 1999).
The present contribution presents a possible function
of the provitamin/previtamin/vitamin D system in al-
gae, plants and fungi, namely as an ultraviolet-B
photoreceptor system. Our discussion is based mostly
on literature data published by other workers. We will
also discuss some own findings in a general way, while
details will be published elsewhere. We shall start
by introducing vitamin D and related compounds and
their formation in various organisms.
Vitamin D has several functions in the human body
and in other terrestrial vertebrates. It was discovered
as the agent which prevents rickets, a disease of the
skeleton (Mellanhy 1918: Steenbock & Black 1924;
Hess & Weinstock 1924), and one of its most impor-
tant functions is still in the regulation of calcium ion
absorption from the food. The only well-documented
case of a mammal not requiring vitamin D from exter-
nal sources or internal production is the damara mole
rat, Cryptomys damarensis (Buffenstein et al. 1991;
Pitcher & Buffenstein 1994a, b), which seems not
to regulate calcium uptake from the intestine (Pitcher
et al. 1992; Pitcher & Buffenstein 1995), although
it can synthesise vitamin D3 if artificially exposed to
sunlight (Pitcher et al. 1994 ). The situation is proba-
bly the same for the related mole-rat Heterocephalus
glaber (Pitcher & Butfenstein 1995), and possibly for
many other burrowing animals feeding from under-
3
ground plant parts, and therefore lacking any obvious
source of vitamin D.
There are two well-known types of vitamin 0, i.e.
vitamin D2 and vitamin D3. Vitamin D1 does not exist;
it turned out that what was first named vitamin D 1 was
a mixture of compounds. In amphibians and reptiles
there exist other, related compounds which probably
have analogous functions.
Vitamins D are formed from provitamins D via
intermediates called previtamins D. The conversion
from provitamin to previtamin is a photochemical
process requiring ultraviolet-B radiation and taking
place with a high quantum yield: for provitamin 02
and provitamin D3 photoconversion quantum yields
of 0.20 to 0.31 have been reported (Pfoerter & Weber
1972; Havinga et al. 1973; Pottier & Russell 1991 );
0.26 being a value often used in various computations.
We thus have the reaction sequences, which are not
enzyme-catalysed:
(I) provitamin D2 (ergosterol) u~ previtamin 02
===} vitamin D2
(2) provitamin D3 (?-dehydrocholesterol) u~B previ-
tamin D3 ===} vitamin D3
Provitamin D3 is also called cholecalciferol.
Even at human body temperature the conversion of
previtamin to vitamin is a slow process, requiring days
to approach completeness. At the lower temperature of
most organisms, the conversion is even slower.
Provitamin D3 (but not D2) is formed in human
skin, and we can form vitamin D3 after exposure to
ultraviolet-B radiation. A well-known food source for
4
Table I a. Contents of provitamins and vitamins D in phy-
toplankton. After Sunita Rao & Raghuramulu (1996) (a)
and Takeuchi et al. ( 1991) (b). Amounts are expressed as
microgram per gram of dry matter.
Source of Provit. D2 Provit. D3 Vit. D2 Vit. D
phytoplankton
Hussain Sagar
(India) (a) 3.9 23.6 0.0525 0.8035
Biwa Lake
(Japan) (b)
August 10.1 14.5 0.043 0.1473
October 2.9 3.6 0.0189 0.0496
December 2.6 3.4 0.0217
vitamin D3 is cod-liver oil; the product first shown to
prevent rickets (Mellanby 1918). The vitamin is not
synthesised by the fish, but by the plankton forming
the beginning of the marine food-chain (Sugisaki et al.
1974, Sunita Rao & Raghuramulu 1996a, b; Takeuchi
eta!. 1991).
Occurence of provitamins D and formation of
vitamins D in algae
Provitamin D2 (ergosterol) is present in many al-
gae from very different groups, but not in all algae.
The following have been shown to contain ergos-
terol: Several species of Chlorella (Chlorophyceae)
(Patterson 1971 ), Chlamydomonas reinhardtii (Ehren-
berg) (Chlorophyceae) (Patterson 1974), Skeletonema
menzelii (Greville) (8acillariophyeeae) (Holick 1989),
Emiliania huxleyi (Lohm.) (Prymnesiophyceae or
Haptophyceae) (Holick 1989), Ochromonas danica
(Wyssotzki) (Chrysophyceae) (Stem et al. 1960; Ger-
shengorn eta!. 1968, and others).
In Table I a we have collected literature data on
provitamins and vitamins D for phytoplankton col-
lected in India and in Japan, and in Table I b results
of our own calculations on these data. The latter table
shows the following:
(I) The ratio of vitamin to provitamin is always higher
for the D3 type than for the D2 type (more than twice
as high). Thus vitamin accumulation from provitamin
D3 is more efficient than formation from provitamin
D2 under these natural conditions. This may at first
seem surprising, since the quantum yield measured for
conversion in solution is almost the same for provita-
mins D2 and D3. Different explanations are possible
Table I b. Ratios of vitamins D to provitarnins D (multiplied by
1000). Code letters for references as in Table Ia.
D3 ratio
Source of D2 ratio x I000 D3 ratio x 1000
D2 ratio
phytoplankton
Hussain Sagar
(India) (b) 13.5 34.0 2.5
Biwa Lake
(Japan) (d)
August 4.3 10.2 2.4
October 6.5 13.8 2.1
December 6.4
for this apparent discrepancy, such as faster conversion
from previtamin to vitamin with less previtamin disap-
pearing in side reactions, or slower breakdown of the
vitamin D3 in vivo. It may somehow be related to the
fact that only vitamin D3 reaches the highest trophic
levels.
(2) More provitamin is converted to vitamin in Hus-
sain Sagar Lake in India (latitude ca 18° N), than
in Lake 8iwa in Japan (latitude ca 35.3° N). The
UV-8 radiation is, of course, stronger at the lower
latitude, and this may be reflected in the accumulation
of vitamin.
(3) ln Japan the vitamin accumulation is less efficient
in December than in August or October, reflecting the
change in UV-8 exposure over the year.
Although the data are limited we propose that de-
terminations of provitamins, previtamins and vitamins
D could be used as an internal dosimeter to evaluate
the exposure of phytoplankton to ultraviolet-8 radia-
tion. Calibration could be carried out with controlled
exposures of the organisms. Determination of radia-
tion exposure under natural conditions is otherwise
almost impossible, as the plankton moves up and down
in the water column. Galkin & Terenetskaya ( 1999)
have discussed the use of provitamin D3 in vitro for
dosimetry, and Webb et a!. ( 1988) have compared
provitamin D3 conversion in vitro with that in skin
under different daylight conditions.
There are no corresponding data for macroalgae
in the literature, but we have found provitamins and
vitamins D2 and D3 in the brown macroalga, Fucus
vesiculosus L. The ratios of vitamin to provitamin
were higher for material from the Swedish west coast,
latitude 58.3° N than for material from the Norwegian
west coast, 68.1 aN (both collected within twelve days

in September). In this case, however, the ratios were
higher for the 02 than for the 03 form.
Effects of provitamin D2 and vitamins D on algae
We would like to refer to an interesting investigation
by Fries ( 1984). She added provitamin 02, vitamin
0 2 or vitamin 0 3 to the growth medium of the brown
macroalga Fucus spiralis. L. At the lowest concen-
tration, I o- 8 M, vitamin 03 (but not the other two
compounds) had a large growth-stimulating effect. At
a tenfold higher concentration, also vitamin 02 had
a growth-stimulating effect, while the stimulation by
provitamin 02 was much smaller.
Occurrence of provitamins and vitamins D in
higher plants
Plants produce provitamins 0, vitamins 0 and related
compounds in their leaves (Napoli et al. 1977; Wasser-
man 1975; Prema & Raghuramulu 1994, 1996; Zucker
eta!. 1980; Rambeck et al. 1981; Horst et al. 1984).
In the case of ergosterol and vitamin 02 one has to
be cautious in assigning substances found in the analy-
sis of plants to synthesis by the plants themselves.
Many plants, among them many grasses, harbour en-
dophytic fungi (Clay 1990; Redlin & Carris 1996;
Siegel eta!. 1987), and fungi regularly produce ergos-
terol as their major sterol. The content of ergosterol
in plant tissue has been used as a measure of fungal
contamination (Gessner & Schmitt 1996).
It is a widespread misconception in the literature
that plants produce only provitamin 02 and vitamin
0 2 (e.g., Buddecke 1980). Often as much provitamin
0 3 and vitamin 03 are produced (Zucker et a!. 1980,
Prema & Raghuramulu 1996). Even I ,25-dihydroxy
vitamin 0 3 has been found in plants (Napoli et al.
1977), as well as a glycoside of this compound, some-
times at concentrations high enough to poison grazing
animals (Wasserman et al. 1976).
We have confirmed a UV-B dependent synthesis of
vitamins 0 2 and 0 3 in the leaves of the tomato plant.
An interesting observation is that the provitamin 03
content of tomato leaves is not reduced by growing
plants under UV-B radiation, although a substantial
amount of vitamin 03 is formed (Table 2). This points
to a feedback mechanism regulating the amount of the
provitamin.
5
Table 2. Contents of provitamins and vitamins D2 and D3 in tomato
(Lycopersicon esculentum Mill). Tomato plants were grow~ in a
urecnhousc with or without UV-B radiation (0.85 kJ plant we1ghtcd
UV-B radiation per m 2 and day).
Organism Micrograms per gram dry weight
Provit. D2 Provit. D3 Vitamin D2 Vitamin D3
Tomato (-UV-B) 1.83 0.61 0 0
Tomato (+UV-B) 2.23 0.76 (J.087 0.28
Recent! y Curino et a!. ( 1998) made the startling
discovery that Solanum glaucophyllum cells are able
to synthesise vitamin 03 and its derivatives in dark-
ness. This is the only documented case of vitamin
0 3 synthesis in the absence of UV-B. Solanum g/au-
cophyllum is a very special plant which accumulates
large amounts of dihydroxy vitamin 03 as a protection
against grazing mammals. Mechanisms for nonphoto-
chemical formation of vitamin 0 have been proposed
by Norman & Norman (1993).
Effects of vitamins D on plants
Vitamin 0 applied to herbaceous and woody plants
stimulates initiation of adventitious roots (Buchala &
Schmid 1979; Jarvis & Booth 1981; Moncousin &
Gaspar 1983 ). Vitamin 03 (I o- 9 M) inhibits root
elongation in Phaseolus vulgaris and promotes ger-
mination of light sensitive lettuce seed in darkness
(Buchala & Pythoud 1988). Thus vitamin 0 has a
number of physiological effects.
Intracellular localisation of provitamin D
In human skin cells provitamin 03 occurs in the outer
cell membrane. After conversion to vitamin D3 an OH
group extends from the membrane to the intercellular
compartment, where it attaches to a carrier protein and
the vitamin is transferred to the blood stream.
In most fungi ergosterol (provitamin D2) is the ma-
jor sterol in the outer cell membrane. Upon irradiation
with ultraviolet radiation under aerobic conditions it
disappears, and the corresponding spectral change can
be observed both in intact yeast cells and in isolated
cell membranes (Arami et al. l997a, b). It is not
known whether the provitamin D present in plants is
located to the cell membranes, but this appears likely,
6
and we have started experiments to find out whether
this is the case or not.
The role of provitamins and vitamins D in plants
Although provitamins and vitamins 0 (both 02 and 03
forms) occur in many, perhaps most, algae and plants,
nothing is known about their possible function.
We would like to propose here a function which
has not been considered by other authors, i.e .. that
the provitamin/vitamin 0 system acts as a sensor for
ultraviolct-B radiation.
Several processes in plants are regulated specif-
ically by ultraviolet-B radiation. The action spectra
for some such processes have been determined and
show peaks at about 295 nm (reviews by Beggs
et al. 1994 and Bjorn 1999). Also some algae can
sense the radiation level: prysemniophytes and di-
noflagellates are able to adjust the content of radiation-
screening mycosporine-like amino acids according to
need (Hannach & Sigleo 1998). The action spec-
trum for pigment formation in a fungus has peak near
295 nm (Hsiao & Bjorn 1982). An action spectrum
for mycosporine induction in a cyanobacterium has a
rather different shape with a peak at 310 nm and a long
tail towards longer wavelength (Portwich & Garcia-
Piche! 2000). In the latter case there is some evidence
for the role of a pterin as photoreceptor chromophore,
and this as also been suggested by several researchers
for the higher plant photoreceptor.
The action spectrum for photochemical conversion
of provitamin D to vitamin D has been determined
only for human skin and in this case also exhibits a
peak at 295 nm (MacLaughlin et al. 1982).
Provitamin D is suitable as a radiation sensor be-
cause of the very high quantum yield for photoconver-
sion. The latter is 0.26 for conversion of provitamin D2
to previtamin D2 at 0 oc both at 254 nm and 313 nm
(Havinga 1973), and the yield for provitamin D3 con-
version is of a similar magnitude (Pottier & Russell
1991 ).
The sterol composition of plant cell mem-
branes affects ATPase activity and proton pumping
(Grandmougin-Ferjani et al. 1997). and in model ex-
periments affect water permeability and ordering of
acyl chains (Schuler et al. 1991 ). In yeast the photo-
chemical removal of ergosterol causes a proportionate
reduction in ATPase activity and changes in the activi-
ties of other plasmamembrane-bound enzymes (Arami
et al. 1997a, b). Thus the effects of ultraviolet-B radi-
ation on photoconvertible sterols such as provitamins
D2 and D3 could form the start of a signal transduction
chain.
Photoinactivation of plant plasma membrane
ATPase has an action spectrum peaking at 290 nm
(Imbrie & Murphy 1982, 1984 ). However, when na-
tive lipid vesicles containing ATPase are investigated,
the action spectrum has much more of a tail towards
longer wavelengths than what is compatible with a
UV-B receptor, and direct UV action on ATPase is
therefore ruled out as a UV sensing reaction.
Infection of a plant with an endophytic fungus
(Cheplick & Clay 1988) increases the content of
ergosterol. If conversion of ergosterol is important
for reactions of plants to ultraviolet-B radiation, one
would presume that infected plants react to ultraviolet-
B radiation in a different way from uninfected plants.
In fact, Newsham et a!. ( 1998) found that in Lolium
perenne a certain treatment with ultraviolet-B de-
creased the number of spikes, the number of seeds,
and the seed weight by 70--75% , but only if the plants
were infected by the fungus Neophytium /olii.
Conclusion
Plants and some algae are able to perceive ultraviolet-
B radiation and regulate chemical processes and mor-
phogenesis in a radiation-dependent manner, and are
thought to have an ultraviolet-B specific photorecep-
tor. Provitamins D2 and D3 are present in leaves of
land plants and in some algae, and are converted, with
a high quantum yield, to previtamins D and vitamins
D upon exposure to ultraviolet-B radiation. The action
spectrum for several ultraviolet-induced phenomena in
plants (Bjorn 1999) and for provitamin D conversion
in human skin peak at the same wavelength. Pho-
todestruction of provitamin D is known to change the
activity of membrane-bound enzymes in yeast cells.
We have pointed to the possibility that provitamins D
act as ultraviolet-B photoreceptors for UV-B induced
regulatory reactions in plants, and proposed that the
proportions between provitamins, previtamins, and vi-
tamins D can be used to evaluate the UV-B exposure
of phytoplankton and plants.
Acknowledgements
We are grateful to the following persons at Lund Uni-
versity: Professor Anders Sodergren and Dr Goran

Bengtsson for letting us use the HPLC equipment at
the Dept. of Chemical Ecology, to Dr Olov Sterner for
advice and use of equipment at the Dept. of Organic
Chemistry 2, and to Prof. Wilhelm Graneli and Dr
Niclas Johansson for advice and help with growth of
planktonic algae in preliminary experiments. Thanks
are due also to Prof. Edna Gran eli and Dr Per Carlsson
at Kalmar University for growing planktonic algae and
to Dr Olof Hellgren and Mrs Eva Olsson for grow-
ing tomato plants at the Phytotron of the Agricultural
University of Sweden, Alnarp. Professor Michael F.
Holick kindly provided us with literature that was
difficult to obtain in Sweden.
The investigation was financed by Lund Univer-
sity, The Royal Swedish Academy of Sciences and
The Swedish Natural Science Research Council. It
was carried out as a complement to the UVAQTER
project sponsored by the European Commission (con-
tract ENV _CT97-0580).
References
Arami, S.. Hada, M. & Tada, M. 1997a. Nern·-UV-induced ab-
sorbance change and photochemical decomposition of ergosterol
in the plasma membrane of the yeast Saccharomvces cerevesiae.
Microbiology 143: 1665-1671.
Arami, S., Hada, M. & Tada. M. 1997b. Reduction of ATPase
activity accompanied by photodecomposition of ergosterol by
ncar-UV irradiation in plasma membranes prepared from Sac-
chammvces cerevesiae. Microbiology 143: 2465-2471.
Beggs, C. J. & Wellmann, E. 1994. Photocontrol of flavonoid
biosynthesis. pp. 733-751. In: Kendrick. R. E. & Kronenberg,
G. H. M. (eds), Photomorphogenesis in Plants, 2nd cd. Kluwer
Academic Publishers, Dordrecht.
Bjorn, L. 0. 1999. UV-B effects: Receptors and targets. pp. 793-
803. In: Singhal, G. S. et a!. (eds), Concepts of Photobiology.
Narosa Publishing House, New Delhi.
Buchala, A. J. & Schmid, A. 1979. Vitamin D and its analogues
as a new class of plant growth substances affecting rhizogenesis.
Nature 2XO: 230-231.
Buchala, A. J. & Pythoud, F. 1988. Vitamin D and related com-
pounds as plant growth substances. Physiol. Plant. 74: 391-396.
Buddecke, E. 1980. Grundriss der Biochemic (6. Auf!.). W. De
Gruyter, Berlin.
Buffenstein, R., Skinner, D. C., Yahav, S. D., Moodley, G. P., Cav-
aleros, M., Zachcn, D., Ross, F. P. & Pettifor, J. M. 1991. Erfect
of oral cholecalciferol supplementation at physiological and sup-
raphysiological doses in naturally vitamin D3 deficient subter-
ranean damara mole rats (Cryptmnys damarensis) . .T. Endocrinol.
131: 197-202.
Cheplick, G. P. & Clay, K. 1988. Acquired chemical defenses of
grasses: the role of fungal endophytes. Oikos 52: 309-318.
Clay, K. 1990. Fungal endophytes of grasses. Annu. Rev. Ecol. Syst.
21: 275-297.
Curino, A., Skliar, M. & Boland, R. 1998. Identification of
7-dehydrocholesterol, vitamin D3, 25(0H)-vitamin D3 and
l ,25(0H)2-vitamin D3 in Solanum glaucophyllum cultures
7
grown in absence of light. Biochim. Biophys. Acta 1425: 485-
492.
Feldman, D., Glorieux, F. H. & Pike, J. W. (eds) 1997. Vitamin D.
Academic Press, New York.
Fries. I"· 1984. D-vitamins and their precursors as growth regulators
in axenically cultivated marine macroalgae. J. Phycol. 20: 62-66.
Galkin. 0. N. & Terenetskaya. T.P. 1999. 'Vitamin D' hiodosimeter:
basic characteristics and potential applications. J. Photochem.
Photobiol. B: Bioi. 53: 12-19.
Gershengorn, M. C.. Smith, A. R. H., Goulston, G., Goad.
L. J., Goodwon, T. W. & Haines, T. H. 1968. The sterols of
Orhromonas danica and Ochromonas mu/hamensis. Biochem-
istry 7: 1698-1706.
Gessner, M. 0. & Schmitt, A. J. 1996. Use of solid-phase extraction
to determine ergosterol concentrations in plant tissue coloniLed
by fungi. Appl. Environ. Microbial. 62: 415-419.
Grandmougin-Perjani, A., Schuler-Muller, I. & Hartmann, M.-A.
1997. Sterol modulation of the plasma membrane H+·ATPasc
activity from corn roots reconstituted into soybean lipids. Plant
Physiol. 113: 163-174.
Hannach, G. & Sigleo, A. C. 1998. Photoinduction oi'UV-absorbing
compounds in six species of marine phytoplankton. Mar. Ecol.
Progr. Ser. 174: 207-222.
Havinga, E. 1973. Vitamin D. example and challenge. Experientia
29: 1181-1193.
Hess, A. F. & Weinstock, M. 1924. Antirachitic properties imparted
to inert fluids and green vegetables by ultraviolet radiation. J.
Bioi. Chern. 62:301-313.
Holick. M. F. 1989. Phylogenetic and evolutionary aspects of vi-
tamin D from phytoplankton to humans. pp. 7-43. In: Pang.
P. K. T. & Schreibman, M. P. (eds), Vertebrate Endocrinol-
ogy: Fundamentals and Biomedical Implications, volume 3.
Regulation of Calcium and Phosphate. Academic Pre". New
York.
Holick, M. F (ed.) 1999. Vitamin D: Molecular Biology, Physiol-
ogy, and Clinical Applications (Nutrition and Health). Humana
Press, Totowa, NJ 07512.
Horst R. L., Reinhardt T. A., Russell J. R. & Napoli J. L. 1984. The
isolation of vitamin D2 and vitamin D3 from Medicafio satim
(alfalfa plant). Arch. Biochem. Biophys. 231: 67-71.
Hsiao. K. C. & Bjorn, L. 0 1982. Aspects of photoinduction and
carotenogencsis in the fungus Vertici/lium aguricinum. Physiolo-
gia Plantarum 54: 235-238.
Imbrie. C. W. & Murphy. T. M. 1982. UV-action spectrum (254-405
nm) for inhibition of a K 1 -stimulated adenosine triphosphatase
from the plasma membrane of Rosa damascena. Photochem.
Photobiol. 36: 537-542.
Imbrie, C. W. & Murphy, T. M. 1984. Photoinactivation of
detergent-solubilized plasma membrane ATPase from Ro.m dam-
ascelw. Plant Physiol. 74: 617-621.
Jarvis, B. C. & Booth, A. 1981. Influence of indole-butyric acid,
boron. mvo -inositol, vitamin Do and seedling age on adventi-
tious root developmant in cutting: of Phoseolus aureus. Physiol.
Plant. 53: 213-218.
MacLaughlin, J. A .. Anderson, R. R. & Holick, M. F. 1982. Spectral
character of sunlight modulates photosynthesis of previtamin D.1
and its photoisomers in human skin. Science 216: 1001-1003.
Mellanby. E. 1918. The part played by an 'accessory factor' in the
production of experimental rickets. J. Physiol. (London) 52: 11-
14.
Moncousin, C. & Gaspar, T. 19X3. Peroxidase as a marker for
rooting improvement of Cynura scolymus L. cultured in vhro.
Biuchem. Physiol. Pflanzen 178: 263-271.
8
Napoli J. L.. Reeve L. E.. Eisman J. A .. Schnoes, H. K. &
DeLuca, H. F 1977. Solanum glaucophvllum as source of
1,25-dihydroxyvitamin D3. J. Bioi. Chern. 252:2580-2583.
Neva, E. 1995. Mammalian evolution underground. The ecological-
genetic-phenetic interfaces. Acta Theriologica. Suppl. 3: 9-31.
Newsham, K. K., Lewis, G. C., Grcenshade, P. D. & McLeod, A. R.
1998. Neotvphodium lolii . a fungal endophyte. reduces the fertil-
ity of Lolium perenne exposed to elevated UV-B radiation. Ann.
Bot. 81: 397-403.
Norman, T. C., Norman, A. W. 1993. Consideration of chemical
mechanisms for the non photochemical production of vitamin D3
in biological systems. Bioorganic Med. Chern. Lett. 3: 1785-
1788.
Patterson, G. W. 1971. The distribution of sterols in algae. Lipids 6:
120-127.
Patterson. G. W. 1974. Sterols of some green algae. Comparative
Biochem. Physiol. 47B: 453-457.
Pfoerter. K. & Weber. J. P. 1972. Photochemie dcr Vitamin D-Reihe.
I. Kinetik und Quantenausbeuten der Ergosterinbestrahlung bei
L=253,4 nm. Helvetica Chimica Acta 55:921-937.
Pitcher. T. & Buffenstein. R. 1995. Intestinal calcium transport in
mole-rats (Cryptomys damarensis and Heterocephalus glaber)
is independent of both genomic and non-genomic vitamin D
mediation. Exp. Physiol. 80: 597-608.
Pitcher. T., Butlcnstein, R .. Keegan, J.D., Moodley, G. P. & Yahav,
S. 1992. Dietary calcium content, calcium balance and mode
of uptake in a subterranean mammal, the damara mole-rat. J.
Nutrition 122: 108-114.
Pitcher, T., Sergeev, I. N. & Buffenstein, R. 1994. Vitamin D
metabolism in the damara mole-rat is altered by exposure to sun-
light, yet mineral metabolism is unaffected. J. Endocrinolol. 143:
367-374.
Pitcher, T., Pettifor, J. M. & Buffenstein. R. 1994. The effect of
dietary calcium content and oral vitamin D3 wpplementation
on mineral homeostasis in a subterranean mole-rat, Cryptom.vs
damarensis. Bone Mineral. 27: 145-157.
Portwich, A. & Garcia-Piche], F. 2000. A novel prokaryotic
UVB photoreceptor in the cyanobacterium Chlorogloeopsis PCC
6912. Photochcm. Photobiol. 71: 493-498.
Pottier, R. H. & Russell, D. A. 1991. Quantum yield of a photo-
chemical reaction. pp. 45-57. In: Valenzano, D.P., Pottier, R. H.,
Mathis. P., Douglas, R. H. (eds) Photobiological Techniques.
Plenum Press. New York. pp. xiv+381.
Prema, T. P. & Raghuramulu, N. 1994. Free vitamin D3 metabolites
in Cestrum diurnumleaves. Phytochemistry 37: 677-681.
Prema, T. P. & Raghuramulu. N. 1996. Vitamin D3 and its metabo-
lites in the tomato plant. Phytochemistry 42:617-620.
Rambeck W. A., Kreutzberg 0., Bruns-Droste C. & Zucker,
H. 1981. Vitamin D-likc activity of Triselum .flavescens. Z.
Pflanzcnphysiol. I 04: 9-16.
Redlin. S. C. & Carris, L. M. (eds) 1996. Endophytic fungi in
grasses and woody plants: Systematics, ecology and evolution.
The American Phytopathological Association (APS Press), St.
Paul, Minnesota.
Schuler, I., Milon, A.. Nakatani, Y.. Ourisson. G., Albrecht, A.-M ..
Beneviste, P. & Hartmann, M.-A. 1991. Differential effects of
plant sterols on water permeability and on acyl chain ordering
of soybean phosphatidylcholine bilayers. Proc. Nat. Acad. Sci.
USA 88: 6926-6930.
Siegel, M. C., Latch, G. C.M. & Johnson, M. C. 1987. Fungal
endophytes of grasses. Annu. Rev. Phytopathol. 25: 293-315.
Steenbock, H. & Black, A. 1924. The induction of growth-
promoting and calcifying properties in a ration by exposure to
ultra-violet light. J. Bioi. Chern. 61: 405-422.
Stern, A. I., Schiff, J. A. & Klein, H. P. 1960. Isolation of ergos-
terol from Euglena gracilis; distribution among mutant strains.
J. Protozoal. 7: 52-55.
Sugisaki, N .. Welcher, M. & Mander. C. 1974. Lack of vitamin D3
synthesis by goldfish (Carassius auratus L.). Comp. Biochem.
Physiol. 49B: 647-653.
Sun ita Rao, D. & Raghurarnulu, N. 1996a. Food chain as origin of
vitamin Din fish. Camp. Biochem. Physiol. 114A: 15-19.
Sunita Rao, D. & Raghuramulu, N. 1996b. Lack of vitamin D3
synthesis in Tilapia rnossamhica from cholesterol and acetate.
Comp. Biochem. Physiol. 114A: 21-25.
Wasserman R. H. 1975. Vitamin D-like substances in Solanum
malacoxylon and other calcinogenic plants. Nutr. Rev. 33: 1-5.
Wasserman, R. H., Henion, J. D .. Haussler, M. R. & McCain,
T. A. 1976. Calcinogenic factor in Solanum malacoxvlon: Ev-
idence that it is I ,25-dihydroxyvitamin D1-glycoside. Science
194: 853-855.
Webb, A. R., Kline, L. & Holick, M. F 1988. Influence of sea-
son and latitude on on the cutaneous synthesis of vitamin D3:
Exposure to winter sunlight in Boston and Edmonton will not
promote vitamin D3 synthesis in human skin. J. Clin. Endocrinol.
Metabolism 67: 373-378.
Zucker, H., Stark, H. & Rambeck, W. 1980. Light-dependeut
synthesis of cholecalciferol in a green plant. Nature 283: 68-69.
 Section 1: General

Antarctic hairgrass <Deschampsia 1111tarcrica) at Leonie Island, maritime Antarctic,

Pollen of Antarctic plants may enable reconstruction of historic UV-B levels.
(Photograph by Jelte Rozema)
 Plant Ecologv 154: 11-26, 200 I.
II
© 2001 Khm·er Academic Publishers.

(Poly )phenolic compounds in pollen and spores of Antarctic plants as

indicators of solar UV-B
A new proxy for the reconstruction of past solar UV-B?

J. Rozema, A. J. Noordijk, R. A. Broekman, A. van Beem, B. M. Meijkamp, N. V. J. de Bakker,

J. W. M. van de Staaij, M. Stroetenga, S. J.P. Bohncke 1, M. Konert 1, S. Kars 1, H. Peat2 ,
R. I. L. Smith 2 & P. Convey 2
Department of Systems Ecology, Free University, 1Department of Quaternary Geology and Geomorpholog_v, De
Boelelaan 1087, 1081 HV Amsterdam, The Netherlands; 2 British Antarctic Survey, Natural Environment Research
Council, Cambridge CB3 OET. UK (E-mail: jrozema@bio. vu.nl)

Key words: Antarctic, Bio-indicator, Colobanthus quitensis, Deschampsia antarctica, Historic UV-B radiation,
Pollen, Polyphenolics, Proxy, Sporopollenin, Stratospheric ozone depletion

Abstract
The morphology, size and characteristics of the pollen of the plant species Antarctic hairgrass (Deschampsia
antarctica, Poaceae) and Antarctic pearl wort ( Colobanthus quitensis, Caryophyllaceae) are described by scanning
electron microscopy and light microscopy. Based on the number of pores the pollen of Colobanthus quitensis is
classified as periporate or polypantorate, while that of Deschampsia antarctica is monoporate.
Pollen of Viciafaba plants, exposed to enhanced UV-B (I 0.6 kJ m- 2 day- 1 UY-BsE) in a greenhouse. showed
an increased content of UV-B absorbing compounds. There was also an increase of UV-B absorbing compounds
in response to exposure to UY-A. By sequential chemical extraction three 'compartments' of UV-B absorbance of
pollen can be distinguished: a cytoplasmic fraction consisting of, e.g., tlavonoids (acid-methanol extractable), a
wall-bound fraction, consisting of, e.g., ferulic acid (NaOH extractable) and aromatic groups in the bioresistant
polymer sporopollenin possibly consisting of, e.g., para-coumaric acid monomers (fraction remaining after ace-
tolysis). The sporopollenin fraction in the pollen of Helleborus Joetidus showed considerable UV-B absorbance
(280-320 nm). There is evidence that enhanced solar UV-B induces increased UV-B absorbance (of sporopollenin)
in pollen and spores of mosses, which may be preserved in the fossil record. As there are no instrumental records
of solar UV-B in the Antarctic before 1970 and no instrumental records of stratospheric ozone over the Antarctic
before 1957, the use of UV-B absorbing polyphenolics in pollen (and spores) as bio-indicator, or proxy of solar
UV-B, may allow reconstruction of pre-ozone hole and subrecent UV-B and stratospheric ozone levels. Pollen and
spores from herbarium specimens and from frozen moss banks (about 5000-10 000 years old) in the Antarctic may,
therefore, represent a valuable archive of historical UV-B levels.

The stratospheric ozone hole over the Antarctic
Stratospheric ozone depletion was first reported only
about two decades ago. Farman and colleagues were
the first to report a 'hole' in the stratospheric ozone
layer occurring over the Antarctic each spring since
1979, see Figure I (Farman et al. 1985). Since the
paper by Molina & Rowland (1974) suggested that
anthropogenic chlorine compounds could disturb the
stratospheric ozone layer, it was rapidly proposed
that the 'ozone hole' could be 'man-made' and that
consequential increases in solar UY-B were a threat
to all life on earth. It was soon determined that
chlorine, derived from emitted CFC's, but also ni-
trous oxide (Johnston 1971 ). methane and carbon
monoxide, could be held responsible for the antarc-
tic stratospheric ozone destruction. With the Mon-
treal Protocol ( 1987) and its later amendments the
12
400 stratospheric ozone depletion
D
350
D D
"'"
:::0
300
c
_2 250
0
Cl
v occurring through natural causes implying for exam-
"0~ 200
u
] 150
0.
~
'@ 100
ii)
50
1900 1 910 1 920 1930 1940 1950 1960 1 970 1980 I 990 2000
Figure I. Measurements of the thickness of the stratospheric
oLOne layer at the Antarctic (Dobson Units) since 1957. The
data refer to springtime (October) average total stratopheric ozone
values in the latitude range 63°-90° for the southern hemi-
sphere (Newsham eta!. 1997). Systematic measurements of solar
UV-B at the Antarctic started in 1970. Modified after SORG
1999. For more data of stratospheric ozone and solar UV-B at
the Antarctic see the web site of the British Antarctic Survey.
http://www. n bs.ac. uk/pub Iic/icd/jds/ozone/
emission of CFC's was banned. The thinning of the
stratospheric ozone layer has been predicted to con-
tinue at least until the year 2000, when loading of
chlorine in the stratosphere will peak (Hofmann 1996;
Hofmann et al. 1997; Madronich et al. 1992, 1993,
1995, 1998; Stolarski et a!. 1992). A gradual recovery
is then predicted, but according to recent authorita-
tive assessments (e.g., UNEP 1998; SORG 1999),
stratospheric ozone depletion has continued and as
yet there are no signs of recovery of the stratospheric
ozone layer. A damaged stratospheric ozone layer and
enhanced solar UV-B radiation on earth may remain
for at least another 50 years (Madronich et a!. 1995).
Warming of the lower stratosphere by the green-
house effect may relate to a cooler upper stratosphere
and lead to a more intensified destruction of
stratospheric ozone (Shindell et a!. 1998)
The need and possibilities for the reconstruction of
past solar UV-B
Measurements of Antarctic stratospheric ozone started
in 1957 and recording of solar UV-B in 1970 (SORG
1999). Over this period springtime stratospheric ozone
measurements in the Antarctic have shown a sustained
decrease since around 1980 (Figure 1). Since no sys-
tematic instrumental measurements of stratospheric
ozone and solar UV-were made before these dates, any
previous variations in the history of the stratospheric
ozone layer are unknown. It is very likely that anthro-
pogenic CFC's have led to current ozone depletion.
There is, however, no information available on the
possibility or frequency of previous analogous events
ple that current reduction of stratospheric zone forms
part of a natural cycle.
Coinciding with the current stratospheric ozone
depletion, there is a continuous gradual increase of
atmospheric carbon dioxide levels, related to global
warming. Together these are included in global
change. In contrast with depletion of stratospheric
ozone and enhanced solar UV-B, knowledge of past
atmospheric C02 and temperature levels is consider-
able. Based on palynological and palaeo-ecological
studies past climates have been reconstructed. Re-
construction of historic C02 levels and related global
temperatures have been possible, via both direct mea-
surements (such as trapped air bubbles in ice cores,
and historical records of atmospheric C02), and based
on indirect evidence from the fossil record (such as
the occurrence of vegetation types -based on palyno-
logical analyses- indicative of a particular climate: the
stomatal index which is correlated with atmospheric
C02; and isotopic analyses such as of 8 18 0 related
to temperature variation. In the latter case there is
differential fractionation between 18 0 and 16 0 during
evaporation and condensation processes (Levesque &
Cwynar 1994; Wagner et a!. 1999; !sarin & Bohncke
1999).
Solar UV-B radiation penetrating to the earth is a
function of the thickness of the stratospheric ozone
layer, which is in turn, among other environmental
factors, a function of (biogenic), atmospheric oxygen
(Berkner & Marshall 1965 ).
In comparison with atmospheric C0 2 , reconstruc-
tion of historic stratospheric ozone and solar UV-B
fluxes is obstructed by several factors, including the
absence of historic (direct) records of atmospheric
oxygen and solar UV-B and difficulties involved in
(indirect) reconstruction of past atmospheric oxygen.
Except Markham et al. (1990), who found that
there was an increase of both UV-B absorbance and
relative flavonoid levels in Bryum argenteum with
antarctic stratospheric ozone levels (Dobson units) de-
creasing from 1971-1980, we are unaware of any
direct or indirect attempt to reconstruct past solar
UV-B. Only indirectly or theoretically past levels of
solar UV-B are considered in studies reconstructing
the early atmosphere, the evolution of the biosphere or

the possibilities of extraplanetary life (Cocke!! 1998,
1999, 2000; Garcia-Piche! 1998; Kasting et al. 1989,
1993; Pierson et al. 1993; Towe 1996).
There is some indirect evidence of the devel-
opment and variation of atmospheric oxygen and
the stratospheric ozone layer during the evolution of
plant life on earth. It is generally accepted that at-
mospheric oxygen has gradually (or abruptly, or with
fluctuations) increased, and that the development of
stratospheric ozone followed proportionally (Berkner
& Marshall 1965; Margulis et al. 1976; Budyko et al.
1987; Rozema et al. 1997, 1999).
Deposition of iron oxide (Fe203) in Red Beds,
dated 2 billion years ago indicated aerobic terrestrial
weathering, and 02 at that time is estimated to have
been 0.001% of the present level. Berkner & Marshall
( 1965) indicate that a stratospheric ozone layer could
only have developed when the atmospheric oxygen
content exceeded 1% level of the present level, about
600 million years ago. For plant life to develop on
land, an oxygen content of I 0% of the present level
was required. This level may have been reached before
the appearance of the first land plants at the begin-
ning of the Silurian, about 430 million years ago. The
present-day level of 21% atmospheric oxygen may
have been reached 350 million years ago. Such evo-
lutionary reconstructions of atmospheric oxygen are
based on assumptions and calculations. rather than
on empirical data. There is no precise information
on past stratospheric ozone and solar UV-B radia-
tion available, leaving only assumptions to exist for
the period before the development of the stratospheric
ozone hole over the Antarctic during the last 30-40
years. We now experience values of 15-30 (up to
60% at the Antarctic, occasionally 30% at the Arc-
tic) stratospheric ozone depletion, during the antarctic
spring, but we do not know whether and when such
ozone depletion has occurred before. Did stratospheric
ozone depletion and recovery occur in the 19th or
20th century or in subrecent times? In other words is
stratospheric ozone depletion a young phenomenon or
did it occur as part of a natural cyclisity during the
holocene record?
The primary aim of this paper is to consider possi-
bilities of reconstruction of past levels of stratospheric
ozone and solar UV-B over historical and subrecent
timescales. We propose linking the observation that
solar UV-B induces UV-B absorbing compounds, in-
cluding pollen and spores, with examination of such
material which may be preserved in the fossil record.
13
Induction of (poly )phenolics in plants by enhanced
UV-B
Solar UV-B radiation may affect plant life directly
or indirectly. There may be some direct effects of
enhanced solar UV-B on photosynthesis and plant pri-
mary production (Caldwell et al. 1998; Hader 1997;
Rozema 1999; Sullivan & Rozema 1999). Some
of the indirect effects relate to physiological and
ecological functions of various protective absorbing
(poly)phenolics, whose production is induced by UV-
B. in addition to other environmental factors. These
form part of the phenyl propanoid pathway (Beggs
& Wellmann 1994; Kubitzki et al. 1987; Rozema
et al. 1999; Meijkamp et al. 1999; Bjorn 1999). So-
lar ultraviolet-B radiation is known to stimulate the
synthesis of the enzymes phenyl alanine ammonia
lyase (PAL) and chalcone synthase (CHS) and other
branch-point enzymes of the phenylpropanoid path-
way (Beggs & Wellmann 1994; Gitz et al. 1997;
Rozema et al. 1999; Meijkamp et al. 1999). PAL cat-
alyzes the transformation of phenylalanine into tran-
scinnamic acid, which is a central intermediate in the
formation of complex phenolic compounds such as
flavonoids, condensed tannins and lignin (Meijkamp
et al. 1999). Polyphenolic UV-B screens such as
flavonoids, are localised in epidermal plant tissue (Day
1993; Hutzler et al. 1998; Karabourniotis et al. 1998;
Schnitzler et al. 1997; Manetas 1999; Van de Staaij
et al. 1995; Stephanou & Man etas 1997). Also, plant
cuticles may attenuate UV-B radiation (Kraus et al.
1997; Martin & Juniper 1970). A further characteris-
tic of these UV-B induced polyphenolics is that they
are not easily degraded. A UV-B induced increase
of lignin in plant litter caused a reduced rate of lit-
ter decomposition (Rozema et al. 1997). A lignin
like compound in plant pollen and spores is sporopol-
lenin, consisting of an aliphatic chain with aromatic
groups (Shaw 1971; Van Bergen et al. 1995). The
monomer para-coumaric acid, a phenolic acid, forms
part of these aromatic groups (Wehling et al. 1989).
Sporopollenin is a major component of pollen grains
remaining after fossilization. In addition to sporopol-
lenin other biomacromolecules remain in the fossil
record such as lignin, cutan. tannins, suberans and
resins. The latter form part of resistant plant tissues
such as cuticles, seed coats, fruit walls and periderm
(Van Bergen et al. 1995; Hemsley et al. 1996).
We hypothesize that enhanced solar UV-B ex-
posure induces UV-B absorbing compounds (in this
14
paper generalized as: polyphenolics) in pollen (higher
plants) and spores (mosses and other lower plants).
Therefore, the second aim of this paper is to assess
whether polyphenolics in pollen and spores, preserved
in the fossil record, can be used as bio-indicators
(proxies, transfer-functions (Isarin & Bohncke 1999)
of past UV- B levels and thus allow a reconstruction of
the palaeo UV-B climate.
In this context, we exposed the crop Vicia faba and
the antarctic native plant species Deschampsia antarc-
tica and Colobanthus quitensis to enhanced UV-B (see
also Rozema et a!. 2001). In addition to growth and
physiological analysis of Vi cia faba (Meijkamp et al.
200 I) and Deschampsia antarctica and Colobanthus
quitensis (Rozema et a!. 2001), we collected pollen
produced in the greenhouse. In case of Vicia faha
the amount of pollen production of UV-B irradiated
plants was sufficient for some chemical analysis of
UV-B absorbing compounds. However, in Deschamp-
sia antarctica and Colobanthus quitensis the small
amount (and biomass) of collected pollen grains ob-
tained allowed only light microscopical and scanning
electron microscopical study of the pollen. We also
collected the pollen from the abundantly !lowering
Hellehorus foetidus in the botanical garden of the
Vrije Universiteit, Amsterdam, which was sufficient
for sequential extraction of UV-B absorbing com-
pounds. With sequential extraction of UV-B absorbing
compounds we aim at quantifying soluble, wall-bound
and sporopolleninous UV-B absorbing compounds in
pollen grains. The third aim of this paper is to eval-
uate in practice whether and how past solar UV-B
levels and stratospheric ozone can be effectively re-
constructed in the antarctic region, where since 1979
an ozone hole has occurred in the antarctic spring pe-
riod. For this purpose we propose a practical research
strategy.
This paper therefore has a threefold nature: (I) we
review literature on UV-B induced polyphcnolics in
pollen and spores, (2) we present morphological char-
acteristics of the pollen of two antarctic higher plants,
and (3) we present measurements on the UV-B ab-
sorbance of pollen and spores and discuss the possi-
bility of using this to reconstruct past UV-B levels.
Material and methods
Collection and cultivation ofDeschampsia antarctica
and Colobanthus quitensis
About twenty tufts each of Deschampsia antarctica
and Colobanthus quitensis with adhering soil were
collected during the antarctic summer period at the
end of January 1999 at Leonie Island (see Rozema
et a!. 200 I). Two days later the tufts were stored in
a climate room (4 °C, relative humidity 75%), PAR
ISO {-Lmol m- 2 s- 1, in Amsterdam, The Netherlands.
Plants were further cultivated in a climate room using
a diurnal cycle of 12 °C, at 300 {-Lmol PAR m- 2 s- 1
from 06:00-20:00 and 4 oc from 20:00 to 6:00. An-
thers from flowering plants were removed and stored
in petri dishes for light microscopy and scanning
electron microscopy analysis.
Cultivation ofVicia faba and UV-B treatment
Nine days after germination of pods, seedlings of
Vicia faba cultivar Minica were grown in a clima-
tized greenhouse (day: 20 °C, Relative Humidity 72%;
burning period PAR lamps (300 f-LillOI PAR m- 2 s- 1)
6:00-20:00 h; night: I 5 °C, 81% R.H.) in 2.6 I
pots filled with garden soil (Jongkind, Aalsmeer,
NL) enriched with 7.8 gram osmocote (controlled re-
lease fertilizer, Grace Sierra, Heerlen, NL). They
were exposed to three radiation regimes throughout
their growing and flowering period (n = 6 plants per
treatment): (a) 0 UV-B/0 UV-A (PAR), (b) 0 UV-
B/+UV-A (UV-A), (c) +UV-A/+UV-B (UV-B). Un-
der all three radiation treatments plants were irradiated
with 300 {-Lmol m- 2 s- 2 PAR (supplied by Philips
400 Watt HPI/T lamps). PAR radiation was measured
with a Li-185b quantum sensor (Li-Cor Inc., Lincoln,
NE, USA). Above UV-A ( +UV-A) irradiated plants,
Philips fluorescent tubes were installed, wrapped in
Mylar foil, absorbing UV-B, but transmitting UV-A.
Fluorescent tubes above UV-B ( +UV-B ) irradiated
plants were wrapped in cellulose acetate foil, transmit-
ting UV-B and UV-A radiation. Plants under the UV-B
treatment received a daily dose of 10.6 kJ m- 2 day- 1
UV-BsE (sec also Meijkamp ct a!. 2001 ). Non-burning
fluorescent tubes were placed above the PAR irra-
diated plants. Mylar foil was replaced weekly and
cellulose acetate foil twice a week to avoid degrada-
tion effects. Plants were watered twice a week. There
were six Vi cia plants per radiation treatment. After 24
days the Vicia plants started to flower, which continued
 15

for about six weeks. The corolla of Vicia faba flow-

ers covers the anthers and stigma, so that the anthers
themselves are not directly exposed to enhanced UV-
B, assuming that the corolla absorbs most of the inci-
dent UV-B (Flint & Caldwell 1983, 1984). Thecas of
Vicia flowers, that started to release pollen grain were
collected with a pair of tweezers, and stored in petri-
dishes at room temperature. Collection of pollen was
continued throughout the six week flowering period,
during which new flowers were formed .

Scanning electron microscopy

The pollen grains were mounted using double sided

adhesive tape and coated with gold for about I 0 min.
Scanning electron microscopy analysis was performed
with a Jeol JSM Scanning microscope with a Sputter
Coating Bio Rad E5005 fac ility, according to methods
described in Chapman ( 1986).

Light microscopy

Pollen material was embedded in glycerin and directly

viewed with the Zeiss Axioskop, with a Sony color
video camera model DXC-151AP and applied theRe-
search Assistant3 software package RVS (Ruijs Visual
Computing BY, Maarsen, NL). Prints were made with
an Epson Stylus Photo-printer (Type 700).
Figure 2. A-B. Scanning electron micrograph ofmonoporate pollen

Sequential extraction of (~f UV-8 absorbing

compounds in pollen grains
For UV-B absorbance analysis pollen grains of Vicia
faba and Helleborusfoetidus, were collected from the
petri-dishes with a pencil and weighed with a Sarto-
rius MP2 microbalance with a standard deviation of
0.00 I mg (I /kg). The mass of pollen weighed per sam-
ple ranged from 50- I 50 !kg for Vi cia faba and from
400- 500 !kg for Helleborus foe tidus. Because insuf-
ficient pollen grains of Vicia faba were available for
sequential extraction, pollen were collected from the
abundantly flowerin g Helleborus foetidus . Ripe an-
thers from ten flowering plants of Helleborusfoetidus
were collected in the botanical garden of the Vrije
Universiteit on March 23, 2000. These were air dried
in petridishes before approximately 500 /kg of Helle-
horus pollen grains per sample was used for sequential
extraction of of UV-B absorbing compounds in pollen
grains.
or Deschampsia aman:tica ( lA), and or periporate (Faeg ri &
Iversen 1964) or polypantoporate pollen (Moore & Webb 1978;
Moore et al. 199 1) Co!obanthus quiten;'is (B). cultivated in a climate
room with 250 !' lllOlm - 2 s- l PAR.
(a) Soluble, cytoplasm ic frac tion (Acid methanol
extracrion)
UV-B absorbing pigments were extracted from pollen
of Vicia faba with a MeOH:HzO:HCI (79:20: I v/v)
solution. Samples (50- I 50 !kg pollen of Vi cia faba ,
400- 500 Jkg of Helleborus jiJetidus) in I ml solu-
tion were heated in a waterbath (90 °C) for 2 hours.
The absorbance, integrated from 280- 320 nm was
measured using a Perkin-Elmer Lambda 15 UV!VlS
spectrophotometer (cf. Visser et al. 1997 ; Rozema
et al. 200 1; and Meijkamp et al. 2001). The solution
was then centrifuged at 3500 rpm ( 15') and the super-
natant was carefully removed, the pellet was air-dried
and used for the NaOH extraction
(b) Wa/1-bound.fraction ( NaOH extraction)
I ml 2 M NaOH was added to the pellet of (a), in a
tube . The tubes were shaken on a tubcshaker, sonifi-
16

cated (5') and after I hour at room temperature, and

occasional intermittent shaking, centrifugated (15',
3500 rev min- 1). 220 111 concentrated HCl was added
to the supernatant to set the pH at l.O. The UV-B
•
absorbance of the supernatant was measured for the
280-320 nm band as described for (a). The remaining
pellet was air dried and used for acetolylis extraction.

(c) Sporopolleninfraction (Acetolysis extraction)

One ml of an acetolysis mixture ( I v/v concentrated
( > 95%) H2S04 : 9 acetic acid anhydrid) was added to
the pellet of (b). The suspension was kept in a boiling
water bath for 1 h and occasionally stirred. The ace-
tolysis residue was centrifuged at 3500 rpm (15') and
washed twice in acetic acid and three times in distilled
water. The dark brown residue (mainly consisting of
sporopollenin) was suspended in glycerol (Xiong et al.
1997). The absorbance of the suspension was mea-
sured from 280- 320 nm and integrated as described
for (a) and (b).
25 mu
Statistical analysis

The data were statistically analysed by one way analy-

sis of variance using Systat 5.1 (Sokal & Rohlf 1995).
Homogeneity of variance was confirmed with the
Bartlett test for homogeneity of group variances. Sig-
nificantly different means (indicated by different let-
ters on figures) were detected with the Tukey HSD
multiple comparison test.

Results

Pollen morphology

The pollen grains of the antarctic grass Deschamp-

sia antarctica are circular and monoporate with
irregularly-spaced grain-like, microverrucatc surface
structures (Figures 2 and 3). Their diameter of the
pollen grains of Deschampsia antarctica is about 25-
30 f.lm . The pore is surrounded by an annulus and
has an operculum. The pollen surface of Deschamp-
sia antarctica is somewhat deflated, which may he
an artefact due to the vacuum to which the pollen 35 um
was exposed during preparation for scanning elec-
tron microscopy. Such shrinking may be avoided by
Figure 3. A-B. Light microscopical images of the pollen of of De-
application of critical point dry ing method.
schampsia antarctica (A) and Colobanthus quiiensis (B), cultivated
The pollen of Colobanthus quitensis has a sur- in a climate room with 300 1-1-mol m- 2 s- 1 PAR.
face with more or less evenly distributed thorn like
structures (spinules). Grain diameter varied between
 17

25-35 tJm. The 25-30 pores are present per pollen

grain, in depressions on the pollen surface and cov-
ered by a granular operculum. In addition, there are
itTegularly spaced small punctures on the surface.
Based on the large number of pores, the pollen grains
of Colobanthus quitensis are classified as periporate
(Faegri & Iversen I 964) or polypantoporate (Moore &
Webb 1978; Moore eta!. I 998).
In contrast with the scanning electron microscopy
images, the content of the pollen grains content tended
to protrude somewhat with light microscopy analysis.
The pollen of Vicia faha have a bean-like shape
with three pores, arranged along the groove of the
colpe (Figures 4 and 5). The pollen surface is covered
with irregularly shaped elevations and depressions.
Grain length varied from 45-50 f.l.lll, and the width
from 24-27 tJm. Both under scanning and light mi-
croscopical images, the pollen content protruded. In
the light microscopical pictures of the pollen of De-
schampsia, Colobanthus and Vicia there appear to be
no obvious differences in the light transparency.

UV-B absorbance in pollen

Vicia faba showed no clear indication that enhanced

UV-B influenced the light transparency and thickness
of the pollen grain walls (Figure 5).
The content of acid-methanol extractable UV-B
absorbing compounds in the pollen of Vicia f(zba in-
creased significantly under exposure to both enhanced
UV-8 radiation alone and under exposure to UV-A
radiation (Figure 6).
Sequential extraction of UV-B absorbing com-
pounds in the pollen of Helleborusfoetidus (Figure 7),
demonstrated that the absorbance (280- 320 nm) of the
sporopollenin fraction was the largest, with interme-
diate UV-B absorbance by the cytoplasmic fraction
(acid methanol extracted) and the wall-bound fraction
giving the least absorbance.

Discussion

We propose to use the studies described to mlttate

discussion of the possibility of using polyphenolics in
pollen and spores as indicators of contemporary levels
of incident solar UV-B, leading to the reconstruction Figure 4. A,R,C. Scanning electron micrographs of Vicia faba
of past UV-B levels based on the quantitative analysis pollen of plants cultivated in a climate room exposed to
of polyphenolics in sub-fossil pollen. 300 J.imol rn- " s- 1 PAR (A); PAR with UV-A , but no UV-B (B)
and PAR with UV-A and 10.6 kJ m- 2 day- 1 UV-B (C).
In doing so we will first justify the advantages of
focussing on plants from antarctic regions. We aim at
18
Vicia faba

''"" .... c
u

8.
40

..... "'E
~ 30
'E
0
=
~
.... ~
•
20

..
u
u
@
-e
~
~
10
:>"'::0
u
~
-~

~
0 10.6

PAR +UV-A/-UV-8 +UV-A/+UV-8

Figure 6. Relative amount of acid-methanol extractable UV-B ab-

sorbi ng compounds in pollen grains of Vicia .fc7ba exposed to
enhanced UV-B radiation ( 10.6 kJ m- 2 day- 1 UV-Bs El- The
absorbance of the extract was scanned from 280--320 nm and in-
tegrated. Mean values of three replications with standard error of
the mean. The difference hetween the UV-A and UV-B treatment
differed significantly (p :0 0.05 ) from the PAR treatment, hut there
was no significant different between the UV-A and UV-B treatment.

Sequential extraction UV-B absorbing compou nds

c 00
E
1?.
,...
~ tiO

j
] 40
~

"'
>
.,
:;)

r
20
E

sporopollenin
Figure 7. UV-B absorbance in the cytoplasmic, wall-bound and
sporopollen fraction of pollen of Helleborus f oetidus, obtained after
sequential extraction. The UV-B absorbance was measured spec-
trophotometrically and integrated from 280--320 nm and expressed
Figure 5. Light microscopical images of Vicia faba pollen of plants
on a per mg pollen basis. Mean values of three replications with
cultivated in a climate room exposed to 300 {tmol m- 2 s- 1 PAR
standard error of the mean.
(A, top); PAR with UV-A, but no UV-B (B, middle) and PAR with
UV-A and 10.6 kJ m-2 day- 1 UV-B (C, bouom).

ecies. Third, we need sensitive and specific analytical

developing a practical research strategy for the recon-
struction of past UV-B levels. To enable this, first, a
detailed description is required of the dose response
relationship between the solar UV-B absorbed and
polyphenolic content in pollen and spores. Second,
palynological and ecological research should be fo-
cussed on pollen and spores of identifiable plant sp
techniques to quantify polyphenolics at the level of
individual pollen and spores. Research to reconstruct
past UV-B should be preferably done in the Antarctic.
Experimental data presented in this paper show
that the pollen of the two resident Antarctic higher
plants Deschampsia antarctica and Colubanthus
quitensis, can be easily identified and that UV-B ab-

sorbing compounds in plant pollen are found in a
soluble cytoplasmic fraction, an insoluble wall-bound
fraction and in an insoluble sporopollenin fraction.
The latter fraction also gives the greatest propor-
tional contribution to absorbance, and is likely to be
preserved in sub-fossil pollen and spores, therefore
offering source material for the the reconstruction of
past UV-B levels. It is as yet uncertain if and to what
extent the NaOH and acetolysis treatment may cause
changes in the UV-absorption properties of the pollen
and spores. Comparison of the UV-B absorbances of
the three sequentially obtained extracts requires care,
since the absorbance of extracted polyphenols is pH
dependent and because the UV-B absorbance of the
acetolysis extract was measured in a glycerol suspen-
sion of the sporopollenin residue, which may show
high turbidity.
On the reconstruction of past solar UV levels:
advantage of research in the Antarctic
The depletion of stratospheric ozone and associated
increased solar UV-B in the Antarctic has occurred in
the austral spring every year since the first report of
the antarctic ozone hole in 1985 (Farman et al. 1985).
More recently, depletion of stratospheric ozone has
also been measured at lower and north polar latitudes,
and most recently (March 2000) the Arctic spring-
time ozone hole was of considerable extent. However,
this is generally much less marked than that of the
Antarctic. At lower latitudes away from the south
pole the detection of thinning of stratospheric ozone is
obstructed by a number of meteorological and climati-
cal factors causing 'natural' variation and fluctuations.
Monitoring programmes away from Antarctic have re-
vealed enormous variations of UV-B irradiance (Pyle
1997). The main justification for focusing studies on
antarctic sites is, therefore, that Antarctic ozone de-
pletion is both greater and more predictable than at
other latitudes. However, we also recognize that any
putative previous depletion may have followed a dif-
ferent geographical pattern from that observed today.
Research on historic levels of UV-B should initially
be confined mainly to Antarctic locations, roughly lo-
cated within the area of the south polar vortex, where
the contemporary occurrence of ozone depletion and
enhanced solar UV-B radiation is most marked. The
reconstruction protocol we propose will of course be
applicable to studies at any latitude at the northern an
southern hemisphere.
19
Pollen and spores
Pollen is very important element in plant ecology. Suc-
cessful reproduction depends amongst various factors
on the success of pollination. To protect the pollen
grain from biotic and abiotic damage it is encapsu-
lated by extremely resistant pollen grain walls, which
aid their preservations as fossils. These pollen walls
consist of an inner layer, the intine, and an outer
layer, the exine. The intine is a cellulose-pectin layer,
which is not preserved during fossilization and is also
removed by modem pollen preparation, such as ace-
tolysis (Traverse 1988). The exine in contrast is very
persistent, and this is what remains preserved in fos-
sil pollen. Its major component is sporopollenin, a
very resistant biopolymer (Jungfermann et al. 1997;
Meuter-Gerhards et al. 1999).
Pollen grains represent small plant structures con-
taining the haploid male gamete nuclei and are sur-
rounded by a double wall, the exine and the intine
(Paxson-Sowders et al. 1997). Pollen represents a vul-
nerable stage in the reproductive cycle of plants espe-
cially while the pollen grains are transported from the
male stamen to the female stigma. There, the pollen
tube may develop through pores in the pollen wall and
penetrate the stigma tissue. The pollen transport from
the anthers to the stigma is usually by wind or water,
but may also be via insects. To ensure its survival,
protection is needed against dehydration, herbivory,
microbial attack, physical and chemical breakdown,
and against radiation damage.
It is likely that the UV-B absorbing properties of
the sporopolleninous wall (p-coumaric acid monomer,
cf., Wehling et al. 1989) help to protect the (vulnera-
ble) pollen protoplasm from UV-B radiation damage,
through its ability to absorb UV-B (280-315 nm) (Fig-
ure 7). We hypothesize that acid methanol extracted
polyphenolic UV-B absorbing pigments (including
soluble ftavonoids), in the pollen protoplasm more
particularly, protect the growing pollen tube, since
this is not surrounded by a pollen wall. Spores of
many lower plants such as mosses, ferns and algae
contain compounds chemically similar to sporopol-
lenin (Xiong et al. 1997; Van Bergen et al. 1995;
Blokker et al. 1999). Like pollen, such spores are
often well preserved in the fossil record (Van Bergen
et al. 1995). For the evaluation of the use of UV-B
absorbing polyphenolics for the reconstruction of his-
toric levels of UV-B we therefore consider spores of
equal importance.
20
Dependent on the flower morphology and struc-
ture, pollen grains developing and ripening on the
parental plant may or may not be exposed to solar
radiation. The corolla of many flowers is opaque to
UV-B (Flint & Caldwell 1983). The corolla of Vi-
cia faba plants probably acts in this fashion as a UV
screen, preventing exposure of pollen. However, the
open structured flowers of Deschampsia antarctica
and Colobanthus quitensis are likely to lead to pollen
on the anthers being exposed to solar UV-B (Haltom
& Greene 1967).
UV-B induced polyphenolics in pollen and spores of
plants: bioindicators of past UV-B, a new proxy for
reconstruction of solar UV-B?
We propose the use of UV-B induced polyphenolics
in pollen and spores of plants for the reconstruction of
past UV-B levels. It is not yet clear whether the content
ofUV-B absorbing compounds in pollen is determined
by the UV-B dose absorbed by the parental plants or
the exposure of pollen grains themselves.
Day & Demchik ( 1996) measured a higher con-
tent of UV-B-absorbing compounds in pollen derived
from plants cultivated under elevated UV-B radiation,
indicating acclimation of the pollen in response to
UV-B stress of the plants. Plants grown in a 'high
UV-B environment' apparently acclimate by providing
their pollen with protective UV-B absorbing polyphe-
nolics in the pollen wall. These UV-B filters prevents
UV-B damage to the DNA and other targets in the
pollen, in addition to photoreactivation and dark re-
pair processes (Jackson & Linskens 1979). They will
also protect pollen from UV-B damage during trans-
port from the anther to the stigma when they are
clearly exposed to solar UV-B. These UV-B-absorbing
compounds are probably of pollen cytoplasmatic ori-
gin, where flavonoids surround mainly the generative
nucleus (Musil 1995; Musil & Wand 1997). This
outermost pollen wall is most probably deposited on
the pollen cell under control of the parental genome
(Rogers & Harris 1969; Traverse 1988). This is done
by specific tissue in the anther: the tapetum, which sur-
rounds the microspore mother cells. Fernando & Cass
(1994) suggest that this tissue releases sporopollenin
precursors and sporopollenin bodies onto the pollen
membrane. If so, it suggests that the polyphenolic
content of the pollen and moss spores is largely de-
termined by the solar UV-B dose to which the parental
plant is exposed during the growing season.
Dose response relationships between solar UV-B
absorption and the content of pollen polyphenolics
The content of polyphenolic compounds in pollen and
spores is likely to be a function of absorption of solar
UV-B integrated over the (summer) growing season of
the plant. In the Antarctic, that is the complete period
in each year, that a plant in the Antarctic is exposed
to solar radiation, including solar UV-B. Therefore,
when plants flower or mosses sporulate, we assume
that the polyphenolic compounds induced in the pollen
and spores reflect the UV-B dose absorbed by the
parental plants during the growing season for that year.
If polyphenolics in pollen and spores are shown to be
good indicators of UV-B and the presence of this sig-
nal in fossil pollen is demonstrated, it may provide a
proxy measure of solar UV-B exposure. Based on the
polyphenolics content of pollen and spores, historical
levels of solar UV-B and the thickness of stratospheric
ozone may be reconstructed.
To prevent damage from solar UV-B radiation, the
pollen wall is effective in screening out more than
80% of the incident ultraviolet radiation (Tevini 1993;
Demchick & Day 1996). Sporopollenin forms part of
the wall of pollen and spores, and contains aromatic
and aliphatic compounds (Jungfermann et a!. 1997;
Meuter-Gerhards et al. 1999; Blokker et al.l999),
absorbs ultraviolet radiation, preventing UV-B reach-
ing the DNA or other sensitive targets. Despite the
very strong and protective pollen wall, a consider-
able fraction of incident UV-B radiation, upto 20%,
may be transmitted by this wall, and thus reaching
into the pollen cell (Demchik & Day 1996). In most
longer-lived pollen grains, DNA is in a dehydrated
state, which is particularly sensitive to ultraviolet-B
irradiance (Musil1995).
To be a useful biological indicator, the induced re-
sponse of polyphenolic production in pollen by solar
UV-B should preferably be sensitive. To be a relevant
indicator of solar UV-B the pollen polyphenolic con-
tent should be able to track solar UV-B changes related
to 0-50% stratospheric ozone depletion. Day & Dem-
chick (1996) reported that (soluble) UV-B absorbing
compounds increased with increased UV-B (simulat-
ing 15% stratospheric ozone depletion) and, similarly,
we found increased UV-B absorbing compounds in
pollen of Viciafaba under enhanced UV-B (Figure 6).
Field and experimental research is required to
quantify and analyse the relationship between the dose
of UV-B radiation absorbed and the content of UV-
B absorbing polyphenolic compounds of the pollen.

Also the induction of polyphenolics by UV-B should
preferably be specific. However, a variety of envi-
ronmental factors, both abiotic and biotic influence
polyphenolics in plants (Manetas 1999). Additionally,
any other factors affecting the polyphenolic content
must be recognised, in order to identify the specificity
of any response. As yet, dose response relationships
between the solar UV-B dose absorbed and the content
of polyphenolics of pollen, are largely unknown.
What evidence is there for the preservation of
UV-B absorbing compounds in the fossil record?
Flavonoids or other phenolic compounds, present in
the cytoplasm of the pollen grains are likely to be
lost in the process of fossilization (Traverse 1988).
However, UV-B absorbing compounds in the exine
of pollen (e.g., para-coumaric acid, cf., Wehling
et al. 1997) and possibly wall-bound hydroxycinnamic
acids such as ferulic acid (Liu et al. 1995; Hemsley
et al. 1996, 1998) will be preserved in the fossil record.
The quantities of aromatic groups in sporopollenin
can be induced by solar UV-B (Hemsley et al. 1996;
Meuter-Gerhards et al. 1999). It appears to be the UV-
B dose which is absorbed by parental plants - and
not the one to which ripe or released pollen or spores
are exposed - which is likely to determine the quality
and quantity of the polyphenolics in the sporopollenin.
UV-B absorbing compounds similar to sporopollenin
in pollen of higher plants, have been found in walls of
spores of mosses and algae by Xiong et al. (1997).
Xiong et al. (1997) state that sporopollenin or re-
lated compounds in pollen of higher plants and spores
of mosses are a major protection mechanism against
the damaging effects of ultraviolet-B radiation. They
also found an increasing sporopollenin content in two
algal species during prolonged exposure to solar UV-
8. Therefore there is an increasing body of evidence
that the UV-B absorbing capacity of sporopollenin
in pollen grains and moss spores can be elevated in
response to enhanced ultraviolet-B irradiance.
Will the content of UV-B absorbing polyphenolics
in pollen and spores be maintained or altered over
time during the process of fossilization of pollen? Will
sporopollenin chemically change during time and fos-
silization? Hemsley et al. (1996) found that sporopol-
lenin is chemically altered by oxidation and diagenesis
and reported that an increase in aromatic compounds
within the exines was found over time. Their report
demonstrates that sporopollenin and aromatic groups
therein are preserved after fossilization. The content
of aromatic groups may have been increased because
of their resistance to degradation. In a litter decompo-
21
sition study the content of lignin generally increased
with time (Rozema et al. 1997). This aspect certainly
merits further investigation.
Pollen and spores of plants: a well protected archive
of past UV-B?
Pollen are known to fossilize quite well (Faegri &
Iversen 1964; Moore & Webb 1978). This is generally
ascribed to the (poly )phenolic nature of sporopollenin,
explaining (part) of the UV-B absorbance, but also
contributing to the resistance to microbial and chemi-
cal decay of pollen (Hemsley et a! 1996). Because the
sporopollenin is located in the outer layer of pollen
(exine) and spores, this part of pollen and spores is
well preserved during fossilization. Based on the mor-
phology (e.g., size and number of pore(s) in pollen,
pollen and spores, may be identified (Figures 2-5).
In an antarctic context, identification and collection
of pollen and spores extracted from cores taken from
moss peat banks is further simplified because of the
occurrence of only two higher plant species in the re-
gion. The majority of the pollen grains produced take
no part in fertilisation, as most pollen is deposited on
the ground, without performing any reproductive task.
Plants produce thousands, even sometimes more than
a billion, pollen grains per flower to ensure success-
ful pollination (Janssen 1974; Traverse 1988). Pollen
therefore provides a good source of fossilizable ma-
terial occurring in great quantities, being deposited
almost everywhere, and containing a very persistant
pollen wall.
We have provided here some evidence that, per-
haps more than in other plant tissue, enhanced solar
UV-B induces soluble UV-B absorbing compounds in
the pollen and spores, as extracted with acid methanol.
In addition we report that the sporopollenin, a highly
resistant biopolymer, which forms part of the exine
of pollen, contains UV-B absorbing compounds. The
UV-B absorbing para-coumaric acid monomer has
been reported to form an important part of sporopol-
lenin. We hypothesize that solar UV-B will also alter
the UV-B absorbance of the sporopollenin, which
will be preserved in the fossil record. Furthermore it
appears that the content of the UV-B induced polyphe-
nolics of the pollen, represents the total dose of solar
UV-B of the growing season to which parental plants
have been exposed. It is as yet unknown, whether ex-
posure of the ripe pollen to solar UV-B, released by the
anthers can influence its UV-B absorbing properties.
22
Archives ofpast UV-B: pollen and spores from
herbarium specimens and antarctic moss peat banks
Two archives of pollen and spore material with poten-
tial for past UV-B level reconstruction can be recog-
nized. The first archive is for the recent past (ca.
1900-2000), via analysis of the content of polyphe-
nolics in herbarium plants specimens collected in the
Antarctic and the second archive is for the subre-
cent period (5000 up to 10 000 years Before Present),
based on dated layers of frozen moss peat banks in the
Antarctic.
Much preserved dried antarctic plant material
(both vascular plants and bryophytes) is available in
the herbarium of the British Antarctic Survey, from
collections made at intervals from the early 1960s to
the present (Peat 1998). There is also material dating
back to ca. 1930. The antarctic plant collections at
the British Museum (Natural History) and the botan-
ical collection at Kew are also accessible. There are
417 records on the database from 1930 or earlier (in-
cluding moss, liverworts and lichens). Spores have
been successfully obtained from herbarium specimens
and used in the study of the reproductive biology of
mosses (Convey & Smith, 1993; Smith 1993, 1994;
Convey 1996, 1997). Herbarium collections exist from
the sub-antarctic locations southwards to the antarc-
tic continent, although in the antarctic area vascular
plants do not occur south of Marguerite Bay on the
Antarctic Peninsula. Spores and pollen may therefore
be obtained both from herbarium specimens and from
contemporary moss turf or cushions and peat cores.
Markham et al. (1990) analysed flavonoids in
leaves of 35 samples of the moss (Bryum argen-
teum) using herbarium specimens collected over the
period 1957-1989 from the Ross Sea area of con-
tinental Antarctica. Dry moss samples were finely
ground and extracted with methanol for UV-visible
absorbance spectrophotometry measurements and for
HPLC analyses. The sum of peak heights of identified
flavonoids was used as a measure of UV-B absorbing
flavonoids. With antarctic stratospheric ozone levels
(Dobson units) decreasing from 1971-1980, there was
an increase of both UV-B absorbance and relative
flavonoid levels in Bryum argenteum.
Bjorn et al. (1997) reported the content of two
flavonoid aglycones (myricetin and quercitin) in the
leaves of 44 herbarium specimens of the (sub )antarctic
Cassiope tetragona, covering a period from 1820--
1997. The plant material was collected in the summer
period from June to August in the neighbourhood of
Abisko, near the north polar circle at the northern
hemisphere.They found no significant change of the
flavonoid content over time. However, ozone depletion
in the Arctic is far less marked that in the Antarctic
(Pyle 1997), and is currently thought to be a recent
phenomenon.
In addition, it is uncertain whether methanol
extractable UV-B absorbing compounds including
flavonoids, are chemically stable during dry storage of
varying duration such as occurs in herbarium collec-
tions. UV-B induced wall-bound phenolic acids (e.g.,
ferulic acid) and aromatic groups in the sporopollenin
are preserved in the fossil record (Van Bergen et al.
1995) and have a larger potential for reconstruction of
historic solar UV-B levels (cf. Table 7).
Moss peat banks
Ancient moss peat banks underlaying living moss oc-
cur at several sites in the maritime Antarctic, generally
forming part of the permafrost layer. They may be at
least up to 5 m deep, and are present on the South
Orkney Islands (especially Signy Island), South Shet-
lands islands (especially Elephant Island) mid-western
antarctic Peninsula (especially near Palmer Station on
Anvers Island, and Litchfield and other islands in
the nearby Argentine Island, Green Island and Cape
Tuxen). Younger, much thinner, moss peat banks are
found in many locations south to Marguerite Bay. The
bottom of moss banks in South Orkney Islands and
Elephant Island have been dated to ca. 5500 yr BP,
indicating continuous growth over that period, while
those farther south are likely to be younger, with the
oldest radiocarbon date being ca. 2500 yr BP. These
frozen moss peat banks form a more or less homo-
geneous amorphous matrix of well-preserved plant
material. Similar-aged buried moss deposits have been
reported from the Vestfold Hills, Eastern Antarctica,
while there may be older remains in lake bed sed-
iments. Moss peat banks have been described by
Ingolfsson et al. ( 1992), Bjorck & Smith (1992)
and Fabiszewski & Wojtun (1997). The two higher
plant species Deschampsia antarctica and Coloban-
thus quitensis, as well as numerous fruiting moss
species, grow in the vicinity of all the moss banks.
In principle the preservation of frozen plant and
moss material in the moss peat banks may also al-
low DNA extraction and quantification of thymine-
thymine dimers caused by exposure to solar UV-B
radiation (Rozema et al. 1997; Caldwell et al. 1998;
Rousseau et al. 1999). If these dimers are preserved in

the frozen plant remainders, they might represent an
additional indicator of past solar UV-B levels.
Dating of pollen and spores
Antarctic plants and mosses in the herbarium of the
British Antarctic Survey and other British collections
are dated. The period of 1920-2000 is covered by
these herbarium specimens.
Layers of the moss peat banks containing pollen
and spores to be collected have been succesfully dated
with the 14 C. The precision of this dating technique
can be further improved by making 'wiggle match dat-
ing' (Kilian et al. 1995; Van Gee! et al. 1998), which
requires a series of 14 C dates to be obtained over a
short section ofthe core which mimicking fluctuations
(wiggles) in the 14 C curve (cf. a tree ring curve). By
matching the form of the wiggles C4 c curve and 14 C
dates from the core) an exact age determination in
calibrated years (calendar years) is obtained without
having to deal with large uncertainties.
On the reconstruction of past solar UV-B
The proposed method of reconstruction of the
stratospheric ozone thickness and solar UV-B lev-
els for the periods 1920-2000 and 3000 (up to
5000-10 000) yr BP upto recent time, may reveal
whether events similar to the recent depletion of the
stratospheric ozone and associated increased solar UV-
B radiation have occurred previously. It will allow
identification both of any natural cycle and of irregular
events, allowing interpretation of possible alterna-
tive natural or man-made causes of such events (e.g.,
volcanic eruptions, other unknown ozone depleting
compounds in the atmosphere or other causes). It may
also make clear whether the II year solar cycle will af-
fect polyphenolics in plants, including pollen (Stuiver
et al. 1991 ).
A reconstructed record of stratospheric ozone and
increased solar UV-B radiation may indicate whether
(or not) and how recovery of stratospheric ozone oc-
curred in the past. The latter observation may provide
a reference point for current ozone recovery scenar-
ios (Madronich ct al. 1998), currently predicting a
restored stratospheric ozone layer around 2050. As yet
(2000) there are no signs of recovery and the reliability
of such predictions is limited.
23
Acknowledgements
We thank Dr Ad Huiskes and Drs Daniela Lud for their
help at the field research site on Leonie Island and the
collection of tillers of Deschampsia antarctica in Feb-
ruary 1999. The research ofJ.R. at Rothera and Leonie
Island (project number 751.499.06) was financed by
Netherlands AntArctica Programme (NAAP) of the
The Netherlands Geosciences Foundation (GOA) of
NWO, which is gratefully acknowledged. The analysis
of UV-B absorbing compounds in pollen and spores is
partially funded by the EU, DGXII Environment and
Climate (ENV4-CT97-0580). The support of British
Antarctic Survey, Cambridge in logistics, transport
and accommodation is greatly acknowledged.
References
Beggs, C.J. & Wellmann, E. 1994. Photocontrol of flavonoid
biosynthesis. Pp. 733-751. In: Kendrick. R. E. & Kronen-
berg, G. H. M. (eds), Photomorphogenesis in Plants. Kluwer
Academic Publishers, Dordrechl.
Berkner, L. V. & Marshall, L. C. 1965. History of major atmospheric
components. Proc. Nat. Acad. Sci. 53: 1215-1226.
Bjorck, S. & Lewis Smith, R. I. 1992. Late Pleistocene and
Holocene glacial history of James Ross Island, Antarctic Penin-
sula. Boreas. 21: 209-222.
Bjorn. L. 0., Callaghan. T., Gehrke, C., Gunnarson, T .. Holmgren,
B .. Johanson, U .. Snogerup, S .. Sonesson, M., Sterner, 0. & Yu,
S.-G. 1997. Effects on subarctic vegetation of enhanced lJV-B
radiation. Pp. 233-246. In: Lumsden, P. J. (ed.), Plants and LV-
B, Responses to Environmental Change. Cambridge University
Press, Cambridge.
Bjcirn. L. 0. & Murphy, T. M. 1985. Computer calculations of solar
ultraviolet radiation at ground level. Physiol. Vcg. 23: 555-561.
Bjorn. L. 0. 1999. Ultraviolet-B radiation, the ozone layer and
ozone depletion. Pp. 21-:17. In: Rozema. J. (ed.). Stratospheric
Ozone Depletion: the Effects of UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers. Leiden.
Blokker. P.. Schouten, S., DeLeeuw. J. W .. Sinnighe Damste, J. S.
& Van den Ende, H. 1999. Molecular structure of the resistant
hiopolymer in zygospore cell Walls of Chlamydommws monoica.
Planta 207: 539-543.
Budyko. M. I .. Ronov, A. B .. Yanshin. A. L. 1987. History of the
Earth's Atmosphere. Springer-Verlag, Berlin.
Caldwell. !VI. M. 1984. Partial inhibition of in vitro pollen germi·
nation hy simulated solar ultraviolct-B radiation. Ecology 65:
792-795.
Caldwell, M. :vi., Bjorn, L. 0 .. Bornman, J. F., Flint. S. D ..
Kulandaivelu, G., Teramura, A. H. & Tevini. M. 1998. Effects
of increased solar ultraviolet radiation on terrestrial ecosystems.
J. Photochem. Photobiol. B. 46: 40-52.
Caldwell, M. M .. Teramura, A. H .. Tevini, M., Bornman. 1. F.,
Bjorn. L. 0. & Kulandaivelu, G. 1995. Effects of increased solar
ultraviolet radiation on terrestrial plants. Ambio 24: 166-173.
Chapman. S. K. 1986. Working with a Scanning Electron Micro-
scope. Pp. 113. Lodgemark Press Ltd, Kent.
24
Cocke II, C. S. 1998. Biological effects of high ultraviolet radiation
on early Earth - a theoretical evaluation. J. Theor. Bioi. 193:
717-729.
Cocke!!, C. S. 1999. Carbon biochemistry and the ultraviolet radi-
ation environments ofF, G, and K main sequence stars. Icarus
141: 399-407.
Cocke! I. C. S. 2000. The ultraviolet history of the terrestrial planets
- implications for biological evolution. Planet. Space Sci. 48:
203-214.
Convey, P. & Lewis Smith, R. I. 1993. Investment in sexual
reproduction by antarctic mosses. Oikos 68: 293-302.
Convey, P. 1996. Reproduction of Antarctic flowering plants.
Antarct. Sci. 8: 127-134.
Convey, P. 1997. Environmental change: possible consequences for
the life histories of antarctic terrestrial niota. Kor. .1. Pol. Res. 8:
127-144.
Crutzen, P. J. 1992. Ultraviolet on the increase. Nature 356: 104-
105.
Day, T. A., Ruhland, C. T. Grobe, C. W & Xiong, F. 1999. Growth
and reproduction of Antarctic vascular plants in response to
warming and UV radiation in the field. Oecologia I I 9: 24-35.
Day, T. A. 1993. Relating UV-B radiation screening effectiveness
of foliage to absorbing-compound concentration and anatomical
characteristics in a divers group of plants. Oecologia 95: 542-
550.
Day, T. A. & Demchik, S. M. 1996. Influence of enhanced UV-
B radiation on biomass allocation and pigment concentrations in
leaves and reproductive structures of greenhouse-grown Brassica
rapa. Vegetatio 127: 109-116.
Dcmchik, S.M. & Day, T. A. 1996. Effects of enhanced UV-B radi-
ation on pollen quantity, quality, and seed yield in Brassica rapa
(Brassicaceae). Am. J. Bot. 83: 573-579.
Edwards, J. A. 1972. Studies in Co/ohanthus quitensis and De-
.r.;chnmpsia nntarctico. V. Distrihution, ecology and vegetative
perfomance on Signy Island. Br. Antarct. Surv. Bull. 28: 11-28.
Fabiszcwski, J. & Wojtun, B. 1997.The occurrence and development
of peat mounds on King George Island (Maritime Antarctic).
Acta Soc. Bot. Pol. 66: 223-229.
Faegri, K. & Iversen, J. 1964. Textbook of Pollen Analysis. Munks-
gaard, Copenhagen, pp. 295.
Farman, .T. C., Gardiner, B. G.& Shanklin, J. D. I 985. Large losses
of total O?one in Antarctica reveal seasonal CLO,!NO,. Nature
315: 207-210.
Feldheim, K. & Conner. J. K. 1996. The ctlects of increased UV-
B radiation on growth, pollination success, and lifetime female
fitness in two Brassica species. Oecologia 106: 284-297.
Fernando, D. D. & Cass, D. D. 1994. Plasmodial tapetum and pollen
wall development in Buwmus umbellatus (Butomaceae). Am. J.
Bot. 81: 1592-1600.
Flint, S. D. & Caldwell. M. M. 1983. Influence on floral optical
properties on the ultraviolet radiation environment of pollen. Am.
J. Bot. 70: 1416-1419.
Flint, S. D. & Caldwell, M. M. 1984. Partial inhibition of in vitro
pollen germination by simulated solar ultraviolet-B radiation.
Ecology 65: 792-795.
Fricker, M. D., Chow, C.-M., Errigton, R. L May, M., Mellor, J.,
Meyer, A. J., Tlalka. M .. Vaux. D. J., Wood, J. & White, N. S.
1997. Quantitative imaging of intact cells and tissues hy multi-
dimentional confocal fluorescence microscopy. Experimental
Biology Online 2, 19, http://link.springer.de.
Garcia-Piche\, F. 1998. Solar ultraviolet and the evolutionary history
of cyanobacteria. Origins Life Evol. Biosphere 28: 321-347.
Git7, D. C., Liu, L. & McClure, J. W. 1997. Phenolic metabolisme,
growth, and UV-B tolerance in phenylalanine ammonia-lyase-
inhibiting red cabbage seedlings. Phytochemistry 49: 377-386.
Greenberg, B. M., Wilson, M. 1., Huang, X. D., Duxbury, C. L., Ger-
hardt, K. E. & Gensemer, R. W. 1997. The effects ofultraviolct-B
radiation on higher plants. Pp. l-33. In: Wang, W., Gorsuch,
J. W. & Hughes, J. S. (eds.), Plants for Environmental Studies.
CRC, Boca Raton, FL
Hader, D.-P. 1997. The Erects of Oone Dpletion on Auatic Eosys-
tems. Academic Press & R.G. Landes Co., pp. 275.
Hemsley, A. R., Scott, A. C., Barrie, P. J. & Chaloner, W. G. 1996.
Studies of fossil and modern spore wall biomacromolecules
using t 3c solid state NMR. Ann. Bot. 78: 83-94.
Hemsley, A. R., Vincent, B., Collinson, M. E. & Griffiths, P. C.
1998. Simulated self-assembly of spore exines. Ann. Bot. 82:
105-109.
Herman, J. R., Bhartia, P. K., Ziemke, J.. Ahmad, Z. & Larko, D.
1996. UV-B increases (1979-1992) from decreases in total zone.
Gcophys. Res. Letters 23: 2117-2120
Hofmann, D. J. 1996. The 1996 Antarctic ozone hole. Nature 383:
129.
Hofmann, D. J., Oltrnans, S J., Harris, J. M., Johnson, B. J. &
Lathrop, J. A. 1997. Ten years of ozone sonde measurements at
the South Pole: implications for recovery of springtime antarctic
ozone. J. Gcophys. Res. 102: 8931-8943.
Haltom, A. & Greene, S. W. 1967. The growth and reproduction of
Antarctic flowering plants. Pp. 323-337. In: Smith, J. E. (ed.).
A Discussion on the Terrestrial Antarctic Ecosystem. Trans. R.
Soc. Bioi. Sci., 777, London.
Huiskes, A. H. L., Lud, D., Moerdijk-Poortvliet, T. C. W. &
Rozema, J. 1999. Impacts of UV-B radiation on antarctic terres-
trial vegetation. Pp. 313-337. In: Rozema, J. (ed.), Stratospheric
Ozone Depletion; the Effects or UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden .
Hutzler P., Fischbach, R., Heller. W., Jungblut, T. P., Reuber, S ..
Schnmitz. R., Veit, M., Weissenbi:ick, G. & Schnitzler, J.-P. 1998.
Tissue localisation of phenolic compounds in plants by confocal
laser scanning microscopy. J. Exp. Bot. 49: 953-965.
Ingolfsson, 0 .. Hjort, C., Bjorck, S. & Lewis Smith, R. I. 1992. Late
Pleistocene and Holocene glacial history of James Ross Island,
Antarctic Peninsula. Boreas 21: 209-222.
!sarin, R. F. B. & Bohncke, S. J. P. \999. Mean July temperatures
during the Younger Dryas in Northern and Central Europe as
inferred from Climate Indicator Plant Species. Quat. Res. 5 I:
158-173.
Jackson, J. F. & Linskens. H. F. 1979. Pollen DNA repair after treat-
ment with the mutagens 4-nitrquinoline-1-oxide. ultraviolet and
near-ultraviolet in·adiation, and boron dependence of repair. Mol.
Gen. Genetics 176: ll-16.
Janssen, C. R. 1974. Verkenningen in de Palynologie. Oosthoek,
Scheltema & Holkema, Utrecht.
Johnston, H. 197!. Reduction of stratospheric ozone by nitrogen
oxide catalysts from supersonic transport exhaust. Science 173:
517-522.
Jungfermann, C., Ahlers, F.. Grote, M., Gubatz, S .. Steuernagel,
S., Thorn, I., Wctzels, G. & Wicrmann, R. 1997. Solution of
sporopollcnin and reaggregation of a sporopollenin-like material:
a new approach in the sporopollenin research. J. Plant Physiol.
151:513-519.
Karabourniotis, G., Kotidis, G., Fasseas, C., Liakoura, V. &
Drossopoulos, I. 1998. Polyphenol deposition in leaf hairs of
Olea europaea (Oiaceae) and Quercus i/ex (Fagaceae). Am. J.
Bot. 85: 1007-1012.

Kasting, J. F., Zahnle, K. J., Pinto, J.P. & Young, A. T. 1989. Sulfur,
ultraviolet radiation, and the early evolution of life. Origins Life
Evol. Biosphere 19: 95-108.
Kasting, J. F., Eggler, D. H. & Raeburn, S. P. 1993. mantle redox
evolution and the oxidation state of the archaean atmosphere. J.
Geol. 10 I: 245-257.
Kilian, M. R., Vander Plicht, J. & Van Geel, B. 1995, Dating raised
bogs: New aspects of AMS CI4 wiggle matching, a reservoir
effect and climate change. Quat. Sci. Rev. 14: 959-966.
Krauss, P., Markstlidter, C. & Riederer, M. 1997. Attenuation of
UV radiation by plant cuticles from woody species. Plant Cell
Environ. 20: 1079-1085.
Kronenberg, G. H. M. Photomorphogenesis in Plants. Kluwer
Academic Publishers, Dordrecht, pp. 733-751.
Kubitzki, K. 1987. Phenylpropanoid metabolism in relation to land
plant origin and diversification. J. Plant Physiol. 131: 17-24.
Kok, S. J., Kristenson, E. M., Gooijer, C.. Velthorst, N. H. &
Brinkman, U. A. Th. 1997. Wavelength-resolved laser-induced
fluorescence detection in capillary electrophoresis, J. Chro-
matogr. A, 771: 331-342.
Liu, L., Gitz, D. C. & McClure, J. W. 1995. Effects of UV-B on
flavonoids, ferulic acid, growth and photosynthesis in barley
primary leaves. Physiol. Plant. 93: 725-733.
Lowry, B .. Lee, D. & Hebant, C. 1980. The origin of land plants: a
new look at an old problem. Taxon 29: 183-197.
Madronich, S. 1992. Implications of recent total atmospheric ozone
measurements for biologically active ultraviolet radiation reach-
ing the earth's atmosphere. Geophys. Res. Lett. 19: 37-40.
Madronich, S. 1993. UV radiation in the natural and perturbed
atmosphere. Pp. 17-69. In: Tevini, M. (ed.), UV-B Radiation
and Ozone Depletion, Effects on Humans. Animals, Plants,
Microorganisms, and Materials. Lewis Publishers. Boca Raton,
FL.
Madronich, S., McKenzie, R. L., Caldwell, M. M. & Bjorn,
L. 0. 1995. Changes in ultraviolet radiation reaching the earth's
surface. Ambio 24: 143-152.
Manetas, Y. 1999. Is enhanced UV-B radiation really damaging
for plants? Some case studies with European mediterranean
plant species. Pp. 251-291. In: Rozema, J. (ed.), Stratospheric
ozone Depletion; the Effects of UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden.
Markham, K. R., Franke, A., Given, D. R. & Brownsey, P. 1990.
Historical Antarctic ozone level trends from herbarium specimen
flavonoids. Bull. Liaison Group Polyphenols 15: 230-235.
Martin, J. T. & Juniper, B. E. 1970. The Cuticles of Plants. Edward
Arnold Publisherrs Ltd, Edinburgh.
Meijkamp, B., Aerts, R., Van de Staaij, J. W. M .. Tosserams, M ..
Ernst, W. H. 0. & Rozema, J. 1999. Effects of UV-B on sec-
ondary metabolites in plants. Pp. 70-99. In: Rozema, J. (ed.),
Stratospheric Ozone Depletion: the Effects of UV-B Radiation
on Terrestrial Ecosystems. Backhuys Publishers, Leiden.
Meijkamp, B. B., Doodeman, G. & Rozema, J. 2001. The reponse
of Vi cia faha to enhanced UV-B radiation under low and near
ambient PAR levels. Plant Ecol. 154: 135-146 (this volume).
Meuter-Gerhards, A., Riegert, S. & Wiermann, R. 1999. Studies on
sporopollenin biosynthesis in Cucurhita maxima (OUCH.) - II.
The involvement of aliphatic metabolism. J. Plant Physiol. 154:
431-436.
Molina, M. & Rowland, F. S. 1974. Stratospheric sink for chlo-
rofluoromethanes: chlorine atom catalyzed destructioin of ozone.
Nature 249: 810-812.
Moore, P. D. & Webb, J. A. 1978. An Illustrated Guide to Pollen
Analysis. Hodder and Stoughton, London.
25
Musil, C. F. 1995. Differential effects of elevated ultraviolet-B radi-
ation on the photochemical and reproductive performances of di-
cotledonous and monocotyledonous arid-environment cphemer-
als. Plant Cell Environ. 18: 844-854.
Musil, C. F. & Wand, S. J. E. 1999. Impacts of UV-B radia-
tion on South African mediterranean ecosystems. Pp. 265-291.
In: Rozema. J. (ed.), Statospheric Ozone Depletion: the Et~
feels of UV-B Radiation on Terrestrial Ecosystems. Backhuys
Publishers, Leiden.
Newman. P. Gleason, J. F., McPeters, R. D. & Stolarski. R. S. 1997.
Anomalously low ozone over the arctic. Geophys. Res. Lett. 24:
2689-2692.
Paxson-Sowders, D. M., Owen, H. A. & Makaroff, C. A. 1997.
A comparative ultrastructural analysis of exine pattern develop-
ment in wild-type Arahidopsis and a mutant defective in pattern
formation. Protoplasma 198: 53-65.
Peat, H. J. (1998) The Antarctic Plant Database: a specimen and
literature based information system. Taxon 47: 85-93.
Peck, L. S. & Brey T. Bomb signals in old antarctic brachiopods.
Nature 380: 207-208.
Pierson. B. K., Mitchell, H. K. & Ruffroberts, A. L. 1993. Chlo-
ro.fiexus auramiacus and ultraviolet radiation - implications
for archaean shallow-water stromatolites. Origins Life Evol.
Biosphere 23: 243-260.
Pyle, J. A. 1997. Global ozone depletion: observations and theory.
Pp. 3-11. In: Lumsden, P. J. (ed.), Plants and UV-B. Re-
sponses to Environmental Change. Cambridge University Press.
Cambridge.
Rogers, C. M. & Harris, B. D. 1969. Pollen exine deposition: a clue
to its control. Am. J. Bot. 56: 1209-1211.
Rothschild, L. J. 1999. The influence of UV radiation on protistan
evolution. J. Eukaryotic Microbial. 46: 548-555.
Rozema, J., Van de Staaij, J., Bjorn, L.-0. & Caldwell, M.
1997. UV-B as an environmental factor in plant life: stress and
regulation. Trends Ecol. Evol. 12: 22-28.
Rozema, J. G. M., Teramura, A. H. & Caldwell, M. M. 1999.
Atmospheric C02 enrichment and enhanced solar ultraviolet-B
radiation: gene to ecosystem responses. Pp. 169-191. In: Luo, Y..
Mooney, H. A. (eds), Carbon Dioxide and Environmental Stress.
Academic Press, San Diego.
Rozema, J. G. M., Tosscrams, M., Nelissen, H. J. M., Van
Heerwaarden. L.. Brockman, R. A. & Flierman. N. 1997b.
Stratospheric ozone reduction processes: enhanced UV-B radi-
ation affects chemical quality and decomposition of leaves of the
dune grassland species Ca/amagrostis epigeios. Plant Ecol. 128:
284-294.
Rozema, J. G. M., Van de Staaij, J. W. M .. Bjorn, L. 0. & Caldwell.
M. M. 1997a. UV-B as an environmental factor in plant life:
stress and regulation. Trends Ecol. Evol. 12: 22-28.
Rozema, J. 1999. Stratospheric Ozone Depletion: the Effects of
Enhanced UV-B Radiation on Terrestrial Ecosystems. Backhuys
Publishers Leiden. pp. 355.
Rozema, J.. Brockman, R .. Lud, D .. Huiskes. A., Moerdijk, T..
De Bakker, N., Meijkamp, B. & Van Beem, A. 2001. Con-
sequences of depletion of stratospheric ozone for terrestrial
antarctic ecosystems: the response of Desclwmpsia amarctica
to enhanced UV-B radiation in a controlled environment. Plant
Ecol. 154: 101-115 (this volume).
Schnitzler, J.P., Jungblut, T. P., Heller, W., Hutzler, P., Heinzmann,
U., Schmelzer, E., Ernst, D., Langebartels, C. & Sandermann,
H. Jr. 1996. Tissue localisation of UV-B screening pigments and
chalcone synthase mRNA in Scots pine (Pinus sy!l·e.vtris L.)
needles. New Phytol. 132: 247-258.
26
Shaw, G. 1971. The chemistry of sporopollenin. Pp. 305-351. In:
Brooks, J., Grant, P.R., Muir, M.D., Van Gijzel, P. & Shaw, G.
ieds), Sporopollenin. Acadamic Press, London.
Shindell. D. T., Rind. D. & Lonergan, P. 1998. Climate change and
the middle atmosphere. Part IV: Ozone response to douhled C02
l Climate II: 895-918.
Smith, R. I. L. 1993. Bryophyte propagule banks: Case study of
an Antarctic J'ellfield soil. Pp. 55-78. In: Miles, J. & Walton,
D. W. H. (cdsJ, Primary Succession on Land. Blackwell, Oxford.
Smith, R. I. L. 1994. Vascular plants as bioindicators of regional
warming in Antarctica. Oecologia 99: 322-328.
SORG. 1999. Stratospheric ozone 1999. United Kingdom
Stratospheric Ozone Review Group. Seventh report. De-
partement of the Environment, Transport and the Regions.
London.
Stephanou, M. & Manetas,Y. 1997. The effects of seasons, ex-
posure, enhanced UV-B and water stress on leaf epicuticular
and internal UV-B absorbing capacity of Cistus creticus: a
Mediterranean field study. J. Exp. Bot. 48: 1977-1945.
Stolarski, R., Bojkov, R., Bishop. L., Zeferos, C., Staehlin, J. & Za-
wodny, J. 1992. Measured trends in stratospheric ozone. Science
256: 342-349.
Stuiver, M., Braziunas, T. F., Becker, B. & Kromer. B. 1991. Cli-
matic. solar, oceanic and geomagnetic influences on Late Glacial
and Holocene atmospheric Ct41Ct2 change. Quat. Res. 35: 1-24.
Tcramura. A. H. & Sullivan, J. H. 1994. Effects of UV-B radiation
on photosynthesis and growth of terrestrial plants. Photosynt.
Res. 39: 463-473.
Tevini, M. 1993a. Molecular biological effects of utraviolet radia-
tion. Pp. 1-15. In: Tevini, M. (ed.), UV-B Radiation and Ozone
Depletion, Effects on Humans, Animals, Plants, Microorgan-
isms, and Materials. Lewis Publishers, Boca Raton.
Tevini, M. 1993b. Effects of UV-B radiation on terrestrial plants.
Pp. 125-153.1n: Tevini, M. (ed.), UV-B Radiation and ozone De-
pletion, Effects on Humans, Animals, Plants, Microorganisms,
and Materials. Lewis Publishers, Boca Raton, FL.
Tevini, M. 1998. Effects of increased solar ultraviolet radiation on
terrestrial ecosystems. J. Photochem. Photohiol. 46: 40-52.
Towe, K. M. 1996. Environmental oxygen conditions during the
origin and early evolution of life. Life sciences: space and Mars
recent results 18: 7-15.
Traverse, A. 1988. Spores and pollen morphology. Pp. 60-116. In:
Traverse, A. (ed.), Paleopalynology. Unwin Hyman. Boston.
UNEP. 1998. Environmental effects of ozone depletion. Assess-
ment, pp. 192.
Van Bergen, P. F., Collinson. M. E., Briggs, D. E. G., De Leeuw,
J. W., Scott, A. C., Evershed, R. P. & Finch, P. 1995. Resis-
tant hiomacromolecules in the fossil record. Acta Bot. Neerl. 44:
319-342.
Van de Staaij, J. W. M., Huijsmans. R., Ernst, W. H. 0. & Rozema,
J. G. M. 1995. The effect or elevated UV-B (280-320 nm) radia-
tion levels on Silene vulgaris: a comparison between a highland
and a lowland population. Environ. Pollut. 90: 357-362.
Van Gee!, B. & Renssen, H. 1998 Abrupt climate change around
2.650 BP in North West Europe. evidence for climatic telecon-
nections and a tentative explanation. Pp. 21-41. In: Jsar, A. S.
& Brown, N. (eds), Water, Environment and Society in Times
of Climatic Change. Kluwer Academic Press. Dordrecht, The
Netherlands.
Vaughan, D. G. & Doake. C. S.M. 1996. Recent atmospheric warm-
ing and retreat of ice shelves on the Antarctic Peninsula. Nature
379: 328-331.
Wagner, F., Bohncke, S. J. P., Dilcher, D. L., Klirschner, W. M.,
Van Gee!, B. & Visscher, H. 1999. Century-scale shifts in Early
Holocene atmospheric C02 concentration. Science 284: 1971-
1973.
Webb, A. R. 1997. Monitoring changes in UV-B radiation. Pp. 13-
30. In: Lumsden, P. J. (ed.), Plants and UV-B, Responses
to Environmental Change. Cambridge University Press, Cam-
bridge.
Wehling, K., Niester, Ch., Boon, J. J., Willemse, M. T. M.
& Wiermann, R. 1989. p-Coumaric acid - a monomer in the
sporopollenin skeleton. Planta 179: 376-380.
Xiong, F., Komenda, J., Kopecky, J. & Nedbal, L. 1997. Strategies
of ultraviolet-B protection in microscopic algae. Physiol. Plant.
I 00: 378-388.
Xiong, F. S., Ruhland, C. T. & Day, T. A. 1999. Photosynthetic
temperature response of the Antarctic vascular plants Coloban-
thus quitensis and Deschampsia antarctica. Physiol. Plant. 106:
276-286.
Ziska, L. H., Teramura, A. H. & Sullivan, J. H. 1992. Physiolog-
ical sensitivity of plants along an elevation gradient to UV-B
radiation. Am. J. Bot. 79: 863-871.
 Section 1: General

Sub-arctic forest dominated by Betula pubescens ssp. tortuosa at Abisko Sweden. At the field sup-
plementation within this ecosystem mass loss from B. pubescens litter decomposing under elevated
UY-B was slightly. bur signilicantly increased and the structure of the fungal decomposer community
significantly changed.
 Plant Ecology 154: 29-36, 200 L
29
© 2001 Kluwer Academic Publishers.

The direct effects of UV-B radiation on Betula pubescens litter

decomposing at four European field sites

Sandra A. Moody 1, Nigel D. Paul 1, Lars Olaf Bjorn 2 , Terry V. Callaghan 3 , John A. Lee4 ,
Yiannis Manetas 5 , Jelte Rozema6 , Dylan Gwynn-Jones 7 , Ulf Johanson 2 , Aris Kyparissis 5 &
Annemiek M. C. Oudejans 6
1Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, LAJ 4YQ, UK; 2 Department of'
Plant Physiology, Lund University, Sweden; 3 Abisko Scientific Research Station, Sweden; 4 Department of' Animal
and Plant Sciences, University of Sheffield, Sheffield, SN I 0, UK; 5 Department of' Biology, Laboratory c1j' Plant
Physiology, University of Patras, Greece; 6 Department of Systems Ecology, Free University, Amsterdam, The
Netherlands; 7 Institute of Biological Sciences, University of' Wales, Aberystwvth, Ceredigion, SJ23 3DA, UK

Key words: Decomposition, Nutrient cycling, Ozone depletion, Photodcgradation, Ultraviolet-B, UV-B

Abstract
A co-ordinated series of field experiments were conducted to consider the effects of elevated UV-B radiation
applied directly to decomposing plant litter. Betula pubescens was decomposed under ambient and elevated UV-B
(simulating a 15% ozone depletion) using outdoor irradiation facilities at Adventdalen, Norway (78° N), Abisko,
Sweden (68° N), Amsterdam, The Netherlands (52° N,) and Patras, Greece (38° N). There was no significant effect
of treatment on mass loss for samples collected after 2, 12 and 14 months decomposition at Amsterdam, or after 4
months decomposition at Adventdalen. Significant reductions in the mass loss of litter decomposing under elevated
UV-B compared to ambient were found at the other 2 sites. The only effect of treatment on litter chemistry during
decomposition was a significant reduction in the N concentration of material at Abisko and a significant increase
in C:N at Patras for litter decomposing under elevated UV-B. Significant differences were found in the structure of
the fungal community decomposing litter in Sweden, the only site to be tested. These data, and the few published
studies of the response of decomposition to UV-B incident on litter suggest that, in the ecosystems and climates
that have been studied, such direct effects are typically confined to the initial stages of decomposition, and are
rather small in magnitude.

Introduction that ground level UV-B radiation may remain above

Halocarbons and other ozone depleting substances are
expected to reach maximal levels in the stratosphere
around the year 2000 (Madronich ct al. 1998). As a
result, the increase in ground level ultraviolet-B radia-
tion (UV-B: 280-315 nm) resulting from stratospheric
ozone depletion should be expected to reach a peak
within the next 50 years. However non- compliance
with the Montreal Protocol, and the possibility of
interactions with greenhouse gases (Shindell et al.
1998), are just two potential situations likely to ensure
pre 1980 values for the foreseeable future.
Concern over stratospheric ozone depletion has
resulted in investigations of a wide range of biolog-
ical and ecological responses to elevated UV-B over
the past two decades. However, the effects of ele-
vated UV-B radiation on the decomposition of plant
litters have been studied only recently (Gehrke et al.
1995: Rozema et al. 1997; Newsham et al. 1997,
1999: Yue et al. 1998). The responses of decompo-
sition to increased UV-B can be considered in terms of
two processes. Effects of UV-B applied during plant
growth on subsequent decomposition are termed 'in-
30
direct' and result, for example, from changes in litter
chemistry. The effects of UV-B incident directly on
decomposing litter are termed 'direct', and are usu-
ally considered in terms of two component processes.
Firstly, elevated UV-B may alter the composition or
activity of the decomposer community (Moody et a!.
1999). Although the responses of the decomposer
community to increased UV-B may be rather complex,
the growth and reproduction on many decomposer
fungi are inhibited by UV-B (Moody eta!. 1999), and
these responses are generally expected to reduce the
rate of decomposition (Gehrke et a!. 1995). Secondly,
elevated UV-B may increase photodegradation, the
direct physico-chemical breakdown of litter (Moor-
head & Callaghan 1994), and so increase the rate of
decomposition (Rozema et a!. 1997).
The study described here investigated the direct ef-
fects of elevated UV-B on decomposition, measured
through mass loss, chemical composition and fun-
gal colonisation of a standard litter (Betula pubescens
Ehrh.) at four contrasting European field sites. This
allowed an initial assessment of whether differences
between previous studies (Gehrke et al. 1995; Rozema
et al. 1997; Newsham et al. 1997, 1999; Yue et al.
1998) have been a function of the different species
used, or contrasting experimental conditions.
Materials and methods
UV-B treatments
Betula pubescens litter was decomposed in outdoor
irradiation facilities at four contrasting European
sites. The sites were Adventdalen, Svalbard, Norway
(78° N, 16° E), Abisko, Sweden (68° N, 18° E), Am-
sterdam, The Netherlands (52° N, 4° E) and Patras,
Greece (38° N, 29° E). All facilities supplied ele-
vated UV-B radiation equivalent to an ozone depletion
of approximately 15%, although exact methodology
varied between sites (see Gehrke et al. 1995; Johan-
son et a!. 1995 (Adventdalen & Abisko); Rozema
et al. 1997, 1999a (Amsterdam); Stephanou & Mane-
tas 1997 (Patras) for details). Briefly, arrays supplying
supplemental UV-B radiation were situated above nat-
ural vegetation at Adventdalen (high arctic tundra),
Abisko (sub-arctic dwarf shrub heath) and Amster-
dam (dune grassland). Local plant species were trans-
planted to soil below arrays for irradiation in Patras.
All sites had four treatment and four control arrays
except Amsterdam which had 8 replicate UV-B and
control plots. Irradiations were continued throughout
the winter months in Patras and Amsterdam. At the
other sites supplemental UV-B was supplied during
the summer period only, due to heavy snow coverage
in winter months.
Plant litter
Betula pubescens litter was collected at senescence
(September-November 1996) from one tree in Lan-
caster, England. Leaves were air dried and stored until
used in the decomposition studies. To reduce vari-
ability, petioles were removed and each leaf cut into
quarters. Samples (0.7 g air dry weight) were sealed
in plastic bags before gamma irradiation (total dose
25 kGy: Isotron, Bradford, UK) to remove any exist-
ing decomposers. Sealed samples were sent to each
of the participating sites where the litter was placed
in litter cylinders prior to decomposition. Cylinders
consisted of a Plexiglas tube (6.4 em internal diameter,
3 em tall) (Rightons, Manchester, UK) which trans-
mitted 72% of plant-weighted UV-B incident on the
cylinder sides. Litter was contained by mesh (I 0 mm
at the top, I mm at the base), which was secured to
the cylinder by stainless steel wire ties. This arrange-
ment ensured free movement of the litter within the
cylinder by wind and rain, allowing all litter to be
exposed to incident radiation. The complete cylinder,
including mesh, ties, etc., reduced radiation incident
on litter lying on the bottom mesh by approximately
9%. Cylinders were secured to the ground beneath ar-
rays, amongst the vegetation naturally found at each
site, using stainless steel pegs. Decomposition exper-
iments commenced at the four sites between I 0-15
June 1997.
Mass loss and chemical analyses
Samples were collected from the field after 2, 12 and
14 months decomposition. At each sample occasion 3
replicate cylinders were taken from each of 4 replicate
treatment and control arrays at all sites except Ams-
terdam, where I cylinder was taken from each of 8
replicate treatment and control plots. Samples were
air dried and sent to Lancaster for analysis. The lit-
ter was briefly washed in distilled water to remove
attached soil before drying at 80 octo determine mass
loss. The material was then ground using liquid ni-
trogen and percentage C and N were assessed using a
Carlo-Erba 1108 Elemental Analyser (Fisons pic, Ip-
swich, UK). Lignin and a-cellulose were determined
using the ADF/permanganate method of Rowland &
Roberts ( 1994). As litter samples at Adventdalen were

disturbed by birds during the first season of the exper-
iment this material was considered suitable for chem-
ical analysis but for not mass loss determinations. To
obtain mass loss data from this site, a proportion of the
litter was removed from the field (21 August 1997), air
dried and stored until June 1998, when it was placed
back in the field to decompose for a further 2 months
during the summer UV-B irradiation (i.e., a total of
four months decomposition under UV-B, without any
period under snow during winter).
Community structure of decomposer fungi
Additional B. pubescens litter samples (0.5 g air dry
weight) were placed in the field to decompose at
the Abisko site. These samples were used to assess
changes in fungal community structure on the decom-
posing B. pubescens litter under ambient and elevated
UV-B. Samples were examined after 2 months (after
summer irradiation treatments), 12 months (after the
winter snow melt- no winter irradiation was supplied)
and 14 months (after a second summer irradiation pe-
riod). One cylinder was taken from each of 3 replicate
treatment arrays and 3 control arrays at each sample
occasion, air dried for 2 days and returned to Lan-
caster for analysis. Litter discs (2 mm diameter) were
cut from leaves and serially washed in 5 changes of
sterile distilled water ( 15 ml) according to Harley &
Waid (1955). Forty replicate discs per litter cylinder
were plated onto Czapek-Dox agar (Oxoid, Unipath
Ltd., Basingstoke, UK) with 0.135 g 1- 1 Rose Ben-
gal (Sigma Chemical Company, Poole, Dorset, UK)
to suppress the growth of bacteria and rapidly spread-
ing fungi. Plates were examined daily for 4 weeks
and the identity of fungal decomposers on each disc
recorded. The isolation frequency of each species was
expressed as a percentage of the total number of iso-
lates recorded from all discs from each replicate litter
cylinder. The identity of fungal species isolated from
litter was confirmed by CABI Bioscience (Egham,
Surrey, UK).
Statistical analyses
In all cases where more than one replicate was taken
per array, statistical analyses have been carried out us-
ing the means of arrays as replicates (all sites except
Amsterdam). All percentage data were arcsine trans-
formed before analysis. All decomposition data were
analysed by two-way analysis of variance (ANOVA)
with time as an independent variable using the GEN-
STAT (version 5) computer package. Any significant
31
treatment x time interactions were further analysed by
one way AN OVA at each sample occasion.
Results
Mass loss
The effect of the duration of decomposition on mass
loss was significant (P<O.OOI) at all sites where re-
peated sampling was possible. There was no signif-
icant direct effect of UV-B treatment on mass loss
from B. pubescens litter decomposing for 14 months
in Amsterdam (Figure 1). The one measurement pos-
sible at Adventdalen also showed no significant of
UV-B effect (% weight remaining after four months
93.4 ± 0.2, 93.4 ± 0.5 ambient and elevated UV-B
respectively). Elevated UV-B applied during decom-
position resulted in a significant (P<0.05) treatment
x time interaction for B. pubescens litter decomposing
in Patras. When analysed at each sample occasion in-
dividually, mass loss after two months decomposition
at Patras was slightly but significantly less in litter ex-
posed to elevated UV-B than in control litter (6.3%,
4.8% mass lost for ambient and elevated UV-B re-
spectively; P <0.001 ). No significant treatment effects
were found after 12 or 14 months decomposition at
Patras. At Abisko mass loss was 3.1 %, 5.9% and 3.7%
less in litter exposed to elevated UV-B than in controls
after 2, 12 and 14 months decomposition respectively
(Figure 1). The main UV-B effect on decomposition
at Abisko was significant (P<0.05) and there was no
significant interaction with time.
Chemical analyses
Analysis of litter chemistry during decomposition
showed significant time effects for lignin and N con-
centration, and C:N ratio at all sites ( P <0.00 I), and
for a-cellulose at Amsterdam (P<O.Ol) and Patras
(P<0.05). No significant effects of UV-B or UV-B x
time were found for a-cellulose or lignin at any site
(Tables 1a and I b). The only significant effects of UV-
B on nitrogen concentration in litter (Table I c) were at
Abisko, where both UV-B and UV-B x time effects
were significant (P <0.05). When analysed at each
sample occasion individually, a significant reduction
in N concentration (12.9%) was found for litter de-
composed under elevated UV-B compared to ambient
after 12 months in the field (Table I c). There were no
significant effects on C:N (Table I d) at any site except
Patras (main UV-B effect P<0.05), where significant
32
--Ambient
100
- - - -Elevated
80
co
=
2
·c; 60
E summer of UV-B treatment (Table 3).
e
Eco
";) 40
~
~
20
0 posing under elevated UV-B, which they attributed to
May-97 Aug-97 Oct-97 Jan-98 Apr-98 Jun-98 Sep-98
Figure I. Percentage weight remaining (± S.E.) for B. pubescens
litter decomposing under ambient (open symbols) and elevated
UV-B (tilled symbols) at Abisko (circles), Amsterdam (squares) and
Patras (triangles) aticr 2, 12 and 14 months. Significant effect of
time at all sites (P<O.OOl). Significant effect of treatment at Abisko
(P <0.05). Significant treatment x time interaction at Patras: signif-
icant reduction in mass loss from litter decomposing under elevated
UV-B compared to controls after 2 months (P<O.OOI). N=4 all
sites expect Amsterdam where N =8.
increases of 5.4%, 8.2% and 7.5% were found for litter
decomposing under elevated UV-B compared to am-
bient after 2, 12 and J4 months, respectively. These
results reflected a tendency for the N concentration to
be lower in litter decomposing under elevated UV-B
compared to ambient UV-B at this site (Table lc). UV-
B treatment had no effect on changes in the content
of nitrogen (Table 2a) or lignin (Table 2b) over the
course of decomposition at any site (Table 2). In all
cases losses of nitrogen were small ( <5% of the initial
content) and while around 50% of initial lignin was
lost after 14 months decomposition at Patras, it was
notable that the content of lignin appeared to increase
at both Amsterdam and Abisko.
Decomposing jim gal community structure at Abisko
The fungal community isolated from B. pubescens
litter after 2 months decomposition was significantly
affected by UV-8 treatment (Table 3). Cladosporium
sp. was significantly more abundant on control lit-
ter (26% isolation frequency compared with 3% in
litter decomposing under elevated UV-B). The isola-
tion frequencies of two species of Penicillium were
also substantially, hut not significantly reduced un-
der UV-B. By contrast Cystodendron sp. and Phoma
herbarum were isolated significantly more frequently
from litter decomposing under elevated UV-8 (25%
and 4% under elevated UV-B compared to 5% and 0%
under ambient UV-B for each species, respectively).
No significant differences in the fungal community
were apparent after 12 months decomposition, after
the winter snow melt or 2 months later after the second
Discussion
Rozema et al. ( 1997) reported significantly increased
mass loss from Calamagrostis epigeios leaves decom-
photodegradation. Other studies of the direct effects of
UV-B on mass loss have shown no significant effects
(Gehrke et al. I 995; Moody et al. 2000), or signifi-
cant, but transient, reductions (Newsham et al. 1997).
In the present field experiment, the effects of elevated
UV-B on B. pubescens litter, when significant (main
UV-B effect or UV-B x time interaction significant
in repeated measures analysis), resulted in decreased
mass loss compared to litter decomposing under am-
bient UV-8 (Figure l ). These significant decreases in
mass loss under elevated UV-B were confined to sites
and/or sample dates when mass loss was less than
approximately 40%, suggesting that the direct UV-B
effects on mass loss may be confined to the initial
phases of decomposition. As noted above, mass loss
from Q. mburlitterdecomposingunderelevated UV-B
was significantly but transiently decreased (Newsham
et a!. 1997). Thus, data available at present tend to
confinn the suggestion (Newsham et al. 1997), that
the most consistent direct etl'ect of increased UV-B
may be to inhibit mass loss early in decomposition,
during what has been defined as the 'initial stage'
of a two-three phase pattern of litter decomposition
(Berg & Staaf 1980). Changes in mass loss are not
readily related to any clear effects of UV-B on litter
chemistry. Although UV-B had some effect on the N
chemistry of litters at Abisko and Patras (Table 2),
significant changes in mass loss at Abisko preceded
significant decrease in N concentration, and at Patras
changes in mass loss were transient, while C:N ratio
was consistently increased under UV-8. Changes in
lignin during decomposition are hard to interpret be-
cause lignin content increased during decomposition
at Amsterdam and Abisko (Table 2). This highlights
the limits on measuring lignin using standard prox-
imate analyses, which are unable to fully separate
lignin from other acid-insoluble residues, not only
 33
Table I. Mean concentrations ±S.E. of a-cellulose. lignin and nitrogen (all as% dry weight), and C:N ratio of Betula
pube>cens litter decomposed in the field at 4 European sites under ambient and elevated UV-B. Chemistry of original
litter before the experiment started and after 2. 12 and 14 months decomposition. NA- data not assessed due to lack
of material. The results of repealed measures ANOVAs are summarised. * and *** represents effects significant at
P <0.0 and P <0.00 1 respectively; ns equals no significant difference. N =4 all sites except Amsterdam where N =8.

UV-B Duration of decomposition A NOVA

treatment 2 months 12 months 14 months Time UV-B TxUV-B

(a) a-cellulose concentration (%).Initial concentration in fresh litter: 13.2 ± 0.07

Adventdalen Ambient 15.2 ± 1.22 14.2 ± 0.39 14.9 ± 0.39

ns ns ns
Elevated 14.0 ± 0.46 14.1 ±0.41 14.5 ± 0.56

Abisko Ambient 15.4 ± 0.15 15.7 ± 0.20 16.4 ± 0.75

ns ns ns
Elevated 14.8 ± 0.46 16.0 ± 0.33 16.0 ± 0.62

Amsterdam Ambient 14.8 ± 0.48 16.5 ± 0.65 NA

Elevated 14.2 ± 0.67 16.9 ± 0.75 NA
*** ns ns

Patras Ambient 12.2 ± 0.20 9.4± 1.65 13.2 ± 0.23

ns ns ns
Elevated 12.0 ± 0.22 11.0 ± 0.41 12.9 ± 1.55

(b) Lignin concentration(%). Initial concentration in fresh litter: 14.0 ± 0.35

Adventdalen Ambient 19.8 ± 0.47 21.8 ± O.:lO 23.4 ± 0.29

Elevated 19.5 ± 0.49 21.6 ± 0.30 2:l.6 ± 0.29
*** ns ns

Abisko Ambient 22.1 ± 0.37 28.3 ± 0.42 34.1 ± 1.01

Elevated 21.4 ± 0.58 25.9 ± 2.78 31.1 ±0.75
*** ns ns

Amsterdam Ambient 33.1±1.77 40.2 ± 0.69 NA

Elevated 33.2 ± 0.45 38.4 ± 0.71 NA
*** ns ns

Patras Ambient 15.1 ±0.13 25.4 ± 1.78 25.8 ± 0.36

ns ns
Elevated 15.2 ± 0.24 26.1 ± 1.09 24.4 ± 1.45

(c) Nitrogen concentration(%). Initial concentration in fresh litter: 1.08 ± 0.05

Adventdalen Ambient 1.21 ± 0.04 1.37 ± 0.05 1.46 ± 0.10

ns ns
Elevated 1.09 ± 0.05 1.38 ± 0.02 1.61 ±0.09

Abisko Ambient 1.27 ± 0.08 1.75 ± 0.02 1.65 ± 0.()3

Elevated 1.32 ± 0.04 1.52 ± 0.04 1.55 ± 0.05
***

Amsterdam Ambient 1.74 ± 0.03 2.20 ± 0.08 2.20 ± 0.06

Elevated 1.69 ± 0.08 2.31 ± O.OJ 2.15 ±0.04
*** ns ns

Patras Ambient 1.06 ± 0.03 1.54 ± 0.04 1.57±007

Elevated 1.02 ± 0.04 1.51 ± 0.08 1.44 ± 0.05
*** ns ns

(d) Carbon:nitrogen ratio. Initial ratio of fresh litter: 44.0 ± 1.92

Advenldalen Ambient 38.5 ± 1.09 32.7 ± 1.09 33.4 ± 0.99

ns ns
Elevated 42.8 ± 1.90 32.2 ± 0.60 33.4 ± 1.89

Abisko Ambient 37.9 ± 2.29 27.6 ± 0.31 28.2 ± 0.66

*** ns ns
Elevated 36.4 ± 1.05 31.9± 1.00 29.9 ± 0.99

Amsterdam Ambient 27.5 ± 0.48 21.6±0.74 20.0 ± 0.48

Elevated 28.6 ± 1.33 20.6 ± 0.26 20.1 ± 0.20
*** ns ns

Patras Ambient 42.4 ± 1.14 23.3 ± 0.57 22.5 ± 0.57

Elevated 44.7 ± 1.97 25.2±0.71 24.2 ± 0.60
*** ns
34
Table 2. Changes in the content of nitrogen and lignin of Betula pubescens litter decomposed in the field at Abisko.
Amsterdam and Patras under ambient and elevated UV-B. Data are expressed as% of the content of litters at the start of
decomposition, '-'indicates that lignin or nitrogen was lost during decomposition, '+'that the content of lignin or N
increased. NA- data not assessed due to lack of material. N = 4 at Abisko and Patras, N = 8 at Amsterdam.

UV-B Duration of decomposition AN OVA

treatment 2 months 12 months 14 months Time UV-B TxUV-B

(a) Change in litter nitrogen content

Abisko Ambient -0.80 ± 0.39 +0.16±011 -0.63 ± 0.15

ns ns ns
Elevated -0.48 ± 0.21 -0.63 ± 0.16 -0.90 ± 0.33

Amsterdam Ambient -0.16 ± 0.24 -2.59± 0.42 -2.67 ± 0.58

*** ns ns
Elevated -0.51 ± 0.23 -2.20 ± 0.18 -3.00 ±0.50

Patras Ambient -0.58 ± 0.18 -3.88 ± 0.98 -4.88 ± 0.56

*** ns ns
Elevated -0.76 ± 0.26 -4.70 ± 0.33 -4.45 ± 0.51

(h) Change in litter lignin content

Abisko Ambient +19.4 ± 1.5 +26.9± 2.2 +40.9 ±4.6
*** ns ns
Elevated +17.0±2.5 +33.1 ± 1.6 +33.6 ± 2.8

Amsterdam Ambient +41.4 ± 5.1 -6.7 ± 8.7 NA

ns ns
Elevated +42.1±4.9 -8.6 ± 3.3 NA

Patras Ambient +3.1 ± 1.7 -25.4± 9.9 -51.9 ± 11.3

*** ns ns
Elevated +3.0 ± l.3 -44.9 ± 3.0 -45.1 ± 7.8

original litter components such as cutin and condensed
tannins, but also 'humic' substances formed during
decomposition (Preston et a!. 1997). By contrast, re-
duced mass loss under elevated UV-B is consistent
with the observed changes in the microbial commu-
nity colonising litter. Although many organisms play
a significant role in decomposition, it is notable that
there was a significant alteration in the fungal com-
munity structure after 3 months UV-B treatment of B.
pubescens litter decomposing at Abisko, the only site
to be examined. These changes were dominated by a
switch in two key species, Cladosporium sp. being
abundant under ambient UV-B while Cystodendron
sp. was more frequently isolated under elevated UV-
B (Table 3). This is the first time such changes have
been noted in a field decomposition experiment where
litter was placed into natural vegetation. However,
Newsham eta!. ( 1997) reported significant changes in
the mycoflora of Quercus robur litter decomposing in
the field that showed close parallels to those we ob-
served here, e.g., a decrease in Cladosporium spp. and
increase in Coelomycetes such as Cystodendron sp.
UV-B has also been shown to alter fungal community
structure on litter of Vaccinium uliginosum decompos-
ing under laboratory conditions (Gehrke et al. 1995).
Such changes are consistent with the marked interspe-
cific variation in fungal responses to UV-B determined
in vitro (Moody et al. 1999). Significant direct ef-
fects of UV-B on the mycoftora of I itters, like those
on mass loss, appear to be transient. Thus, there were
no significant differences in the fungi isolated from B.
pubescens litter decomposed for 12 and 14 months at
Abisko (Table 3). Similarly, several of the significant
direct UV effects on fungi colonising Q. robur litter
(Newsham et a!. 1997) were confined to the initial
sampling occasion. Alterations in the fungal commu-
nity have been associated with reduced respiration
from litters under elevated UV-B (Gehrke eta!. 1995;
Moody et a!. 2000). In both these studies, respiration
in litter exposed to increased UV-B was significantly
reduced only early in decomposition.
Thus, transient changes in microbial community
of litters appears to be a rather consistent response
to increased UV-B during decomposition, and may
contribute to decreases in mass loss. However, micro-
bial respiration per se is not the only route of mass
loss from litters, and changes in this component may
be masked by, for example, leaching of soluble com-
 35
Table 3. Percentage isolation frequency of fungi co Ionising decomposing B. pubescens Etter after 2 months, 12
months and 14 months decomposition under ambient (Am b) or elevated (Elev) UV-B radiation in the field at Abisko.
Data were analysed using one-way ANOVA. *and** indicate differences at P<0.05 and P<ll.OI respectively: ns
-non-significant. N = 3.

Fungal species % Isolation frequency

2 months 12 months 14 months
Am b. Elev. p Am b. Elev. I' Am b. Elev. p

Astermna microspermum 6 20 ns 5 ns 0 2 ns
Cystodendron sp. 25 ** 5 11 ns 3 ns
Cladosporium sp. 26 3 3 ns 7 8 ns
Penicillium spinulosum 9 0 ns 6 2 ns 2 4 ns
Penicillium sirnplicissirnurn 7 ns 16 22 ns 22 22 ns
Cladosporium herharum 4 ns 4 4 ns 4 7 ns
Mucor hiema/is 11 ns 7 8 ns
Phoma nehulosa 8 5 ns 8 ns
Phoma herbarum 0 4 ** 7 I ns

pounds, fragmentation and consumption by soil fauna References

or, perhaps, photodegradation. The changing balance
between these various processes of mass loss during
the progress of decomposition, plus the normal suc-
cession of decomposer fungi, may limit the duration
of direct UV-B effects. While there is rather consistent
evidence that increased UV-B during decomposition
can significantly alter the fungal community colonis-
ing litter, the resulting inhibition of mass loss appears
to be transient and rather small in magnitude (a few
percent in this and previous studies). However, the
mechanisms proposed here may assume far greater
significance in certain plant species or ecosystems (cf.,
Paul et al. 1999; Rozema et al. l999h ), for example
where 'standing dead' is a significant component of
decomposition. Thus, such mechanisms merit further
investigation, both in the context of ozone depletion
and the consequences of seasonal and geographical
variation in UV-B.
Acknowledgments
We are grateful to EU (Contract ENV4 CT96 0208)
and DETR (PECD 7/12/21) for financial support. We
would also like to thank Debra Hurst and Jane Martin
for chemical analyses and Philip Smith for technical
support.
Berg. B. & Staaf. H. 1980. Decomposition rate and chemical
changes of Scots pine needle litter II innuence of chemical
composition. Ecol. Bull. 32: 373-390.
Gehrke, C.. Johanson, U., Callaghan, T. Y., Chadwick, D. &
Robinson, C. H. 1995. The impact of enhanced ultraviolet-
B radiation on litter quality and decomposition processes in
Vaccinium leaves from the Subarctic. Oikos 72: 213-222.
Harley. J. L. & Waid. J. S. 1955. A method of studying active
mycelia on living roots and other surfaces in soil. Trans. Brit.
Mycol. Soc. 38: 104-118.
Johanson, U., Gehrke, C., Bjorn, L. 0., Callaghan, T. Y. & Sones-
son, M. 1995. The effects of enhanced UV-B radiation on a
sub-arctic heath ecosystem. Ambio 24: 106-111.
Madronich, S., McKenzie, R. L.. Bjorn, L. 0. & Caldwell. M. 1998.
Changes in biologically active ultraviolet radiation reaching the
Earth's surface. J. Photochem. Photobiol. B46: 5-19.
Moody. S. A .. Newsham, K. K., Ayres, P. G. & Paul, N.D. 1999.
Variation in the responses of litter and phylloplane fungi to UV-R
radiation (290-315 nm). Mycol. Res. 103: 1469-1477.
Moorhead & Callaghan, 1994. Effects of increasing ultraviolet B
radiation on decomposition and soil matter dynamics: a synthesis
and modelling study. Bioi. Fert. Soils 18: 19-26.
Newsham, K. K .. McLeod, A. R .. Roberts, J. D., Greenslade, P. D.
& Emmett, B. A. 1997. Direct effects of elevated UV-B radiation
on the decomposition of Quercus mbur leaf litter. Oikos 79: 592-
602.
Paul, N .. Callaghan, T. Y., Moody. S., Gwynn-Jones.D .. Johan-
son,U. & Gehrke,C. 1999. UV-B impacts on decomposition and
biogeochemical cycling. Pp. 117-133. In: Rozema. J. (ed.),
Stratospheric Ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden.
Preston, C. M .. Trofymow, J. A., Sayer, B. G. & Niu. J. 1997. 13 c
nuclear magnetic resonance spectroscopy with cross-polarization
and magic-angle spinning investigation of the proximate-analysis
fractions used to assess litter quality in decomposition studies.
Can. J. Bot. 75: 1601-1613.
36
Rowland, A. P. & Roberts, J. D. 1994. Lignin and cellulose frac-
tionation in decomposition studies using acid-detergent fibre
methods. Commun. Soil Science Plant Anal. 25: 269-277.
Rozema, J .. Tosserams, M., Nelissen, H. J. M., Van Heerwaar-
den, L., Brockman, R. A. & Flierman, N. 1997. Stratospheric
ozone reduction and ecosystem processes: enhanced UV-B radi-
ation affects chemical quality and decomposition of leaves or the
dune grassland species Calamagrostis epigeios. Plant Ecol. 128:
284-294.
Ro7.ema, J., Kooi, B., Brockman, R. A. & Kuiper, L. 1999a. UV-B
radiation and litter decomposition (of a dune grassland ecosys-
tem), a modelling approach. Pp. 135-157. In: Rozema, J. (ed.),
Stratospheric Ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden.
Rozema, J., Oudejans, A., Hauter, N., Schoonheim, H., Walraven,
I., Van 't Klooster, C., Van de Staaij, J., Tosserams. M .. De
Bakker, N., Van Beem. A., Stroetenga, M., Brockman, R., Van
Heerwaarden, L., Nelissen, H. & Aerts, R. 1999b. Responses of
plants tram a dune grassland ecosystem in the Netherkands to
solar UV-B: UV-B filters and supplementation experiments. Pp.
203-225. Ro7.ema, J. (ed.), Stratospheric Owne Depletion: the
Effects of Enhanced UV-B Radiation on Terrestrial Ecosystems.
Backhuys Publishers, Leiden.
Shindcll, D. T., Rind, D. & Lonergan, P. 1998. Increased polar
stratospheric ozone losses and delayed eventual recovery ow-
ing to increasing greenhouse-gas concentrations. Nature 392:
589-592.
Stephanou, M. & Manetas, Y. 1997. The effects of seasons, expo-
sure, enhanced UV-B radiation and water stress on leaf epicutic-
ular and internal UV-B absorbing capacity of Cis/us creticus: a
Mediterranean tield study. J. Exp. Bot. 48: 1977-1985.
Yue, M., Li, Y. & Wang, X. 1998. Effects of enhanced ultraviolct-B
radiation on plant nutrients and decomposition of spring wheat
under field conditions. Environ. Exp. Bot. 40: 187-196.
 Section 2: Terrestrial Plants and Terrestrial Ecosystems

Fl uorescent tubes above dune grassland vegetation to expose Ca/anwgrostis epigeios and Carex arenaria to cn h~nced
U V-B. (Photograph by l Rozema)
 Plant Ecology 154: 39--48, 200 I.
39
© 200 I KIL!Wer Academic Publishers.

The reduction of aboveground Calamagrostis epigeios mass and tiller

number by enhanced UV-B in a dune-grassland ecosystem

A.M. C. Oudejans, A. Nijssen, J. S. Huls & J. Rozema

DepartmentofSystems Ecology. Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands

Key words: Calamagrostis epigeios, Carex arenaria, Dune-grassland ecosystem, Enhanced UV-B radiation,
Reduced plant mass accumulation, Stratospheric ozone depletion, Tillering

Abstract
Since early May 1997 dune-grassland vegetation in the Netherlands has been exposed to enhanced levels of
ultraviolet-B (UV-B) radiation. Expected increases in the amount of biologically effective UV-B (UY-BsE) upon a
reduction of the stratospheric ozone layer with 15% were calculated and artificially supplemented.
In June and September 1998, above- and below ground vegetation samples were taken. Of the dominant species
Calamagrostis epigeios and Carex arenaria aboveground mass accumulation, leaf weight (LW), leaf area (LA),
specific leaf area (SLA) and tiller number were assessed separate from the remaining vegetation.
The results of our study indicate alterations in the vegetation structure due to UV-B supplementation. In June,
a significant reduction due to UV-B supplementation in number of tillers and aboveground dry weight per soil
area unit was found for C. epigeios. As C. epigeios is the most dominant species of the dune-grassland, these
effects indicate a change in vegetation structure due to UV-B enhancement. Indications of UV-B effects on other
parameters, such as the number of tillers of C. arena ria and aboveground plant dry weight of the group of species
other than C. epigeios and C. arenaria, may also represent changes in vegetation structure. The LA and LW data
show the same patterns as the mass accumulation trends. No significant UV-B effects on the SLA of the species or
of the total vegetation could be found.
Trends in patterns of species dry weight accumulation and partitioning of dry weight between species groups are
different in June and September. This may indicate seasonal dependence ofUV-B responses. Also, the consistently
reducing trend in total and aboveground plant dry weight may indicate deleterious effects of UV-B on total plant
matter accumulation. Possible causes of observed trends and effects are discussed.

Introduction fects on growth parameters such as leaf area, internode

Enhanced ultraviolet-B (UV-B) radiation due to
stratospheric ozone depletion may have far-reaching
consequences for ecosystem structure and function-
ing (e.g., Caldwell et al. 1995, 1998; Sullivan 1997;
Rozema et al. 1997a, 1999b ). In the past. research
effort has mainly stressed the damaging effects of
UV-B radiation on plant physiology and morphology,
varying from DNA damage (Stapleton et al. 1997)
and damage to the photosynthetic apparatus, proteins
and membranes (Tevini & Teramura 1989; Jansen
eta!. 1998) to destruction of photosynthetic pigments
(Tevini et al. 1991; Strict & Porra 1992). In addition,
enhanced UV-B radiation often results in negative ef-
length and axillary shoot production (Biggs et a/.
1981; Teramura 1983; Murali & Teramura 1986a;
Musi11996).
However, many indoor as well as outdoor studies
do not show any effect of enhanced UV-B radiation
on photosynthesis and plant performance (e.g., Ziska
et a/. 1993; Van de Staaij 1994: Fiscus & Booker
1995: Dillenburg eta!. 1995). In fact, plant species and
even cultivars of the same species vary greatly in their
response to UV-B: negative, neutral as well as posi-
tive effects on plant performance have been reported
(Biggs eta!. 1981; Teramura 1983; Krupa & Kickert
1989; Sullivan et al. 1992; Dai et a!. 1994; Musil
1995; Tosserams & Rozema 1995; Tosserams et a/.
40
1996, 1997). This suggests that some plant species
may be well adapted to UV-B radiation while others
are not.
In ecosystems, differential sensitivity to UV- B
radiation may influence competitive interactions be-
tween plant species. possibly leading to changes in
species composition and an altered distribution of nat-
ural vegetations (Tevini & Tcramura 1989). At present
it is not clear to what extent direct UV-B damage
plays a regulating role in the structure and function-
ing of natural ecosystems. Enhanced UV-8 radiation
has been shown to cause DNA damage (Stapleton
et a/. 1997; Rousseaux et a/. 1999), destruction of
photosynthetic pigments (Tevini et al. 1981) and re-
duced primary production (Yuan et al. 1998) under
field conditions. However, instead of effects on plant
growth and primary production, as often found in lab-
oratory and greenhouse studies, field studies in most
cases only show alterations in plant morphology or
secondary metabolism upon UV-8 enhancement (Sul-
livan 1997; Rozema et al. 1997a, c, 1999b). Low
photosynthetically active radiation (PAR) levels of-
ten used in indoor studies may lead to exaggerated
UV-B responses (Warner & Caldwell 1983; Mirecki
& Teramura 1984 ), the main reasons being increased
effectiveness of repair mechanisms and enhanced pro-
duction of UV-B absorbing compounds at higher PAR
levels (Rozema et al. 1997a).
Indirect physiological and ecological conse-
quences of UV-B effects on morphogenesis and sec-
ondary metabolism may lead to changes in the struc-
ture and functioning of ecosystems in general and of
dune-grassland ecosystems in particular (see Rozema
et al. 1997a, b, 1999a). An important mechanism of
plants to avoid direct damage of UV-8 to key targets
is through wavelength selective filtering of UV-B by
phenolics, such as flavonoids, localized in epidermal
cells or throughout the tissue (see, e.g., Day et al.
1992; Schnitzler et a!. 1997; Hogue & Remus 1999;
Meijkamp et al. 1999). Apart from serving as a UV-B
screen, accumulation of polyphenolic compounds may
mediate several ecosystem-level responses to UV-B.
For example, alterations in plant chemistry could af-
fect decomposition and nutrient cycling (Gehrke eta/.
1995; Rozema et al. 1997b; Paul et al. 1999), water
relations (Drilias et a/. 1997; Man etas et al. 1997),
insect herbivory (McCloud & Berenbaum 1994), and
plant-pathogen interactions (Paul 1997). In addition,
mutualistic plant-soil fungi relations may be affected
by stratospheric ozone depletion (Klironomos & Allen
1995; Van de Staaij eta/. 1999).
Apart from ecosystem-level effects mediated by
UV-B effects on plant chemistry, species-specific
changes in morphology, probably mediated by pho-
toreceptors (Stapleton 1992; Balian~ eta/. 1995; Ros
& Tevini 1995), can alter the competitive balance be-
tween different species in a community. This may
eventually result in a changed species composition of
the vegetation (Caldwell 1997). Some of the plant
morphogenic parameters that may change due to UV-
B are plant height, leaf area, leaf thickness, tiller
number and shoot-to-root ratio.
In the past, UV-B responses of several wild
plant species from a dune-grassland ecosystem in the
Netherlands have been examined both indoors (Tosser-
ams & Rozema 1995; Tosserams el al. 1996, 1997,
Oudejans, unpublished results) and outdoors (Tosser-
ams et a/. 1997). These studies indicate little effect
of enhanced UV-B radiation on growth, morphology
and secondary metabolism of dune-grassland species
such as C. epigeios. Unlike C. epigeios, Gwynn-Jones
and Johanson (1996) found Calamagrostis purpurea
growth and the number of Calamagroslis lapponica
tillers to he negatively affected by enhanced UV-8
under greenhouse conditions.
The number of studies concerning effects of UV-B
enhancement on plants in their natural environment is
limited (Sullivan & Rozema, 1999). As plants function
differently in a vegetation community than in isola-
tion, field research on a community level is needed to
evaluate possible consequences ofUV-B enhancement
for the structure and functioning of ecosystems (e.g.,
Norton et al. 1999). Furthermore, environmental con-
ditions in the field, such as nutrient availability (Murali
& Teramura 1985, 1987; Musil & Wand 1994; Deck-
myn & Impens 1997; Ernst et al. 1997; Tosserams
eta/. 2001) and water availability (Tevini et al. 1983;
Murali & Teramura 1986b; Balakumar et a/. 1993;
Drilias eta/. 1997), may influence UV-8 responses of
plants. Multiple season studies as performed by Sulli-
van & Teramura (1992), Gwynn-Jones et al. (1997)
and Newsham et a/. ( 1999) may help in predicting
long-term effects of enhanced UV-8 radiation on agri-
cultural and natural ecosystem functioning (Sullivan
& Rozema 1999).
This ecosystem study aims at assessing long-term
effects of enhanced UV-B radiation levels on some
aspects of vegetation structure, species mass accumu-
lation, species morphology and total plant mass ac-
cumulation in semi-natural dune-grassland vegetation
in the northwestern coastal zone of the Netherlands.
The grassland vegetation is dominated by the grass

Calamagrostis epigeios and to a lesser extent by Care.r
arenaria. Some plant species present are Oenothera
biennis, Rubus caesius, Plantago lanceolata, Festuca
rubra and Agrostis capillaris. The vegetation is dense
and a humic layer is absent in the sandy, calcareous
soil. The dune-grassland ecosystem is described in
more detail by Rozema eta!. ( 1999a).
Dune-grassland vegetation has been exposed to
enhanced levels of UV-B radiation from May 1997.
simulating a degree of stratospheric ozone depletion of
15%. Because of the natural spectral background radi-
ation present in field studies (see McLeod 1997) and
the use ofUV-A controls (see Newsham eta/. 1996),
relatively realistic responses of individual species and
the vegetation as a whole can be obtained. This paper
describes a vegetation sampling experiment in which
part of the above- and bclowground dune-grassland
vegetation treated with or without additional UV-B
was harvested in June and September 1998.
Material and methods
Experimental design
At the field site. the vegetation of eight plots mea-
suring I x 2 m was treated with enhanced doses
of ultraviolet-B (UV-B) radiation. Eight other plots
served as controls. Eight wooden frames (4.8 x 1.8
x 1.8 m), each carrying I 0 Philips 40W /12 fluores-
cent tubes placed 40 em apart, were placed on the
dune grassland site in 1996. The frames were posi-
tioned in two rows of four on an area of 12 x 20 m.
In order to produce a stable output. the fluorescent
tubes were pre-burned for at least 100 h before in-
stalling them 1.5 m above the soil surface. UV-B
transmitting Plexiglas plates could be placed directly
under the tubes. These were used as filter carriers for
either cellulose diacetate (CA: absorbs all radiation
below 290 nm) or Mylar (absorbs all radiation be-
low 313 nm) filter material. The CA filter material
was used for the enhanced UV-B radiation treatment
in order to prevent energy-rich ultraviolet-C (UV-C)
radiation emitted by the lamps to reach the vegetation.
Because the vegetation treated with enhanced UV-B
radiation also received additional ultraviolet-A (UV-
A) radiation emitted by the lamps, the vegetation in
the control plots was also treated with extra UV-A ra-
diation by means of filtering the lamp light with Mylar
foil, which absorbs UV-C and UV-B, but no UV-A ra-
diation. Due to photo-degradation of the material. CA
41
and Mylar filters were replaced after 50 and I 00 h of
irradiation respectively.
Each wooden frame was divided into two by means
of a metal plate in the soil, dividing the rooting zone
under the frame into two halves. In this way, two
plots were created underneath each frame: an ambient
UV-B control plot and an enhanced UV-B plot. In or-
der to avoid UV-B radiation from UV-B treated plots
to reach the vegetation in the adjacent control plots.
Mylar screens were vertically stretched in the middle
of each frame. Because shading caused by the frames
and lamps was apparent, control and UV-B plots were
alternately chosen. This way the UV-B and the control
treatment were shaded to the same extent.
UV-B enhancement
UV-B closes emitted by the lamps covered with CA
were measured at vegetation height with a double-
monochromator spectroradiometer (Optronic Model
OL 752, Orlando, FL, USA). Biologically etTec-
tive UV-B (UV-BRrJ doses were calculated using the
plant action spectrum of Caldwell ( 1971 ). The addi-
tional UV-B radiation was supplied in a square-wave
mode (sec McLeod 1997). As the natural UY-BsEdose
changes throughout the year, the burning time of the
lamps was adjusted every other week. Supplementary
doses mimicking a depletion of the ozone layer with
159( were calculated using the program of Bjorn &
Murphy ( 1985). The monthly long-term ( 1971-1993)
average stratospheric ozone thickness measured at the
KMI in Ukkel (Belgium, 51 o N latitude) was used
for the model calculations. The treatment was started
early May 1997.
Han·est
ln June and September 1998. two random samples
were taken from each plot in the following way: the
aboveground plant material of two ring-shaped areas
with a diameter of 4 ern was cut otT in each plot. af-
ter which the belowgrouncl plant material was taken
out by hammering a piece of PVC pipe of the same
diameter into the soil. The biomass bclowgrouncl was
cleared of sand, dried and weighed. It was not possible
to sort the belowground material on a species level.
Aboveground plant material was further separated into
biomass and litter mass of C. epigeios, C. arena ria and
the remaining vegetation.
42

a b c
Ca/amagrostis epigeios Carex arenaria remaining vegetation
(/)
(/) 0,8
"'
E
c:N" 0,6
"' E:
a.
-o?! 0,4
*
§ 0
0 C)
.><
~
~~ 0,2
>
0
{l 0,0
June Sept. June Sept. June Sept.
FiRure 1. Effects of enhanced UV-B radiation on aboveground dry weight production (biomass and litter mass) on soil area basis of dune grass-
land vegetation. The vegetation was exposed to ambient solar UV-B radiation (black bars) or enhanced levels of UV-B radiation representing
a depletion of the ozone layer with 15% (grey bars). Vegetation was harvested after more than a year of treatment in June and September
(Sept.) 1998. The aboveground plant matter of Calamagrostis epigeios (a) and Carex arena ria (b) was harvested separate from the remaining
vegetation (c). The significant treatment effect indicated with * had a p-value of 0.03. All data presented are means of X plms ± SEM .

a b
Calamagrostis epigeios Carex arenaria
3
(/)

~ 00
-.;::; 2 * I
.
0
0
~

<ll(\J

n
J:),

II ~
E
:J
g
c:

0
June Sept. June Sept.
Figure 2. Elfects of enhanced UV-B radiation on the amount of tillers of Ca!amagrostis epi[ieios (a) and Carex arena ria (b) per soil area unit
in a dune-grassland vegetation. The vegetation was exposed to ambient solar UV-B radiation (black bars) or enhanced levels of UV-B radiation
representing a depletion of the ozone layer with 15% (grey bars). Vegetation was harvested after more than a year of treatment in J une and
September (Sept.) 1998. The signilicanttreatmenl ~ rr~cl indicated with * had a p-valu~ of0.03. All data presented are m~ans of 8 plots± SEM.

Statistics of C. epigeios tillers was also significantly reduced

Because of the matched pairs design of the experi-
ment, treatment effects were tested with a two-tailed
paired comparison t test, using the SPSS Base 8.0
computer program. Data derived from two plots of the
same lamp-frame were regarded as matching pairs.
Results
In June, the total aboveground plant mass (including
litter mass) of C. epigeios was significantly reduced
due to the enhanced UV-B treatment (Figure 1a). At
this time aboveground biomass and aboveground lit-
ter mass of this species showed the same trend when
considered separately (Table I). Aboveground mass
differences between C. epigeios treated with and with-
out extra UV-B were not significant in September
(Figure I a, Table I). Figure 2a shows that the number
by UV-B in June. Treatment differences in the same
direction were present in September as well, but no
significant effects on biomass or tillering could be
detected that time (Figures Ia, 2a).
Additional UV-B radiation did not seem to affect
the aboveground plant mass (including litter mass) ac-
cumulation of C. arena ria (Figure I b, Table I). In
both harvesting periods, the aboveground biomass as
well as the aboveground litter mass of this species was
on average slightly higher in the enhanced UV-B plots
compared to measurements in control plots (Table I).
Figure 2b shows a trend in the number of C. arena ria
tillers to increase due to UV-8 enhancement in June,
although no significant diflerence could be detected.
For the remaining plant species a trend towards a
reduced aboveground plant mass (biomass and litter
mass) due to the UV-8 treatment can be observed in
September, but not in June (Fig ure I c, Table I). For
 43
Table /_ Effects of enhanced UV-B radiation on above- and belowground plant dry weight accumu-
lation (biomass and litter mass) on a soil area basis in a dune-grassland ecosystem. The vegetation
was exposed to ambient solar UV-B radiation or enhanced levels of UV-B radiation representing a
depletion of the ozone layer with 15%. Vegetation was harvested after more than a year of treatment
in June and in September. The aboveground plant matter of Ca/amagrostis epigeios and Co rex are-
na ria was harvested separate from the remaining vegetation. All data represent means of 8 plots ±
SEM. None of the treatment differences is statistically significant.

Variable (kg m- 2 ) June September

Control Enhanced UV-B Control Enhanced UV-B

Total plant mass 2.98 ± 0.21 2.78 ± 0.30 3.26 ± 0.23 3.08 ± 0.18
Aboveground biomass 0.49 ± 0.09 0.39 ± 0.06 O.S:l ± 0.13 0.43 ± 0.10
Aboveground litter mass 0.60 ± 0.10 o.5o ±om 0.89 ± 0.15 0.73±0.13
Belowground plant mass 1.82 ± 0.20 1.89 ± 0.15 1.85 ± 0.12 1.96±0.10
Shoot: root ratio 0.68 ± 0.11 0.52 ± 0.09 0.80 ± 0.22 0.62 ± 0.13
Aboveground biomass 0.24 ± 0.08 0.10 ± 0.04 0.34 ± 0.10 0.22 ± 0.08
Calanwgroslis epigeios
Aboveground litter mass 0.23 ± 0.07 0.16 ± 0.04 0.27 ± 0.10 0.23 ± 0.09
Calamagrostis epigeios
Aboveground biomass 0.13 ± 0.04 015±0.04 0.11 ± 0.04 0.12 ± 0.05
Carex arena ria
Aboveground litter mass 0.06 ± 0.02 ().06 ±0.02 0.09 ± 0.03 0.09 ± 0.04
Carex arena ria
Aboveground biomaS> 0.12 ± 0.04 0.14 ± 0.05 0.16 ± 0.05 0.10 ± o.m
remaining vegetation
Aboveground litter mass 0.31 ± 0.05 0.27 ± 0.08 0.52 ± 0.06 0.41 ± 0.11
remaining vegetation

this species group no statistically significant treatment
effects could be detected.
No statistically significant etlects on the partition-
ing (percentages) of aboveground plant mass (total
mass, biomass and litter mass) between C. epigeios,
C. arenaria and the remaining vegetation could be
detected (data not shown). Trends were however sim-
ilar to the trends descrihed above, with a reduction
of C. epigeios in June and of plant species other than
C. epigeios and C. arena ria in September.
Total plant mass was not significantly affected by
enhanced UV-B radiation (Table I). However, both
in June and in September total plant mass accumula-
tion as well as total aboveground biomass and litter
mass parameters show a trend towards reduction due
to enhanced UV-B or at least a change in this direction
(Table I).
The belowground plant mass was not affected in
June, but seemed to be slightly stimulated due to en-
hanced UV-B radiation in September (Tahle I). Con-
sequently, the shoot/root ratio of the vegetation treated
with additional UV-B radiation was on average lower
than the shoot/root ratio of the vegetation in the control
plots, especially in September (Table I). As indicated
in the table, none of the above mentioned differences
are statistically significant.
Leaf area and the leaf weight measurements show
the same trends as described above for the total above-
ground plant mass of the vegetation and the ditlerent
species groups (data not shown). Treatment effects
were, however, not significant. The specific leaf area
(SLA) of the total vegetation as well as of the sepa-
rate groups was not significantly affected by the UV-B
treatment and no clear pattern could be observed (data
not shown).
Discussion
The results of this ecosystem study indicate that en-
hanced levels of UV-B radiation can lead to transient
changes in the structure of dune-grassland vegetation.
This conclusion is mainly based on the statistically
significant reduction due to enhanced UV-B in above-
ground plant mass accumulation (Figure I a) and tiller
number per unit soil area (Figure 2a) found for Calam-
44
agrostis epigeios in June. Since C. epigeios is the most
dominant species in the dune-grassland, these changes
will be important for the structure of the grassland
community as a whole. Other trends observed upon
UV-B supplementation, such as an increase in the
number of Carex arenaria tillers in June (Figure 2b)
and a decreased aboveground plant mass (total mass,
biomass and litter mass) of the plant species other than
C. epigeios and C. arena ria in September (Figure lc;
Table I) may represent an effect on vegetation struc-
ture as well, although treatment differences were not
statistically significant.
An important aspect of the vegetation structure is
the species composition. Trends in aboveground mass
partitioning between species groups (data not shown)
are similar to the above described trends observed
in absolute aboveground plant mass. However, these
treatment differences were not statistically significant.
Possibly, the observed tendencies may represent real
UV-B effects, obscured by the relatively high 'natural'
variability between vegetation samples in comparison
to pots with individual plants or species. In mixed
species stands often a very large number of replica-
tions is needed in order to detect treatment effects
of UV-B radiation on vegetation structure (see, e.g.,
Norton et al. 1999).
The different response pattern observed in June
and September might indicate seasonal variability in
the effects of UV-B enhancement. One of the factors
possibly causing this variability is nutrient availability,
which may have been higher in June than in Septem-
ber due to exhaustion of nutrients during the growing
season. Nutrient availability is known to affect plant
sensitivity to UV-B (see, e.g., Tosserams et al. 2001).
Seasonal variation in the solar spectrum and/or other
climatic factors as temperature, precipitation etc. may
also cause seasonal variation in UV-B responses (see
Gwynn-Jones et al. 1999b ). As the UV-B sensitivity
of plants may depend on the growth stage (Murali &
Teramura 1986a; Teramura et al. 1991; Musil & Wand
1994; Correia et al. 1998), differences in sensitivity
due to differences in physiological development may
also have mediated seasonal variability in treatment
effect in our study.
In accordance with most other plant-community
studies performed so far (e.g., Barnes et al. 1988; Fox
& Caldwell 1978; Norton et al. 1999), no significant
effect of UV-B enhancement on total plant mass ac-
cumulation was found. In contrast, Yuan el al. ( 1998)
observed a reduction in biomass accumulation due to
UV-B enhancement in a monoculturc of spring wheat
under field conditions. The authors argued that this
effect was at least partly due to enhanced intraspecific
competition and self-thinning, increasing the number
of dead shoots. In our study, enhanced UV-B radiation
may have affected C. epigeios tiller starvation as well.
We might speculate that the consistency in the trends
in total plant mass, aboveground plant mass, above-
ground biomass and aboveground litter mass (Table I)
indicates deleterious effects of UV-B supplementa-
tion on the productivity of the vegetation. Logically,
apart from possible effects on starvation, enhanced
UV-B radiation may also have reduced the number of
C. epigeios tillers by affecting the amount of tillers
produced.
In theory, the observed reduction of aboveground
C. epigeios mass due to enhanced UV-B radiation may
have been caused directly by damaging effects of UV-
B radiation on C. epigeios physiology. However, in
previous experiments direct damage caused by UV-
B enhancement could not he detected in this species,
even under relatively low PAR levels (Tosserams &
Rozema 1995; Tosserams et al. 1997). In fact, most
studies on agricultural and natural ecosystems fail to
show any damaging effects; alterations in morphologi-
cal characteristics and/or enhanced production of UV-
B absorbing secondary metabolites (e.g., Ziska et al.
1993; Dillenburg eta/. 1995; Fiscus & Booker 1995)
are, however, often found. Photo-morphogenic effects
of UV-B may influence the competitive balance he-
tween species, which could lead to changes in canopy
structure, light interception and calculated stand pho-
tosynthesis (Barnes et al. 1996). Barnes et al. (1988)
showed that interspecific variation in length-growth
responses to enhanced UV-B radiation could alter the
competitive balance between wheat and wild oat in the
field. Length measurements of tillers grown in similar
grassland vegetation as examined here may indicate a
reduction in length growth due to UV-B enhancement
for C. epigeios (Rozema et al. 1999a; Oudejans, un-
published results). Possibly, reduced length-growth of
C. epigeios may have changed competition for light in
favor of one or more species in this dense vegetation,
of which length-growth was not, or less, impaired by
the treatment.
In contrast, Fox & Caldwell ( 1978) argued that
shifts in competitive advantage might be due to UV-B
effects on plant density, as alterations in plant density
may also influence competition for light. Because the
number of C. epigeios tillers was significantly reduced
in June, plant density may have been a major factor in

the reduction of aboveground C. epigeios plant mass
in this period.
A common response to UV-B enhancement is
increased tillering of monocotyledons. for instance
Deschampsia antarctica (Rozema el a!. 200 I). and
increased branching of dicotyledons, for instance faha
bean (Meijkamp et al. 2001 ). However, in this paper
we report a reduced tiller number of C. epigeios due
to UV-B enhancement. This finding contradicts with
greenhouse experiments with individual C. epigeios
plants in pots as well: no effect of UV-B enhancement
on tiller number was found (Tosserams & Rozema
1995; Tosserams et a!. 1997). Agricultural grass
species as spring wheat (Yuan eta!. 1998) and rice
(Teramura et a!. 1991) show a similar contradiction
between results of greenhouse and field studies.
Discrepancies between the results of pot-plant
studies and the results of ecosystem-level experiments
as mentioned above may be due to many factors, such
as differences in climatic circumstances, spectral ir-
radiance, growth conditions, duration of exposure to
UV-B and growth stage. These factors can greatly
modify UV-B responsiveness in plants (Teramura eta/.
1991; Gwynn-Jones eta/. 1999b). Furthermore, re-
duction of C. epigeios growth may also have been
mediated by community-level interactions occurring
in the dune-grassland vegetation under consideration.
For instance, Gwynn-Jones et a!. ( 1999a) stated that
the exposure of a specific plant species to UV-B
might be influenced by the phenology, productivity
and structure of other species within the canopy. Ten-
tatively, the contrasting effects of UV- B enhancement
on the number of tillers of C. epigeios (Figure 2a)
compared to C. arena ria (Figure 2b) in June may be
taken to indicate that competitive interaction played a
role in our study.
Interactions between trophic levels are also consid-
ered to contribute to ecosystem-level UV-B responses
(e.g., Rozema et al. 1997b, 1999b; Paul eta!. 1999).
For example. negative effects of enhanced UV-B on
plant root infestation with YAM fungi, which are
mostly beneficial for plant growth. seemed present
for C. epigeios in similar vegetation as described here
(Van de Staaij et al. 200 I). Negative effects of UV-
B on mutualistic fungi may have been related to the
negative etlects on C. epigeios observed in this study.
In conclusion, the negative effects of UV-B en-
hancement on C. epigeios clearly indicate that vege-
tation structure has been affected in this study. More-
over, UV-B effects seem to be seasonally dependent:
different trends were observed in June and September
45
(Figures I and 2; Table I). However, the mecha-
nisms behind these effects have not been revealed
by this study. It might be argued that direct damage,
photo-morphogenic changes and indirect UV-B ef-
fects on ecosystem processes and interactions may all
have more or less accounted for the observed effects
and trends. Examining different physiological. chemi-
cal and morphological responses in similar ecosystem
studies may provide more information about UV-B
effects on plants in their natural environment. Apart
from plant responses alone, research of long-term UV-
B etlects on ecosystem processes as litter degradation
and other trophic interactions may help in providing a
realistic scenario of the consequences of stratospheric
ozone depletion for natural ecosystems.
Acknowledgements
We wish to thank all people who contributed to this
paper. Special thanks are due to Hans Nelissen, who
helped with the design and build-up of the lamp sys-
tem. We are also grateful for the large amount of field
data gathered by Irma Walraven. Adri van Beem and
Martin Stroetenga. Extra thanks are due to Adri van
Beem for the many hours of maintenance he spent
in the field. In addition, we would like to thank the
mechanics and electronics departments of the Vrije
Universiteit for technical assistance, especially Rob
Stoevelaar, Flip de Kriek, Peter Maas, Daan van
Marum and Martien van Vilsteren. The efforts of
Prof. Dr M. A. P. A. Aerts and Prof. Dr W. H. 0.
Ernst, who commented on the manuseript(s), were
also greatly appreciated. Furthermore, we would like
to thank Dr M. E. Keizer for the many useful sugges-
tions regarding structure and grammatical correctness
of the text. The research for this paper was part of
the UVECOS research consortium. financed by the
EC (contract ENV4-CT96-0208), which is gratefully
acknowledged. Finally, we thank the PWN for grant-
ing us access to the dune area and for permitting us to
carry out our experiments on their territory.
References
Balakumar. T.. Hani Babu Vincent. V. & Paliwal. K. 1993. On the
interaction of UV-B radiation (280-:J 15 nm) with water stress in
crop plant>. Physiol. Plant. 87: 217-222.
Ballarc. C. L.. Barnes. P. W. & Hint. S. D. 1995. Inhibition of
hypocotyl elongation by ultraviolct-B radiation in de-etiolated
tomato seedlings. 1. The photoreceptor. Physiol. Plant. 93: 584-
592.
46
Barnes, P. W., Jordan, P. W., Gold, W. G., Flint, S. D. & Caldwell,
M. M. 1988. Competition, morphology and canopy structure
in wheat (Triticum ae>tivum L.) and wild oat (Avena jiauna
L.) exposed to enhanced ultraviolet-B radiation. Funct. Ecol. 2:
319-330.
Barnes, P. W., Balian~, C. L. & Caldwell, M. M. 1996. Photomor-
phogenic effects of UV-B radiation on plants: consequences for
light competition. J. Plant Physiol. 148: 15-20.
Biggs, R. H., Kossuth, S. V. & Teramura, A. H. 1981. Response of
19 cultivars of soybean to ultraviolet-B irradiance. Physiol. Plant.
53: 19-26.
Bjorn, L. 0. & Murphy, T. M. 1985. Computer calculations of solar
ultraviolet radiation at ground level. Physiol. Veg. 23: 555-561.
Caldwell, M. M. 1971. Solar UV irradiation and the growth and de-
velopment of higher plants. Pp. 131-177. In: Giese, A. C. (cd.),
Photophysiology, Vol. 6. Academic Press, New York.
Caldwell, M. M. 1997. Alterations in competitive balance. Pp.
305-315. In: Lumsden, P. J. (ed.), Plants and UV-B: Re-
sponses to Environmental Change. Cambridge University Press,
Cambridge.
Caldwell, M. M., Tcramura, A. H., Tcvini, M., Bornman, J. F.,
Bjorn, L. 0. & Kulandaivelu, G. 1995. Effects of increased solar
ultraviolet radiation on terrestrial plants. Ambia 24: 166-173.
Caldwell, M. M., Bjorn, L. 0 .. Bornman, J. F., Flint, S. D., Ku-
landaivelu, G., Teramura, A. H. & Tevini, M. 1998. Effects of
increased solar ultraviolet radiation on terrestrial ecosystems. J.
Photochem. Photobiol. B46: 40-52.
Correia, C. M., Areal, E. L. V., Torres-Pereira, M. S. & Torres-
Pereira, J. M. G. 1998. Intraspecific variation in sensitivity to
ultraviolet-B radiation in maize grown under field conditions. I.
Growth and morphological aspects. Field Crop Res. 59: 81-89.
Dai, Q., Peng, S., Chavez, A. Q. & Vergara, B. S. 1994. Intraspe-
cific responses of 188 rice cultivars to enhanced UVB radiation.
Environ. Exp. Bot. 34: 433-442.
Day. T. A., Vogelmann, T. C. & DeLucia, E. H. 1992. Are some
plant life forms more effective than others in screening out
ultraviolet-B radiation° Oecologia 83:513-519.
Deckmynn, G. & Impens, 1. 1997. Combined effects of enhanced
UV-B radiation and nitrogen deficiency on the growth, composi-
tion and photosynthesis of rye (SeGJ/e cerea/e). Plant Ecol. 128:
235-240.
Dillenburg, L. R., Sullivan, J. H. & Teramura, A. H. 1995. Leaf
expansion and development of photosynthetic capacity and pig-
ments in Liquidambar styracijfua (Hamamelidaceae)- effects of
UV-B radiation. Am. J. Bot. 82: 878-885.
Drilias, P., Karabuurniotis, G., Levizou, E., Nikolopoulos, D.,
Petropoulou, Y. & Manetas, Y. 1997. The effects of enhanced
UV-B radiation on the Mediterranean evergreen sclerophyll Ner-
ium oleander depend on the extent of summer precipitation.
Austr. J. Plant Physic!. 24: 301-306.
Ernst, W. H. 0., Van de Staaij, J. W. M. & Nelissen, H. J. M. 1997.
Reaction of savanna plants from Botswana on UV-B radiation.
Plant Ecol. 128: 162-170.
Fiscus, E. L. & Booker, F. L. 1995. Is increased UV-B a threat to
crop photosynthesis and productivity? Photosynl. Res. 43: 81-
92.
Fox, F. M. & Caldwell, M. M. 1978. Competitive interaction in plant
populations exposed to supplementary ultraviolet-B radiation.
Oecologia 36: 173-190.
Gehrke, C., Johanson, U., Callaghan, T. V., Chadwick, D. &
Robinson, C. H. 1995. The impact of enhanced ultraviolet-
B radiation on litter quality and decomposition processes in
Vaccinium leaves tfom the Subarctic. Oikos 72: 213-222.
Gwynn-Jones, D. & Johanson, U. 1996. Growth and pigment pro-
duction in two subarctic grass species under four different UV-B
radiation levels. Physiol. Plant. 97: 701-707.
Gwynn-Jones, D., Lee, J. A., Callaghan, T.V. & Sonesson, M. 1997.
Etlects of enhanced UV-B radiation and elevated carbon dioxide
concentrations on subarctic forest heath ecosystems. Plant Ecol.
128: 242-249.
Gwynn-Jones, D., Lee, J., Johamon, U., Phoenix, G., Callaghan, T.
& Sonesson, M. 1999a. The response of plant functional types
to enhanced UV-B radiation. Pp. 173-185. In: Ro7.ema, J. (ed.),
Stratospheric ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden.
Gwynn-Jones, D., Johanson, U., Phoenix, G., Gehrke, C.,
Callaghan, T., Bjorn, L. 0., Sonesson, M. & Lee, J. 1999b. UV-
B impacts and interactions with other co-occuring variables of
environmental change: an arctic perspective. Pp. 187-20 I. In:
Rozema, J. (ed.), Stratospheric Ozone Depletion; the Effects of
Enhanced UV-B Radiation on Terrestrial Ecosystems. Backhuys
Publishers, Leiden.
Hoque, E. & Remus, G. 1999. Natural UV-screening mechanisms
in Norway spruce. Photochem. Photobiol. 69: 177-192.
Jansen, M.A. K., Gaba, V. & Greenberg, B. M. 1998. Higher plants
and UV-B radiation: balancing damage, repair and acclimation.
Trends Plant Sci. 3: 131-135.
Klironomos, J. N. & Allen, F. N. 1995. UV-B mediated changes
on below-ground communities associated with the roots of Acer
sacharum. Funct. Ecol. 9: 923-930.
Krupa, S. V. & Kickert, R. N. 1989. The greenhouse effect: Im-
pacts of ultraviolet-B (UV-B) radiation, carbon dioxide (C02)
and ozoe (03) on vegetation. Environ. Pollu. 61:263-393.
Manetas, Y., Petropoulou, Y., Stamatakis, K., Nikolopoulos, D.,
Levizou, E., Psaras, G. & Karabourniotis, D. 1997. Beneficial
effects of enhanced UV-B radiation under field conditions: im-
provement of needle water relations and survival capacity of
Pinus pinea L. seedlings during the dry Mediterranean summer.
Plant Ecol. 128: 100-108.
McCloud, E. S. & Berenbaum, M. R. 1994. Stratospheric ozone
depletion and plant-insect interactions: Effects ofUV-B radiation
on foliage quality of Citri.1· jamhhiri for Trichoplusia ni . .1. Chem.
Ecol. 20: 525-539.
McLeod, A. R. 1997. Outdoor supplementation systems for stud-
ies of the effects of increased UV-B radiation. Pp. 78-92. In:
Rozema, J., Gieskes, W. W. C., Geijn, S. C. and de Boois,
H. (eds), UV-B and Biosphere. Kluwer Academic Publishers,
Dordrecht, The Netherlands.
Meijkamp, B .. Aerts, R., Van de Staaij, J., Tosserarns, M., Ernst, W.
& Rozema, J. 1999. Effects of UV-B on secondary metabolites in
plants. Pp. 71-99. In: Rozema, J. (ed.), Stratospheric Ozone De-
pletion; the Etlects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden.
Meijkamp, B. B., Doodeman, G. & Rozema, J. 2001. The response
of Vicia faba to enhanced UV-B radiation under low and near
ambient PAR levels. Plant Ecol. 154: 135-146 (this volume).
Mirecki, R. M. & Teramura, A. H. 1984. Effects of ultraviolet-
B radiation on soybean. V. The dependence of plant sensitivity
on the photosynthetic photon flux density during and after leaf
expansion. Plant Physiol. 74: 475-480.
Murali, N. S. & Teramura, A. H. 1985. Effects of UV-B irradiance
on soybean. VL Influence of phosphorus nutrition on growth and
flavonoid content. Physiol. Plant. 63:413-416.
Murali, N. S. & Teramura, A. H. 1986a. Effects of supplemen-
tal ultraviolet-B radiation on the growth and physiology of
field-grown soybean. Environ. Exp. Bot. 26: 233-242.

Murali, N. S. & Teramura, A. H. 1986b. Effectiveness of UV-B
radiation on the growth and physiology of field-grown soybean
modified by water stress. Photochem. Photobiol. 44: 215-219.
Murali, N. S. & Teramura, A. H. 1987. Insensitivity of soy-
bean photosynthesis to ultraviolet-B radiation under phosphorus
deficiency. J. Plant Nut. 10:501-515.
Musil, C. F. 1995. Differential effects of elevated ultraviolet-B
radiation on the photochemical and reproductive performances
of dicotyledonous and monocotyledonous arid-environment
ephemerals. Plant Cell Environ. 18: 844-854.
Musil, C. F. 1996. Accumulated effect of elevated ultraviolet-B
over multiple generations of the arid-environment annual Di-
morphotheca sinuata DC. (Asteraceae). Plant Cell Environ. 19:
1017-1027.
Musil, C. F. & Wand, S. J. E. 1994. Differential stimulation of
an arid-environment winter ephemeral Dimorphoteca p!ul'ialis
(L.) Moench by ultraviolet-B radiation under nutrient limitation.
Plant Cell Environ. 17: 245-255.
Newsham, K. K., McLeod, A. R., Greenslade, P. D. & Emmett.
B. A. 1996. Appropriate controls in outdoor UV-B supplementa-
tion experiments. Global Change Bioi. 2: 319-324.
Newsham, K. K., Greenslade, P. D., Kennedy. V. H. & McLeod.
A. R. 1999. Elevated UV-B radiation incident on Quercus robur
leaf canopies enhances decomposition of resulting leaf litter in
soil. Global Change Bioi. 5: 403-409.
Norton, L. R., McLeod, A. R., Greenslade, P. D., Firbank. L. G.
& Watkinson, A. R. 1999. Elevated UV-B radiation effects on
experimental grassland communities. Global Change Bioi. 5:
601-608.
Paul, N. 1997. Interactions between trophic levels. Pp. 317-339. In:
Lumsden, P. J. (ed. ), Plants and UV-B Responses to Environmen-
tal Change. Cambridge University Press, Cambridge.
Paul, N .. Callaghan, T., Moody, S .. Gwynn-Jones, D., Johan-
son, U. & Gehrke, C. 1999. UV-B impacts on decomposition
and biochemical cycling. Pp. 117-133. In: Rozema, J. (ed.),
Stratospheric Ozone Depletion; the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers.
Lei den.
Ros, J. & Tevini, M. 1995. Interaction of UV radiation and IAA
during growth of seedlings and hypocotyl segments of sunflower.
J. Plant Physiol. 146: 295-302.
Rousseaux, M. C., Balian!, C. L., Giordano, C. V., Scoplel. A. L..
Zima, A. M., Szwarcberg-Bracchitta, M., Searles, P. S., Cald-
well, M. M. & Diaz, S. B. 1999. Ozone depletion and UVB
radiation: Impact on plant DNA damage in southern South
America. PNAS 96?
Rozema, J., Staaij, J. van de & Tosserams, M. 1997a. Effects
of UV-B radiation on plants from agro-ecosystems and nat-
ural ecosystems. Pp. 213-232. In: Lumsden. P. J. (ed.). Plants
and UV-B: Responses to Environmental Change. Cambridge
University Press, Cambridge.
Rozema, J., Tosserams, M .. Nelisscn, H. J. M .. Heerwaarden,
L. van, Brockman, R. A. & Flierman, N. 1997b. Stratospheric
ozone reduction and ecosystem processes: enhanced UV-B radi-
ation affects chemical quality and decomposition of leaves of the
dune grassland species Calamagrostis epigjos. Plant Ecol. 128:
284-295.
Rozema, J., Van de Staaij, J. & Tosserams, M. 1997c. UV-B as an
environmental factor in plant life: stress and regulation. Trends
Ecol. Evol. 12: 22-28.
Rozema, J., Oudejans, A., Houter, N., Schoonheim, N .. Walraven,
1., Van 't Klooster, C., Van de Staaij. J ., Tosserams, M., De
Bakker, N., Van Beem, A., Stroetenga. M., Brockman, R .. Van
Heerwaarden, L .. Nelissen, H. & Aerts, R. 1999a. Responses of
47
plants from a dune grassland ecosystem in the Netherlands to
solar UV-B: UV-B filtration and supplementation experiments.
Pp. 203-225. In: Rozema, J. (ed.), Stratospheric Ozone De-
pletion: the Effects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden.
Rozema, J. 1999b. UV-B radiation and terrestrial ecosystems:
processes, structure and feed-back loops. Pp. 101-114. In:
Rozema, J. (ed.), Stratospheric Ozone Depletion; the Effects of
Enhanced UV-B Radiation on Terrestrial Ecosystems. Backhuys
Publishers, Leiden.
Rozema, H.. Brockman, R.. Lud, D., Huiskes, A., Moerdijk.
T.. De Bakker, N .. Meijkamp, B. B. & Van Beem, A. 2001.
Consequences of depletion of stratospheric ozone for terrestrial
antarctic ecosystems: the response of Desclwmpsia antarctica
to enhanced UV-B radiation in a controlled environment. Plant
Ecol. 154: 101-115 (this volume).
Schnitzler. J. P.. Jungblut, T. P.. Feicht. C .. Kofferlcin. M .. Lange-
bartels. C., Heller, W. & Sandermann. H. 1997. UV-B induction
of flavonoid biosynthesis in Scotch pine (Pinus .\Tlvestris L.)
seedlings. Trees Struct. Funct. II: 162-168.
Stapleton, A. E. 1992. Ultraviolet radiation and plants: burning
questions. Plant Cell 4: 1353-1358.
Stapleton. A. E., Thornber. C. S. & Walbot, V. 1997. UV-B com-
ponent of sunlight causes measurahle damage in field-grown
maize (Zea mays L.): developemental and cellular hetrogeneity
of damage and repair. Plant Cell Environ 20: 279-290.
Strict, A. & Porra, R. J. 1992. Alterations in pigment content in
leaves of Pisum sativum atier exposure to supplementary UV-B.
Plant Cell Physiol. 33: 1015-1023.
Sullivan, J. H. 1997. Effects of increasing UV-B radiation and at-
mospheric C02 on photosynthesis and growth: Implications for
terrestrial ecosystems. Plant Ecol. 128: 194--206.
Sullivan. J. & Rozema. J. 1999. UV-B etl"ects on terrestrial plant
growth and photosynthesis. Pp. 39-57. In: Rozema. J. (ed.).
Stratospheric Ozone Depletion: the Etl"ects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden.
Sullivan, J. H. & Teramura, A. H. 1992. The effects ofultraviolet-B
radiation on loblolly pine. I. Growth, photosynthesis and pigment
production in greenhouse-grown seedlings. Trees Struct. Funct.
6: 115-120.
Sullivan, J. H .• Teramura, A. H. & Ziska. L. H. 1992. Variation in
UV-B sensitivity in plants from a 3.000-m elevational gradient in
Hawaii. Am. J. Bot. 79: 737-743.
Teramura. A. H. 1983. Effects of ultraviolet-B radiation on the
growth and yield of crop plants. Physiol. Plant. 58: 415-427.
Teramura. A. H., Ziska, L. H. & Sztein. A. E. 1991. Changes in
growth and photosynthetic capacity of rice with increased UV-B
radiation. Physiol. Plant. 83: 373-380.
Tevini, M. & Teramura, A. H. 1989. UV-B etl"ects on terrestrial
plants. Photochem. Photobiol. 50: 479-487.
Tevini. M .. Iwanzic. W. & Thoma. U. 1981. Some effects of
enhanced UV-B irradiance on the growth and composition of
plants. Planta !53: 388-394.
Tevini. M., Iwanzik. W. & Teramura, A. H. 1983. Effects of UV-
B radiation on plants during mild water stress. II. Effects on
growth. protein and flavonoid content. Z. Pflanzenphysiol. 109:
435-448.
Tevini. M .. Braun, J. & Fieser, F. 1991. The protective function
of the epidermal layer of rice seedlings against ultraviolet-B
radiation. Photochem. Photobiol. 53: 329-333.
Tosserams, M. & Rozema, J. 1995. Effects of ultraviolet-B radiation
(UV-B) on growth and physiology of the dune grassland species
Calamagrostis epigeios. Environ. Pollut. 89: 209-214.
48
Tosserams, M., Pais de Sa, A. & Rozema, J. 1996. The effect of
solar LJV radiation on four plant species occurring in a coastal
grassland vegetation in the Netherlands. Physiol. Plant. 97:731-
739.
Tosserams, M., Magendans, G. W. H. & Rozema, J. 1997. Differ-
ential effects of elevated ultraviolet-B radiation on plant species
from a dune grassland ecosystem. Plant Ecol. 128: 266-283.
Tosserams, M., Smet, J., Magendans, E. & Rozema, .1. 2001.
Nutrient availability influences UV-Fl sensitivity of Plantago
/anceo/nta. Plant Ecol. 154: 157-168 (this volume).
Van de Staaij. J. W. M. 1994. The response of the cereals maize
(cv LG II) and wheat (cv Obelisk) to enhanced levels of UV-B
radiation; a comparison of a glasshouse and a field experi-
ment. Pp. 55-66. In: Enhanced solar ultraviolet-B radiation:
consequences for plant growth. PhD thesis.
Van de Staaij, J., Rozema . .1. & Aerts, R. 1999. The impact of solar
UV-B radiation on mutualistic plant/micro-organism interactions
at the soil-root interface. Pp. 159-171. In: Rozema, J. (ed.),
Stratospheric Ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Baekhuys Publishers,
Lei den.
Van de Staaij, J., Rozema. J. van Beem, J. & Aerts, R. 2001.
Increased solar UV-B radiation may reduce infection by arbuscu-
lar mycorrhizal fungi (AMF) in dune grassland plants: evidence
from five years of field exposure. Plant Ecol. 154: 169-177 (this
volume).
Warner, C. W. & Caldwell, M. M. 1983. Intluence of photon
!lux density in the 400-700 nm waveband of inhibition of
photosynthesis by UV-8 (280-320 nm) irradiation in soybean
leaves: seperation of indirect and immediate effects. Photochem.
Photobiol. 38:341-346.
Yuan, L., Ming. Y & Xunlin, W. 1998. Effects of enhanced
ultraviolet-8 radiation on crop structure, growth and yield com-
ponents of spring wheat under field conditions. Field Crops Res.
57: 253-263.
Ziska, L. H., Teramura, A. H., Sullivan, J. H. & McCoy, A.
1993. Influence of ultaviolet-B (UV-B) radiation on photosyn-
thetic and growth characteristics in field-grown cassava (Manihot
esculentum Crantz). Plant Cell Environ. 16: 73-79.
Section 2: Terrestrial Plants and Terrestrial Ecosystems

The spotted leaves of l'u/monaria officina /is. (Photograph by A . Gaberscik)

 Plant Ecologv 154: 51-56, 2001.
51
© 2001 Kluwer Academic Publishers.

The influence of enhanced UV-B radiation on the spring geophyte

Pulmonaria of.ficinalis

Alenka Gaberscikl.2, Mateja Novak 1, Tadeja Trost 1, Zdenka Mazej 1, Mateja Germ 1 & Lars-
OlofBjom3
1National Institute of Biology & 2 Department of Biology, Biotechnica/ Faculty, University 1J{ Ljubljana,
Vecna pot I 1 I, I 00 I Ljubljana, Slovenia; 3 Lund Universit>; Department of Plant Physiology, Box 117, SE-221 00
Lund, Sweden

Key words: Photochemical efficiency of PSll, Pigments, Protective compounds, Radiation. Understorey plant,
UV-B

Abstract
Pulmonaria officina/is is an understorey spring geophyte, which starts its vegetative period before full foliation
of the tree storey. During its early growth phase it is exposed to full solar radiation, therefore the enhanced UV-
B radiation could present a threat to this species. An outdoor experiment in which potted plants were exposed
to below ambient, ambient, and above ambient (corresponding to 17% ozone reduction) UV-B radiation, was
conducted in order to evaluate the radiation effects. The amount of photosynthetic pigments and photochemical
efficiency of PSH were not affected, but the amount of UV-B absorbing compounds was lower in plants grown
under reduced UV-B. This change was measurable after only fourteen days in reproductive shoots, while in the
vegetative shoots, it was not detectable until after three months. The leaves of P. officina/is arc variegated and the
light green spots became less transparent to PAR under enhanced UV-B. The results reveal that under simulated
17% ozone depletion the harmful effects of UV- B on the measured parameters were negligible.

Introduction mona ria iJfficinalis thrives in understoreys of decidu-

The thinning of the ozone layer and the concomi-
tant increase in UV-B radiation on the Earth's surface
may influence the biota, because of the absorption
of UV-B radiation by important biomolecules. Plants
respond to UV-8 radiation in a species-specific way
(Caldwell et al. 1994 ), balancing the potential dam-
age and the induction of protective mechanisms. The
most frequent response to enhanced UV-B radiation
is a production of various secondary substances, pri-
marily UV-B absorbing compounds (Day et al. 1992,
1996), and morphogenetic changes (Greenberg et al.
1996). Changes in the secondary metabolism of plants
could alter the interspecific relations among organisms
by influencing the throughflow of energy and cycling
of nutrients in the ecosystem.
Until now most investigations have been carried
out with higher plants thriving in open places. Pul-
ous forests. Its vegetative period starts early in spring
and it efficiently uses a brief period before leaves de-
velop on trees, exhibiting a relatively high activity.
This time is a temporal window for a UV-8 impact
because it coincides with the reproduction of P. offici-
na/is and plants seem to be more vulnerable to UV-8
in the reproductive phase (Tevini & Teramura 1989).
The exposure to full sun under the enhanced UV-8
radiation could present a threat to this species. An
outdoor experiment with supplemental UV-B radiation
simulating a 17% ozone reduction was carried out in
order to determine the effects. The changes in pho-
tochemical efficiency of PSII as well as the amount
of photosynthetic pigments and UV-B absorbing com-
pounds were monitored.
52
Materials and methods
Plant material
Rhizomes of P. ofjicinalis were collected in the for-
est ncar the city of Ljubljana (46°04' N, 14°31' E)
and planted into the pots. In the early vegetative sea-
son, i.e., by the end of March, they were exposed to
three UV treatments in the Botanical garden in Ljubl-
jana. The experiment lasted three months. During the
experiment plants were watered regularly.
UV-B simulation
A UV-B supplementing system as described by Bjorn
& Teramura (1993) was used. Three different treat-
ments were applied: (1) simulation of 17% ozone
depletion (UV-B( +))using Q-Panel UV-B 313 lamps,
filtered with cellulose diacetate filters, which cut out
UV-C, (2) reduced level of UV-B radiation (UV-B(-))
using Mylar foil, which cuts out tradiation below
about 318 nm wavelength (Gehrke et al. 1996) and
(3) control: ambient radiation with Q-Panel UV-B
313 lamps filtered with Mylar foil, to include the ef-
fects of the UV-A radiation. The lamps were timer
controlled. The times for simulating 17% ozone de-
pletion were calculated and adjusted weekly using the
programs made by Bjorn & Murphy (1985), taking
into account the generalized plant action spectrum of
Caldwell (1968). UV-B biologically effective doses
(UV-BsE) are presented in Figure 1. Ambient UV-
B radiation was measured using the European Light
Dosimeter Network (ELDONET) measuring system
which also monitors UV-A radiation and PAR. The
data obtained from the system represent unweighted
UV-B, which was then converted to plant weighted
UV-B using a modification of the program of Bjorn
& Murphy ( 1985).
Chlorophyll fluorescence measurements
The potential and actual photochemical efficiencies of
PSII were measured using fluorometer OS-500 (Opti-
Science, USA). The potential or maximal photochem-
ical efficiency of PSII was evaluated as Fvf Fm ratio.
Before the measurements, plants were kept in cuvettes
in the dark for 15 minutes at ambient temperatures.
Photochemical efficiency of PSII under actual light
conditions is the photosynthetic quantum yield (<!>),
which is described by the equation <P = (F;n- F)/ F,;,.
In our experiment yield was measured under full light
conditions (from 1500 to 2000 f.imol m- 2 s- 1) and
under prevailing ambient temperatures. The measure-
ments were carried out on 6 randomly chosen plants in
the third week and after three months.
Photosynthetic pigments and UV-B absorbing
compounds
Carotenoids and chlorophylls a and b were deter-
mined at different stages of development following the
procedure described by Jeffery & Humphrey (1975).
The basic procedure for UV-B absorbing compounds
followed the method described by Caldwell (1968).
UV-B absorbing compounds were extracted from fresh
homogenised plant material with methanol : distilled
water: HCl = 79:20: 1. Crude extracts were cen-
trifuged and the absorbance of supernatants was mea-
sured in the range from 280-320 nm. The absorbance
values were integrated and corrected for the weight of
the sample (about 0.1 g DW).
Leaf transmittance
The penetration of PAR through leaves was measured
in dark green parts and in light green spots using the
LI-1 000 (Ll-COR, USA) quantum sensor.
Statistical analyses
The significance of the differences among treatments
was tested by Student's two-tail t-test.
Results
We followed the changes during two growth phases of
P. officina/is in the period when plants were exposed
to fu!J sun. In the first phase at the beginning of the
experiment, plants developed reproductive shoots, and
simultaneously vegetative rosettes developed. The re-
sults of the measurements of photochemical efficiency
and yield are presented in Figure 2. Only a small neg-
ative effect on yield was found in vegetative shoots
under enhanced UV-B radiation.
By the end of the experiment, the leaves were
examined for morphological features, but there was
no evidence of effects of UV-B treatment on tri-
chome density or length. or on specific leaf mass
(Table 1). The highest trichome density was found
in control plants. The only significant effect of UV-B
radiation was an increase in the content UV- B absorb-
ing compounds under control and enhanced UV-B in
comparison to reduced UV-B (Figure 3).
 53

10

9
~
'0
·a
.....
8

:'"':: 7

......
l:l
0 6

...."' 5 ~~
"'.,
pbn ent
H 4
IQ
' 3
~
2

April May June

Figure I. Daily UV-BsE radiation doses for P. officina/is during treatment (supplement- UV-BBE radiation per day corresponding 17ck of
ozone depletion, natural- daily measurements by ELDONET instrument converted into UV-BBE).

Tahle I. Some morphological properties of the leaves of P. officina/is under different

treatments at the end of the experiment (SE: standard error. n = 16).

Treatment Leaf No. of SE Trichome SE Speci~c leaf SF.

surface trichomes length weight
mm - g dm - 2
_)
flln

UV-B(-) Abaxial 1.68 0.11 849 37 0.502 0.026

Adaxial 1.89 0.15 615 26
Control Abaxial 2.39 0.11 709 19 0.441 0.026
Adaxial 2.42 0.12 573 16
UV-B(+J Abaxial 1.68 0.15 719 32 0.465 0.040
Adaxial 1.47 012 555 14

The leaves of P. officina/is like many understorey Discussion

plants are variegated. The dark green lamina is spot-
ted with light green spots. Chlorophyll a and h,
carotenoids and UV-B absorbing compounds were
very similar in dark and light green areas, even though
the total amount of chlorophyll was somewhat lower
in light green spots (Figure 4). Figure 5 reveals no
differences between the dark and light areas with re-
spect to UV-B absorbing pigments. The comparison of
different treatments revealed slight but nonsignificant
differences. In spite of that, the light green spots be-
came less transparent for PAR under enhanced UV-B
conditions (Figure 6).
The growth of P. officina/is has two distinct phases. In
the first phase, which occurs early in spring when the
canopy is still open , plants develop the reproductive
shoots. When the reproduction period is concluded,
the intensive growth of the vegetative rosette starts,
which enlarges the assimilation area (Masarovicova
& Elias 1980). The photosynthetic activity of newly
grown leaves during the year supports the reproduc-
tion potential in the following year. Increased UV-B
therefore influence the success of this species primar-
ily during early spring when the plants are exposed to
full solar radiation.
The measurements of photochemical efficiency of
PSII do not demonstrate only damage to PSII , but they
54

0,9 0,9
Cau1is a Rose~ a Rosette
0.8
a a a a . 0,8
a a
0.7 a 0,7
a a
0,6 a a a 0,6
b
E a a
Lt. 0,5 I I 0,5 "C
'Lt.> 0.4
Ql
;;::
0.4
0,3 0,3
0,2 02
0,1 0,1
0 ~

m
0
~0
.
m
~

m
e"if :!:
m
~
m
0
~
.!,.
m
0

~ u >
::I
>
::)
0
u >
::)
>
::I
0
u >
::I

In the third v,.oeek After three rrx:>nths

Figure 2. Photochemical efficiency ofPSII (grey column) and yield (white column) measured in the leaves of P (){ficinalis in the 3rd week and
after three months of treatment (vertical bars represent the standard error (n = 7- 8), significance of the differences among treatments, :::: 0.05).

1800
Caulis Ro~tte Ro~tte Ro~tte Ro~tte Rosette b
1600
Vl
"C a b
c: bi a Ia a a
::J 1400
a a
0
a. a a
b i a a~
E
o ~
1200
r+ a a
u ~
0.0
.5 ::I
·c: 1000
f
.D....: a
1-. Cll
0 1-. 800
Vl ~

.D
ell 600
Ill
>
::J 400

200

0
3 5 7 11 12
Week
Figure 3. UV· B absorbing compounds in the leaves of P (){ficinalh during the treatment fro m the beginning of April to the end of June (white -
UV· B (- ), light gray - control, dark gray - UY· B (+), vertical bars represent the standard error (n = 4 ), significance of the differences among
treatments :::: 0.05).
 55

5 5
Spot Dark green Spot Dark green Spot Dark green
a an area area area
a
4 a 4
a
~ I ~
0 a a
Q
00
I a a 00
' a 30:o
00
_§_
3
b
I a s
!J)
;;., a I :2
.I; 0
0.. lb
0 2 a b 2 ~
I..
b b 0'--
0
£ co
u u

0 0
lN-8 (-) Cor1UOI lN-8{-tt
ll·eatrrent
Figure 4. The amount of chlorophyll a (while) and b (lighl grny) and carolcnoids (dark grny) in ligh1 green -pols and dark green pans of 1he
leaves of P. afficinalis ill Ihe end of cxperimcm (vertical har' represenl 1he slandard error (11 = 7-8). s ignificance of 1he difference among
1rea1mcn1 _ ~ 0.05 ).

72((X)

§ b b
, ...
r--
b .--- ,--.- 6
g 1500 b
r'-

.
a .---
" 5 00

1
§ l (XX)
a
r---
r+ l
~ r-
ba
r-
oc
bb
bb r-
~
:e "
r-

~ 500

"'>
::>
0
UV-B(·) Cor<rol UV-B(•l 0
Treatrr.enl IN-8(·) WB( •)
T~U1EIU
Figure 5. Tolal :rmounl of -8 absorbing compound in lighl
green spots (white) and dark green pans (grey) of lhe leav"" of P. Figure 6. PAR penelrmion lhrough lighl green spots (while) and
officina/is Ullhc end of expcrimem (venical bar repre entlhe stan- dark green pan - (grny) of Ihe leaves of P. officinoli< llfter different
=
dard crrur (11 8). ' ignilicance oflhe d ifferences among 1rem mems. treatments (vertical bar repre em the tandard err r (11 = 8). ig-
~ 0.05). nificance of 1hc diffcrenc•s compnring .<pOlS and green are«< (fir<l
sign) and among treaunem (second sig n).~ 0.05).

are also recognised as a powerful way of assessing

the effect of vario us environmental stresses (Ogren &
Oquist 1985). In our experimental plants the F,,j F111
ratio and yield showed no changes which would indi-
cate harmful infl ue nces of UV-8 radiation . Veit et al.
(1996) report that the synthesis of UY-8 absorbing
compounds prevents damage to PSII . In P officina/is
the relative amounts varied during plant development.
A slight. but not significant enhancement in compari-
son to the ambient UY-8 level was observed in June.
The comparison with plants grown under a reduced
leve l of UV-8 radiation revealed a significant increase.
Chlorophyll and carotenoid contents in leaves of
P (~fjicina lis did no t vary sig ni ficantly among differen t
treatments. It has been reported that in some cases UV-
56
B and UV-A radiation can stimulate the chlorophyll
and carotenoid synthesis (Rau & Schrott 1987).
Under elevated UV-B light green spots on leaves
became less transparent to PAR, which is probably
due to a structural change in the mesophyll, since the
pigment content was not affected.
We conclude that on the one hand enhanced UV-
B radiation corresponding to 17% ozone depletion
affects neither photochemical efficiency of PSII, nor
photosynthetic pigments in P officinalis, even though
it is exposed to full solar radiation in the period before
the tree foliation. On the other hand the content of UV-
B absorbing compounds was increased by enhanced
UV-B radiation.
Acknowledgements
This research is part of the project 'The role of UV-
B radiation on aquatic and terrestrial ecosystems: an
experimental and functional analysis of the evolution
of protective and adaptive mechanisms in plants', En-
vironment and Climate, PL 970637. The financial
support is gratefully acknowledged.
References
Bjorn, L. 0. & Murphy. T. M. 1985. Computer calculation of solar
ultraviolet radiation at ground level. Physiol. Vcg. 23: 555-561.
Bjorn, L. 0. & Teramura. A. H. 1993. Simulation of daylight Ul-
traviolet radiation and effects of ozone depletion. Pp. 41-71. In:
Young, A. R. (eel.), Environmental UV Photobiology. Plenum
Press. New York.
Caldwell. M. M. 1968. Solar ultraviolet radiation as an ecological
factor for alpine plants. Ecol.l Monogr. 38: 243-268.
Caldwell, M. M. & Flint S. D. 1994. Stratospheric ozone reduc-
tion, solar UV-B radiation and terrestrial ecosystems. Climatic
Change 28: 375-394.
Day, T. A .. Vogel mann, T. C. & Delucia, E. H. 1992. Are some plant
life forms more effective than others in screening out ultra violet
-B radiationo Oecologia 92: 513-519.
Day, T. A., Howells, B. W. & Ruhland, C. T. 1996. Changes in
growth and pigment concentrations with leaf age in pea under
modulated UV-B radiation field treatments. Plant Cell Environ.
19: 101-108.
Gehrke, C., Johanson, U., Gwynn-Jones, D.. Bjorn, L. 0.,
Callaghan, T.V. & Lee, J. A. 1996. Single and interactive effect-
sot' enhanced ultraviolet-B radiation and increased atmospheric
C02 on terrestrial and suharctic ecosystems. Ecol. Bull. 45:
192-203.
Greenberg B. M., Wilson M. 1., Gerhardt K. E. & Wilson K. E.
1996. Morphological and physiological respoonsc of Brassica
napus to ultraviolet-B radiation: Photomodification of Ribulose-
! ,5-biphosphate carboxylase/oxydase and potential acclimation
processess. J. Plant Physiol. 148: 78-86.
Jeffery, S. W. & Humphrey, G. F. 1975. New spectrophotomet-
ric equations for determining chlorophylls a, b, c I and c2
in higher plants, algae and natural phytoplankton. lliochcm.
Physiol. Pflanzen 167: 191-194.
Masarovicova, E. & Elia, P. 1980. Chlorophyll content in leaves of
plants in a Oak-Hornbeam Forest. Photosynthetica 14:580-588.
Ogren, E. & Oquist G. 1985. Effects of drought on photosynthe-
sis, chlorophyll fluorescence and photoinhibition susceptibility
in intact willow leaves. Planta 166: 380--388.
Rau, W. & Schroll, E. L. 1987. Blue light control of pigment biosyn-
thesis. Pp. 43-64. In: Senger, H. (ed.), Blue Light Responses:
Phenomena and Occurrence in Plants and Microorganisms I.
CRC Press, Boca Raton. FL.
Tevini, M. & Teramura, A. H. 1989. UV-B effects on terrestrial
plants. Photochem. Photobiol. 50: 479-487.
Veil, M., Bilger, W., Miihlbauer, T., Brummet, W. & Wlinter, K.
1996. Diurnal changes in ftavonoids. J. Plant Physiol. 148: 478-
482.
 Section 2: Terrestrial Plants and Terrestrial Ecosystems

Anthemis arvem·is L. individuals in the experimental site. Insert: a SEM view of rhe ligulate ftorer surface of plants grown
under enhanced UV-B radiation. (Photograph by Y Manetas)
 Plant Ecology 154: 59-64, 200 I.
59
© 2001 Kluwer Academic Publishers.

The growth, flower properties and demography of Anthemis arvensis

exposed to enhanced UV-B radiation

Y. Petropoulou, 0. Georgiou, G. K. Psaras & Y Manetas

Section of Plant Biology, Department of' Biology, University of' Patras, Patras 265 00, Greece (E-mail:
Y.Manetas@upatras.gr)

Key words: Anthemis arvensis, Floret surface, Flower demography. Flower properties, UV-B radiation

Abstract
The winter annual species Anthemis arvensis L. (Asteraceae) was grown for 3.5 months in the field under ambient
or ambient plus supplemental UV-B radiation, simulating a 15% ozone depletion over Patras (38.3° N, 29.1 o E).
Enhanced UV-B radiation had no effect on the methanol extractable UV-B absorbing capacity of leaves, pheno-
logical and morphometric parameters of anthesis (flowering time, anthesis duration, head life span, number of
heads per plant, number of tubular and ligulate florets per head, area per ligulate floret). Concerning the optical
properties of heads, enhanced UV-B radiation had no significant effect on the extractable absorbance of both floret
types nor on the spectral reflectance of the tubular florets. However. under UV-B supplementation the white ligulate
florets exhibited a slight, statistically significant decrease of reflectance in the visible region of the spectrum. This
may be due to structural changes of the floret surface, since microscopic examination under SEM revealed the
papillae of the adaxial epidermal cells to be swollen. The above ground dry mass measured at plant harvest was not
aflected but a significant increase in root biomass (and accordingly in root/shoot ratio) was observed. We conclude
that Anthemis arvensis is resistant against UV-B radiation damage. The possible consequences of UV-B induced
structural changes on floret epidermis are discussed.

Introduction the present investigation, we extend our observations

Many plants, belonging to various growth forms,
have been examined in the field for possible effects
of enhanced UV-B radiation on growth. In the ma-
jority of the cases the effects were subtle or non
significant (Bjorn et al. 1997; Rozema et al. 1997;
Sullivan & Rozema 1999). Mediterranean plants, in
particular, are very resistant against UV-B radiation
damage (Manetas 1999). However, the plants in-
vestigated under Mediterranean field conditions were
mainly evergreens possessing long-lived leaves for all
seasons. These leaves are rich in phenolics (Wand
1995) and phenolics arc considered as potent UV-B
screening agents, attenuating the harmful UV-B radia-
tion before reaching sensitive UV-B targets (Caldwell
et al. 1983). The few Mediterranean plants exam-
ined, having an annual above-ground part, develop and
maintain leaves during the summer and, accordingly
are considered highly adapted to UV-B radiation. In
by studying the effects of enhanced UV-B radiation
on Anthemis arvensis, a winter annual plant whose
growth occurs during the low UV-B radiation period.
Although the effects of enhanced UV-B radia-
tion on growth may be negligible. reproduction can
be considerably affected (Gwynn-Jones et al. 1997;
Grammatikopoulos et al. 1998; Musil & Wand 1994;
Stephanou & Manetas 1998). Altered flower phe-
nological patterns may perturb the necessary syn-
chronization with the activity of pollinators. Since
reflectance both in visible and UV region of the
spectrum is used by the pollinators for flower lo-
calization, a possible change in optical properties
may atlect insect behaviour (Proctor & Yeo 1996).
Changes in flower colour (tint) are also possible, since
in most of the cases the visible (anthocyanins) and
UV (flavonoids, simple phenolics) optical properties
are due to the presence of substances stemming from
60
the phenylpropanoid biosynthetic pathway, which is
induced by UV-B radiation (Rozema et al. 1997).
These possibilities have been examined only in Cistus
creticus (Stephanou et al. 2000) and the absence of
any UV-8 radiation effect was attributed to the short
(ephemeral) duration of its flowers. From this point of
view, A. arven.~is has the advantage of a rich blossom
with long-lasting inflorescences, allowing a detailed
demography and facilitating the study of flower optical
properties.
Materials and methods
Plant material and growth conditions
Anthem is arvensis L. is a winter annual species (Aster-
aceae), with head type inflorescences. The female,
periferically arranged ligulate florets are white and the
bisexual tubular florets located in the central part of
the head are yellow. The shoot is a rosette with small,
pinnatifid leaves.
Young seedlings were carefully excavated from
their natural environment around the Patras Univer-
sity campus in late January 1997. The seedlings were
placed in small plastic pots with local soil and trans-
ferred to a small open nursery in the vicinity of the
experimental site. Their growth rate was recorded up
to 15 February and 72 similar seedlings were selected
for further experimentation. Selection was based on
their leaf number and rosette diameter.
The experiment was initiated by placing the pots
under the appropriate control and UV-B frames, which
were located in a horizontal, shade free area with
no reflecting objects around. A distance of 200 em
between the frames and a 20 em, horizontal, metal
shield at the height of the outermost UV-B tubes were
enough to exclude all supplemental UV-8 radiation
from the UV-B frames to reach the plants in the con-
trol frames, as shown by preliminary measurements
of UV-B irradiance. A total of eight frames (plots)
was used, four controls (ambient UV-8 radiation) and
four UV-B (ambient plus supplemental UV-8 radia-
tion), with nine seedlings per plot. The experiment was
terminated on 2 June by harvesting the plants.
Growth conditions have been described in detail
previously (Manetas et al., 1997). In brief, UV-8 ra-
diation was given by 6 Q-Panel UV-B 313 fluorescent
tubes per frame, located 20 em apart from each other
and suspended approximately 170 em above the plant
apex. The tubes were wrapped with 0.1 mm thick cel-
lulose acetate which was 'pre-burnt' for 12 h in the
laboratory with the same tubes and replaced after 20 h
of field tube function to avoid changes in its UV-B
transmission properties. In control frames, the tubes
were replaced by white, plastic tube effigies of the
same diameter. In this way, shading under control and
UV-B frames was similar (less than I0% attenuation
of PAR on a daily basis). The total daily difference in
UV-A radiation between the control and UV-8 plots
was less than 1% (see Manetas et al., 1997, for details).
We adopted a square wave mode system to control UV-
8 radiation since resources constrained a modulated
system. The lamps were on and off in two steps cen-
tered at solar noon, with absolute spectral irradiance at
plant height (measured with a calibrated OL 752 Op-
tronic, Orlando FL spectroradiometer) weighted with
the generalized plant action spectrum normalized at
300 nm (Caldwell 1971 ). This was used in conjunc-
tion with the computer program of Bjorn & Murphy
(1985) in order to calculate the duration the lamps
should be on each day. Calculations were based on a
15% ozone depletion scenario over Patras (38.3° N,
29.1 o E) and cloud free conditions. During the whole
experimental period (lasted for I 08 days), I 0 days
were cloudy with a mean maximum photon fluence
rate of 660 11m ole m~ 2 s~ 1. Meteorological data were
taken from a Skye Mini Met (Wales, UK) located
nearby the experimental plots. Lamp duration was
modified monthly in order to follow the natural march
of ambient UV-8 change.
Plants received I 00 ml (March and early April) or
200 ml (mid-April to early-June) of water every other
day plus the natural precipitation.
Sampling techniques
Inflorescences of the same age were collected in the
morning and immediately transferred to the labora-
tory for analysis. Sampling was performed two times,
on 7th and 20th of May. On each sampling date 2
inflorescences per plot (i.e., 8 inflorescences per treat-
ment) were examined. Accordingly, during the whole
experimental period, a total of 32 inflorescences was
analysed. Since head life span decreased with time
(with the same pattern under ambient and enhanced
UV-8 radiation), care was taken to collect on each
sampling date inflorescences which had covered about
equal proportion of their life duration. Sampling for
microscopic observation under SEM was performed
once during the experimental period and a total of
18 inflorescences (9 inflorescences per treatment) was
used.
 61

Measurements
50
During the flowering period, the 'open' inflorescences ~
were visually counted and tagged daily, to study the
40
phenological parameters of anthesis. For each inflo- ~

I
0

rescence the starting date (ligulate florets unfolded and ai

u ~-·-CON
c . - 0 - UV-8.
oriented perpendicularly to the head disk) and the end- (1l 30
ing date (central tubular florets were opened) were uQ)
0::::
recorded. Q)
cr
Optical properties of inflorescences were exam- 20

ined with an Optronic (Orlando, FL) system, com-
posed of a OL 752-1 OU (200 W tungsten coiled-coil
filament) light source stabilised through an OL 65 pre-
cision current source, a Taylor type integrating sphere
(IS 1000) and an OL 752 spectroradiometer. The spec-
tral reflectance of the white ligulate and the yellow
tubular florets was measured separately. The diameter
of the exit port of the integrating sphere was adjusted
in order to accommodate either a number (usually 3-4)
of detached ligulate florets or a head (all tubular florets
still attached to the inflorescence, hut ligulate florets
removed) and with the minimum spatial distortion.
UV-B absorbing compounds were assessed from
absorption spectra of methanolic extracts. Leaves and
ligulate florets were extracted in boiling methanol:
H20:HCI (90:1:1, v:v) for 10 min (Day eta!. 1994).
In the case of tubular florets, since they contain yel-
low pigments (probably carotenoids) which could be
destroyed by acidification and boiling, the following
extraction procedure was adopted: 50 florets from each
inflorescence were put in a glass test tube and im-
mersed in liquid nitrogen for 10 min. Subsequently
4 ml of absolute methanol were added and the tubes re-
mained at 4 oc for ~ 18 h. In all cases a Shimadzu U V-
160 A (Kyoto, Japan) recording spectrophotometer
was used.
The area of ligulate florets was measured with an
ADC AM 100 leaf area meter (Hertfordshire, Eng-
land).
For scanning electron microscopic (SEM) observa-
tions, positive replicas of ligulate florets were prepared
according to Wenzel et a!. (1997): a dental impres-
sion material (Sandro-sil, Germany) was applied to the
adaxial surface of the florets. After setting, the mate-
rial was peeled off, washed, air dried and filled with
Durcupan ACM (Fluka). After polymerization the
negative moulds were peeled. The positive resin repli-
cas were mounted on stubs, sputter coated with gold
and examined using a Jeol 6300 SEM. Micrographs
were recorded as video prints.
1
10
0
~
300 400 500 600 700
wavelength, nm
Figure /. Spectral reflectance of intact ligulate florets. The values
are means from two sampling dates and four plots per treatment
(n = 8), with two individual inflorescences measured per plot
(see Materials and Methods). Rewlts l'rom the two sampling dates
were similar. Differences were statistically significant only for the
560-700 nm region.
At final harvest, each plant was divided into stems,
flowers and below ground parts and the dry mass was
measured after drying at 80 oc for 24 h to constant
weight.
Statistics
Levels of significance in the differences between treat-
ments (ambient and enhanced UV-B radiation) were
analyzed by one-way ANOVA (SPSS 8.0). Data were
not transformed. The statistical unit was the experi-
mental plot (11 = 4). Within the plots, the correspond-
ing parameters were measured on each plant and the
average value was calculated. Data for each plot were
normally distributed. In the case of ligulate florets,
differences between treatments in the number of epi-
dermal cells and the spectral reflectance were analyzed
by paired t-test.
Results
Enhanced UV-B radiation had no effect on shoot or
head dry mass, yet a significant increase in root and,
accordingly, to root/shoot ratio was evident (Table I).
Finally, supplemental UV-B radiation had no effect on
the methanol extractable UV-B absorbing capacity of
the leaves (data not shown for brevity).
62
Table 1. Effects of enhanced UV-B radiation on biomass allocation in control and
UV-B treated plants. Values are means± SO from four plots with nine plants per plot.

D. W. per plant, g CON uv %change p

Inflorescences (heads) 1.736 ± 0.167 1.814 ± 0.119

Shoot (minus heads) 2.436 ± 0.296 2.380 ± O.D25
Root 0.418 ± 0.007 0.495 ± 0.052 +18.4 0.027
Total 4.591 ± 0.415 4.688 ± 0.136
Root/shoot 0.103 ± 0.010 0.122 ± 0.012 +18.5 0.050

Table 2. Effects of enhanced UV-B radiation on some II oral properties. Values are means± SD from four plots
per treatment with nine plants per plot. CON: ambient UV-B, UV: enhanced UV-B.

Parameter CON uv 0/r, change p

No of heads plant- 1 30.24 ± 2.98 31.00 ± 1.67

Head I ife span, days 17.17±10.08 17.34 ± 10.98
No of tubular florets hcad- 1 260.5 ± 23.2 248.2 ± 36.0
No of ligulate florets head- 1 14.19 ± 0.12 14.69 ± 0.94
Area per ligu1atc floret, mm 2 21.66 ± 1.05 21.63 ± 2.84
No of epidermal cells per unit ligulate surface 104.2 ± 11.6 117.9 ± 12.4 +13.1 0.028

As shown on Table 2, supplemental UV-B ra-
diation had no significant effect on the number of
inflorescences and florets, the area of ligulate florets
and on head life span. Anthesis started at mid-March,
peaked during the first 15 days of May and lasted up to
early-June. Flower life span progressively decreased,
being ~30 days in March and ~5 days in late May, but
the pattern of decrease was independent of UV-B sup-
plementation. A decreasing trend was also observed in
the number of ligulate florets per head and particularly
in the area per ligulatc floret, yet this decrease was
again irrespective of UV-B enhancement.
UV-B radiation had no effect on the spectral ab-
sorbance of methanol extractable material from both
floret types (data not shown). In vivo assessment of
their optical properties revealed that tubular florets
absorb intensively in the UV-8, UV-A and blue but
reflect strongly above 500 nm. No significant differ-
ences on the in vivo spectral reflectance of tubular flo-
rets were observed between control and UV-B treated
plants (data not shown). Ligulate florets absorb only in
the UV and, being white, reflect strongly in the visible
region of the spectrum. However, under supplemental
UV-B radiation, ligulate florets exhibit a slight, yet sta-
tistically significant (P < 0.05) decrease in reflectance
between 560-700 nm (Figure 1). Since the extracts
of these florets do not absorb in this region of the
spectrum, the observed reflectance difference could
not be attributed to chemical changes. Alternatively, it
could be related to structural surface alterations. Ex-
amination of the surfaces under SEM revealed that
the external walls of the adaxial epidermal cells are
papillate. However, the epidermis of UV-B treated
samples possess a significantly higher number of cells
per unit area (Table 2). In addition, the papillae of the
UV-B treated florets were considerably swollen and
in contact to each other, while in control florets they
were thinner and cone-shaped, leaving a more or less
flat interpapillae area covering about 40% of the total
epidermal surface (Figure 2).
Discussion
UV-B induced changes in biomass allocation are not
uncommon (Rozema et al. 1997). The preferential in-
crease in root biomass (Table 1) was also found in one
out of two pea varieties studied by Gonzalez et al.
( 1998b ). An increased root/shoot ratio may improve
the leaf water relations and lead to higher growth rates.
This was not observed in the case of A. arvensis, prob-
ably because the plants were well watered during the
whole experimental period. Under natural precipita-
tion, A. arvensis may suffer moderate water stress

Figure 2. A SEM view of the surface of ligulate florets grown under ambient (a) or ambient plus supplemental (b) UY-B radiation.
towards the end of its growing period (late spring),
during which anthesis takes place.
It is evident from the results of this investigation
that the phenology and demography of anthesis in
A. a111ensis were not affected by supplemental UV-B
radiation. Some previous laboratory and glasshouse
studies have reported considerable effects (either posi-
tive or negative) on the reproductive performance of
various plants (Demchik & Day 1996; Feldheim &
Conner 1996; Musil & Wand 1994; Musil 1995).
Under field conditions, Sullivan & Teramura ( 1990)
found no effects on seed yield in soybean. How-
ever, increases in flower production were observed
in Vaccinium myrtilus (Gwynn-Jones et a!. 1997) and
Mentha spicata (Grammatikopoulos el a!. 1998). We
have therefore to conclude that UV-B radiation ef-
fects on flowering are species-specific. Our results also
showed an absence ofUV-B radiation effects on flower
colour, although the long duration of A. an;ensis flow-
ers allowed ample time for UV-B radiation to induce
an acceleration in phenolic production . We have to as-
sume that colour production in this plant is not UV-B
radiation limited, as it was the case in the ephemeral
flowers of C. creticus (Stephanou et al. 2000).
Structural alterations on leaf surfaces induced by
UV-B radiation have been reported for trichome den-
sity (Barnes et a!. 1996) and the configuration of
epicuticular waxes (Cen & Bornman 1993). In addi-
tion, changes of surface structures of Cladonia mitis
podetia, observed more frequently since the 1970s,
were, at least partly, ascribed to UV-B radiation en-
hancement (Heide-Jorgensen & Johnsen 1995). In our
case, the increase in the number of epidermal cells per
unit ligulate surface occurred in the absence of any
63
effect on total ligulate surface area (Table 2). There-
fore, it can be regarded as a UV-B radiation effect on
cell division. Both decreases (Gonzalez et a!. 1998a)
and increases (Staxen & Bornman 1994) in leaf cell
division rates by UV-B radiation have been reported.
Concerning the swelling of epidermal papillae, it is
difficult to speculate both for the reasons or the possi-
ble consequences of this change. However, the surface
structure of a plant organ can modify the behaviour of
visiting insects (sec Schoonhoven et al. 1998 and the
literature therein).
Tn conclusion, the overall results of this investi-
gation indicate that enhanced UV-B radiation is not
harmful for A. a111ensis. However, the subtle changes
in ligulate Ooret re Oectance and the increase of the
root/shoot ratio may have an impact on plant repro-
duction.
Acknowledgement
We are grateful to the Commission of the European
Communities for fin ancial support.
References
Barnes, J.D., Percy, K. E., Paul. N. D. Jones, P., McLaughlin, C. K. .
Mullineaux. P. M., Creissen, G. & Wellburn, A. R. 1996. The
influence of UV-B radiation on the physicochemical nature of
tobacco (Nicoriana at bacum L.l leaf surfaces. J. Exp. Bot. 47:
99- 109.
Bjorn, L. 0 ., Callaghan, T. Y., Johnsen, 1., Lee, J. A. , Manetas. Y. ,
Paul, N. D., Sonesson, M., Wellburn, A. R., Coop, D.. Heide-
Jorgensen. H. S., Gehrke, C. , Gwynn-Jones, D., Johanson. U..
Kyparissis. A. , Levizou, E .. Nikolopoulos, D., Petropoulou, Y. &
64
Stephanou, M. 1997. The effects of UV-B radiation on European
heathland. Plant Ecol. 128: 252-264.
Bjorn. L. 0. & Murphy, T. M. 1985. Computer calculation of solar
ultraviolet radiation at ground level. Physiol. Yeg. 23: 555-561.
Caldwell, M. M. 1971. Solar UV radiation and the growth and de-
velopment of higher plants. Vol. 6, pp. 131-177. In: Giese, A. C.
(ed.), Photophysiology. Academic Press, New York.
Caldwell, M. M., Robberecht, R. & Flint, S.D. 1983. Internal filters:
prospects for UV-acclimation in higher plants. Physiol. Plant. 58:
445-450.
Cen. Y. P. & Bornman J. 1993. The etl'ect of exposure to enhanced
UV-B radiation on the penetration of monochromatic and poly-
chromatic UV-B radiation in leaves of Brassica napus. Physiol.
Plant. 87: 249-255.
Day, T. A .. Howells, B. W. & Rice, W. J. 1994. Ultraviolet ab-
sorption and epidermal-transmittance spectra in foliage. Physiol.
Plant. 92: 207-218.
Demchik, S. M. & Day, T. A. 1996. Effect of enhanced UV-B radi-
ation on pollen quantity, quality, and seed yield in Brassica rapa
(Brassicaceae). Am. J. Bot. 83: 573-579.
Fendheim, K. & Conner, J. K. 1996. The effects of increased UV-
B radiation on growth, pollination success, and lifetime female
fitness in two Brassica species. Oecologia I 06: 284-297.
Gonzalez, R. Mepsted, R. Wellburn, A. R. & Paul, N. D. 1998a.
Non-photosynthetic mechanisms of growth reduction in pea
(Pisum sativum L.) exposed to UV-B radiation. Plant Cell En-
viron. 21: 23-32.
Gonzalez, R., Wellburn. A. R. & Paul. N.D. 1998b. Dose responces
of two pea lines to ultraviolet-B radiation (280-315 nm). Physiol.
Plant. 104: 373-378.
Grammatikopoulos. G., Karousou, R., Kokkini, S. & Manetas, Y.
1998. Differential effects of enhanced UV-B radiation on repro-
ductive effort in two chemotypes of Mentha spicata under field
conditions. Austr. J. Plant Physiol. 25: 345-351.
Gwynn-Jones. D., Lee, J. A. & Callaghan, T. V. 1997. Effects of
enhanced UV-B radiation and elevated carbon dioxide concen-
trations on a sub-arctic forest heath ecosystem. Plant Ecol. 128:
242-249.
Heide-.Torgensen H. S. & Johnsen I. 1995. Analysis of surface struc-
tures of Cladonia mitis podetia in historic and recent collections
from Greenland. Can. J. Bot. 73: 457-464.
Manetas, Y. 1999. Is enhanced UV-B radiation really damaging for
plants? Some case studies with European MediteTI'anean species.
pp. 251-263. In: Rozema, J. (ed.), Stratospheric Ozone Deple-
tion; the Effects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden, The Netherlands.
Manetas, Y., Petropoulou, Y., Stamatakis, K., Nikolopoulos, D.,
Levizou, E., Psaras, G. & Karabourniotis. G. 1997. Beneficial
effects of enhanced UV-B radiation under field conditions: im-
provement of needle water relations and survival capacity in
Pinus pinea L. seedlings during the dry MediteTI'anean summer.
Plant Ecol. 128: 100-108.
Musil, C. F. 1995. Differential effects of elevated ultraviolet-B
radiation on the photochemical and reproductive performances
of dicotyledonous and monocotyledonous arid-environment
ephemerals. Plant Cell Environ. 18: 844-854.
Musil, C. F. & Wand, S. J. E. 1994. Differential stimulation of
an arid-environment winter ephemeral Dimorphotheca pluvialis
(L.) Moench by ultraviolet-B radiation under nutrient limitation.
Plant Cell Environ. 17: 245-255.
Proctor, M., Yeo, P. & Lack, A. 1996. The Natural History of
Pollination. Harper Collins Publishers, London.
Rozema, J., Van de Staaij, J., Bjorn, L. 0. & Caldwell, M. 1997. UV-
B as an environmental factor in plant life: stress and regulation.
Trends Ecol. Evol. 12: 22-28.
Shoonhoven, L. M., Jenny, T. & Van Loon, J. J. A. 1998. Insect-
Plant Biology: from Physiology to Evolution. First edition.
Chapman & Hall, London.
Staxen, I. & Bornman J. F. 1994. A morphological and cytological
study of Petunia hybrida exposed to UV-B radiation. Physiol.
Plant. 91: 735-740.
Stephanou, M. & Manetas, Y. 1998. Enhanced UY-B radiation in-
creases the reproductive effort in the McditeTI'ancan shrub Cistus
creticus under field conditions. Plant Ecol. 134: 91-96.
Stephanou, M., Petropoulou, Y., Georgiou, 0. & Manetas, Y. 2000.
Enhanced UV-B radiation, flower attributes and pollinator behav-
iour in Cistus creticus: a Mediterranean tield study. Plant Ecol.
147: 165-171.
Sullivan, J. H. & Rozema, J. 1999. UV-B effects on teTI'estria1 plant
growth and photosynthesis. pp. 39-57. In: Rozema, J. (ed.),
Stratospheric Ozone Depletion; the Effects of Enhanced UV-
B Radiation on TeTI'estrial Ecosystems. Backhuys Publishers,
Leiden, The Netherlands.
Sullivan, J. H. & Teramura, A. H. 1990. Field study of the in-
teraction between solar ultraviolct-B radiation and drought on
photosynthesis and growth in soybean. Plant Physiol. 92: 141-
146.
Wand. S. J. E. 1995. Concentration ofultraviolet-B radiation absorb-
ing compounds in leaves of a range of fynbos species. Vegetatio
s
116: 1-61.
Wenzel, C. L., Chandler, P. M., Cunningham, R. B. & Passioura,
J. B. 1997. Characterization of the leaf epidermis of barley
(Hordeum vulgare L. 'Himalaya'). Ann. Bot. 79: 41-46.
Section 3: Arctic and Antarctic Plants and Ecosystems

Field UV-B exposure frames based in Abis ko, Swede n (68° N). (Photograph by D. Gwynn-Jones)
 Plant Ecology 154: 67-73, 2001.
© 2001 Kluwer Academic Pub/i,her,.
67

Short-term impacts of enhanced UV-B radiation on photo-assimilate

allocation and metabolism: a possible interpretation for time-dependent
inhibition of growth

D. Gwynn-Jones
Institute (~f Biological Sciences, University of Wales, Aberystwvth, Ceredigion. SY23 3DA. Wales, UK
(E-mail: DYJ@aher.ac.uk)

Key words: Growth, Maintenance respiration. Protection, Repair, Source-sink relations, UV-B

Abstract
To test the hypothesis that plant source-sink relations are important in determining response to UV-B radiation,
a short-term (45 d) field experiment was conducted at Abisko Scientific Research Station, Abisko, Sweden
(68° N). Tillers of the grass Calamagrostis purpurea were grown outdoors at levels of UV- B radiation representing
25% ozone depletion. Growth, respiration. photo-assimilate allocation and UV-B protective compounds were
subsequently measured.
There were no significant effects of enhanced UV-B on total plant dry weight, leaf area, Shoot: Root ratio, leaf
weight ratio, leaf area ratio, specific leaf area, tiller number per plant or blade thickness of this species. However,
the amount of UV-B absorbing compounds and respiration rates were significantly increased in young and mature
leaves. Increases in leaf respiration were accompanied by alterations in plant carbohydrate allocation at enhanced
UV-B. The amount of soluble root carbohydrates was reduced following UV-B exposure. Enhanced UV-B also
caused increases in the soluble sugar : starch ratio of young leaves, the stem and total aboveground biomass.
The importance of source-sink relations and constitutive versus induced defense are discussed in relation to UV-B
response.

Introduction constitutive or induced protection against and/or re-

A large number of experiments world-wide have ad-
dressed the impacts of enhanced UV-B radiation (290-
315 nm) on plant growth (e.g., Bjorn et a!. 1997;
Rozema et a!. 1997; Caldwell and Flint 1994; Tevini
1993, 1994; Krupa & Kickert 1989). Plant species
(and groups) vary considerably in their response to
UV-B, depending on experimental set up, treatment
regimes and duration (see, Gwynn-Jones eta!. 1999;
Weih eta!. 1998; Middleton & Teramura 1994; Tevini
1994; Warner & Caldwell 1983 ). Regardless of such
factors, several published (and unpublished) studies
have shown evidence of plant resistance to UV-B ra-
diation (Krupa & Kickert 1989; Rozema et a!. 1995;
Newsham et a!. 1996; Gwynn-Jones et a!. 1997;
Liakoura ct a!. 1999; Manctas 1999) possibly via
pair of UV-B damage. Protection against UV-B can
involve alterations in cuticle (Drilias eta!. 1997; Stein-
muller & Tevini, 1985) and leaf thickness (Johanson
et a!. 1995a) and/or increased production of UV-B
protective pigments (Van de Staaij eta!. 1995; Cen
& Bornman 1990). In the event of protective mecha-
nisms failing to shield the genome and photosynthetic
machinery against UV-B, repair mechanisms arc re-
lied upon (e.g., Takeuchi et a!. 1993). Most plant
species are thought to have adequate repair capacities
(photoreactivation-photorepair) to deal with projected
increases in UV-B (Taulavuori et al. 1998; Beggs
et al. 1986). Nevertheless, one crucial factor to such
tolerance is the duration of exposure. as longer-term
studies show evidence of cumulative plant damage
68
(e.g., Johanson et a!. 1995b; Sullivan & Teramura
1992).
Provision of resources may be crucial for mainte-
nance of protection and repair, following UV-B expo-
sure. Photo-assimilate allocation and availability are
therefore important in determining a plant response
to UV-B radiation. Here it is hypothesized that cu-
mulative reduced growth at enhanced UV-B could be
interpreted as carbohydrate resource limitation over
long time scales.
Materials and method
Plant material and treatment
Calamagrostis purpurea (Trin.) were separated as
tillers during late May 1997 from stock mother plants
collected from the Abisko region, northern Sweden
(68° N) during August of the previous year. Each tiller
was planted in a 15 em pot (3.41) containing local soil
(1.63 mg N g- 1 dry weight and 0.23 mg P g- 1 dry
weight). Following two weeks of acclimation to re-
potting, the tillers were selected for uniformity and
placed under the different UV-B treatments. Plants
were watered daily and randomised within treatments
on a weekly basis.
The methods of Johanson et a!. (1995a) were
adopted to enhance UV-B radiation using frames
(2.5 x 1.3 x 1.5 m high) each with 6 fluorescent
lamps (Q-PANEL UVB-313, Cleveland, OH, USA).
The models developed by Bjorn & Murphy (1985) and
Bjorn & Teramura (1993) were used to calculate the
daily increase in UV-B radiation resulting from a 25%
ozone depletion under clear sky conditions.
Harvesting
Tillers were harvested for leaf area and dry weight par-
titioning following 45 days exposure to the treatments
on 29 July 1997. Leaf area was determined in each
case using aLI-COR 3100 leaf area meter (LICOR
Cor Inc, Lincoln, NE, USA) and the dry weight of
the separated plant components determined after dry-
ing at 60 oc for 48 h. Relative growth rate was not
determined, as there were an insufficient number of
established plants for a time zero harvest at the on-set
of the experiment.
UV-B absorbing compounds
Powdered leaf material of a known weight (approx-
imately 10 mg) was ground in 10 ml of acidified
methanol (79% MeOH : 20% H2 0 : I% HCI). Follow-
ing extraction the samples were then centrifuged for
10 min at 1600 rpm. UV-B protective pigments were
determined by scanning the extract for absorbance
between 280-320 nm using a spectrophotometer (Shi-
madzu UV-160, Japan). Pigment content was assessed
by the area under the absorbance curve per dry weigh
(g) of tissue extracted (as described by Gwynn-Jones
& Johanson 1996).
Carbohydrate analysis
In order to minimise any diurnal effects on carbohy-
drate content all dry weight harvests were performed
between 12:00 and 14:00 (GMT). Plant material was
rapidly killed by placing it in a drying oven at 80 °C
for 1 h, followed by a drying period of 48 h at 60 °C.
Dried material was ground into a powder (particle size
<I mm) and extracted in 5 ml 95% ethanol solution
and maintained at 60 oc for 24 h. The extract was
decanted and the remaining material extracted in a
50 mol m- 3 acetate buffer adjusted to pH 4.5 with
sodium hydroxide. Plant material was ground with a
pestle and mortar in 4 ml of the buffer and incubated
for 2 h at 25 oc. The sample was filtered through a
Whatman glass microfilter (GF/A, 2.1 em) on a sinter
glass funnel under vacuum, a further I ml of buffer
was used to wash the filter. The two 5 ml extracts
were combined and frozen prior to analysis for soluble
carbohydrates.
For extraction of starch, the residue was suspended
in 4 ml of the acetate buffer adjusted to pH 4.8, to
which 0.2 ml of an amyloglucosidase (from Rhizopus,
Sigma) solution (0.2 g in 100 ml water, representing 5
units). The sample was incubated at 55 °C for 24 h,
cooled, centrifuged and frozen prior to analysis. A
reagent blank containing 0.2 ml amyloglucosidase in
4 ml of the buffer was also prepared, since the enzyme
extract also contained sugar. Carbohydrates in the two
final extracts were assayed by the phenol sulphuric
acid method according to Dubois et a!. (1956).
Measurement of dark respiration
Dark respiration of young and mature leaves of a stan-
dard area (I cm 2) were determined polarographically
at 15 oc according to the methods of Bingham and
Farrar (1988). Values were expressed on a dry weight
 69

4,----------------,----------------~ Table I. Dry weight partitioning, leaf thickness and tiller number
Young leaves Mature leaves in Calamagrostis purpurea at the end of the experiment. The values
are expressed as a mean with standard error in brackets. F -ratio
values are shown in each case and significance expres-;ed ao.; NS Not
significant (n = 24 in each case).

Natural UV-B Enhanced UV-B F-value

Total DW(g) 1.44 (0.13) 1.47(0.15) 0.002NS

Total leaf area (cm 2 ) 32.40 (2.15) 33.48 (3.09) 0.037NS
Shoot : root ratio 2.23 (0.10) 2.39 (0.47) o.soNs
Leaf area ratio 24.10 (1.11) 24.20 ( 1.93) 0.16'1S
Leaf weight ratio 0.47 (0.03) 0.47 (0.02) 0.21 'IS
Leaf thickness (mm) 0.24 (0.01) 0.24 (0.01) 0.23NS
Fifiure I. UV-B absorbing compounds (expressed as the arbitrary
Tiller number 13.66 (1.24) 13.13 (0.98) o.osNs
units - integrated area under the absorbance curve between 280 and
320 nm per dry weight) in young and mature leaves following plant
exposure to natural (Control) and Enhanced UV-B radiation for a
period of 45 days. Significant effects are expressed as * P < 0.05.
*** p < 0.001 (n = 12 in each case).
Young leaves Mature leaves

basis following subsequent drying of the leaf tissue at ***

60 oc for 48 h.

Statistical treatment of data

In each case, a one way analysis of variance was

used to test for the effects of UV-B on each parameter
(SPSS for Windows, SPSS Inc., Chicago, IL, USA).
Transformation of data was performed when data were
Figure 2. Leaf respiration rate (expressed on a dry weight basis as
not of normal distribution. All data presented are the
fJ- mol h 1 g- 1 dry weight) measured at the end of the experiment
non-transformed values. in young and mature leaves following plant exposure to natural
(Control) and Enhanced UV-B radiation for a period of 45 days.
Significant effects are expressed as*** P < 0.001 (11 = 6 in each
case).
Results

Results in Table I, show no significant (P > 0.05) Discussion

effects of enhanced UV-B on the dry weight, leaf area,
partitioning (Shoot: Root ratio, Leaf weight ratio, Leaf
area ratio, Specific Leaf area), tiller number or blade
thickness of this species. However, the amount of
UV-B absorbing compounds (280-320 nm) were sig-
nificantly increased in young ( P < 0.001) and mature
leaves (P < 0.05) (Figure I).
Effects were also observed on root soluble carbo-
hydrate content, which was significantly ( P < 0.05)
reduced at enhanced UV-B (Table 2). Exposure to en-
hanced UV-B also caused increases in soluble sugar:
starch ratio in young leaves ( P < 0.05), the stem
(P < 0.001) and overall in the shoot (P < 0.001)
(Table 2). Such changes in the allocation of above-
ground carbohydrates might account for the significant
( P < 0.001) stimulation of respiration observed in
both young ( P < 0.00 I) and mature leaves (Figure 2).
The current study on C. purpurea contrasts with our
previous indoor study on the same species (Gwynn-
Jones & Johanson 1996), as plant dry weight was not
inhibited by enhanced UV-B radiation (Table 1). This
species is therefore tolerant to short-term exposure to
enhanced UV-B under more realistic outdoor condi-
tions. Measurements of leaf UV- B absorbing pigments
and leaf respiration rates (young and mature) suggest
induced leaf protection and metabolism at enhanced
UV-B (Figure 2).
Increases in the amounts of UV protective com-
pounds have been commonly shown in the literature
(Van de Staaij et al. 1995; He et al. 1993; Santos
et al. 1993; Dai et al. 1992; Ziska and Teramura 1992;
Teramura et al. 1991; Tevini et al. 1991 ), while a
stimulation in leaf respiration has previously been ob-
70
Table 2. Soluble carbohydrate (CHO) and starch content (SCH- mg g- 1 dry weight)
in the plant components of Calamagrostis purpurea. The values are expressed as a
mean with standard error in brackets. Significant effects are expressed as * P < 0.05,
** P < 0.01 (n = 6 in each case).

Parameter Treatment Young leaves Mature Stem Shoot Root

leaves

Soluble Control 45.1 41.7 62.5 110.4 37.5

CHO (mgg- 1) (3.9) (5.9) (6.3) (7.5) (4.3)
Enhanced UV 53.2 45.7 85.2 140.6 26.0
(5.8) (3.8) (16.0) (16.0) (3.5)*

Starch (SCH) Control 125.9 104.5 165.8 333.0 118.0

(mgg-1) (15.0) (6.7) (21.0) (34.0) (7.6)
Enhanced UV 116.0 99.3 131.8 293.5 101.5
(15.2) (3.3) (11.1) (17.5) (10.3)

Total CHO Control 171.0 146.2 228.0 443.4 155.4

(mgg-1) (17.4) (12.5) (26.7) (40.7) (10.7)
Enhanced UV 169.3 145.0 216.9 434.4 127.2
(20.6) (6.0) (26.3) (33.1) (13.6)

SCH: Soluble Control 0.37 0.39 0.38 0.33 0.32

(0.03) (0.03) (0.02) (0.02) (0.03)
CHOratio Enhanced UV 0.47 0.46 0.62 0.47 0.26
(0.02)* (0.39) (0.08)** (0.02)** (0.02)

served (Ziska et al. 1991; Brandle et al. 1977; Sisson
& Caldwell 1976) but not discussed.
From this evidence, it is hypothesized that a stimu-
lation of leaf respiration represents increased resource
demands for protection and repair (cuticular thick-
ening, flavonoid biosynthesis and photoreactivation).
The stimulation of respiration in non-growing ma-
ture leaves (Figure 2) supports this view as it can
be used to reflect maintenance respiration (Amthor
1984 ). Maintenance respiration can be closely corre-
lated with plant nitrogen content (Ryan 1991, 1995)
and may account for stimulation of nitrogen com-
monly observed in leaf tissue/litters at enhanced UV-B
(Gwynn-Jones 1999).
Marked changes in the carbohydrate allocation
between root and shoot of C. purpurea with UV-B
exposure also provide supportive evidence for this hy-
pothesis (Table 2). The soluble carbohydrate: starch
ratio was higher in young leaves, the stem and overall
in the shoot, whilst the amount of soluble carbohy-
drates within the roots was reduced at enhanced UV-B.
The results partially agree with a previous study by
Phoenix et al. (1999), a long-term stimulation of solu-
ble leaf carbohydrates was observed in the dwarf shrub
Vaccinium ulginiosum, although root and rhizome
carbohydrates were not measured. The findings of
both studies might be explained by increased respira-
tory demand in the leaves influencing photo-assimilate
allocation.
The production of UV protective secondary
metabolite compounds (via the shikimic acid pathway)
is dependent on the availability of carbohydrates from
photosynthesis and storage (see Matsuki 1996 and
literature there in). Similarly carbohydrates are also
required to provide respiratory energy for protection,
maintenance (and repair) of plant activity and structure
(Farrar 1989). Carbohydrate availability and alloca-
tion could therefore play a pivotal role in the trade-off
between growth and UV-B defense in plants. Such
a relationship has previously been discussed in the
context of defense against herbivory (Matsuki 1996;
Harper 1989; Jonasson et al. 1986) but not UV- B.
This presents an interesting analogy given the sim-
ilarities between UV-B and herbivory defenses (i.e.,
induction of phenolics, tannin, cellulose and ligni-
fication, see Gwynn-Jones 1999). However, UV-B
also incurs additional respiratory costs in terms of re-
pair following 'DNA damage, direct photosynthesis
damage, membrane changes, protein destruction and

hormone inactivation' (Tevini 1989 & 1991 as cited
by Stapleton 1992).
Prolonged investment of assimilates into UV tol-
erance and repair would inevitably affect growth (cu-
mulatively) and/or reproductive output (Feldheim &
Conner 1996; Johanson et al. l995a; Sullivan & Ter-
amura 1992; Rau et al. 1988). The experiment on C.
purpurea provides some supportive evidence for this
view and emphasizes the importance of investigating
protection, maintenance and repair processes even if
the growth of a species is initially unatlected by UV-B.
In an earlier review (Gwynn-Jones et al. 1999)
we found that plant groups varied in their negative
growth responses to UV-8, with moss species gener-
ally being most sensitive to exposure. It was suggested
that species with high constitutive and induced levels
of UV-B absorbing compounds (phenolics) have the
highest tolerance to UV-B, coincidentally they also,
potentially have the highest photo-assimilate and ni-
trogen reserves. Indeed, shrub species showing cumu-
lative long-term damage (Johanson et al. l995a) have
large below-ground reserves (>80% biomass, Jonas-
son 1982), as do tree species (Sullivan and Teramura
1992), especially when compared to moss species
which are sensitive to enhanced UV-B over shorter
time durations (Gehrke 1998, 1999).
From evidence above, I propose that time depen-
dent UV-B response can be partially explained by the
photo-assimilate balance between maintenance, re-
pair and growth processes rather than accumulated
UV-B damage. Plant groups with effective constitu-
tive and induced UV-B protection (e.g., trees) and/or
large assimilate reserves (e.g., dwarf shrubs) would
be expected to show longer-term damage responses. It
can be further hypothesised that species with a poor
protective capacity and assimilate availability (e.g.,
mosses) would respond negatively and rapidly to UV-
B radiation. Such an argument could also be turned
around with regards to assimilate investment. The
'value of a leaf' (Harper 1989) is higher and therefore
'better' defended in plant species that retain leaves for
longer, such species also general! y have larger translo-
catory pathways and assimilate reserves (e.g., dwarf
shrubs).
This manuscript not only emphasizes the need to
consider source-sink relations in the context of plant
UV-B response, it also highlights the need to address
interactions with belowground processes in more de-
tail. A reduced root soluble carbohydrate pool in C.
purpurea at enhanced UV-B had no short-term effects
on the growth of this species. However, differences
71
at the physiological (reduced root capacity and nutri-
ent uptake) and ecological scale might be anticipated
over longer periods of exposure. Klironomos & Allen
( 1995) showed that the soil microbial population could
be influenced by root-derived substrates of Acer sac-
charum exposed to enhanced UV-B. Similarly, Van
de Staaij et al. (2000) report reduced infection by
arbuscular mycorrhizeal fungi by enhanced UV-B in
dune grasslands plants. A reduced allocation of car-
bohydrates to the roots and ultimately to rhizosphere
via UV-B could therefore have long-term ecological
implications.
Adaptive capacity, resource availability and inter-
action with the rhizosphere might all be used to inter-
pret individual plant responses to UV-B radiation. In
this context, I propose that cumulative long-term dam-
age in many cases may represent resource depletion
(of carbohydrates and leaf nitrogen), and an inabil-
ity to sustain protection and repair. To test the above
hypothesis, further studies are proposed, address-
ing UV-B impacts on protection and repair in rela-
tion to growth, respiration and carbohydrate/nitrogen
turnover in range of plant groups grown under natural
conditions.
Acknowledgements
I am indebted to the European Community (Contract
EV5V-CT910032) for funding the current UV-B ini-
tiative at Abisko. Gratitude is also shown to Christina
Castleden for her field assistance during a Nuffield
Foundation bursary (Ref. AT/100/97/0084). Thanks
are also expressed to the British Ecological Society for
a small project grant for partly funding a field assis-
tant (Mr. Nicholas Money). Finally, I am grateful to
Profs. Terry Callaghan and John Lee, for discussion of
results, logistic support and excellent coordination of
the UVECOS, EC consortium.
References
Amthor, J. S. 1984. The role of maintenance respiration in plant-
growth. Plant Cell Environ. 7 (8): 561-569.
Beggs. C. 1., Schneider-Ziebert. U. & Wellman, E. 1986. UV-
B radiation and adaptive mechanisms in plants, Pp. 235-250.
In: Won·est. R. C. & Caldwell, M. M. (eds), Stratospheric
Ozone Reduction. Solar Ultraviolet Radiation and Life, Vol. I.
Springer-Verlag, Berlin.
Bingham. I. J & farrar, J. E 1988. Regulation of respiration in rooh
of barley. Physiologia. Plantarum 73: 278-285.
72
Bjorn, L 0. & Murphy, T. M. 1985. Computer calculations of solar
ultraviolet radiation at ground leveL Physiol. Vegetale 23: 555-
561.
Bjorn, L 0. & Teramura, A. H. 1993. Simulation of daylight ultra-
violet radiation and effects of ozone depletion. Pp. 41-7 L In:
Young, A. R., Bjorn., L 0., Moan., J. & Nultsch, J. (eds),
Environmental UV Photobiology, VoL L Plenum Press, New
York.
Bjorn, L 0., Callaghan, T. V., Johnsen, J., Lee, J. A., Manetas, Y.,
Paul, N. D., Sonesson, M., Wellburn, A. R., Coops, D., Heide-
Jurgensen, H. S., Gehrke. C., Gwynn-Jones, D., Johanson, U.,
Kyparissis, A., Levizou, E., Nikolopoulos, D., Petropoulou, Y. &
Stephanou, M. 1997. The effects of UV-B radiation on European
heathland species. Plant EcoL 128 (1-2): 252-204.
Brandle, J. R., Campbell, W. F., Sisson, W. B. & Caldwell, M. M.
1977. Net photosynthesis, electron transport capacity, and ultra-
structure of Pisum sativum L exposed to ultraviolet-B radiation.
Plant Physiol. 60: 165-169.
Caldwell, M. M. & Flint, S.D. 1994. Stratospheric ozone reduc-
tion, solar UV-B radiation and terrestrial ecosystems. Climatic
Change 28: 375-394.
Cen, Y. & Bornman, J. F. 1990. The response of bean plants to UV-B
radiation under different irradiances of background visible light.
J. Exp. Bol. 41: 1489-1495.
Dai, Q .. Coronel, V. P., Vergara. B. S., Barnes, P. W. & Quin-
tos, A. T. 1992. Ultraviolet-B radiation effects on growth and
physiology of four rice cultivars. Crop Sci. 32: 1269-1274.
Drilias, P., Karabourniotis. G., Levizou, E., Nikolopoulos. D.,
Petropoulou, Y. & Manetas, Y. 1997. The effects of enhanced
UV-B radiation on the Mediterranean evergreen sclerophyll Ner-
ium oleander depend on the extent of summer precipitation.
Austr. J. Plant Physiol. 24: 301-306.
Dubois, M., Giles. K. A., Hamilton, 1. K., Rebus, P. A. & Smith,
F. 1956. Colorimetric method for the determination of carbohy-
drates and related chemicals. Anal. Chern. 248: 3441-3445.
Farrar, 1. F. 1989. The carbon balance of fast-growing and slow-
growing species. Pp. 241-245. In: Lambers, H (ed.), Causes and
Consequence of Variation in Growth Rate and Productivity of
Higher Plants, Vol. 1. SPB Academic Publishing, The Hague.
Feldheim, K., Conner, J. K. 1996. The effects of increased UV-B
radiation on growth, pollination success, and lifetime fitness in
two Brassica species. Oecologia 106: 284-297.
Gehrke, C. 1999. Impacts of enhanced ultraviolet-B radiation on
mosses in a subarctic heath ecosystem. Ecology 80 (6): 1844-
1851.
Gehrke. C. 1998. Effects of enhanced UV-B radiation on
production-related properties of a Sphaf!,num fuscum dominated
subarctic bog. Funct. Ecol. 12 (6): 940-947.
Gwynn-Jones, D. & Johanson, U. 1996. Growth and pigment pro-
duction in two subarctic grass species under four different UV-B
irradiation levels. Physiol. Plant. 97:701-707.
Gwynn-Jones, D., Lee, J. A. & Callaghan, T.V. 1997. The effects of
UV-B radiation and elevated carbon dioxide on sub-Arctic forest
ecosystems. Plant Ecol. 128: 242-249.
Gwynn-Jones. D., Johanson, U., Phoenix, G. K., Callaghan, T. V.,
Sonesson, M. & Lee, J. A. 1999. The responses of plant func-
tional types to enhanced UV-B. Pp. 187-201. In: Rozema.
J. (ed.), Stratospheric Ozone Depletion. The Effects of En-
hanced UV-B Radiation on Terrestrial Ecosystems. Backhuys
Publishers, Leiden.
Gwynn-Jones, D. 1999. UV-B and herbivory. Ecol. Bull. 47: 77-83.
Harper, 1. L. 1989. The value of a leaf. Oecologia 80 ( 1): 53-58.
He, J., Huang, L-K., Chow, W. S., Whitecross, M. 1. & Anderson,
J. M. 1993. Effects of supplementary ultraviolet-B radiation on
rice and pea plants. Austr. J. Plant Physiol. 20: 129-142.
Johanson, U., Gehrke, C., Bjorn, L. 0. & Callaghan, T.V. 1995a.
The effects of enhanced UV-B radiation on the growth of dwarf
shrubs in a subarctic heathland. Funct. Ecol. 9:713-719.
Johanson, U., Gehrke, C .. Bjorn. L. 0., Callaghan, T. V & Sones-
son, M. 1995b. The effects of enhanced UV-B radiation on a
sub-arctic heath ecosystem. Ambia 24: I 06-111.
Jonasson, S. 1982. Organic matter and phytomass in three Nmth
Swedish tundra sites, and some adjacent tundra areas. Holarctica
Ecol. 5: 367-375.
Jonasson, S .. Bryant J. P., Chapin, F. S. Ill. & Andersson, M.
19H6. Plant phenols in relation to variations in climate and rodent
grazing. Am. Nat. 128 (3): 394-408.
Klironomos, J. N & Allen, M. F. 1995. UV-B mediated changes
on below ground communities associated with the roots of Acer-
Saccaharum. Funct. Ecol. 9 (6): 923-930.
Krupa, S. V. & Kicker!, R.N. 1989. The greenhouse effect: impacts
of Ultraviolet-B (UV-B) radiation, carbon dioxide (C02), and
ozone (03) on vegetation. Environ. Poll. 61:263-393.
Liakoura, V. Staurianakou, S.. Liakopoulos, G., Karabournio-
tis, G .. & Manetas, Y. 1999. Effects of UV-B radiation on
Olea europaea: comparisons between a greenhouse and a field
experiment. Tree physiology.
Manetas, Y. 1999. Is enhanced UV-B radiation really damaging for
plants? Some case studies with European Mediterranean species
(in press). In: Rozema, J. (ed.). Stratospheric Ozone Deple-
tion: the Effects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden.
Matsuki, M. 1996. Regulation of plant phenolic synthesis: from
biochemistry to ecology and evolution. Austr. J. Bot. 44: 613-
634.
Middleton, E. M .. , Teramura, A. H. 1994. Understanding photo-
synthesis, pigment and growth-responses induced by UV-B and
UV-A irradiances. Photochem. Photobiol. 60 (I): 38-45.
Newsham, K. K., Mcleod, A. R., Greenslade, P. D. & Emmett, B. A.
1996. Appropriate controls in outdoor UV-B supplementation
experiments. Global Change Bioi. 2: 319-324.
Phoenix, G. K., Gwynn-Jones, D., Callaghan, T. V. & Lee, J. A.
2000. The impacts of UV-B radiation on the regeneration of a
sub-arctic heath community. Plant Ecol. 146 (1): 67-75.
Rau, W., Hoffman, H., Huber-Wilier, A., Mitzke-Schnabel, U.,
Schroll, E. 1988. Die Wirkung von UV-B auf fotoregulierte En-
twicklungsvorgange bei Pflanzen, Gesellschaft ftir Strahlen-und
Umweltforschung mbH, Mtinchen AbschluB-bericht.
Rozema, J., Tosserams, M. & Magendans, E. 1995. Impact of
enhanced solar UV-B radiation on plants from terrestrial ecosys-
tems. In: Zwerver, S., Van Rompay, R. S. A. R., Kok, M. T. J.
& Berk, M. M (eds), Climate Change Research: Evaluation and
Policy Implications. Elsevier Science, Amsterdam.
Rozema, J., Van de Staaij, J., Bjorn. L. 0. & Caldwell, M. M.
1997. UV-B as an environmental factor in plant life: stress and
regulation. Trends Ecol. Evol. 12: 22-28.
Ryan, M. G. 1991. Etfects of climate change on plant respiration.
Ecol. Appl. 1(2): 157-167.
Ryan, M.G. 1995. Foliar maintenance respiration of sub-alpine and
boreal trees and shrubs in relation to nitrogen-content. Plant Cell
Environ. 18(7): 765-772.
Van de Staaij, J. W. M., Ernst, W. H. 0 .. Hakvort, H. W. .1. &
Rozema, 1. 1995. Ultraviolet-B (290-320 nm) absorbing pig-
ments in the leaves of Silene vulgaris: their role in UV-B
tolerance. J. Plant Physiol. 147: 75-80.

Santos. !., Almeida, J. M. & Salcma, R. 1993. Plants of Zea
mays L. developed under enhanced UV-B radiation. I. Some
ultrastraetural and biochemical aspects. J. Plant Physiol. 141:
450--456.
Sisson, W. B. & Caldwell, M. M. 1976. Photosynthesis. dark
respiration, and growth of Rummex pat entia L. exposed to ultra-
violet irradiance (288 to 315 nanometers) simulating a reduced
atmospheric ozone column. Plant Physiol. 58: 563-568.
Stapleton. A. E. 1992. Ultraviolet radiation and plant : Burning
questions. Plant Cell 4: 1353-1358.
Steinmliller, D. & Tevini, M. \9~5. Action of ultraviolet radiation
(UV-8) upon cuticular waxes in some crop plants. Planta 164:
557-564.
Sullivan. J. H. & Teramura, A. H. 1992. The effects of ultraviolet-
B radiation on loblolly pine 2. Growth or field grown seedlings.
Trees Strucl. Funct. 6: 115-120.
Takeuchi. Y., Ikeda, S. & Kasahara. H. 1993. Dependence on wave-
length and temperature of growth inhibition induced by UV-8
irradiation. Plant Cell Physiol. 34:913-917.
Taulavuori, E., mickman, M .. Taulavuori. K., Gwynn-Jones, D ..
Johanson, U., Laine, K., Callaghan. T., Sonesson. M. & Bjorn.
L. 0. 1998. Long-term exposure to enhanced UV-B radiation
in the suh-arctic does not cause oxidative stress in Vaccinium
mvrti/lus. New Phytol. 140: 691-697.
Teramura, A. H., Sullivan, J. H. & Lydon, J. 1990. Effects of UV-B
radiation on soybean yield and seed quality: a 6-year field study.
Physiol. Plant. 80: 5-11.
Teramura. A. H., Ziska, L. H. & Sztein, A. E. 1991. Changes in
growth and photosynthetic capacity of rice with increased UV-B
radiation. Physiol. Plant. 83(3): 373-380.
73
Tcvini, M. 1993. Etlccts of UV-B radiation on terrestrial plants.
Pp. 125-153. In: Tevini, M. (cd.), UV-B Radiation and 01.one
Depletion: Etlects on Humans, Animals, Plants, Microorganisms
and Materials, VoL I. Lewis Publishers. London.
Tevini, M. 1994. UV-B effects on terrestrial plants and aquatic
organisms. Progressive Bot 55: 174-190.
Tevini. M .. Braun, J. & Fieser, G. 1991. The protective func-
tion of the epidermal layer of rye seedlings against ultraviolet-B
radiation. Photochem. Photobiol. 53(3 ): 329-333.
Warner. C. W. & Caldwell M. M. 1983. Influence of photon tlux
density in the 400-700 nm waveband on inhibition of photosyn-
thesis hy UV-8 (280-320 nm) irradiation in soyhean leaves: Sep-
aration or indirect and immediate effects. Photochem. Photobiol.
41: 1489-I495.
Weih, M .. Johanson, t:. & Gwynn-Jones, D. \998. Growth and
nitrogen utilization of mountain birch (Betula puhescens ssp.
tortuosa) as atlectcd by ultraviolet radiation (UV-A and UV-13)
under lahoratory and outdoor conditions. Trees Struct. Funct. 12:
201-207.
Williams. K., PercivaL F., Merino. J. & Mooney, H. i\. 1987. Esti-
mation of tissue construction cost from heat of combustion and
organic nitrogen content. Plant Cell Environ. I0: 725-734.
Ziska, L. H .. Teramura. A. H. & Sullivan, J. H. 1991. Physiolog-
ical sensitivity of plants along an elcvational gradient to UV-B
radiation. Am. J. Bot. 79(8): 863-871.
Ziska, L H. & Tcramura, i\. H. 1992. C02 enhancement of growth
and photosynthesis in rice (Orv:a sativa) - Modilication hy
increased ultraviolet-b radiation. Plant Physiol. 99 (2): 473--481.
 Section 3: Arctic and Antarctic Plants and Ecosystems

An array of cloches in a vegetatio n of Stereocou!on olpinum on Leonie Island, Antartica . (Photograph hy A. H. L. Huiskes )
 Plant Ecology 154: 77-86, 200 L
77
© 200 I Kluwer Academic Publishers.

Field research on the effects of UV-B filters on terrestrial

Antarctic vegetation

A H. L. Huiskes, D. Lud & T. C. W. Moerdijk-Poortvliet

Netherlands Institute of Ecology, Centrefor Estuarine and Coastal Ecology, P.O. Box 140,
4400 AC Yerseke, The Netherlands (E-mail: huiskes@cemo.nioo.knaw.nl)

Key words: Alga, Antarctica, Grass, Lichen, Moss, Ozone depletion, Photosynthetic efficiency, UV-B filters,
UV-B radiation, UV-B supplementation

Abstract
Patches of vegetation of six common species growing on Leonie Island (67°35' S, 68°20' W), Antarctic Peninsula
region were covered with either UV-B transparent perspex screens or UV-B absorbing screens. Uncovered plots
served as a control. Temperature and relative humidity were monitored during the austral summer under and outside
the screens. The mean effective PSII quantum efficiency showed significant differences among the species, but not
between the UV-B treatments. It was concluded that the temperature and the moisture status of the vegetation
obscured any possible influence of UV-B treatment on the tteffectivc PSII quantum efficiency. he usefulness of
various UV-B exclusion and supplementation methods used to study the influence of UV-B in the field is discussed.

Introduction in the Antarctic atmosphere, by anthropogenic sub-

Emissions of chlorofluorocarbons into the atmosphere
have led to a depletion of the stratospheric ozone layer
since the 1980's (Farman et al. 1985) (Figure 1a). As a
result, the level of solar UV-B radiation (280-315 nm)
reaching the Earth's surface has increased (Frederick
& Snell 1988; Frederick et a!. 1989; Blumthaler &
Ambach 1990; Kerr & McElroy 1993) (Figure lb).
Enhanced UV-B radiation is known to affect both the
structure and functioning of plants (Caldwell et al.
1989). One of the processes likely to be affected, is
photosynthesis (Teramura & Sullivan 1994; Rozema
et a!. 1997b ).
As the depletion of the stratospheric ozone layer
is maximal over the Antarctic. the relative enhance-
ment of UV-B radiation has been, and still is, maximal
in this area (SCOPE 1992). Therefore, studies on
the effect of enhanced solar UV-B radiation on plants
can be carried out there under ambient circumstances,
without supplementation, using lamps. Studies in this
region have also the advantage that the atmosphere
is very little polluted, and absorption of solar UV-8
stances is negligible.
In order to study the possible differences in re-
sponse to enhanced UV-8 radiation between individ-
ual species from the same community, field exper-
iments are executed in a collaborative research pro-
gramme by Dutch and British scientists in the Antarc-
tic Peninsula region. In this research programme, main
emphasis is put on the influence of enhanced UV-8
radiation on the process of photosynthesis. The study
focused on three lichens, a moss, a terrestrial alga and
an angiosperm, all occurring in the coastal zone of
the Antarctic Peninsula. The assessment of possible
damage to this process as well as the ways in which in-
dividual plant species may protect themselves against
it are being studied.
Unlike other studies in which the level of UV-8 ra-
diation is artificially enhanced by arrays of UV lamps
(e.g., McLeod 1997; Rozema eta!. 1997a), this study
focuses on the attenuation of solar UV-8 radiation by
means of filter systems (so-called cloches) of different
type of acrylic plastic (PERSPEX).
78
·~ b Wavelength (nm)
J§r_~~pr'j~0~l117;~~}JG~
~1150<1
'ii 1000
1157·1972
0
Figure 1. (a) Monthly mean ozone levels (in Dobson units) at the
British research station f'araday (since 1990 the Ukrainian base Ver-
nadsky, 65° S, 64° W). (h) UV-BsE radiation levels calculated from
the ozone data in (a), using the BAS Radiation Transfer Programme,
kindly provided by Dr. B. Gardiner and weighted with the General
Plant Action Spectra used in this programme, following Green et al.
(1974).
Figure 2. An array of cloches in a vegetation of Turgidoscu/um
complicarulwn on Leonie Island, Antarctica (photograph: A.H.L.
Huiskes).
In this paper measurements on photochemical ef-
ficiency of photosystem II (PSII) using chlorophyll
a fluorescence techniques (Schreiber et al. 1988;
Bolhar-Norde nkampf et al. 1989; Krause & Weis
199 1; Schroeter 1994) made outside and underneath
the perspex screens are reported, using the effective
PSII quantum efficiency (!::1 F I f~, =
(F,~, - Fr) / F~ )
(Genty et a!. 1989) as a measure.
UOE.C2
..- 1.60E..02
·e
c
"~ 1.2ClE·02 • Lamp
~ - UV·absorbing
€ 8.00E.oJ
w UV·transparen~
ii
.E 4.00E..o3
o.ooe.oo r=::::--~~-~='-------'
280 320 3110
Figu re 3. lrradiance (in W m- 2 nm- 1) measured under a calibra-
tion lamp (Bentham CL6 wi th OSRAM Xenophot HLX 24V I SOW
light bulh), with a Bentham OM 150 spectroradio meter. Thick line:
light sensor underneath GS233 acrylic plastic (UV-B absorbing).
Thin line: light sensor underneath GS 2458 acrylic plastic (UV-B
transparent): circles: no acrylic plastic.
Materials and methods
Study area
The field research is carried out at Leonie Island
(67°35' S, 68°20' W), situated in Ryder Bay some
8 km SW of Rothera Point (Adelaide Island, Antarc-
tic Peninsula). The north side of the island is usually
free of snow during the austral summer, with a vege-
tation of lichens, mosses, terrestrial algae (e.g., Prasi-
ola crispa) and the angiosperm species Deschampsia
antarctica and Colohanthus quitensis. The south side
of the island is permanently covered by ice and snow.
The species di versity of the, in places dense, vege-
tation on the island is high compared to other areas
in the same region,which is probabl y due to the posi-
tion of the island: in front of a series of glaciers on
Adelaide Island which reflect the sunlight, resulting
in a milder climate, especially in sheltered sites (R. I.
Lewis Smith, pers. comm.).
Field sites
Acrylic plastic screens (so-called cloches) (Figure 2)
were placed in six vegetation types each domi-
nated by one of the following species: Deschampsia
antarctica (Angiospermae, Poaceae), Sanionia unci-
nata(= Drepanocladus uncinatus) (Musci), Prasiola
crispa (Algae, Ulvales), Turgidosculum complicatu-
lum (=Mastodia tesselata) (Lichenes), Stereocaulon
alpinum (Lichenes), and Usnea antarctica (Lichenes).
In each vegetation type two similar sets of cloches
were installed in January I 997. In addition, two vege-
tated plots of 50 em x 50 em were marked with dots
 79

Deschampsia antarctica (Poaceae) a Turgldiusculum complicatutum (Lichenes) d

0.8 0.8
0.7 0.7
- 0.6 -E 0.6
ff_ 0.5 LL 0.5
~ f 0.4
-E
0.4
0.3 -·E 0.3
!:!:.. 0.2 ~ 0.2
0.1 0.1
0 ~~~LL~~~~~~~~~~~~~~ 0 ~~~LL~~~~~~~--~~~~~~

San/on/a uncinata (Musci)

b Stereocauton alpinum (Lichenes)
e
0.8 0.8
0.7 0.7
-E 0.6 -E 0.6
u. 0.5 u. 0.5
~
-·E
0.4 ~ 0.4

I
0.3 -E 0.3
!!:. !!:.

II
0.2 0.2
0.1 0.1
0

Prasio/a crispa (Algae)

c Usnea antarctica (Lichenes)
f
0.8 0.8
0.7 0.7
-E 0.6 0.6
-E
u. 0.5 u. 0.5
~
-·fE
0.4
-·E 0.3
0.4
0.3
!!:. 0.2 !!:. 0.2
0.1 0.1
0

Figure 4. Mean effective PSI! quantum efficiency (D. F / F~ 1 I of six plant species in light adapted state in field plots covered with ditlerent types
of perspex cloches. The values for the replicate plots arc neighbouring each other.

of white paint, to serve as control plots. The cloches
measured 30 em x 50 em. One smaller side of the
cloche was resting on the surface, the other one was
supported by a 10 em x 30 em piece of acrylic plastic,
rendering a wedge shaped design, facing a direction
between NE and NW (Figure 2). Four types of cloches
were used: Two were ofUV-B transparent (PMMA GS
2458, Ri:ihm, Darmstadt Germany), one with sides,
rendering a completely closed cloche in which the
temperature was above ambient and one without sides
with a temperature inside the cloche close to ambi-
ent. The two other types, also a closed and an open
type, consisted of UV-B absorbing acrylic plastic (GS
233, Ri:ihm, Darmstadt, Germany). The UV-B absorb-
ing perspex also filters out part of the UV-A radiation
(Figure 3).
The cloches were assembled using all-weather UV
resistant transparent tape (Sellotape type 1433 PE).
The cloches were fixed to the rocky substrate by
strips of glass fiber reinforced tape affixed to the cor-
ners of the cloche with holes punched into the loose
ends through which rockpins were stuck which were
subsequently wedged into fissures in the rock.
80

e
100
a -.-
el;'
90
so
'S 70
·e 60
"
J:
50
40
~
:--~.·\ ... ~. ,.., . '\/\.·\ :. /' .... i
30

..·\:'
20
·.·· ~~ ~ 10
0 L---~--~--~--~--~--~--~--~
.....
<QI
Date
Date
Figure 5. Daily maximum (dotted line), mean (solid line) and min-
imum (dashed line) temperature under ambient circumstances (a),
b under open screens (b), and under closed screens (c). Daily maxi-
mum (dotted lines), mean (solid lines) and minimum (dashed lines)
% relative humidity under ambient circumstances (d) and under a
closed screen (e). The data are recorded in an Usnea antarctica
:'-
:'
.. ·'.,: ,. vegetation during the austral summer of 1998/99.
j \
'
Field measurements

Three to four weeks after the cloches were placed

in the field, the effective PSII quantum efficiency
Date ((F:, - Ft)j F~1 ), was measured when the plants were
in a light adapted state, using a chlorophyll fluo-
rescence meter (PAM-2000, Walz GmbH, Effeltrich,
c Germany). At least 15 individual chlorophyll-a fluo-
'•
rescence measurements were made under each cloche
'• and in each control plot spaced in random order over
~ :~; : I
the total surface area of the vegetation.
~· ' . Temperature and relative humidity under and out-
side the cloches were measured every ten minutes
using thermocouples (copper-constantan) and relative
humidity sensors (HMP 35AC, Vaisala Oyj, Vantaa,
Finland). The temperature sensors were placed in the
vegetation, about I em above the rock surface, the
Date humidity sensor abouts 3 em above the rock surface.

100 d Statistical analysis of the data

l : A nested analysis of variance, using the vanatwn

l;' 70
'S 60
between replicate cloches within each species and
'E 50 cloche type as the error term against which the effect
"
J: 40 of species, cloche (or treatment) and their interac-
~ 30
~ 20 tion was tested, was performed on a subset of the
~ 10 data. In this ANOVA the measurements in the con-
0 L---~--~~~~--~--~--~----L
trol plots were compared with the measurements in
.q;".. ,..... OJ" ........ ..,Of" ..,... .... Of.. ..,fll..
the cloches of UV-B transparent perspex without sides,
Date being the type most similar in environmental circum-
stances to the control plot. A similar nested ANOVA
was performed on the measurements underneath the
 81

four types of cloches. For these analyses the SYSTAT 0.6

package was used (Wilkinson 19R8). Mean and stan- *
dard deviation of the mean were calculated for each 0.5
set of measurements.
0.4
-E
Results !:!::
~ I
0.3
Figure 4(a-f) shows the average values· of ( F'm - F) -E
_ t
1
I F111 about one month after deployment of the cloches. !:.. 0.2 **
Differences in the effective PSII quantum efficiency
between the species occur and the effective PSll quan- 0.1
tum efficiency measured in replicate cloches differs
strongly for some species (Figures 4(c), 4(d) and 4(f)). 0.0
No significant differences could be detected he-
Before After
tween measurements made underneath the open trans-
parent cloches (the cloche type closely resembling Spraying
ambient circumstances) and the control sites for any Fixure 6. Box-and-whisker plot of 20 measurements on effective
of the species studied (Table I). Nor did the effective PSII quantum efficiency (( F,;,- F1 )/ F,;,) measured in Usnea an/Ore-
PSII quantum efficiency measured underneath the dif- tim in light adapted state before and after spraying with tap water.
ferent types of cloche show any significant differences The length or the box represents the interquartile range. The hor-
izontal division in the median value. The vertical lines are drawn
(Table 2). to the largest resp. the smallest observation within 1.5 interquartile
Figure 5(a-f) shows the daily means, the maxima ranges of the group. Ohservations heyond these limits are shown
and minima of ambient temperature and the tempera- individually.
ture in open and closed cloches and the daily means,
the maxima and minima of the ambient relative humid-
Usnea antarctica, under ambient circumstances. The
ity and the relative humidity in closed cloches. It can
effective PSII quantum efficiency of the thalli var-
be clearly seen that temperature and relative humidity
ied considerably as can be seen in a box-and-whisker
fluctuations in the cloches are larger than the ambient
plot (Figure 6). After these measurements the thalli
fluctuations and that the temperature fluctuations in
were sprayed with tap water and after 30 min chloro-
the open cloches are smaller than in the closed cloches.
phyll a fluorescence was measured again on the same
thalli. The variation in (F' - F, )IF' was then de-
creased and the median va{~e increas;d. These results
Discussion
are supported by Schroeter ( 1994 ), who found that the
The differences in mean effective PSII quantum ef-
(F,;, - F1) IF,;, could vary strongly between thalli of a
single lichen species depending on their moisture sta-
ficiency between the two replicate cloches and plots
tus, which in turn depends on the moisture availability
(Figure 4) are most likely caused by differences in
which may vary considerably between microhabitats
moisture status of the vegetation due to the way in
of the species.
which the measurements were taken. In order not to
The microclimate in the cloches differs from that
disturb the vegetation already in the first field sea-
outside the cloches. Temperature and relative humidity
son, the measurements were taken under existing en-
m the cloches are more variable than under ambient
vironmental circumstances, the vegetation under the
circumstances. With respect to temperature, the vari-
cloches was not compensated for any loss of precip-
ation in the cloches is greater on sunny days, than on
itation. Time of day (mid- afternoon) and cloudiness
overcast days (cf., the data for 15 January-16 Febru-
were relatively similar during measurements, but the
ary, respectively the data for 22 December-IS January
variation in moisture status among as well as within
of Figure 5(c)). Also the variation in relative humidity
the plots was probably large.
is greater in the cloches: the maximum daily values arc
Support for this hypothesis may be found in a sim-
higher and the minumum daily values are lower (Fig-
ple experiment performed afterwards. Chlorophyll a
ures 5(d) and 5(e)). The daily mean temperature and
fluorescence measurements were taken on the lichen
82
Table I. Analysis of variance of measurements of effective PSII quantum efficiency in vege-
tation plots dominated by different species and covered by UV-B transparent perspex cloches
without sides and not covered with a cloche (control).

Source Sum of DF Mean F-ratio

squares square

'Cloche type' 0018 0.018 0.057"'

'Species' 6.255 5 1.25 I 4.037**
'Species' x 'cloche type 0.478 5 0.096 0.309"'
Error term:
'Clochcrcplicatc' {'species' {'cloche type'}} 3.719 12 0.310
Error within replicates 4.495 325 0.014

Table 2. Analysis of variance of measurements of effective PSI! quantum efficiency in vege-

tation plots dominated by different species and covered by UV-B transparent or UV-B opaque
pcrspcx cloches, which are either open or closed at the sides.

Source Sum of DF Mean F-ratio

squares square

'Cloche type 0.596 4 0.149 0.605 11 '

'Species' 18.834 5 3.767 15.284***
'Species x 'cloche type' 2.151 20 0.108 0.436"'
Error term:
'Clochereplicate' {'species' {'cloche type'}} 7.394 30 0.246
Error within replicates 15.058 826 0.018

relative humidity in the cloches are higher than am-
bient. Because the vegetation underneath the cloches
grows on soil as a substrate evaporation of mois-
ture from the soil renders a higher humidity in these
cloches. When the vegetation underneath the cloches
grows on a rocky substrate, evaporation is low and the
humidity underneath the cloches is lower than ambi-
ent. As a result of Sanionia uncinata (Musci) shows a
higher mean effective PSII quantum efficiency inside
the cloches as compared to the control sites (Fig-
ure 4(b)), but, e.g., Usnea antarctica (Lichen) (Fig-
ure 4(f)) shows a lower mean effective PSII quantum
efficiency (see also Huiskes ct a!. 1999).
As in the measurements of (F:n - F1 ) / F~, no sig-
nificant influence of UV- B could be shown, this could
mean that the effect of attenuation of ambient UV-B is
obscured by other environmental factors, as moisture
availability, temperature fluctuation, and fluctuation in
relative humidity. We believe that moisture availability
is the overriding factor, obscuring the possible effect
of UV-B exclusion. Also other authors (Day et a!.
1999; Baker et a!. 1997) do not show a clear effect
of UV-B on the vegetation when comparing ambient
(enhanced when compared to pre-ozone hole times)
and below-ambient exposure to UV-B. But although
the plants under study may not show any effect in their
effective PSII quantum efficiency (Allen et al. 1999;
Searles et a!. 1999), other changes may have occurred
in the plants which can be attributed to enhanced
ambient UV-B radiation, such as accumulation of pro-
tective pigments or other screening and quenching
compounds. This is presently under study.
In an experimental study in a climate room
Rozema eta!. (200 I) found reduced length growth and
increased tillering in Deschampsia antarctica under
2.5 and 5 kJ m- 2 day UV-BBE· In the cloche exper-
iment in the field the grass Deschampsia antarctica
does not show an effect of ambient UV-B. However,
the presence of the 'greenhouse effect' of the cloches
results in a high primary production. This is shown
in Figures 7 (a) and (b). The pictures were taken in
exactly the same cloche, one year apart (sec also Lud
et al., 2001).
The results of the first season of a long-term ex-
periment prompt a discussion on the usefulness of of
the design of field experiments. Field studies on the ef-
 83

Figure 7. Development of the vegetation in comrol plots (a. b) and unde rneath perspex cloches (c, d). (a) Photograph of a control plot dominated
by the terrestrial alga Prasiola crispa, take n in February 1997. (b) Photograph of the same plot taken February 1998. All species in the
photograph have grown over the year 1997, butt he relati ve abundance of the species has not c hanged. (c) Photograph of the vegetation under a
transparent cloche with sides, dominated by the terrestrial alga Prusiolo crispo, taken in February 1997. (d) Photograph of the same vegetation
taken in February 1998. The algal vegetation has changed into a vegetation do minated by Sanionia uncinllfo. with Deschampsia antarClico
invading.
84
feet of enhanced UV-B radiation are usually executed
using two opposite experimental designs: supplemen-
tation of the UV-B radiation using fluorescent UV-B
lamps or attenuation of the UV-B radiation using
certain types of transparent filters.
Supplementation systems for studies on the ef-
fect of enhanced UV-B radiation have been reviewed
in detail by McLeod ( 1997). Two systems are used:
switched or square-wave systems and modulated sys-
tems, capable of varying the radiant flux from the
fluorescent lamp to supply UV- B radiation that is
more proportional to ambient levels.
UV-B attenuation systems are simple to construct
and maintain. These systems usually comprise a set of
screens filtering the ambient sunlight by absorbing the
UV-B and/or the UV-A components of the solar spec-
trum, depending on the filter material used. Studies
on the influence of UV-B radiation using absorption
techniques take into account that absorption of the UV
component of the solar spectrum has an effect on the
organisms or biota studied, and it is intrinsically as-
sumed that this effect will be opposite to the effect
of enhancing UV-B radiation. In the experiments the
effects of different types of filters are compared and
the effect of the UV-B transparent filter is interpreted
as a UV-B effect.
Various designs of screens are used. Sheets of
polycarbonate (filtering out UV-A and UV-B) and or
Mylar (UV-B only) mounted in frames (Jackson &
Seppelt 1997) also sheets of UV-transparent and UV-
absorbing acrylic plastic are used. These have the ad-
vantage that they are sturdy and can be left unattended
for prolonged periods of time (Wynn-Williams 1996;
Huiskes et al. 1999). More elaborate cylindrically
shaped screens are also used (Day et al. 1999). These
last screens filter the solar radiation effectively, espe-
cially in areas like the Antarctic where the solar zenith
angle is low, without affecting climatic circumstances,
especially moisture conditions too much. Therefore,
the influence ofUV-B radiation is less obscured by the
effect of changes in moisture availability (see above).
UV-B attenuation systems, as applied in the
present study, show climatic constraints, in Antarctic
vegetation especially with respect to moisture avail-
ability and moisture regulation. Moisture availability
should be optimized at least prior to measurements
or samplings. However, a regular water supply to the
vegetation under the screens might lead to problems
with fungal infection as evapotranspiration is affected
(Bacherau 1997).
Despite these problems, the use of screening sys-
tems is usually based on decisions balancing the
shortcomings against the advantages of using simple
systems in the field.
UV-B attenuation systems do not have the short-
comings of square-wave supplementation systems
such as a disturbed spectral balance. In that respect
they are a better tool to study the effects of ambi-
ent UV-B radiation. The UV-B/PAR ratio under the
screens is less affected, and this undisturbed spectral
balance may make UV-B exclusion manipulation more
realistic than in supplementation systems (Teramura
1980; Cen & Bornman 1990; Deckmyn et al. 1994;
Santas et al. 1997). Another artifact can be introduced
when solar irradiance is high; effects of photoinhibi-
tion and photodamage to PSII reaction centres may be
observed, which are not a primary detrimental effect
ofUV-B (Bakeretal. 1997).
Recently, we attempted to use an even simpler
method. In stead of using attenuation or supplementa-
tion systems, we monitored the natural variation in the
effective PSII quantum efficiency and the possible pro-
duction of pigments and other screening and quench-
ing compounds along natural gradients. A problem
with this method is that the effects might not be species
specific. In nature high levels of UV-B irradiation al-
ways coincide with high PAR levels and, depending on
the microsite, also with temperature, which may result
in differences in moisture availability.
Acknowledgements
The results described in this paper form part of the
BRUVA- programme (Biotic Responses to UV-B Ra-
diation in Antarctica), a joint British-Dutch research
programme in which the British Antarctic Survey,
Cambridge, UK, the Anglia Polytechnic University,
Cambridge, UK, the Free University, Amsterdam, NL,
and the Netherlands Institute of Ecology, Centre for
Estuarine and Coastal Ecology, Yerseke, NL, collabo-
rate. The Dutch part of the programme is funded by
the Netherlands AntArctic Programme (NAAP), ad-
ministered by the Earth and Life Sciences Division of
the Netherlands Organisation for Scientific Research
(NWO), which is gratefully acknowledged.
We thank the British Antarctic Survey, Cambridge,
UK for giving us hospitality and logistic support at
the research station Rothera during our stay in Antarc-
tica. We also thank the population at Rothera Research
Station for their friendship and support.

Brian Gardiner and Timothy Martin are thanked
for their help with the calculation of UV-B radia-
tion from ozone data and for letting us use the BAS
Radiation Transfer Model (BASRTM).
References
Allen. D. J.• Nogucs. S. & Baker. N. R. 1999. Ozone depletion and
increased UV-B radiation: is there a real threat to photosynthesis''
J. Exp. Bot. 49: 1775-1788.
Bacherau, F. 1997. Effets de I' exclusion selective du rayonncment
solaire (visible et UV) de haute altitude sur Ia biochimie et Ia
physiologic de divers modeles vegctaux: Piswn sativwn L. (pais
cultivc), Sedum album L. (orpin blanc) et Cetraria islwulica
IL.) Arch. (lichen tcrricole). Thesis, University Jouseph Fourier,
Grenoble, 175 pp.
Raker, N. R., Nogues, S. & Allen, D . .1. 1997. Photosynthesis and
photoinhibition. Pp. 95-111. Tn: Lumsden. P. .1. led.), Planh
and UV-B, Responses to Environmental Change. Camhridge
University Press, Cambridge.
Blumthaler, M. & Ambach, W. 1990. Indication of increasing solar
ultra-violet-B radiation flux in Alpine regions. Science 248: 206-
208.
Bolhar-Nordenkampf, H. R., Long, S. P., Baker, N. R., bquist, G.,
Schreiber, U. & Lechner. E. G. 1989. Chlorophyll fluorescence
as a probe of the photosynthetic competence of leaves in the field:
a review of current instrumentation. Funct. Ecol. 3:497-514.
Caldwell, M. M .. Teramura, A. H. & Tevini, M. 1989. The chang-
ing solar ultraviolet climate and the ecological consequences for
higher plants. Trends Ecol. Evol. 4: 363-367.
Cen, Y. P. & Bornman, J. F. 1990. The response of bean plants to
UV-B radiation under different irradiance of background visible
light. J. Exp. Bot. 41 : 1489-1495.
Day, T. A., Ruhland, C. T., Grobe, C. W. & Xiong, F. 1999.
Growth and reproduction of Antarctic vascular plants in response
to warming and UV radiation reductions in the field. Oecologia
119: 24-35.
Deckmyn, G., Martens, C. & Tmpens, I. 1994. The importance of the
ratio UV-B/photosynthetic active radiation (PAR) during leaf de-
velopment as determining factor of plant sensitivity to increased
UV-B irradiancc: effects on growth, gas exchange and pigmen-
tation of bean plants (Phaseolus mlgaris cv. Label). Plant, Cell
Environ. 17: 295-301.
Farman, J. C., Gardiner, B. G. & Shanklin, .1. D. 1985. Large
losses of total ozone in Antarctica reveal seasonal ClO,/NO,
interaction. Nature 315: 207-210.
Frederick, J. E. & Snell, H. E. 1988. Ultraviolet radiation levels
during the Antarctic spring. Science 241: 438-440.
Frederick, J. E., Snell, H. E & Haywood, E. K. 1989. Solar ultra-
violet radiation at the earth's surface. Photochem. Photobiol. 50:
443-450.
Genty. B., Briantais, J.-M. & Baker, N. 1989. The relationship be-
tween the quantum yield of photosdynthetic electron transport
and quenching of chlorophyll fluorescence. Biochim. Biophys.
Acta 990: 87-92.
Green, A. E. S .. Sawada, T. & Shettle. E. P. 1974. The mid-
dle ultraviolet reaching the ground. Photochem. Photobiol. 19:
251-259.
Huiskes, A. H. L., Lud, D, Moerdijk-Poortvliet, T. C. W. &
Rozema, J. 1999. Tmpact ofUV-B radiation on antarctic terres-
trial vegetation. Pp. 313-337. In: Rozema, J. (ed.), Stratospheric
85
Ozone Depletion; the Effects of Enhanhced UV-B Radiation on
Terrestrial Ecosystems. Backhuys Publishers, Lciden.
Huiskcs, A. H. L., Lud, D. & Mocrdijk-Poortvliet, T. C. W. 2000.
Responses to UV-B radiation in terrestrial antarctic vegetation.
Pp. 252-257. In: Davison W. et al. (cds), Antarctic Ecosystems:
Models for Wider Understanding. Caxton Press, Christchurch.
Jackson, A. E. & Seppelt. R. D. 1997. Physiological adaptations to
freezing and UV radiation exposure in Prasiola crispa, an Antarc-
tic terrestrial alga. Pp. 226-233. In: Battaglia, B .. Valencia. J.
& Waltun, D. H. W. (eds). Antarctic Communities: Species,
Structure and Survival. Cambridge University Press. Cambridge.
Ken. J. B. & McElroy, C. T. 1993. Evidence for large upward trends
of ultraviolet-B radiation linked to ozone depletion. Science 262:
1032-1034.
Krause, G. H. & Weis, E. 1991. Chlorophyll fluorescence and pho-
tosynthesis: the basics. Ann. Rev. Plant Physiol. Plant Mol. Bioi.
43: 313-349.
Lud, D., Huiskcs, A. H. L.. Moerdijk, T. C. W. & Rozema. J. 2001.
The etlects of altered levels of UV-B radiation on an Antarctic
grass and lichen. Plant Ecol. 154: 87-99 (this volume).
McLeod, A. R. 1997 Outdoor supplementation systems for studies
of the effects of increased UV-B radiation. Plant Ecol. 128: 78-
92.
Rozema, J., Van de Staaij J. W. M. & Tosserams M. 1997a. Effects
of UV-B radiation on plants from agro- and natural ecosystems.
Pp. 213-232. ln: Lumsden, P. J. (ed.). Plants and UV-B, Re-
sponses to Environmental Change. Cambridge University Press,
Cambridge.
Rozema, J., Van de Staaij, J., Bjorn, L. 0. & Caldwell. M.
l997b. UV-B as an environmental factor in plant life: stress and
regulation. Trends Ecol. Evol. 12: 22-28.
Rozema, J., Brockman, R .. Lud, D., Huiskcs, A .. Mocrdijk,
T., De Bakker, N., Mcijkamp, B. B. & Van Beem, A. 2001.
Consequences of depletion of stratospheric ozone for terrestrial
antarctic eco5.ystems: the response of De.Khampsio antarctica
to enhanced UV-B radiation in a controlled environment. Plant
Ecol. 154: 101-115 (this volume).
Santas, R., Koussoulaki, A. & Hader, D.-P. 1997. In assessing
biological UV-B etfects. natural fluctuations of solar radiation
should be taken into account. Plant Ecol. 128: 93-97.
Schreiber. U., Bilger, W .. Klughammer, C. & Neubauer, C. 1988.
Application of the PAM fluorometer in stress detection. Pp. 151-
155. In: Liehtenthalcr, K. H. (ed.). Applications of Chlorophyll
Fluorescence. Kluwer Academic Publishers, Dordrecht.
Schroeter. B. 1994. In situ photosynthetic differentiation of the
green algal and the cyanobacterial photobiont in the crustosc
lichen Placopsis contortuplicota. Occologia 98: 212-220.
SCOPE 1992. Effects of increased ultraviolet radiation on global
ecosystems. Proceedings of a workshop arranged by the Sci-
entific Committee on Problems of the Environment (SCOPE),
Tramariglio, Sardinia, 47 pp.
Searles, P. S., Flint, S. D .. Dfaz, S. B .. Rousseaux, M. C., Ballarc,
C. L. & Caldwell, M. M. 1999. Solar ultraviolct-B radiation in-
fluence on Sphagnum bog and Curex fen ecosystems: first field
season findings in Tierra del Fuego, Argentina. Global Change
Bioi. 5: 225-234.
Teramura, A. H. 1980. Effects of ultraviolet-B irradiance on soy-
bean. Interaction between ultraviolet-B and photosynthetically
adive radiation on net photosynthesis, dark respiration, and
transpiration. Plant Physiol. 65: 483-488.
Teramura. A. H. & Sullivan .1. H. 1994. Effects ofUV-B radiation on
photosynthesis and growth of terrestrial plants. Photosynt. Res.
39: 463-473.
86

Wilkinson, L. 1988. SYSTAT, the system l'or statistics. Systat Inc., and cryptogams. Pp. 243-257. In: Weiler, C. S. & Penhale, P. A.
Evanston. (eds), Ultraviolet Rradiation in Antarctica: Measurements and
Wynn-Williams, D. D. 1994. Potential effects or ultraviolet radiation Biological Effects. Antarctic Research Series, Vol. 62.
on Antarctic primary terrestrial colonizers: cyanobacteria, algae
Section 3: Arctic and Antarctic Plants and Ecosystems

A mini UV-B lamp system over the coastal lichen Turp,idosculum comp/icawlum on Leonie Island,
Antarctica. (Photograph .1. Rozema)
 Plant Ecology 154: 89-99, 200 l.
89
© 200 I Kluwer Academic Publishers.

The effects of altered levels of UV-B radiation on an Antarctic grass and

lichen

D. Lud 1, A, H. L. Huiskes 1, T. C. W. Moerdijk 1 & J. Rozema 2

Netherlands Institute of Ecology 1Centre for Estuarine and Coastal Ecology, P 0. Box I 40,
4400 AC Yerseke, The Netherlands (E-mail: lud@cemo.nioo.knaw.nl); 2 Department of Systems Ecolof?y,
Faculty of Biolog)~ Vrije Universiteit, De Boelelaan I 087, 108 I HV Amsterdam, The Netherlands

Key words: Antarctica, Carotenoids, Deschampsia antarctica, Photosynthesis, Turgidosculum complicatulum,

UV-B radiation, UV-B supplementation

Abstract
We report a long-term experiment on the photosynthetic response of natural vegetation of Deschampsia antarctica
(Poaceae) and Turgidosculum complicatulum (Lichenes) to altered UV-B levels on Leonie Island, Antarctica.
UV-B above the vegetation was reduced by filter screens during two seasons. Half of the screens were transpar-
ent to UV-A and UV-B (ambient treatment) or absorbing UV-B and part of the UV-A (below-ambient treatment).
Half of the wedge- shaped filters had side walls leading to an enhancement of the daily mean temperature in summer
by 2-4 °C, simulating rising mean air temperature on the Antarctic Peninsula. The other half of the filters were
without side walls resulting in close-to-ambient temperature underneath. Plots without filters served as controls.
UV-B supplementation of an extra 1.3 kJ UV-BBE was achieved using UV-mini-lamp systems during I 5 days
in the second season.
We found no evidence that altered incident UV-B levels and temperature had an etfect on maximum photosys-
tem II efficiency ( Fv I Fm) and effective photosystem II efficiency ( <"> F I Fm ') in both species. UV-B reduction did
not influence contents of chlorophyll, carotenoids and methanol-soluble UV absorbing compounds in D. antarctica.
Flowering shoot length of D. antarctica was not affected by UV- B reduction. Temperature enhancement tended
to result in longer inflorescence axes. Results of two austral summer seasons of UV- reduction in natural stands of
D. antarctica and T. complicatulum suggest that current ambient levels of UV-B do not have a direct effect on the
photosynthetic performance and pigment contents of these species. Cumulative effects on growth have not been
recorded after two years but can not be excluded on a longer term.

Introduction means of ozone column depth leading to increased in-

The effects of global climate change are most pro-
nounced in polar regions. This also holds for the
Western coast of the Antarctic Peninsula. In this region
the annual mean air temperature has been increasing
by 0.2 oc to 0.6 °C per decade between 194 7 to 1990
(King 1994) with the relative temperature increase be-
ing higher in the southern part of the Antarctic Penin-
sula. During periods of ozone depletion in the austral
spring ozone column depths decline to I 00 Dobson
Units (DU) in October. Ozone column depths in Jan-
uary and February recently are lower than long-term
cident UV-B radiation during the austral summer as
well (Jones & Shanklin 1995).
Doses of incident UV-B are highest on sunny
days from mid November through to the beginning of
December. Daily doses weighted with the plant ac-
tion spectrum (Caldwell 1971; Green et al. 1974) at
Rothera Research Station range between 4 kJ m- 2 d -I
with occasional extreme values of up to 8 kJ m- 2 d- 1
in November and December (estimated from measure-
ments with a Bentham scanning spectro-radiometer).
Searles et al. ( 1999) report similar values of daily
doses for a field site in Tierra del Fuego.
90
0.020
o Lamp
-IN-opaque
0,016
~-
E 0.012
~
:;.
·;;;
c
2 0.008
.5
0.004
0.000 4------==--===-------~
280 320 360 400
Wavelength [nm]
Fir<w·e I. Transmission spectra of transparent perspexTM (PMMA
GS 2458) and opaque pcrspcxTM (GS 233) measured with a
Bentham spectroradiometer using a calibration lamp. The lamp
spectrum has been added for comparison.
Field studies on the influence of climate change
on natural vegetation in the Maritime Antarctic are
rare. Research is often focused on the only two na-
tive phanerogam species occurring in Antarctica: the
grass Deschampsia antarctica Desv. and the pearl-
worth Colobanthus quitensis (Kunth) Bartl. Little in-
formation is available about possible consequences of
climate change for cryptogam species.
Low temperatures limit the growth of higher plants
in Antarctica. There have been several recent reports
about increases in population size of higher plants.
Fowbert & Smith ( 1994) and Smith ( 1994) consider
warmer and more extended summers to be the rea-
son for rapidly expanding populations of C. quitensis
and D. antarctica in the Argentine Islands. The num-
ber of established seedlings of C. quitensis has been
found to be positively correlated with mean summer
temperatures on Anvers Island (Grobe et al. 1997).
A long-term study conducted on the effects of UV-
reduction and temperature increase on C. quitensis and
D. antarctica near Palmer Station, 64°46' S revealed
that warming and altered levels of UV-B did affect
both species: Day et al. ( 1999) found that natural am-
bient levels of UV-B reduce growth in both species
with the effects being more pronounced in the second
than in the first year. The field site of the study reported
here is situated on Leonie Island ca. 4 deg of latitude
further South of Palmer Station. Montiel et al. ( 1999)
exposed transplants of D. antarctica and C. quitensis
at Leonie Island to different UV-environments for 7
days and concluded from their findings that current
ambient summer levels ofUV-B did not affect the pho-
tosynthetic activity of these species. As growth rates of
plants in Antarctica are low (e.g., Rozema et al. 2001 ),
there is hence a need for long-term studies to assess the
effects of climate change on Antarctic vegetation.
Lichens are the dominant elements in many ter-
restrial Antarctic ecosystems (Green et al., 1999),
where relative changes of UV-B are most pronounced
(Madronich et al., 1995). However, knowledge of the
performance of lichens under altered levels of UV-B is
limited.
The lichen Turgidosculum complicatulum Kohlm.
et Kohlm. (formerly Mastodia tesselata Hookf et
Harv.) is restricted to polar regions (Redon, 1985).
The dark, foliose thalli of T complicatulum usually
inhabit exposed rocks influenced by birds (Gremmen
et al. 1994; Olech 1990) often close to the shoreline
(Huiskes et al. 1997). Reports on the responses of
lichens to UV-A and UV-B radiation are conflicting.
Swanson & Fahselt (1997) found UV-A to enhance the
concentration of phenolic substances in Umbilicaria
americana Poelt et Nash whereas UV-B decreased
these concentrations in field and laboratory experi-
ments. Investigating samples of U. americana taken
along a natural gradient of UV-exposure, Swanson
et al. ( 1996) found a negative effect of UV- B on phe-
nolic compounds. Increased incident UV-B radiation
might also have indirect effects on lichens as it has
been suggested to enhance susceptibility of Clado-
nia mitis Sandst. to fungal infection in Greenland
(Heide-J¢rgenson & Johnson 1995). Further research
is needed to develop more insight in the possible adap-
tive strategies of lichens to UV-B radiation. Here we
report a field experiment on the photosynthetic re-
sponse of D. antarctica and T complicatulum to UV- B
radiation set up for a period of four years in January
1997. The data of the austral summer 1997/1998 and
1998/1999 are presented in this paper.
Material and methods
Field site
The field experiment described was conducted on
Leonie Island (67°35' S, 68°20' W) in Marguerite Bay,
8 km south of the British Research Station Rothera
(see Huiskes et al. 2001 and Rozema et al. 2001 ).
The north side of the island sustains a cryptogam-
dominated vegetation of lichens, mosses and terrestrial
 91
0.8 a
0.6
·e
-
LL
LL
<1
0.4

0.2

filter type control transparent transparent, sided opaque opaque, sided

0.8 b

0.6 ...-- ...-- ...-- ,..-

-
E
LL
0.4
>
LL
0.2

0.0
filter type control transparent transparent, sided opaque opaque, sided

0.8 c
0.6
·e
LL
- 0.4
LL
<1
0.2

filter type control transparent transparent, sided opaque opaque, sided

- -
0.8
d
0.6

-
E
LL
0.4
>
LL
0.2

0.0
filter type control transparent transparent, sided opaque opaque, sided

Figure 2. Mean maximum PSII quantum efficiency ( Fv I F111 ) and mean effective PSII quantum efficiency. .6. F I F111 1 • of Deschampsia antarctica
and Turgidosculum complicatulum in field plots covered with ditlcrent types of perspex screens. The white columns represent Fv I F111 measure-
ments, the hatched columns represent measurements of .6.F I F111 '.Error bars show standard deviations of means (n=3). (a) and (b) mark graphs
of D. antarctica of the austral summer 1998, (c) and (d) graphs of the austral summer 1999. (e) and (f) mark graphs ofT complicatu/um of the
austral summer 1999.

algae as well as D. antarctica and C. quitensis. In
places, D. antarctica forms extensive swards, whereas
C. quitensis is comparatively rare. The soil underneath
Deschampsia vegetation in bird influenced locations
is generally nutrient rich (Smith 1984, 1985), pH
values in the soil-like substrate range between 6-7,
in accumulated peat, values are considerably lower
(Smith 1984). T. complicatulum grows on exposed
rocks on Leonie Island with high nutrient input from
birds and is common on exposed habitats just above
the shoreline (Gremmen et al. 1995).
Filter experiments and microclimate measurements
In total 12 filter screens per species measuring 30 x
50 em were placed above natural vegetation of D.
antarctica (Poaccae) and T. complicatulum (Lich-
enes). Some results obtained for other species have
already been described by Huiskes et al. (2000).
Four different types of screens were used: (i) UV-
transparent perspex (PMMA GS 2458, Rbhm, Darm-
stadt, Germany), which transmitted 85% of the in-
cident biologically effective UV-B radiation (Green
et al. 1974; Caldwell 1971) without side walls,
92
0.8 e
0.6
-E
....u.. 0.4
u..
<I
0.2

0.0
filter type control transparent transparent, sided opaque opaque, sided

0.8

0.6
E
u..
0.4
>
u..
0.2

0.0
filter type control transparent transparent, sided opaque opaque, sided

Figure 2. Continued.

(ii) UV-transparent perspex with side walls, (iii) UV-B
opaque perspex (GS 233, Rohm, Darmstadt, Ger-
many) which attenuated 10% of the incident bio-
logically effective radiation without side walls and
(iv) UV-B opaque perspex TM with side walls. Trans-
mission properties of the two types of perspex™ used
are shown in Figure I. The etfect of the screens on
the microclimate underneath is discussed in detail by
Huiskes et al. (200 I). Between 22 December 1998
and IS February 1999, mean daily temperature under
screens with side walls was 2.5 oc higher than outside
temperature, mean daily temperature under screens
without side walls was 1.8 oc higher than outside
temperature. The temperature differences were most
pronounced under screens with side walls in exposed
positions on sunny days (see Huiskes et al. 2001 ). The
C02 concentration under open sided screens did not
differ from outside air, the C02 concentration under
screens with side walls was slightly lower than out-
side air (less than I 0%) during the day. Three sets of
screens were constructed per species. For each set a
plot of 30 em x SO em was not covered and served as
control.
Temperature and relative humidity in the vege-
tation underneath the screens have been monitored
throughout the year in one representative set of cloches
with sensors connected to a micrometeorological sta-
tion as described elsewhere (Huiskes et al. 1999), dur-
ing the summer season additional data on temperature
and humidity in vegetation underneath and outside
various screens have been collected using Tiny Talk II
mini- dataloggers TM (Orion components, Chichester,
UK).
Measurement of solar radiation
Spectral global irradiance was measured with a Ben-
tham DMISO scanning spectroradiometer (Bentham
Instruments, Reading, England) at Rothera Research
Station. Scans from 280 nm to 600 nm with a 0.5 nm
step size and a resolution of I nm were recorded at
half-hourly intervals between 8:00 and 19:00. These
scans were used to calculate UV-irradiance and bio-
logically effective dose. Daily doses were estimated
on the basis of half-hourly scans.
UV-supp/ementation
Above natural vegetation of D. antarctica as well as T.
complicatulum eight mini-lamp-arrays were installed
in January 1999 in order to supply additional UV-
B. The mini lamps consist of UV-ftuorescent tubes
(Vilbert-Lourmat, Philips, The Netherlands, 44 em
length, 3 em diameter) mounted in a perspex TM hous-
ing on metal legs (see Rozema et al. 1997 as well as
Rozema et al. 2001 for detailed !description). Filters
were mounted into the housing underneath the lamps
in order to create the different conditions of irradia-
tion. Per species, four lamp-arrays were set up, two
with a mylar foil filter and two with a cellulose acetate
filter below the lamp resulting in ambient UV-B (con-
trol) and above- ambient UV-B levels, respectively.
The lamps burned for 1.5 h each day during a two-
week period starting on 25 January in order to supply

an extra biologically effective dose of 1.3 kJ m- 2 d- 1
UV-B (Caldwell 1971; Green eta!. 1974). The lamp
spectrum was recorded using a Bentham scanning
spectroradiometer with the sensor at 40 em from the
lamps. The distance between lamps and vegetation
was ca. 40 em.
Measurements in the field
Underneath the screens and in the respective control
plots, the effective PSII quantum efficiency, ~ F / Fm 1 ,
as well as the maximum PSIT quantum efficiency
(Fv/ Fm. with the plants dark adapted) was mea-
sured with a chlorophyll-a fluorescence meter (mini-
PAM, Walz GmbH, Effeltrich, Germany) in February
1998 and January 1999. Per plot, 15 chlorophyll-
fluorescence measurements of ~F / F11 ,' and F.,/ F111
were recorded in randomly chosen positions. The
effective PSII quantum efficiency was recorded un-
der natural light conditions, and then maximum PSII
quantum efficiency was measured under a black cloth
after the plots were allowed to become dark-adapted
for20 min.
Prior to the measurements, the vegetation was
moistened with tap water for a few days and again
one hour before measurements took place to reduce
variation in photosynthetic activity due to differences
in water content (Huiskes eta!. 200 I).
Samples were taken from the plots after measure-
ments to assess water content which was found to be
similar for all treatments (77 ±2% in D. antarctica and
74 ± 5% in T. complicatulum) and considered as non-
limiting in both species.
Plots of one set were measured after one another as
quickly as possible. Therefore environmental condi-
tions were assumed to be constant between replicates
and treatments.
The same parameters were recorded under the
lamp-arrays. Effective and maximum PSII quantum
efficiency were measured before the lamps were
switched on at the beginning of the two week period
of UV-supplementation.
At the end of the 15-day period of supplemen-
tation, the etfective PSI! quantum efficiency was
recorded before lamps were switched on. It was
recorded again with the lamps switched on, after the
lamps had been burning for one hour. After the lamps
had been switched off, maximum PSI! efficiency was
measured on dark adapted samples.
93
Growth
As growth of T. complicatulum is very slow, growth
analysis was conducted in the plots of D. antarctica
only. ln March 1999, at the end of the second sea-
son, 15 flowering shoots were harvested per plot. The
length of the inflorescences, dry weight of dead, living
and reproductive parts were measured and the number
of caryopses per inflorescence was counted.
Additionally, several hundred caryopses were har-
vested from under the open-sided screens and I 0
batches of 200 caryopses (caryopses including palea
and lemma) were weighted additivcly on a balance
with the precision of 0.0 I mg (Sartorius R200D,
Sartorius, Gottingen, Germany).
Chlorophyll, carotenoid\· and methanol soluble
UV-absorbing compounds
Six samples of green leaf material of D. antarctica per
plot were collected on 22 January 1999 in the after-
noon. At 14:00, daily incident UV irradiance was at its
maximum, with UV-B irradiance being 0.97 W m- 2
and UY-A irradiance being 40.71 W m- 2 . A daily
dose of 3.5 kJ m- 2 UV-BsE (Caldwell 1971; Green
eta!. 1974) was estimated from half hourly scans mea-
sured with the Bentham spectroradiometer at Rothera
Research Station.
The same amount of samples was taken of T. com-
plicatulum on 25 January 1999 in the afternoon. At
13:00 incident UV-8 irradiance was 0.64 W m- 2 , UV-
A irradiance was 28.60 W m- 2 (daily maximum),
the estimated daily dose of UV-B 8 E (Caldwell 1971;
Greenetal. 1974)was 1.7kJm 2 d-'-
Five samples were directly frozen in liquid nitro-
gen at the field site and subsequently stored at -80 oc
until analysis. The remaining sample was used to as-
sess water contents. Fresh and dry weight (after freeze
drying for 72 h) was determined to have an estimate of
water content of the samples.
Chlorophyll and carotenoids were determined us-
ing HPLC following the method of Wright et a!.
( 1991 ). The pigments were extracted from frozen
material ( -80 °C) after grinding in liquid nitrogen
with methanol (96%) and ammonium acetate (4% ).
MgC03 was added to extracts of T. complicatu-
lum to buffer lichen acids (Schroeter et al. 1995).
UV-absorbing compounds were measured in the di-
luted chlorophyll extracts scanning from 280 nm to
700 nm on a UVIKON 940 spectrophotometer (Kon-
tron, ZUrich, Switzerland).
94
0.8 dayO day15 0.8
Statistical analyses
0.6 • t
• • 0.6
E
For statistical analyses of the data Statistica 98 (Stat- E
LL
LL
0.4 0.4;:
soft Inc., 1998) was used. The chlorophyll fuorescence a: LL

data of the UV-filter experiment were tested using 0.2 0.2

ANOVA with screen type and UV-B as factors and

0.0 0.0
non-covered plots as isolated control. lt\)~.'0 9¢--'0\0~... ,..\)~.'0 e(~,,''O\e~\
For the pigment data, t-tests were used. Shoot UV- treatment
length data were tested using the Mann-Whitney U-
test as data were not normally distributed, the weigbts
of caryopses and dry weight proportions were tested
with the Mann-Whitney U-test as well. The chloro- 0.8
dayO day15 0.8
day 15
phyll fluorescence data of the UV-B supplementation lamps off Iampson lamps off
0.6 0.6
experiment were not tested statistically as the number i: i:
? "
LL LL
0
of replicates was too small (n=2).
Treatment effects were considered significant at
;;: 0.4
<1 " t t 0.4 ~
<1

0.2 0.2
the P < 0.05 level.
0.0 0.0

Results UV- treatment

Fif<llrf 3. Mean maximum PSI! quantum efficiency (Fvl F111 ) and

Chlorophyllfluorescence measurements of UV filter
experiment
The results of chlorophyll fluorescence measurements
in D. antarctica plots are shown in Figure 2a-d.
Neither the effective photosystem II quantum ef-
ficiency nor the maximum photosystem II quantum
efficiency was influenced by the treatments during the
1998 summer season. For light adapted measurements
of effective PSII quantum efficiency the F and p val-
ues were 0.178 and 0.682, respectively (screen-type)
and 0.013 and 0.911, respectively (UV-trcatment). The
F and p values for dark adapted measurements of
maximum PSTT quantum efficiency were 0.159 and
0.698, respectively (screen-type) and 0.212 and 0.655,
respectively (UV-treatment).
The same results were found for the 1999 data
recorded in D. antarctica stands witb F and p for
the effective PSTT quantum efficiency being 3.170 and
0.1 05, respectively (screen-type) and 0.018 and 0.896,
respectively (UV-treatment). F and p values of max-
imum PSII quantum efficiency were 1.579 and 0.238
(screen-type) and 1.1 12 and 0.316 (UV-treatment).
The values obtained 1999 for T. complicatulum
shown in Figure 2e an 2f were much lower than the
values for D. antarctica. In natural vegetation of T.
complicatulum, no significant influence of UV-B or
screen type on quantum efficiency of photosynthesis
could be detected. F and p values for effective PSII
quantum efficiency were 1.502 and 0.248, respectively
mean effective PSII quantum efficiency. f'..F I F,n'. of Deschampsia
antarctica in field plots under UV-Iamps. Circles represent mea-
surements in vegetation under lamps supplying additional UV-B
radiation (cellulose acetate), triangles represent measurements in
vegetation under lamps supplying no additional UV-B radiation
(mylar). Means and standard deviations of IS individual measure-
ments underneath two pairs of lamps arc given (n=2). (a) shows
the values of F, I Fm in dark adapted vegetation underneath the
lamps at the beginning (day 0) and at the end (day 15) of the
UV-supplementation period. (b) shows the values of f'..F I F111 ' in
light adapted vegetation underneath the lamps at the beginning (day
0) of the UV-supplementation period and at the end of the period
(day 15). recorded while the lamp' were switched on (lamps on)
and while the lamps were switched off (lamps off).
(screen-type) and 0.286 and 0.604, respectively (UV-
treatment). For maximum PSII quantum efficiency, F
and p values were 4.R06 and 0.053 (screen-type) and
0.431 and 0.526 (UV-treatment).
UV-supplementation
Figure 3 shows the results of the chlorophyll flu-
orescence measurements in the D. antarctica lamp
plots. The results of the UV-supplementation exper-
iments for T. complicatulum are shown in Figure 4.
In D. antarctica (Figure 3a) and T. complicatulum
(Figure 4a), the maximum photosystem II quantum ef-
ficiency values recorded prior to UV-supplementation
and at the end of the supplementation period remain
unchanged.
The values for t;.F I F;n' measured in the lamp plots
with the lamps switched on are more variable in both
 95

contents nor chlorophyll-b contents. Contents of fJ-

0.8 dayO day15 0.8

0.6 0.6 carotene, lutein, neoxanthin and violaxanthin were not

...E ...E affected by UV-treatment. Zeaxanthin was present in
~ 0.4 0.4-;:
...
ol:
0.2
! y
• t
0.2
some samples, but not always in amounts which could
be quantified (data not shown).
UV-absorption of methanol extracts remained un-
o.o
.-r~-~
•
.
.._ .; lit~~·'& .~'0-.,P"
0.0
changed by treatments in both species .
UV- treatment

Growth of Deschampsia

Flowering shoot length, including reproductive parts,

0.8 0.8
measured in the experimental plots (n=3) ranged from
b dayO day15 day 15

lamps off lamps on

0.6
...e
ii: 0.4
...
•
'
0.2 0 'V
0 §
o.o
uv- treatment
Figure 4. Mean maximum PSII quantum efficiency ( F,, I F111 ) and
mean effective PSII quantum efficiency. D.F I F111 ', of Turgidoscu-
lum comp/icatulum in field plots under UV-lamps. Circles represent
measurements in vegetation under lamps supplying additional UV-B
radiation (cellulose acetate), triangles symbolize measurements in
vegetation under lamps supplying no additional UV-B radiation
(mylar). Means and standard deviations of 15 individual measure-
ments underneath two pairs of lamps are given (n=2). (a) shows
the values of Fv I Fm in dark adapted vegetation underneath the
lamps at the beginning (day 0) and at the end (day 15) of the
UV-supplementation period. (b) shows the values of D.FI F111 ' in
light adapted vegetation underneath the lamps at the beginning (day
0) of the UV-supplementation period and at the end of the period
(day 15), recorded while the lamps were switched on (lamps on)
and while the lamps were switched off (lumps off).
D. antarctica and T. complicatulum (Figures 3b and
4b, respectively). Due to low number of replicates,
no conclusions can be drawn about significance of the
differences.
Chlorophyll, carotenoids, methanol soluble
UV-absorbing pigments
39 mm in a control plot to 145 mm in a plot cov-
lamps off
0.6
ered by an opaque cloche with side walls (Table 3). In
...e the monospecies stand of D. antarctica, shoot lengths
ii:
0.4
... were highest, whereas in the other vegetation type
0.2
shoots were shorter. The UV-treatment did not lead
0.0 to significant differences in shoot length ( U = 13.
p = 0.485 ). The shoots under both types of screens
were longer when compared to uncovered control
plots. The difference between shoots from control
plots and shoots from open-sided plots was not sig-
nificant (U = 6, p = 0.548). The length of shoots
under sided screens differed significantly from shoots
in control plots (U = I, p = 0.048) with the shoots
from under sided screens being 35% longer.
Weight (!f caryopses
Ten sets of 200 caryopses (caryopses plus palea and
lemma) each were weighed from uncovered control
plots, transparent and opaque filters without side walls
(n=3). The weights, expressed per caryopse, were
similar for all treatments. In control plots the average
weight was 0.134 mg ± 0.006 mg, in plots cov-
ered with transparent and opaque screens, the average
weight was 0.144 mg ± 0.002 mg and 0.138 mg ±
0.00 I mg respectively. The weights of the caryopses
were not significantly different between treatments
(U = 6, p = 0.548 for UV-B treatment and U = 5,
p = 0.381 for screen-type).

Reducing ambient levels of UV-B did not alter the
chlorophyll content of green shoots of D. antarc-
tica after two seasons (Table I). Also the ratio of
chlorophyll a to chlorophyll b remained unchanged.
The contents of tJ-carotene, lutein, neoxanthin and
violaxanthin did not respond to the UV-treatment.
The same results were found forT. complicatulum,
chlorophyll contents and carotenoid contents (Table 2)
of the lichen were lower than values found for D.
antarctica. UV-treatment did not affect chlorophyll-a
Proportion (!f reproductive biomass
The proportions of reproductive biomass to living bio-
mass on a dry weight basis of the 15 shoots harvested
per plot ranged from 2.58 in a control plot to 0.72
under a sided screen. The means of 15 shoots har-
vested per plot were highest in control plots (2.27 ±
0.20) and lowest in sided screen plots ( 1.62 ± 0. 12
and 1.51 ± 0.14 in sided opaque and sided trans-
parent screen plots, respectively). The proportions
96
Tuh/e I. Chlorophyll and carotenoid contents (ug g- 1) in green leaves of Deschampsia antarctica on a fresh-
weight basis. Five samples were harvested from underneath the different types of filter screens. The values
represent means and standard deviation of replicate sets of screens (n=3), F and p values of UY-treatrnent
effect are given.

Control Transparent Transparent Opaque Opaque, F p

sided sided

Chorophyll a 977 ± 65 834 ± 79 874 ± 26 901 ± 124 886 ± 114 0.106 0.753
Chorophyll h 319 ± 25 270 ± 2H 284 ± 9 293 ± 44 2HH ± 41 0.045 0.838
fi-carotene 70±4 61 ±6 64 ± 2 66± 10 64±9 0.169 0.691
Lutein 136 ± 7 123 ± 13 130± 5 138 ± 22 128 ± 16 0.081 0.078
Ncoxanthin 33 ±I 29 ± 2 30± I 31 ± 3 30± 3 0.007 0.935
Yiolaxanthin 58± 8 41 ±9 45 ±3 48 ± 13 47 ± 13 0.760 0.409

Table 2. Chlorophyll and carotenoid contents (ug g- 1) in lobes of Turgidosculum complicatulum on a fresh-
weigt basis. Five samples were harvested from underneath the different types of filter screens. The values
represent means and standard deviation of replicate sets of screens (n=3). F and p values of UY-trcatment
effect are given.

Control Transparent Transparent. Opaque Opaque. F p

sided sided

Chorophyll a 500 ± 130 504 ± 125 405 ± 212 344 ± 89 542 ± 76 0.014 0.909
Chorophyll h 167 ± 49 186 ± 45 151 ± 74 124 ± 68 218 ± 62 0.003 0.959
tJ -carotene 21 ±6 16±5 13 ±7 14±6 25 ±5 1.640 0.229
Lutein 77± 19 77± 18 56± 20 50± 31 98 ± 28 0.246 0.631
Neoxanthin 26 ±4 20±4 18±7 15 ±9 28 ± 8 0.208 0.658
Yiolaxanthin 47 ± 10 38 ± 11 30 ± 13 27 ± 19 52± 17 0.378 0.553

of reproductive biomass to living biomass of shoots
under screens without sides were 1.83 ± 0.23 and
1.87 ± 0.42 for transparent and opaque screen plots
respectively. Under filter screens with side walls, the
shoots comprised significantly less reproductive bio-
mass per living shoot biomass than in control plots
(U = 0, p = 0.023), the difference in reproduc-
tive biomass proportions between shoots from under
screens without sides and shoots from control plots
were not significant ( U = 2, p = 0.095). UV- B
treatment did not affect the proportion of reproductive
biomass ( U = 16, p = 0.818).
Discussion
The results of our field study suggest that there is
no direct influence of UV-B radiation on effective
and maximum PS II quantum efficiency in D. antarc-
tica and T complicatulum. Neither UV-B reduction
nor UV-B supplementation resulted in any detectable
changes in photosynthetic activity of these species in
our study. However, although we could not detect any
changes in photosynthesis and pigmentation in our
experiments, the low level of replication used (n=3
and 2 in UV filter and supplementation experiments,
respectively) would have constrained our ability to de-
tect subtle changes in plant performance. Allen et al.
( 1998) recently reviewed published data on effects
of UV-B on photosynthesis and concluded that rising
UV-B levels did not significantly affect photosynthetic
productivity in natural vegetations. Inhibition of pho-
tosynthesis could only be found at high doses of UV-B
or when proportions of PAR, UV-A and UV-B were
not realistic (Allen et al. 1998). This also corrobo-
rates findings of Montiel et al. ( 1999), who performed
short term experiments on D. antarctica on Leonie Is-
land. Tosserams & Rozema ( 1995) examined the effect
of UV- B radiation on a temperate grassland species
in the field and similarly found photosynthesis to be
unaffected.

Table 3. Shoot length (mtn) of individual shoots or Desc/wmpsia (/1/[(//"Ctica in the three sets
of experimental plots with ditrerent vegetation types after two years of UV-reduction. Values
represent means and standard deviations of 15 flowering shoots harvested per plot (11 = 3 ).
Vegetation Control
type
Monospecies stand 62 ±II
Mixed vegetation 4X ± 7
Mixed vegetulion 66 ± 10
Chlorophyll fluorescence parameters have been
used in various species to monitor UV-response: Born-
man & Sundby-Emanuelsson (1995) found a decrease
in Fvl F111 in Brassica napus after exposure to UV-
B, the effect being more notable with simultaneous
high light treatment. Nogues & Baker ( 1995) re-
ported that neither 6. F I Fm' nor F, I F111 decreased in
response to UV-B despite the fact that there was an
effect on photosynthesis measured as gas exchange
in pea leaves. This indicates that both chlorophyll
fluorescence parameters are comparatively insensitive
to UV-B radiation. The authors concluded that the
primary target of UV-B is not necessarily photosys-
tcm II. They assigned the decline in gas exchange
rates to a reduction in enzyme concentrations. Field
measurements of gas exchange in D. antarctica and
T complicatulum performed during the first season
and results from laboratory experiments on T com-
plicatulum suggest that there is no effect of UV-B
on gas exchange in these species. Bachereau & Asta
( 1997) did not find any effects of the reduction of am-
bient UV-B levels on photosynthetic gas exchange in
the lichen Cetraria islandica (L.) Ach. Their gas ex-
hange measurements were performed under artificial
light without UV-component, while measurements of
effective and maximum photosynthetic quantum effi-
ciency presented in this study were recorded under the
different UV-irradiation environments.
In the present study we did not examine possible
changes of the response to UV-B during the season.
The levels of incident UV-B radiation are relatively
low in January as compared to November levels. But
during some years snow cover on Leonie Island per-
sists until November. Most of the Deschampsia field
sites of the study reported here were covered in snow
in November during 2 out of 4 years.
UV- B radiation is known to reduce shoot extension
in higher plants (Liu et al. 1995: Day et al. 1999).
Gwynn-Jones & Johanson ( 1996) found a negative ef-
97
Transparenl Transparenl, Opaque Opaque.
sided sided
61 ± 16 113±21 lOS± 13 124 ±IS
67 ± 7 l)l ± 14 62± II 72 ± 23
62 ± 8 63 ±IX 63 ± 10 70 ±S
feet of UV-B on the growth of two subarctic grass
species. Rozema et a!. (200 I) discovered a negative
effect on growth of D. antarctica under controlled con-
ditions. The variability of our data on shoot length
might indicate that growth in natural vegetation is
influenced by other factors such as temperature and
competition.
Growth underneath sided filter screens was en-
hanced when compared to screens without side walls.
The microclimate was altered underneath the filter
screens (temperature and humidity. sec Huiskcs et al.
200 I). The temperature is in an Antarctic habitat prob-
ably the most important effect. As our field site is
close to the southern limit of D. antarctica (Smith &
Poncet 1987), warming is likely to affect growth in
D. antarctica positively. The temperature optimum for
photosynthesis in D. antarctica is broad. between 5
and 20 oc (Edwards & Smith 1988: Montiel et al.
1999). but field air temperatures during summer days
are often suboptimal.
Convey (1996) found the total biomass of tillers
of D. antarctica to he higher in specimens from sub-
antarctic locations as compared to Maritime Antarctic
locations. These findings also suggest that temperature
enhancement increases growth of this species.
During long-term experiments it has been shown
that there are cumulative etlects of UV-B radiation
on growth and sexual reproduction of D. antarctica
(Day et al. 1999). Even though we did not find an
effect of the treatments on seed weights, the screens
seemed to create favourable conditions for establish-
ment of Deschampsia seedlings. Under two of the
screens about 30 seedlings were detected. This might
be partially due to temperature enhancement under-
neath the screens, supporting findings of Fowbert &
Smith ( 1994) and Grobe et al. ( 1997). Taking into
consideration that the growth rates of Antarctic species
are low, from our results after two growing seasons,
longer term etlects of enhanced UV-B radiation on
98
photosynthesis, growth and reproduction cannot be
excluded.
Repeated exposure to UV-B decreases the negative
response (Dring et al. 1996). The results of our sup-
plementation experiments might add to this finding,
as no influence of additional UV could be detected
on Fv/ F111 • Organisms which have naturally been pre-
adapted to ambient levels of UV-B such as the sam-
ples from our field experiment might not respond to
realistic changes in UV-B radiation at all.
Pre-adaptation might also be a reason for why the
negative effects of UV-B on growth or photosynthe-
sis found in greenhouse experiments are not observed
in field experiments. Post & Larkum (1993) could
not induce the formation of UV-absorbing pigments
in Prasiola crispa, the phycobiont of T. complicat-
ulum, collected in the field after samples were ex-
posed to different UV-environments under controlled
conditions.
Ruhland & Day ( 1997) found the UV-absorption of
methanol extracts of field samples of D. antarctica to
be comparatively high. Hence attenuation of UV-B ra-
diation at the leaf surface might be very efficient in this
species. Before the start of our field experiments, the
plants and lichens underneath the screens had all been
exposed to ambient UV-B levels. Growth in lichens
is so slow that the proportion of newly grown material
since the beginning of the experiment would have been
neglibile.
Comparing the proportion of xanthophyll cycle
pigments to total carotenoids, Streb et al. ( 1997) found
higher proportions of xanthophyll cycle pigments in
alpine species as compared to lowland species. D.
antarctica contains a considerable proportion of vi-
olaxanthin, which might be an adaptation to high
irradiances during the austral summer. Our findings
suggest that there is no influence of UV-B reduction
on chlorophyll and carotenoids, which corroborates
the chlorophyll fluorescence data confirming that pho-
tosystem II is not influenced by manipulations of the
UV-B environment. Bachereau & Asta (1997) could
not detect an effect of UV-reduction on chlorophyll
and carotenoid concentrations in Cetraria islandica,
but they did find a positive effect of UV-B on lichen
phenolics.
Whether the supplementation of UV-B affected
pigment contents in D. antarctica and T. complicat-
ulum as well as the possible effect on secondary
substances in the lichen will be investigated in the
future.
Acknowledgements
The results described in this paper form part of the
BRUVA (Biotic Responses to UV-B Radiation in
Antarctica) programme. This is a joint British-Dutch
research programme with participation of the British
Antarctic Survey, Cambridge, UK, the Free Univer-
sity, Amsterdam, The Netherlands, the University of
Groningen, The Netherlands and the Netherlands In-
stitute of Ecology, Centre for Estuarine and Coastal
Ecology, Yerseke, The Netherlands. This study has
been funded by the Netherlands Antarctic Programme
(NAAP), administered by the Research Council for
Earth and Life Sciences (ALW) part of the Nether-
lands Organisation for Scientific Research (NWO),
which is gratefully acknowledged. The authors thank
the people at Rothera Research Station for their friend-
ship and support. This is publication 2685 of the
Netherlands Institute of Ecology, Centre for Estuarine
and Coastal Ecology, Yerseka.
References
Allen, D. J., Nogues, S. & Baker, N. R. 1999. Ozone depletion and
increased UV-B radiation: is there a real threat to photosynthesis?
J. Exp. llot. 49: 1775-1788.
Bachereau, F. & Asta, J. 1997. Effects of solar ultraviolet radia-
tion at high altitude on the physiology and the biochemistry of a
terricolous lichen (Cetraria is/andica (L.) Ach.). Symbiosis 23:
197-217.
Bornman, J. F. & Sundby-Emanuelsson, C. 1995. Response of
plants to UV-ll radiation: some biochemical and physiological
effects. Pp. 245-262. In: Smirnoff, N. (ed.). Environment and
Plant Metabolism. Bios Scientific Publishers, Oxford.
Caldwell, M. M. 1971. Solar ultraviolet radiation and the growth
and development of higher plants. Pp. 133-177. In: Giese, A. C.
(ed.), Photophysiology, Volume 6, Academic Press, New York.
Convey, P. 1996. Reproduction of Antarctic flowering plants.
Antarctic Sci. 8: 127-134.
Day. T. A., Ruhland, C. T., Grobe, C. W. & Xiong, F. 1999.
Growth and reproduction of Antarctic vascular plants in response
to warming and UV radiation reductions in the field. Oecologia
119: 24-35.
Dring, M. J., Makarov, V., Schoschina, E .. Lorenz, M. & Luning,
K. 1996. Influence of ultraviolet-radiation on chlorophyll fluores-
cence and growth in different life-history stages of three species
of laminaria (phaeophyta). Mar. Bioi. 126: 183-191.
Edwards, J. A. & Smith, R. I. L. 1998. Photosynthesis and respira-
tion of Co/obanthus quitensis and Deschampsia antarctica from
the maritime Antarctic. Brit. Antarctic Survey Bull. 81: 43-63.
Fowbert, J. A. & Smith, R. I. L. 1994. Rapid population increases
in native vascular plants in the Argentine Islands, Antarctic
Peninsula. Arctic Alpine Res. 26: 290-296.
Green, A. E. S., Sawada, T. & Shettlc, E. P. 1974. The mid-
dle ultraviolet reaching the ground. Photochem. Photobiol. 19:
251-259.
Green, T. G .. Schroeter, B. & Sancho. L. G. 1999. Plant life in
Antarctica. Pp. 495-543. In: Pugnaire, F. I. & Valladares. F.

(cds), Handbook of Functional Plant Ecology. Marcel Dekker.
New York.
Gremmen, N. J. M., Huiskes, A. H. L. & f'rancke, .l. W.
1994. Epilithic macrolichen vegetation of the Argentine Islands.
Antarctic Peninsula. Antarctic Sci. 6: 463-471.
Gremmen, N.J. M., Huiskes, A. H. L. & Francke. J. W. 1995.
Standing crop of the coastal macro lichen Masrodia resselaw. and
its relationship to nutrient concentrations. on Petermann Island.
Antarctica. Lichenologist 27: 3X7-394.
Grobe. C. W., Ruhland. C. T. & Day. T. A. 1997. A new population
of Co/o/Janlhus quilensis ncar Arthur Harbour. Antarctica: Cor-
relating recruitment with warmer summer temperatures. Arctic
Alpine Res .. 29: 217-221.
Gwynn-Jones. D. & Johanson. U. I <)<)6. Cirowth and pigment pro-
duction in two subarctic grass species under four different UV-B
irradiation levels. Physiol. Plant. 97: 701-707.
Hcide-Jprgenson, H. S. & Johnsen. I. 1995. Analysis of sur-
face structures of C/adonia milis podetia in historic and recent
collections from Greenland. Can. J. Bot. 73: 457-464.
Huiskes, A. H. L.. Gremmen. N. J. M. & Francke. J. W. 1997.
Monphological effects on the water balance on Antarctic foliose
and fruticose lichens. Antarctic Sci .. 9: 36-42.
Huiskes. A. H. L., Lud, D .• Moerdijk-Poortvliet. T. C. W. &
Rozema. J. 1999. Impact of UV-B radiation on Antarctic terres-
trial vegetation. Pp. 313-337. In: Rozema. J. (ed.). Stratospheric
oOzone Depletion; the Effects of Enhanced UV-B Radiation on
Terrestrial Ecosystem. Backhuys Publishers. Lcidcn.
Huiskes, A. H. L., Lud, D. & Moerdijk-Poortvlict. T. C. W. 2000.
Responses to UV-B radiation in terrestrial Antarctic vegetation.
Pp. 252-257. In: Davison. W. et. al. (cds). Antarctic Ecosystems:
Models for Wider Understanding. Caxton Press, Christchurch.
Huiskes. A. H. L.. Lud, D. & Mocrdijk-Poortvliet. T. C. W.
2001. Field research on the etlects of UV-B lilters on terrestrial
antarctic vegetation. Plant Ecol. 154: 75-86 (this volume).
Jones. A. E. & Shanklin. J. D. 1995. Continued decline of total
ozone over Halley. Antarctica. since 1985. Nature 376: 409-411.
King, J. C. 1994. Recent climate variability in the vicinity of the
Antarctic Peninsula. Int. .1. C:lim. 14: 357-36<).
Liu. L. Gitz. D. C. Ill & McClure. J. W. 19<J5. Etlects ol UV-B
on flavonoid, fcrulic acid. growth and photosynthesis in harley
primary leaves. Physiol. Plant. 93: 725-733.
Madronich. S .. McKenzie, R. L.. Caldwell. M. & Bjorn. L. 0. 1995.
Changes in ultraviolet radiation reaching the earth's surface.
Ambia 24: 143-152.
Montiel. P., Smith, A .. Kciller, D. 1999. Photosynthetic responses
of selected Antarctic plants to solar radiation in the Southern
maritime Antarctic. Polar Res. (in press).
Nogues, S. & Baker, N. R. 1995. Evaluation of the role of damage
to photosystem II in the inhibition of C02 assimilation in pea
leaves on exposure to UV-B radiation. Plant Cell Environ. I X:
781-787.
Olech. M. 1990. Preliminary studies on ornithocoprophilous lichens
ol the Arctic and Antarctic regions. Proc. NIPR Symp. Polar
Bioi. 3: 218-223.
Post. A. & Larkum. A. W. D. 1993. UV-ahsorbing pigments,
photosynthesis and UV exposure in Antarctica: comparison of
terrestrial and marine algae. Aquatic Bot. 45: 231-243.
99
Redon. J. 1'185. Liquenes Antarctic<JS. Instituto Antm1ico Chileno.
Santiago. 123 pp.
Rozema. J .. Van de Staaij, J .. Meijkamp. B .. Lud. D .. Mo-
erdijk. T. & Huiskes. A. 1997. Stratospheric ozone depletion
and effects of enhanced UV-B radiation on terrestrial antarc-
tic plants: the methodology of a mini-UV-B supplementation
system. Circumpolar J. 12: 43-48.
Rozema. J.. Brockman. Lud, D .. Huiskes. A.. Moerdijk. T.. De
Bakker. N .. Mcijkamp. B. B. & Van Beem. A. 2001. Con-
sequences of depletion of stratospheric ozone for terrestrial
antarctic eco~ystems: the response of Desclwmpsia antarctica
to enhanced UV-B radiation in a controlled environment. Plant
Ecol. 154: 101-115 (this volume).
Ruhland. C. T. & Day. T. A. 1997. Leaf screening characteristics of
Antarctic vascular plants. Bull. Ecol. Soc. Am. 7R: 305.
Schroeter. B .. O!ech. M .. Kappen. L. & Heitland. W 1995.
Ecophysiological investigations of Usnea onturctica in the mar~
itime Antarctic. I. Annual microclimatic conditions and potential
primary production. Antarctic Sci. 7: 251-260.
Searles. P. S .. Flint. S. D .. Diaz. S. B .. Rousseaux. M. C., Ballare.
C. L. & Caldwell. M. M. 1999. Solar ultraviolet-B radiation in-
fluence on Sphagnum bog and Care.-r J'en ecosystems: first lleld
season tindings in Tierra del fuego. Argentina. Global Change
Bioi. 5: 225-2.14.
Smith. R. I. L. 1984. Terrestrial plant biology of the sub-Antarctic
and Antarctic. Pp. 61-162. In: Laws. R. M. (ed.). Antarctic
Ecology. volume I. Academic Press. London.
Smith. R. I. L. 1985. Nutrient cycling in relation to biological pro-
ductivity in Antarctic and sub-antarctic terrestrial and freshwater
ecosystems. P.p. 138-155. In: Siegried. W. R .. Condy. P.R. &
Laws. R. M. (eds). Antarctic Nutrient Cycles and Food Web.
Springer-Verlag. Berlin.
Smith. R. I. L. 1994. Vascular plants as bioindicators of regional
warming in Antarctica. Oecologia 99: 322-328.
Smith. R. I. L. & Poncet. S. 1987. f)eschampsia anTarctica and
Co/ohantlws quitcnsis in the Terra Firma Islands. Brit. Antarctic
Survey Bull. 74: 31-35.
Statsort. Inc. 1998. Statistica for windows. Stat Soft. Tulsa.
Streb. B .. Feierahend . .1 .• & Bligny. R. 19<J7. Resistance to pho-
toinhihition of photosystem II and catalase and antioxidativc
protection in high mountain plants. Plant Cell Environ. 20:
1030· 1040.
Swanson. A. & Fahsell. D. 1997. Effects of ultraviolet on polyphe-
nolics of Umhilicar;a amerhww. Can. J. Bot. 75: 284-289.
Swanson. A .. Fahselt, D. R. & Smith. D. 1996. Phenolic levels
in Umhi/icariu americonu in relation to enzyme polymorphism,
altitude and sampling date. Lichcnologist. 2H: 331-3.19.
Tosscrams. M. & Rozema . .1. 1995. Effects of ultraviolet- B radi-
ation (UV-B) on growth and physiology of the dune grassland
~pecies Ca/anwgrostis epigeios. Environ. Pollut. 89: 209-214.
Wright. S. W.. Jetlery. S. W .. Mantoura. R. F. C.. Llewellyn. C. A ..
Bjiirnland, T., Repeta. D. & Wclschmeycr. N. 1991. Improved
HPLC method for the analysis of chlorophylls and carotenoids
from marine phytoplankton. Mar. Ecol. Progress Ser. 77: 183-
196.
 Section 3: Arctic and Antarctic Plants and Ecosystems

A mini UV-B lamp system installed over Deschampsia anwrctica ur a north-faced slope of rocks at Island Leonie. At the
background the polar ocean with icebergs and glaciers at the horizon . (Photograph by J. Rozema)
 Plant Ecolot;y 154: 103-115,2001,
103
© 200 I Kluwer Academic Publishers.

Consequences of depletion of stratospheric ozone for terrestrial Antarctic

ecosystems: the response of Deschampsia antarctica to enhanced UV-B
radiation in a controlled environment

Jelte Rozema 1, Rob Broekman 1, Daniela Lud 2 , Ad H, J. Huiskes 2 , Tanja Moerdijk 2, Nancy de
Bakker 1, Barbara Meijkamp 1 & Adri van Beem 1
1Department of' Systems Ecology, Faculty 1!/' Biology, Vrije Unil·ersireit, De Boelelamz 1087, 1081 HV Amsterdam,
The Netherlands; 2 CEMO, Yen·eke, Tlze Netherlands

Key words: Antarctica, Climate change, Deschampsia antarctica, Ecosystem, Ozone depletion, UV-B,
UV-B supplementation

Abstract
Mini UV lamps were installed over antarctic plants at Leonie Island, Antarctic peninsula, and shoot length
measurements of Deschampsia antarctica were performed during the austral summer January-February 1999.
We studied the response of the antarctic hairgrass, Deschampsia antarctica to enhanced UV-B. In a climate
room experiment we exposed tillers of Deschampsia antarctica, collected at Leonie Island, Antarctic peninsula, to
ambient and enhanced levels of UV-B radiation. In this climate room experiment with 0, 2.5 and 5 kJ m- 2 day- 1
UV-B 8 E treatments we observed that length growth of shoots at 2.5 and 5 kJ m- 2 day- 1 UV-B 8 E was markedly
reduced compared to 0 kJ m- 2 day- 1 UV-BsE . In addition, there was an increased number of shoots and increased
leaf thickness with enhanced UV-B. The Relative Growth Rate (RGR) was not affected by UV-B, possibly because
reduced shoot length growth by enhanced UV-B was compensated by increased tillering. Light response curves of
net leaf photosynthesis of plants exposed to 5 kJ m - 2 day- 1 UV- BsE did not differ from those exposed to 0 kJ
m- 2 day- 1 UV-BBE· The content of UV-B absorbing compounds of plants exposed to increasing UV-B did not
significantly change.
Mini UV-B lamp systems were installed in the field, to expose the terrestrial antarctic vegetation at Leonie
Island to enhanced solar UV-B. In that study, the increment of shoot length of tagged plants of Deschampsia
antarctica during the January-February 1999 at Leonie Island. was recorded and compared to shoot length growth
under controlled conditions.
The consequences of enhanced UV-B radiation as a result of ozone depletion for the terrestrial antarctic
ecosytems are discussed.

Introduction
Depletion of stratospheric ozone and terrestrial
antarctic research
Since the publication of dramatic depletion of
stratospheric ozone over the Antarctic (Farman et al.
1985), there has been extensive scientific interest
in stratospheric ozone depletion (Madronich 1992;
Madronich et al. 1995; Roscoe et al. 1997) and the
consequences of enhanced solar UV-B radiation for
the terrestrial biota. From 1985 until now, there has
been and there is serious depletion of stratospheric
ozone over the Antarctic, particularly during the
antarctic spring (Farman et al. 1985; Gleason et al.
1992; Stolarski eta!. 1992; Caldwell et al. 1998). Low
stratospheric ozone values may occur over the Antarc-
tic, e.g .. 147 Dobson Units at Vernadsky station and
even lower values (I 00 Dobson Units) recorded at the
BAS base Rothera, occurred in 1997-1998. Antarc-
tic terrestrial biota are therefore periodically exposed
to enhanced solar UV-B (Lubin & Frederick 1991;
104
Hoffmann 1996; Hoffmann et al. 1997; Roscoe et al.
1997; Huiskes et al. 200 I). Rousseaux et al. ( 1999)
reported solar UV-B induced DNA damage in plants of
the austral tip of South America. This area is regularly
exposed to decreased ozone levels under the influence
of the Antarctic vortex. This study demonstrates that
variations in solar UV-B levels caused by ozone fluc-
tuations have an impact on plant DNA damage density
in the field.
Enhanced solar UV-B radiation as a result of
stratospheric ozone depletion may cause damage to
plants, although UV-B protective mechanisms can
mitigate these effects (Caldwell et al. 1989; Post &
Larkum 1993; Rozema et al. 1997b). UV-B damage
such as the occurrence of thymine-thymine dimers in
DNA may be repaired by the enzyme DNA photo lyase
(Bjorn 1996). The activity of this enzyme is tem-
perature limited, however, and antarctic plants may
therefore be sensitive to enhanced UV-B because of
low antarctic temperatures. Plants may further pro-
tect themselves from UV-B damage by morphogenic
changes and the development of UV-B absorbing
screens (Tosserams & Rozema 1995; Rozema et al.
1997b; Jansen et a!. 1998; Bornman 1999; Meijkamp
et al. 1999).
Since UV-B radiation may cause damage to mem-
branes, DNA (e.g., Burna et a!. 1995) and various
other cellular structures and processes, it has been ex-
pected that plant and crop growth would suffer from
enhanced solar UV-B radiation (Caldwell et al. 1989;
Rozema et al. 1997b ). Responses of plant species from
various latitudes and from low and high UV-B envi-
ronments to UV-B have been reported. Many of these
experimental studies of UV-B effects on plants relate
to crop plants exposed to enhanced UV-B in indoor
controlled environment experiments (Fiscus & Booker
1995; Rozema et al. 1997b; Jansen et al. 1998). The
responses of natural plants from natural ecosystems to
enhanced UV-B have been reported in relatively few
studies (Rozema et al. 1997a; Caldwell et al. 1998).
So far, no direct effects of enhanced UV-B in lowering
primary production of terrestrial ecosystems have been
found (Caldwell et al. 1998).
Enhanced UV-B and antarctic ecosystems
There are some reports on the consequences of ozone
depletion on the marine antarctic biota (e.g., Kar-
entz et al. 1991 a, b; Behrenfeldt et al. 1993: Vernet
et al. 1994; Vincent & Quesada 1994, 1997), but it
is remarkable that research on the consequences of
the ozone hole for the terrestrial antarctic biota is
scanty. This may partially be explained by the fact
that there exists only limited terrestial antarctic veg-
etation, mainly at the maritime Antarctic (Holdgate
1994 ). Day eta!. ( 1999) report on the responses of De-
schampsia antarctica and Colobanthus quitensis to a
reduction of ambient solar UV-B by UV filter systems
at Palmer station at the Antarctic Peninsula. In this
report it is demonstrated that growth of Deschamp-
sia antarctica under ambient antarctic solar UV-B is
reduced compared with plots where part of the solar
UV-B was filtered out.
The vegetation of the terrestrial ecosystem at
Leonie Island, as part of the maritime Antarctic, con-
sists of a few species of lichens, mosses and only two
phanerogamic species.
The distribution of Deschampsia antarctica at the
maritime Antarctic extends to 68°42' S, 67°31' W,
at Marguerite Bay (Smith & Poncet 1987) and the
plants of Deschampsia antarctica at Leonie Island
represent the almost southern population at the mar-
itime Antarctic. The occurrence and ecology of
this antarctic grass species Deschampsia antarctica
Desv. (Gramineae) and the herb Colobanthus quitensis
(Kunth) Bart. (Caryophyllaceae) have been described
earlier (Holtom & Greene 1967; Moore 1970; Smith
1982; Poncet & Smith 1985). Deschampsia antarctica
was first described at the Antarctic at the South Orkney
Islands in 1823 (Edwards 1972) and since that time,
both antarctic phanerogams have been reported from
many antarctic locations (Holtom & Greene 1970;
Greene 1967; Edwards & Smith 1994; Smith 1996;
Convey 1996, 1997). Outside the maritime Antarc-
tic Deschampsia antarctica and Colobanthus quiten-
sis occur in South America: the Falkland Islands,
Tierra del Fuego and at the Andean Mountains up to
about a latitude of 34° S (Convey 1996). There is in-
creasing evidence that the distribution of both higher
plant species has been markedly extended as a result
of global warming (Fowbert & Smith 1994; Smith
1994; Convey 1997), temperature has increased 1-
1.5 oc during the last decades in the maritime antarctic
(Smith 1994; Convey 1997).
In this paper the response of the antarctic grass
Deschampsia antarctica from Leonie Island (67"36' S,
68°21' W, maritime Antarctic) (Figure I) to en-
hanced solar UV-B is described. This research forms
part of a larger Dutch-British project: Biotic Re-
sponses to UV-B radiation in Antarctica (BRUVA), in
which responses of lichens, mosses and higher plants
from Leonie Island to solar UV-B are being studied
 105

0 2 3 5km ROTH ERA

I Aesear~ S~lion -~~~-~,\.:?'·;~)

RYDER BAY BritiSh Antorcuc Survey ·<:·:· KILLINciBECK
··· .... . ISLAND

.....1€?
········...
LEONIE
~: ~1.5
.... ,. .. :·
ISLAND ...~\ '.U....· ..
·.... :LAGOON
ISLAND

LEONIE

IS LANDS

::.
O studyArea
C::J [{IMPET
ISLAND

68' 20' W 68° 10'W

....
Bellinghausen

Figure I. Geographical map indicating the Antarctic peninsula and Adelaide Island (I). Adelaide Island with Rot hera (2). research station of
the British Antarctic Survey and (3 ) Leonie Island with the study area where the mini UV lamps we re installed.
106
(Huiskes eta!. 1997a, b; see Huiskes eta!. 2001; Lud
eta!. 2001 ). In the Antarctic summer of 1998-1999 we
installed mini UV-B lamp systems to expose lichens,
mosses and phanerogams to enhanced UV-8. In this
way responses of terrestrial antarctic plants to ambient
and above ambient solar UV-B in the field can be made
(see Lud eta!. 2001 ).
Tillers of the grass Deschampsia antarctica, col-
lected at Leonie Island, were cultivated in a climate
room in Amsterdam, The Netherlands, to study the
response of the antarctic grass Deschampsia antarc-
tica to enhanced UV-B in a controlled environment
( 12 °C). To our knowledge, this paper is the first report
of the response of the antarctic grass Deschampsia
antarctica to experimentally enhanced UV-B radiation
in a controlled environment (cf. McLeod 1997).
Material and methods
Characterization c~f environmental conditions at
Leonie Island
Leonie island (Figure I) is surrounded by the polar sea
and massive glaciers. Leonie Island lies in a radiation
'window' close to the Hurley and Turner glaciers on
Adelaide Island. Some plant growth is possible locally
on north faced, sheltered coastal rocky sites where ice
melts and water percolates or accumulates. The moss
Drepanocladus uncinatus (now: Sanionia uncinata)
is likely to be the colonizer of such moist, sheltered
coastal sites, where sea spray, and the faeces of marine
mammals, pinguins and skuas represent a significant
supply of nutrients. Tufts of Deschampsia antarctica
may occur on or in mats of Drepanocladus uncinatus.
Thus, plant growth of Deschampsia antarctica
and Colobanthus quitensis is locally possible on such
sites in the maritime Antarctic, but not on continental
Antarctic sites.
The southern most distribution of Deschampsia
antarctica extends to Marguerite Bay, somewhat south
of Leonie Island.
A rough indication of the mean monthly air tem-
perature at Leonie Island is -0.3 ( °C) (November),
0.1 ( °C) (December), 2.1 ( °C) (January), 0.0 ( °C)
(February) and -1.6 ( oq (March) measured at nearby
Rothera Point in 1994 and 1995 (Figure 1) (Convey
1996). At the study site at Leonie Island, temperatures
may be considerably higher. On sunny austral summer
days, air temperature may rise to 8 oc and temper-
atures within the tufts may be 15 oc around midday
(Huiskes et a!. 2001 ). PAR may be up to 1800-2000
~mol m- 2 s- 1 around noon. On cloudy days it drops
to about 600 ~mol m- 2 s- 1 at midday. Precipitation in
the austral summer at Leonie Island is about 200 mm,
most of which is available to the terrestrial plants, be-
cause of the above zero temperatures during the day
(Huiskes eta!. 2001 ).
Installation of' mini UV-8 lamp systems at Leonie
Island
Mini UV-B lamp systems technically described in
Rozema eta!. ( 1997c), see also front page of this pa-
per, were installed over the terrestrial alga Prasiola
crispa, the lichen Mastodia tesselata (now: Turgid-
iusculum complicatum), and the grass Deschampsia
antarctica. Responses of these plants to enhanced UV-
B in the field are being reported by Lud et al. (200 I).
The electrical supply of the mini fluorescent tubes was
by 12 V batteries. The UV radiation was filtered by a
strip of cellulose acetate (enhanced UV-B treatment),
or by a strip of mylar (no extra UV-B: ambient UV-
B treatment). The fluorescent tubes were switched on
from 12:00-13:30. Based on the distance between the
lamps and the Antarctic plants (height of the plants
was about 15 em) and the radiation spectrum (mea-
sured with an OL 752 Spectroradiometer) of the UV
lamps measured in a climate room (8 oc) at the Vrije
Universiteit, Amsterdam, and the application of the
GPAS action spectrum (Caldwell 1971 ), it was calcu-
lated that an UV-BsE dose at the soil surface of 2.4 kJ
m- 2 day- 1 UV-BsE was supplemented. No spectro-
radiameter was available on Leonie Island and only
measurements with an UVX meter could be done in
the field, the UV-B output of the fluorescent lamps
may have been lower at Leonie on windy days with an
air temperature of 0 oc than in the 8 oc climate room.
Reduced output of fluorescent tubes at low tempera-
ture has been demonstrated and discussed by Johanson
& Zeuthen ( 1998).
Plant cultivation and controlled environment
experiment
About twenty tufts of Deschampsia antarctica with
adhering soil were collected during the antarctic sum-
mer period at the end of February 1998 at Leonie
Island by A. H. L. Huiskes and D. Lud and stored in
a cool box at 0-4 °C. After transport by the R. R. S.
Bransfield of the British Antarctic Survey, the arrival
of the plants was May 15, 1998 in Amsterdam. The
tufts were then stored in a climate room (4 oc day and
 107

night, relative humidity 75%), PAR 150 JLmol m- 2 Deschampsia antarctica It•
s- 1. At the start of the experiments, the temperature in
the climate room was set at 12 oc day and night. For Qa Q a
g. o•
about 2 months there was no substantial new growth
of leaves and shoots and existing leaves and shoots
s
~
Qb

¢· 9 b ¢b
J
~ b
senesced. In September 1998 new shoots and leaves Qb
~b
Qb b
started to develop and old senescent leaves were re-
moved. We divided the tufts into units of about I 0 00 ¢· ~:¢ b be
c l!.c

.<:: Q• ab b

a:~
developing shoots and potted these into pots, 7.2 x "' a D 0 kJ m-2 day-1

7.2 em and 8 em high. These pots were filled with
commercial garden soil (Jongkind BV Aalsmeer, NL),
mixed with vermiculite and with osmocote controlled
release fertilizer (2 g per liter soil; N:P:K:Mg:Fe,
13:13:13:3:2, Grace Sierra Int., Heerlen, NL).
UV-B treatments in a controlled environment
PAR light was provided by Sylvania F36W/133 cool
white TL tubes (length 120 em), supplying 150 JLmol
m- 2 s- 1 PAR at the plant level, measured with aLI-
185b quantum sensor (LI-COR Inc., Lincoln, NE,
USA). UV-B radiation was obtained with Philips TL
12/40 fluorescence tubes. A section of 60 em of these
tubes was wrapped in mylar foil (Dupont Industries,
USA) absorbing radiation below 313 nm, representing
the 0 kJ m- 2 day- 1 UV-BsE treatment; the following
section of 60 em of the tube was wrapped in cellulose
acetate (Tamboer & Co Chemie B.V., Haarlem, NL),
representing the 2.5 kJ m- 2 day- I UV-BsE treatment.
Mylar and cellulose acetate foil was replaced every
week. The distance between the UV fluorescent tube
and the plants was 70 em. A 5 kJ m- 2 day- 1 UV-
BsE treatment was obtained by reducing the distance
between UV fluorescent tube and the plants to 40 em.
The sections under the UV tubes were vertically sepa-
rated by mylar foil. The distance between the TL tubes
for PAR light and the plants was about 20 em for all
three UV-B treatments.
With the UV-B levels used in this experiment
the 2.5 kJ m- 2 day- 1 UV-BsE treatment may simu-
late austral summer ambient solar UV-B values, and
5 kJ m- 2 day- 1 UV-BsE represents an above am-
bient austral summer solar UV-BsE value (see Lud
et al. 2001). It is difficult, however, to indicate how
much stratospheric ozone depletion is being simu-
lated by this value, since stratospheric ozone over
the antarctic shows a drop in the antarctic spring and
fluctuates dramatically too. The spectral irradiance of
the fluorescent tube was measured with an OL 752
Spectroradiometer (Optronics Laboratories, Orlando,
FL, USA). The biologically effective UV-B dose of
0 2.5 kJ m-2 day-1
1!::. 5 kJ m-2 day-1
3
days
0 20 40 60 80
Figure 2. Effect of increased UV-B radiation on the shoot length of
Deschampsia antarctica. Since there were no significant differences
in shoot length between the pots, the data per UV-B treatment were
pooled. The effect ofUV-B on shoot length is statistically significant
(p:;:0.05) after 28 days of exposure to the UV-B treatments. Average
values of 80 replicates (8 tagged shoots per tuft. l 0 pots per treat-
ment) with standard error of the mean. Means with different letters
are statistically significantly different (p:;:0.05).
the three UV-B treatments were measured using a
UVX meter (with a UV-X 31 sensor, San Gabriel
CA, USA) calibrated against the OL 752 Spectra-
radiometer and using the Generalized Plant Action
Spectrum (GPAS) (Caldwell 1971 ), normalized at
300 nm. The burning period of the UV fluoresent tubes
was from 12:00-15:00 h. The PAR level was main-
tained 150 JLmol m- 2 s- 1at the plant level for 24 h
in the climate room. During the antarctic summer at
Leonie Island PAR levels peak around noon, but the
number of hours suitable for net photosynthesis will in
general at least comprise the period from 04:00-22:00,
i.e., 18:00 h. Edwards (1972) assessed the number
of 'usable daylight' hours at Signy Island (60°43' S,
45° 38' W) to be about 17 h. This will be about 20 h
for the more southerly Leonie Island.
For each UV-B treatment there were 16 pots placed
in 2 white plastic trays (18 x 18 x 15 em). Pots were
watered twice a week with demineralized water, but
too moist conditions were avoided. Pots were rotated
within the plastic trays weekly to avoid differences in
exposure to PAR and UV-B.
Shoot length measurements
At the start of the UV-B irradiation experiment in the
climate room (end of October 1998), shoots of tufts
of Deschampsia antarctica were tagged with coloured
rings. These rings were placed at the base of individ-
ual shoots. In the field at Leonie Island similar length
108
measurements of leaves and shoots of Deschamp-
sia antarctica were performed for a comparison with
shooth length growth in the controlled environment
experiment. Shoot length measurements were done
weekly with a ruler from the base of the shoots.
Harvest
After 90 days of exposure to UV-B, plants of De-
schampsia antarctica were harvested. Final measure-
ments of shoot length and number of shoots per pot
were made. None of the plants harvested had devel-
oped flowers or fruits. Leaf area was measured using
a Licor 3100 area meter (Li-Cor. Inc., Lincoln, Ne-
braska, USA). Root systems were carefully detached
from the garden soil by rinsing with demineralized wa-
ter and dried between filter paper before fresh weight
was determined. Samples of fresh shoots were taken
and stored in plastic tubes at -80 oc for the determina-
tion of UV-B absorbing compounds. Plant tissue was
dried overnight in a ventilated stove (80 °C), until dry
weight was measured.
UV-B absorbing compounds
UV-B absorbing pigments were extracted from frozen
( -80 °C) leaf material of plants harvested at the end
(90 days after start) with a MeOH:H20:HCI (79:20: I
v/v) solution. Samples (I 0 mg leaf tissue in 5 ml solu-
tion) were heated in a waterbath (90 oq for 2 h. The
absorbance, integrated from 280-320 nm was mea-
sured using a Perkin-Elmer Lambda 15 UV/VIS spec-
trophotometer (cf., Visser et al. 1997 and Meijkamp
et al. 2001).
Carbon and nitrogen analysis
Carbon and nitrogen of dried leaf material were deter-
mined by gas chromatography using a Perkin Elmer
2400 Series II CHNS/0 analyser.
Photosynthesis measurements
Photosynthesis was measured using an infra-red gas
analyser (LCA3) and PLC (Parkinson Leaf Chamber)
with a 2.5 x 2.5 em window (Analytical Develop-
ment Co., Hoddesdon, Herts, UK). The measure-
ments were done on the youngest fully developed
leaves in a climatized room ( 18 °C). Prior to the mea-
surements the gas analyzer was calibrated against a
347 ppm C02 concentration (Hoek Loos Medical
gases). The gas flux was 150 ml min- 1. The PLC
was cooled by a Peltier element at a temperature of
14.5-15.5°C. Edwards and Smith (1988) report that
the optimum temperature for photosynthesis of De-
schampsia antarctica is between I 0 oc and 15 oc.
Cooling to a lower temperature than 14.5-15.5°C in
our equipment, would imply condensation within the
gas tubes. Relative Humidity of the air entering the
leaf cuvette was maintained at 68%. The leaf area
of Deschampsia antarctica clipped in the PLC was
calculated by multiplying total leaf length and leaf
width.
Light response curves were constructed by irradi-
ating leaves of Deschampsia antarctica with a fixed
HPIIT 400 W Philips lamp and varying the distance
between this light source and the plants. Light inten-
sity was increased from 0 to 2000 llmol m- 2 s- 1 in
20 steps, readings were taken when the C02 uptake
had become stable. Net photosynthesis was calculated
according to Von Caemmerer and Farquhar ( 1981 ).
Statistical analysis
The data were statistically analysed by one way analy-
sis of variance using Systat 5.1 (Sokal & Rohlf 1995).
Data of the growth analysis parameters were log-
arithmically transformed to obtain homogeneity of
variance, analysed with the Bartlett test for homo-
geneity of group variances. Significantly different
means (different letters) were detected with the Tukey
HSD multiple comparison. test Using Systat 5.2, the
light response curves of net leaf photosynthesis were
fitted with the non-linear model: A = Amaxll -
e( -<PL/ Amaxl J - Rd (Goudriaan 1982). where A = net
leaf photosynthesis, Amax = leaf photosynthesis at
light saturation, rp = Quantum yield, L = PAR light
intensity, Rd = dark respiration.
Results
Controlled environment experiment
During the growth experiment in the climate room,
shoot length increased from 3.8 em to a shoot length of
6.5-9 em after 90 days (Figure 2). Shoot length after
90 days at 0 kJ m- 2 day- 1 UV-BsE was 9 em, and
with 2.5 and 5 kJ m- 2 dar 1 UV-BsE respectively,
shoot length was reduced to 7 and 6.5 em at the end
of the experiment. The difference in shoot length of
plants exposed to 2.5 and 5 kJ m- 2 day- 1 UV-BsE
became statistically different from the shoot length at
0 kJ m- 2 dar 1 UV-BsE after 28 days. The Tukey
 109
Leaf thickness Deschampsia antarctica 10 Deschampsia antarctica
200

N
0
u
~

i:
•o•
• ••
•
~
•
0
.• •~

--!' 0 ~ ~ • •
=· It
a 0 iO
~
§_ a ____:___
·~
rx1' ..
~
•
~ 100 'E,..,
:s ~
] ..<=
p,
0 0 kJ m-2 day-1

"'"
~ -2 • 5 kJ m-2 day-1

"
z
-4
0 1000 2000

2.5
kJ rn-2 day-!
Figure 3. Effect of increased UV-B radiation on the leaf thickness
of the leaves of Deschampsia antarctica measured after 90 days of
exposure to the UV-B treatments. Values arc means of 100 leaves
per UV-B treatment with standard error of the mean. Means with
different letters arc significantly different (p:::0.05).
200 Deschampsta an arctica
Number of shoots
b Deschwnpsia antarctica plants after 90 days of exposure to differ-
PAR (~-tmol m-2 s-1)
Figure 5. Light response curves of Deschampsia anrarctica plant"
exposed to 0 and 5 kJ m- 2 day- 1 UV-BsE. Net leaf photosynthesis
at each light intensity of three plants per UV-B treatment arc shown.
There are 60 readings per UV-B treatment. Net leaf photosynthesis
was measured of an intact leaf forming parl of a tuft growinll: in
a well moisturcd pot. clipped into the~ PLC in the ADC-infr;red
gasanalyscr.
Table I. Effect of UV-B radiation on some growth parameters of

T ent UV-B treatments based on fresh weight measurements. Average

values of 10 replicates (one tuft per pot) with standard error of the
mean. There were no statistically signicant effects of UV-B on any
of these parameters.

±
100

+ RGR (g kg- 1 day- 1)

LJ\R (m 2 kg- 1)
0

60.4 ± 3.5
24.1 ±0.5
25

60.3 ± 4.0
14.1 ±0.3
66.1 ± 2.5
17.9 ± 0.1
5

SLA (m 2 kg- 1) 50.8 ± 0.6 38.6 ± 0.4 42.7±0.1

LWR 0.46 ± 0.07 0.36 ± 0.06 0.42 ± 0.02

OkJ/m2/day 2.5kJ/m2/day 5kJ/m2/day

Figure 4. The effect of enhanced UV-B radiation on the number

of shoots of Deschampsia antarctica per pot. Values are means of
10 replicates (counted per pot) with standard error of the mean,
measured after 90 days of exposure to UV-B treatments.
HSD multiple comparison test indicated that the dif-
ference of shoot length between the 5 and 2.5 kJ m- 2
day- 1 UV-BsE irradiated plants, was significant from
day 40-60 but not for the period 60-SO days.
While shoot length decreased with enhanced UV-
B, leaf thickness and the number of shoots per pot
increased significantly (Figures 4 and 5). For both
these morphological parameters the values at 5 kJ m- 2
day- 1 UV-BnE differ from the values at 2.5 and 0 kJ
m- 2 day-! UV-BBE· The number of shoots per pot
increased from 60-70 at 0 and 2.5 kJ m- 2 day- 1
UY-BsE to about !50 at 5 kJ m- 2 day- 1 UV-B 13 E.
Analysis of some growth parameters (Table I),
shows that the Relative Growth Rate was not signif-
icantly affected by enhanced UV-B radiation. Neither
the Leaf Area Ratio (LAR) nor the Leaf Weight Ra-
tio (LWR) change significantlly with enhanced UV-B
radiation. There is a trend (non-significant, p = 0.09)
that the Specific Leaf Area (SLA) is decreased with el-
evated UV-B. This is in line with the observed increase
of leaf thickness with enhanced UV-B (Figure 3).
110
Tahle 2. Effects of UV-B treatments (0, 2.5 and S k.T m- 2
day- 1 UV-BRE) on the UV-B absorbance of acid-methanol
extracts of fresh leaf tissue of Deschampsia antarctica. The
absorbance of the leaf extract was scanned from 280-320 nm
and integrated. Average values of 10 replications (one tis-
sue sample per pot) with standard etTor of the mean. There
was no statistically significant difference between the UV-B
treatments.
0 2.5 5 unambiguously indicating a C3 status of the carboxy-
UV-BBE UV-BsE UV-BsE
(k.T m- 2 day- 1) (k.T m- 2 day- 1) (k.T m- 2 day- 1)
1.86 ± 0.08 1.93 ±0.18 1.80 ± 0.34
Leaf photosyntheis
Curves fitted to the light response data (Figure 5) of
the 0 and 5 kJ m- 2 day- 1 UV-BnE treatment with the
non-linear model:
with r 2 values of 0.965 and 0.942, respectively, pro-
vided estimates of the parameters Amax. ¢ and Rd.
Based on the 95% probabilty intervals calculated
around these parameters, it was concluded that en-
hanced UV-B did not significantly affect quantum
yield of photosynthesis (¢ ), light saturated net leaf
photosynthesis (A max) and dark respiration (Rd).
There was no significant effect of enhanced UV-B
on the absorbance (280-320 nm) of methanolic ex-
tracts of leaf tissue harvested after 90 days of exposure
to the UV-B treatments (Table 2). There was no signif-
icant difference between the UV-B treatments in the
carbon and nitrogen content or the C!N ratio (9. I ±
0.9) of the shoot. The average content of carbon of the
shoot tissue was 37.1 ± 2.5%. The average nitrogen
content of the shoot tissue was 4.1 ± 0.4%.
Leaf photosyntheis and 8 13 C analysis
There was no difference between the light response
curves of net leaf photosynthesis. The curves are based
on the photosynthesis measurements of leaves of De-
schampsia antarctica attached to a well watered potted
tuft, which had been exposed to 0 or 5 kJ m- 2 day- 1
UV-BnE treatment for 80 days. There was no indi-
cation that any of these parameters was affected by
enhanced UV-B (Figure 5).
In fact, the light response curves in Figure 5 do not
show saturation of leaf photosynthesis at 2000 f.Lmol
m- 2 s- 1 . Absence of light saturated photosynthe-
sis is a physiological characteristic of C4 plants. To
check the C3 or C4 C02 fixation status of Deschamp-
sia antarctica , two samples of aerial parts of shoots
of Deschampsia antarctica, collected January 27 at
Leonie Island were analysed for the 13 C/ 12 C isotope
ratio. The 8 13 C values were -27.5%o and -28.7%r,
lation of Deschampsia antarctica (ef., Ehleringer &
Osmond 1991 ).
Shoot length measurements at Leonie Island
Length measurements of leaves and shoots of De-
schampsia antarctica were performed in the field at
Leonie Island in January and February 1999. During
the antarctic summermonth January 1999, there was
little loss of tags (less than 5%) as a result of strong
winds or by the activity of skuas. By the end of the
antarctic summer, February 28, 1999, most tags had
been blown away after heavy stormy weather. Of the
remaining 11 plants with tags, length growth of the
shoots during a period of 60 days varied from 0 to
38 mm with a median of 7 mm. For another group
of plants followed for 40 days, length growth varied
between 0 and 30 mm with a median of 3 mm. The
average shoot length of Deschampsia plants measured
at Leonie is about 5 em, which is less than the shoot
length of plants reached in the pots in the controlled
environment experiment.
Discussion
Response of Deschampsia antarctica to enhanced
UV-B radiation
There is considerable natural variation of stratospheric
ozone thickness over the antarctic, both in the short-
term (within the antarctic spring and summer) and in
the long-term (years). The dose of ambient solar UV-B
during the austral summer (December 21) was calcu-
lated to be 3.5 kJ m- 2 day- 1 UV-BBE· This is reduced
to 2 kJ m- 2 day- 1 UV-BnE at the end of the austral
summer at Leonie Island (Green et a!. 1974, 1980;
Bjorn & Murphy 1985, see Lud et a!. 200 I). In the
short antarctic growing season (from December un-
til the end of February at Leonie Island), a reduced
ozone thickness of less than I00 Dobson Units and
a UV-BnE value of 6.8 kJ m- 2 day- 1 UV-BnE was
recorded December 5, 1998 at Rothcra (Lud, per-
sonal communication). The UV-B levels used in the

controlled environment experiment cover part of this
natural range of antarctic UV-B levels.
While shoot length was significantly reduced with
elevated UV-B from about 9 em (0 kJ m- 2 day- 1 UV-
BsE) to about 6.5 and 6.0 em at 2.5 and 5 kJ m- 2
day- 1 UV-BsE, the number of shoots per pot at 5 kJ
m- 2 day- 1 UV-BsE was about doubled compared to
the number of shoots per pot at 2.5 and 0 kJ m- 2 day 1
UV-BBE· Also, the thickness of the shoots and leaves
was enhanced at 5 kJ m- 2 day- 1 UV-BsE compared
to 0 kJ m- 2 day- 1 UV-BsE and 2.5 kJ m- 2 day- 1
UV-BBE· The counteracting effects of elevated UV-B
on these two morphogenetic parameters (shooth length
and number of shoots) help to explain why the RGR
was not significantly affected. We are aware that the
results relate to a controlled environment study with a
relatively low PARIUV-B ratio. Low PARIUV-B ratios
may lead to an overestimation of UV-8 induced dam-
age, since DNA photolyase may operate at a low level,
with reduced growth as a consequence (Jansen et a!.
1998). In the Antarctic summer PAR fluxes peak up
to 2000 ~-tmol m- 2 day- 1 which may occur for some
hours around midday (see Huiskes eta!. 200 I). How-
ever, since the PAR level of 150 ~-tmol m- 2 s- 1 was
maintained for 24 h (according to the antarctic summer
light conditions), we regard the daily PAR/UV-8 doses
applied fairly realistic. At the moment we run a growth
experiment with Deschampsia antarctica cultivated at
300 ~-tmolm- 2 s- 1 PAR, exposed to enhanced UV-
B levels and at a day temperature of 8 oc and a night
temperature of 4 °C.
Reduced length growth of Deschampsia antarctica
with enhanced UV-8 may he caused by damage of
UV-B to a cellular structure (e.g., membrane, DNA)
or a cellular process (a.o. electron transfer or car-
boxylation, see Sullivan & Rozema 1999) or by a
signal derived from a UV-B photo-receptor. There
is increasing evidence for the existence of a spe-
cific UV-B photoreceptor, which is involved in pho-
tomorphogenesis, growth regulation and the induc-
tion of UV-B-absorbing compounds (Bjorn 1999). A
possible chromophore in this UV-B photoreceptor is
a tetrahydropterin derivative (Bjorn 1999; Bjorn &
Wang 2001). Alternatively, a UV-B stimulated ef-
fect on a wallbound, insoluble phenolic compound
such as ferulic acid, crosslinked in the cell wall ma-
trix, constraining cell expansion and leaf elongation
may be held responsible for decreased shoot length
growth (Liu et al. 1995; Day et a!. 1999: Bornman
1999). Growth inhibition may also relate to degrada-
tion of auxin and the formation of growth inhibiting
Ill
IAA photoproducts (Ros & Tevini 1995). In addition
a disturbed balance of plant hormones under elevated
UV-8 may relate to increased branching. Increased
branching can be explained by lost apical dominance
of dicotyledonous plants. Based on controlled en-
vironment experiments Tosscrams ct a!. (200 I) and
Meijkamp ct al. (200 1), report increased branching in
Viciafaba exposed to enhanced UV-B. The increase of
the number of shoots of Deschampsia antarctica with
enhanced UV-8 is in line with increased branching
of Deschampsia fiexuosa exposed to enhanced UV- B
simulating 25% ozone depletion and with 1300 f1 mol
m- 2 s- 1 PAR, as found by Gwynn Jones (Gwynn
Jones, personal communication).
Our shoot length growth measurements of De-
schampsia antarctica plants during the antarctic sum-
mer at Leonie Island (67°36' S, 68°21' W) indicated a
median length increment of about 0.7 em after a period
of 60 days. Day et al. ( 1999) report shoot length incre-
ment of Deschampsia antarctica cumulative over the
antarctic summer season at Palmer Station (64°46' S,
64°04' W) of 0.9-1.7 em. The higher shoot length
increment recorded at Palmer Station compared to
Leonie Island is probably caused by the relatively
milder climate of that more northerly antarctic loca-
tion. When UV-B was reduced to below ambient UV-8
by filters, shoot length increment was enhanced (Day
et al. 1999), which is in accordance with our results
in this paper. Shoot length growth of Deschampsia
antarctica plants at both maritime antarctic locations
is much less than measurements in the climate room
at 12 °C, where shoot length increment was 4.5 em
during a period of 90 days. This implies that extrap-
olation of findings of the climate room experiments to
the antarctic should be done with care. Growing De-
schampsia in nutrient enriched soil may partly explain
the long shoots developed in the controlled environ-
ment, compared to shoot length at Leonie Island.
However the nutrient and nitrogen input in the natural
habitat of Deschampsia (bird and mammal faeces, sea
spray) leads to a high nitrogen of shoots ( 1-2% with
values of 2.88% and 3.16% close to a sku a nest (Smith
1996 ), not so much lower that the nitrogen content
we measured in the controlled environment cultivated
Deschampsia plants (4% ).
Enhanced UV-8 did not affect the content of UV-
B absorbing compounds in the leaves. Similar results
were reported by Day et al. ( 1999) for Deschampsia
antarctica, and hy Tosserams & Rozema ( 1995) and
Tosscrams ct al. ( 1997) for dune grassland plants and
by Visser et a!. ( 1997) for a crop species. However,
112
the leaf content of UV-B absorbing compounds repre-
sents an important UV screen and one should conclude
that enhanced UV-B induces no further increase of the
content of UV-B absorbing compounds. One should
bear in mind that the Deschampsia antarctica plants
in the present study were grown from tillers collected
at the antarctic peninsula, which have been exposed to
high solar UV-B levels. High levels of UV-B absorb-
ing compounds may not only be linked with elevated
UV-B but also with the nutrient supply of plants (see
Tosserams et al. 200 I) and other biotic and abiotic
stress conditions (see Levizou & Man etas 200 I).
It has been assumed that enhanced UV-B affects
the epicuticular waxes of grasses, thereby reducing
cuticular transpiration. However, Day et al. ( 1999) for
Deschampsia antarctica, and Pilon et al. (1999) for a
number of low land and alpine Poa species, found no
UV-B effects on epicuticular waxes.
Photosynthesis and enhanced UV-B
There was no significant effect of enhanced UV-B
on the light response curves of Deschampsia antarc-
tica (Figure 5). Highest leaf net photosynthesis was
about 7 f.Lmol C0 2 m- 2 s- 1 reached at a PAR level
of 2000 f.Lmoi m- 1 s- 1. There was however no sat-
uration of leaf photosynthesis at 2000 f.Lmol m- 2
s- 1.These values of net leaf photosynthesis are sim-
ilar to those reported by Edwards & Smith ( 1988).
Edwards & Smith ( 1988) report the temperature op-
timum for net leaf photosynthesis to be between 10 oc
and 22 oc. In fact, their data of net photosynthesis
indicate a temperature optimum of 12.5 °C. The ab-
sence of a UV-B effect on net leaf photosynthesis is
in line with the result that the concentration of UV-
B absorbing compounds did not differ between the
three UV-B treatments (Table 2). Similarly, Montiel
et al. ( 1999), found no effect of varying UV-B levels
on leaf photosynthesis, neither they found saturated
photosynthesis at high irradiance, which is in line
with our results given in Figure 5. The 8 13 C value of
shoot material collected at Leonie (-28.0%o, clearly
demonstrates Deschampsia antarctica to be a C3 plant
(cf., Ehleringer & Osmond 1991 ). Apparently the non
saturated leaf photosynthesis at 2000 f.Lmol m- 2 s--l
indicates that Deschampsia antarctica can effectively
utlilize such high PAR irradiances, occurring in the
antarctic summer.
The temperature under which leaf photosynthesis
were done was about 15 °C, which is slightly above
the temperature optimum ( 12.5 oq for photosynthesis
reported for the antarctic grass Deschampsia antarc-
tica, as reported by Edwards & Smith ( 1988). At first
sight such a high temperature optimum (12.5 oq for
leaf photosynthesis appears to be remarkable for an
antarctic plant species. While average daytime air tem-
perature of Deschampsia antarctica on Signy Island in
January varies between 2.5 and 5 °C, the temperature
in Deschampsia antarctica tufts may rise up to 27 °C
(12:00 h) on a sunny day (Edwards 1975). On Leonie
Island mean day time air temperature of Deschampsia
antarctica sites was varying between 0 oc and 4 oc
and mean day time temperature in the tufts may be
15 oc on sunny days (Huiskes et al. 1999; Huiskes
et al. 2001).
On the other hand, this relatively high tempera-
ture optimum for photosynthesis of the antarctic grass
Deschampsia antarctica, as reported by Edwards &
Smith (1988), helps to explain the increased occur-
rence and distribution of Deschampsia antarctica with
the temperature increase as a result of global warming
(Smith 1994; Fowhert & Smith 1994; Convey 1996).
Xiong et al. (1999) also report an optimal leaf
temperature for Pn of around I 0 oc of Deschamp-
sia antarctica plants. Measurements of P11 (whole
canopy) by Xiong et al. (1999), were done on the
Deschampsia antarctica plants growing near Palmer
Station with temperature maintained at 4-5 °C. Tem-
perature responses of P11 showed that relatively high
temperatures are responsible for depressions in P11 and
not visible irradiance (PAR). Low Pn at supraoptimal
temperature (25 °C), appeared to he due to tempera-
ture enhanced respiration. Holtom & Greene (1967)
have shown that diurnally alternating temperatures
(with low night time temperatures) are favourable for
the growth of Deschampsia antarctica. Dark respira-
tion at high night temperatures, may cause release of
C02, hampering growth of Deschampsia antarctica.
Growth and photosynthetic measurements reported in
our paper, relate to Deschampsia antarctica plants cul-
tivated for more than three months at a temperature
of 12 °C, higher than used hy Xiong et al. (1999).
Perhaps these Deschampsia plants had acclimated
to 12 °C in the climate room. These Deschampsia
showed continuous length growth during the 90 days
of the experiment and looked healthy. It may be that
dark respiration rates after such acclimation have been
down regulated after a stay at 12 °C. Another differ-
ence between our single leaf photosynthesis measure-
ments and those of Xiong et al. 1999), is that Xiong
et al. (1999) measured whole canopy photosynthesis,
of which an unknown, but may be significant part con-
sisted of soil and root respiration. Also, attenuation of

PAR radiation may have occurred in the canopies of
Deschampsia antarctica, affecting the photosynthetic
measurements of Xiong eta!. ( 1999).
In recent papers (Caldwell ct al. 1995: Fiscus &
Booker 1995; Bjorn 1996; Kim et al. 1996; Rozema
et al. 1997; Caldwell et a!. 1998; Allen et al. 1998;
Jansen et al. 1998) it has been concluded that ozone
depletion represents no threat to the photosynthesis
and primary productivity of terrestrial biota. This does
not rule out that increased solar UV-B does not affect
plant growth of terrestrial ecosystems. The results of
this paper indicate that marked effects of enhanced
UV-B leading to reduced shoot length may be com-
pensated by UV-B induced branching. Such UV-B
induced morphogenic changes, along with UV-B ef-
fects on the biochemistry of secondary compounds
(Post & Larkum 1993; Rozema et al. 1997a: Bornman
1999; Meijkamp et a!. 1999) may have far reaching
ecological consequences affecting ecosystem structure
and function.
Terrestrial antarctic ecosystems offer unique pos-
sibilities of studying ecosystem responses to en-
hanced UV-B radiation. First because plants of ter-
restrial antarctic ecosystems are directly exposed to
stratospheric ozone depletion and second because of
the relatively simple structure of the antarctic ecosys-
tem. There are no more than two phanerogamic plants;
heterotrophic plants are absent and animal groups such
as rodents, insect herbivores etcetera are lacking (see
Smith 1996 and Convey 1996, for a more detailed
discussion). Also, the content of UV-B-absorbing
compounds, such as ftavonoids and other polypheno-
lic compounds, in present and historic plant material
of antarctic or subantarctic regions may help to re-
construct levels of stratospheric ozone in the past
(Markham eta!. 1990; Rozema eta!. 200 I). Therefore
research on these unique, vulnerable terrestrial antarc-
tic ecosystems may help to understand responses of
the terrestrial biota to stratospheric ozone depletion.
Acknowledgements
The research described on the development and instal-
lation of UV mini lamp systems in particular (project
number 751.499.06) was financially made possible by
Netherlands AntArctica Programme (NAAP) of the
The Netherlands Geosciences Foundation of NWO,
which is gratefully acknowledged. We thank Drs R.
Schorno (NAAP-GOA-NWO) for administrative sup-
port. The controlled environment experiment is par-
tially funded by the EU, DGXII Environment and
Climate, ENV4-CT97-0580).
113
The support of British Antarctic Survey, Cam-
bridge in logistics, transport and accomodation is
highly appreciated. We are indebted to Dr Ron Lewis
Smith, Mr Andy Rossaak and Paul Geissler (BAS,
Cambridge and Rothera) for providing information
and technical support. We thank the complete staff and
colleagues at Rothera research station for their support
and cooperation.
References
Allen. D. J., Nogues, S. & Baker, N. R. 199X. Ozone depletion and
increased UV-B radiation: is there a real threat to photosynthesis?
J. Exp. Bot. 49: 1775-1788.
Behrenfeld. M .. Hardy, J., Gucinsky, H .. Hanneman, A, Lee II,
H & Wanes, A. 1993. Effects of Ultraviolet-B radiation on pri-
mary production along latitudinal transsects in the South Pacific
Ocean. Mar. Environ. Res. 35: 349-363.
Bjorn, L. 0. 1996. Etlects of ozone depletion and increased UV-B
on terrestrial ecosystems. Int. J. Environ. Studies 5 I: 217-243.
Bjorn, L. 0. 1999. UV-B etlccts: receptors and targets. Pp. 821-832.
In: Singhal, G. S. et al. (eds). Concepts in Photohiology: Photo-
synthesis and Photornorphogenesis. Narosa Publishing House.
Bjorn. L.O. & Murphy, T.M. 1985. Computer calculations of solar
ultraviolet radiation at ground level. Physiol. Veg. 23:555-561.
Bj<irn, L. 0. & Wang, T. 2001. Is provitamin D a UV-B receptor in
plants') Plant Ecol. 154: 1-R (this volume).
Bornman, J. F. 1999. Localisation and functional significance of
tlavonoids and related compounds. Pp. 59-69. In: Rozema, J.
(ed.). Stratospheric Ozone Depletion: the Effects of Enhanced
CV-B Radiation on Terrestrial Ecosystem. Backhuys Publishers,
Leiden.
Burna, A. J. G., Van Hannen, E. J., Roza, L. Vcldhuis, M. J. W. &
Gieskes, W.W.C. 1995. Monitoring ultraviolet-B induced dam-
age in individual diatom cells by immunofluorescent thymine
climer detection. J. Phycol. 31: 314-321.
Caldwell, M. M. 1971. Solar UV irradiation and the growth and
development of higher plants. Pp. 131-177. In: Giesen. A. C.
(cd.), Photophysiology, Vol. 4. Academic Press, London.
Caldwell, M. M., Camp, L. B., Warner, C. W. & Plint, S. D.
19R6. Action spectra and their key role in assessing biological
consequences of solar UV-B radiation change. Pp. R7-lll. ln:
Won·est, R. C. & Caldwell, M. M. (eds), Stratospheric Ozone
Reduction, Solar Ultraviolet Radiation and Plant Life. NATO
AS! Series, Vol. G8. Springer-Verlag, Berlin.
Caldwell, M. M., Teramura, A. H., Tevini, M. 1989. The chang-
ing solar ultraviolet climate and the ecological consequences for
higher plants. Trends Ecol. Evol. 4: 363-367.
Caldwell. M. M., Bjorn, L. 0., Bornman, J. F., Flint, S. D., Ku-
landaivelu. G .. Terarnura, A. H. & Tevini, M. 1998. Effects of
increased solar ultraviolet radiation on terrestrial ecosystems. J.
Photochem. Photobiol. B46: 40-52.
Convey. P. 1994. Photosynthesis and dark respiration in antarctic
mosses- an initial comparative study. Polar Bioi. 14: 65-69.
Convey, P. 1996a. Reproduction of Antarctic plants. Ant. Sci. 8:
127-134.
Convey. P. 1996b. Overwintering strategies of terrestrial inverte-
brates in Antarctica- the significance of flexibility in extremely
seasonal environments. Eur. J. Entomol. 93: 489-505.
Convey, P. 1997. Environmental change: possihle consequences lor
the life histories of antarctic terrestrial biota. Kor. J. Pol. Res. 8:
127-144.
114
Convey. P. & Lewis Smith, R. I. 1993. Investment in sexual
reproduction by antarctic mosses. Oikos 68: 293-302.
Day, T. A., Ruhland, C. T.. Grobe, C. W & Xiong, F. 1999. Growth
and reproduction of Antarctic vascular plants in response to
warming and UY radiation in the field. Oecologia 119: 24-35.
Edwards, J. A. & Smith, R. I. L 1988. Photosynthesis and respira-
tion of Ca/abanthus qui tens is and Deschampsia antarctica from
the maritime Antarctic. Br. Antarct. Surv. Bull. 81: 43-63.
Edwards, J. A. 1972. Studies in Colobanthus quilensis and De-
schampsia antarctica. Y. Distribution, ecology and vegetative
pcrfomance on Signy Island. Br. Antarct. Surv. Bull. 28: 11-28.
Ehleringer. J. R. & Osmond, B. 1991. Stable isotopes. Pp. 281-
300. In: Pearcy. R. W. et al. (eds). Plant Physiological Ecology.
Chapman and Hall, London.
Farman, J. C., Gardiner, B. G. & Shanklin, J. D. 1985. Large
losses of total ozone in Antarctica reveal seasonal CLO,!Nox
interaction. Nature 315:207-210.
Fiscus, E. L. and Booker, F. L. 1995. Is increased UV-B a threat to
crop photosynthesis and productivity? Photosynt. Res. 43: 81-
92.
Fowbert, J. A. & Smith, R. I. L. 1994. Rapid population increases
in native vascular plants in the Argentine Islands. Antarctic
Peninsula. Arct. Alp. Res. 26: 290-296.
Gleason, J. F., Bhartia, P. K., Herman, J. R., McPeters, R., Newman.
P.. Stolarski, R. S., Flynn, L., Labow, G., Larko, D.,Seftor, C ..
Wellemeyer, C., Komhyr, W. D., Miller, A. J. & Planet, W. 1993.
Record low global ozone in 1992. Science 260: 523-526.
Goudriaan, J. 1982. Potential production processes. Pp. 98-113. Tn:
Penning de Vries. F. W. T. & Van Laar. H. H. (eds), Simulation
of Plant Growth and Crop Production. Pudoc, Wageningen.
Green, A. E. S., Sawada, T. & Shettle, E. P. 1974. The middle
ultraviolet reaching the ground. Photochem. Photobiology 19:
251-259.
Green. A. E. S., Cross, K. R. & Smith, L. A. 1980. Improved
analytic characterisation of ultraviolet skylight. Photochem. Pho-
tobiol. 31: 59-65.
Greene, S. W. 1970. Studies in Colabanthus quitensis and De-
schampsia antarctica. I. Introduction. Br. Antarct Surv. Bull. 23:
19-24.
Greene, S. W. & Haltom. A. 1971. Studies in Co/ohanthus quiten-
sis and Deschampsia alltarctica. III. Distribution, habitats and
performance in the Antarctic botanical zone. Br. Antarctic Surv.
Bull. 26: 1-29.
Gwynn-Jones, D. & Johanson, U. 1996. Growth and pigment pro-
duction in two subarctic grass species under four different UV-B
irTadiation levels. Physiol. Plant 97: 701-707.
Gwynn-Jones, D., Bjorn, L. 0., Callaghan, T.V., Gehrke, C. Johan-
son, U., Lee, J. A. & Sonesson, M. 1996. Effects of enhanced
UV-B radiation and elevated concentrations of COz on a sub-
arctic heathland. Pp. 197-207. Tn: Korner, Ch. & Bazzaz, F. A.
(eds), Carbon Dioxide, Populations and Communities. Academic
Press, San Diego.
Gwynn-Jones, D., Lee, J. A., Callaghan, T. V. & Sonesson, M. 1997.
Effects of enhanced UV-B radiation and elevated carbondioxide
concentrations on subarctic forest heath ecosystems. Plant Ecol.
128: 242-249.
Hotinann, D.J. 1996. The 1996 Antarctic ozone hole. Nature 3H3:
129.
Hofmann, D. J., Oltmans, S. J., Harris, J. M., Johnson, B. J. &
Lathrop, J. A. 1997. Ten years of ozone sonde measurements at
the South Pole: implications for recovery of springtime Antarctic
ozone. J. Geophys. Res. I 02: 8931-8943.
Holdgate, M.W. 1964. Terrestrial ecology in the maritime Antarctic.
Pp. 181-194. In: Carrick, R., Holdgate, M. & Prevost, J. (eds),
Biologic Antarctique. Hermann, Paris.
Haltom, A. & Greene, S.W. 1967. The growth and reproduction of
Antarctic flowering plants. Pp. 323-337. ln: Smith, J. E. (ed.),
A Discussion on the Terrestrial Antarctic Ecosystem. Trans. R.
Soc. Rio!. Sci. 777.
Huiskes, A. H. L., Gremmen. N. J. M. & Francke, J. W. 1997a.
Morphological effects on the water balance of Antarctic foliose
and fruticose lichens. Ant. Sci. 9: 36-42.
Huiskes, A. H. L., Grcmmen, N.J. M. & Francke, J. W. 1997b.
The delicate stability of lichen symbiosis: comparative studies
on the photosynthesis of the lichen Mastodia tesse/ata and its
free-living phycobiont, the alga Prasiola crispa. Pp. 234-240.
Tn: Battaglia, B .. Valencia, J. & Walton, D. W. H. (eds), Antarc-
tic Communities: Species, Structure and Survival B. Cambridge
University Press, Cambridge.
Huiskes, A. H. L.. Lud, D., Moerdijk-Poortvliet, T. C. W. &
Rozema, J. 1999. Impact of UV-B radiation on Antarctic terres-
trial vegetation. Pp. 313-337. In: Rozema, J. (ed.), Stratospheric
Ozone Depletion; the Effects of Enhanced UV-B Radiation on
Terrestrial Rcosystem. Backhuys Publishers, Leiden
Jansen, M. A. K. Gaba, V. & Greenberg, B. 1998. Higher plants
and UV-B radiation: balancing damage, repair and acclimation.
Trends Plant Sci. 3: 131-135.
Johanson, U. & Zeuthen. J. 1998. The ecological relevance of UV-
B enhancement studies is dependent on tube type, tube age,
temperature and voltage. J. Photochem. Photobiol. R: Bioi. 44:
169-174.
Karentz, D., Cleaver, J. E. & Michel, D. L 1991a. Cell survival
characteristics and molecular responses of Antarctic phytoplank-
ton to UV-B radiation. J. Phycol. 27: 326-341.
Karentz, D., McEuen, F. S., Land, M. C. & Dunlap, W. C. 199lh.
Survey of mycosporine-like amino acid compounds in Antarctic
marine organisms: potential protection from ultraviolet exposure.
Mar. Bioi. 108: 157-166.
Kim, H. Y., Kobayashi, K., Nouchi, I. & Yoneyama, L. 1996.
Enhanced UV-B radiation has little clfect on growth, 8 13 C val-
ues and pigments of pot-grown rice (Orvza sativa) in the field.
Physiol. Plant 96: 1-5.
Lud, D., Huiskes, A. H. L., Moerdijk, T. C. W. & Rozema, J. 2001.
The effect of altered levels of UV-B radiation on an Antarctic
grass and lichen. Plam Ecol. 54: 87-99 (this vomume).
Lubin, D. & Frederick, J. E. 1991. The UV radiation of the Antarctic
Peninsula: the roles of ozone and cloud cover. J. Appl. Meteorol.
30: 478-493.
Levizou, E. & Man etas, Y. 200 I. Combined effects of enhanced UV-
B radiation and additional nutrients on growth of two Mediter-
ranean plant species. Plant Ecol. 154: 179-186 (this issue).
Madronich. S. 1992. Implications of recent total atmospheric ozone
measurements for biologically active ultraviolet radiation reach-
ing the earth's atmosphere. Geophys. Res. Lett. 19: 37-40.
Madronich, S., McKenzie, R. L., Caldwell, M. & Bjorn, L. 0. 1995.
Changes in ultraviolet radiation reaching the earth's surface.
Ambio 24: 143-152.
Marchant, H.J. 1997. Impacts of ozone depletion on Antarctic or-
ganisms. Pp. 367-374. ln: Battaglia, B., Valencia, J. & Walton,
D. W. H. (eds), Antarctic Communities: Species, Structure and
Survival B. Cambridge University Press, Cambridge.
Markham, K. R., Franke, A., Given, D. R. & Brownscy, P. 1990.
Historical Antarctic ozone level trends from herbarium specimen
flavonoids. Bull. Liaison Groupe Polyphenols 15: 230.
McLeod, A. R. 1997. Outdoor supplementation systems for stud-
ies of the effects of increased UV-B radiation. Pp. 78-92. Tn:

Rozema, J.• Gieskes. W. W. C.. Van de Geijn. S. C.. Nolan. C.
& De Boois. H. (eels), liV-B and Biosphere. Kluwer Academic
Publishers, Dordrecht, The Netherlands.
Mcijkamp, B .. Aerts, R .. Van de Staaij, J., Tosscrams, M .. &
Rozema, J. 1999. Effects of UV-B on secondary metabolites in
plants. Pp. 71-99. In: Rozema. J. (ed.), Stratospheric ozone De-
pletion: the Etlects of Enhanced UV-B Radiation on Terrestrial
Ecosystem. Backhuys Publishers. Leiden.
Meijkamp, B. B .. Doodeman, G. & Rozema, J. 2001. The response
of Vicia faba to enhanced UV-B radiation under low and near
ambient PAR levels. Plant Ecol. 54: 135-146 (this volume).
Montiel, P., Smith, A.& Keiller, D. 1999. Photosynthetic responses
of selected Antarctic plant to solar radiation in the southern
maritime Antarctic. Polar Res. 18: 229-235.
Moore, 0. M. 1970. Studies on Co/oba111hus quilensis and De-
schampsin antarcticu. II. Taxonomy. distrihution and relation-
ships. Br. Ant. Surv. 23: 63-80.
Pilon, 1. 1., Lambers, H .. Baas, W.. Tosserams, .\1., Rozema. J. &
Atkin, 0. K. 1999. Leaf waxes of slow-growing alpine and fast-
growing lowland Poa species: inherent differences and responses
to UV-B radiation. Phytochem. 50: 571-580
Post, A. & Larkum, A. W. D. 1993. !!V absorbing pigments.
photosynthesis and UV exposure in Antarctica: Comparison of
terrestrial and marine algae. Aq. Bot. 45: 231-243.
Ros, J. & Tevini. 1995. Interaction of UV-radiation and IAA dur-
ing growth of seedlings and hypocotyl segments of sunflower. J.
Plant. Physiol. 146: 295-302.
Roscoe, H. K., Jones. A. E. & Lee. A. M. 1997. Midwinter start
to Antarctic ozone depletion: evidence from observations and
models. Science 278: 93-96.
Rousseaux. M. C .. Balian,, C. L, Giordano, C. Y., Scopcl. A.
L.. Zima, A. M .. Szwarcbcrg-Bracchitta. '\1., Searles, P. S ..
Caldwell, M. M. & Diaz. S. B. 1999. Ozone depletion and
UVB radiation: Impact on plant DNA damage in southern Somh
America. Proc. Nat. Acad. Sci. USA 96: 15310-15315.
Rozema, 1., Van de Staaij, L Bjiirn, L.-0. & Caldwell, M.
1997a. UV-B as an environmental factor in plant life: stress and
regulation. Trends Ecol. Evol. 12: 22-28.
Rozema, J., Tosserams, M., Nelissen. H. J. M .. Hccrwaardcn,
L. v., Broekman, R. A. & Flierman. N. 1997b. Stratospheric
ozone reduction and ecosystem proce:-.ses: enhanced UV-B radi-
ation affects chemical quality and decomposition of leaves of te
dune grassland species Calamagrostis e!pigeios. Plant Ecol. 12X:
284-295.
Rozema, J., Van de Staaij, J. & Tosserams. M. 1997c. Effects
of UV-B radiation on plants from agro-ecosystems and natural
ecosystems. Pp. 213-232. In: Lumsden P.J. (cd.). Plants and UV-
B: Responses to Environmental Change. Cambridge University
Press, Cambridge.
Rozema, J., Gicskcs, W. W. C., Van de Geijn, S. C .. Nolan. C. &
De Boois, H. 1997d. UV-B and Biosphere. Kluwer Academic
Puhlishers. Dordrecht. The Netherlands.
Rozema, 1.. Van de Staaij, J., Mcijkamp, B.. Lud, D ..
Moerdijk. T. & Huiskes, A. 1997c. Stratospcric ozone depletion
and effects of enhanced UV-B radiation on terrestrial antarc-
tic plants: the methodology of a mini-UV-B supplementation
system. Circumpolar J. 12: 43-48.
Rozema, J., Noordijk. A. 1., Broekman. R. A., Van Beem. A.,
Meijkamp. B. M .. De Bakker, Y.. Van de Staaij, J. W. M.,
Stroetenga. M .. Bohnckc. S. J.P.. Konert, M., Kars, S., Peat, H ..
SMith, R.I. L. & Convey. P. 200 I. (Poly)phenolic compounds in
pollen and spores of antarctic plants as indicators of solar UV-B.
Plant Ecol. 154: 9-26 (this vomume).
115
Smith. R. I. L. 1985. Farthest south and highest occurrences of
vascular plants in the Antarctic. Polar Res. 21: 170-183.
Smith, R. I. L. 1994. Vascular plants as bioindicators of regional
wam1ing in Antarctica. Oecologia 99: 322-328.
Smith, R.I. L. 1996. Terrestrial and Freshwater Biotic Components
of the Western Antarctic Peninsula. Ant. Res. Ser. 70: 15-59.
Smith. R. I. L. & Poncct. S. 1985. New southernmost record for
Antarctic flowering plants. Polar Res. 22: 425-427.
Smith. R. I. L. & Ponce!, S. 1987. Deschampsia wllarctica and
Co/ol>wllhus quilensis in the Terra Firma Islands. Bull. Br.
Antarct. Surv. 74: 31-35.
Smith. R. I. L. & Stephenson, C. 1975. Preliminary growth stud-
ic:-. on Festuca cmllraria and Deschampsia antarctica on South
Georgia. Br. Antarct. Surv. Bull. 41142: 59-75.
Sakal. R. R. & Rohlf. F. .1. 1995. Biometry. The principles and prac-
tice of statistic~ in biological research. 3rd edition. Freeman &
Co. San Francisco. USA.
Stolarski. R .. Bojkov. R .. Bishop, L., Zcferos, C .. Staehlin. J. & Za-
wodny, J. 1992. Measured trends in stratospheric ozone. Science
256: 342-349.
Sullivan, J. & Rozema, .1. 1999. UV-B effects on terrestrial plant
growth and photosynthesis. Pp. 39-57. In: Rozema. J. (ed.).
Stratospheric Ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden.
Teramura, A. H. & Ziska. L. H. 1996. Ultraviolet-B radiation and
photosynthesis. l'p. 435-450. In: Baker, X R. (ed. ), Photo-
synthesis and the Environment. Kluwer Academic Puhlishers.
Dordrecht. The Netherlands.
Tosserams, M. & Rozema, J. 1995. Effects of ultraviolet-B radiation
(UV-B) on growth and physiology of the dune grassland species
Calomagroslis epigeios. Environ. Poll. 89: 209-214.
Tosscrams. M .. Pais de Sa, A. & RoLema. J. 1996. The effect of
solar UV radiation on four plant species occurring in a coastal
grassland vegetation in The Netherlands. Physiol. Plant. 97: 731-
739.
Tosserams, M. Magcndans, G. W. H. & Rozema, .1. 1997. Differ-
ential effects of elevated ultraviolt-B radiation on plant species
from a dune grassland ecosystem. Plant Ecol. I 2H: 266-2H I.
Tosserams, M .. Visser. A., Groen, M. Kalis. G .. Magendans. E.
& Rozema. J. 200 I. Combined etlects of C0 2 concentration and
enhanced UV-B radiation offaba bean. Plant Ecol. 154: 195-210
(this vomume).
Verne!, M., Brody, E. A., Holm-Hansen. 0. & Mitchell B. G. 1994.
The response of Antarctic Phytoplankton to Ultraviolet Radia-
tion: Absorption. Photosynthesis. and Taxonomic Composition.
1\nt. Res. Ser. 62: 143-158.
Vincent. W. F. & Quesada. A. 1994. Ultraviolet Radiation Ef-
fects on Cyanobacteria: Implications for Antarctic Microbial
Ecosystems. Ant. Res. Ser. 62: 111-124.
Vincent, W.F. & Quesada, A. 1997. Microbial niches in the polar
environment and the escape from UV radiation in non-marine
habitats. Pp. 388-395. In: Battaglia, B .. Valencia. J. & Walton.
D. W. H. (eels). Antarctic Communities: Species. Structure and
Survival. Cambridge University Press. Cambridge.
Visser, A . .1., Tosserams . .\1 .. Groen. M. W .. Kalis, G .. Kwant, R ..
Magendans. G. W. H. & Rozema, J. 1997. The combined effect
of C02 and supplemental UV-B radiation on faba bean. 3. Leaf
optical properties, pigments, stomatal index and epidermal cell
density. Plant Ecol. 128: 20H-222.
Xiong, S., Ruhland, C. T. & Day, T. A. 1999. Photosynthetic
temperature response of the Antarctic vascular plants Colohm1-
thus quilcnsis and Deschmnpsia ontarctica. Physiol. Plant. I06:
276-286.
Section 4: Interactions of UV-B Radiation with other Factors of
Terrestrial Environments

Rosmarinus officina/is (L. ), an aromatic, Mediterranean herb grown in the field.

 Plant Ecology 154: 119-122, 200 I.
119
© 2001 Kh<Wer Academic Publishers.

Reduction of ambient UV-B radiation does not affect growth but may
change the flowering pattern of Rosmarinus officinalis L.

George Grammatikopoulos, Periklis Drilias, Aris Kyparissis, Yiola Petropoulou &

Yiannis Manetas
Laboratory of Plant Physiology, Department olBiology, University (Jf'Patras, GR-26500 Patras, Greece
(E-mail: y.manetas@upatras.gr)

Key words: Flowering, Growth, Rosmarinus officina/is, UV-B radiation

Abstract
The effects of sub-ambient levels of UV-B radiation on the shrub Rosrnarinus officina/is L. were investigated in a
field filtration experiment in which the ambient UV-B was manipulated by a combination of UV-B transmitting and
UV-B absorbing filters. As a result, the plants were receiving near-ambient or drastically reduced UV-B radiation
doses. Drastic reduction of UV-B radiation had no effect on mean, total and maximum stem length, number of
stems per plant, dry mass of leaves, stems and roots and leaf nitrogen and phenolic contents. However, flowering
was more pronounced under reduced UV-B radiation during the winter period which coincides with ascending
ambient UV-B radiation. In contrast, during autumn and early winter, a period which coincides with descending
ambient UV-B radiation, flowering was unaffected by reduced UV-B radiation. We can conclude that natural UV-B
radiation does not affect growth of Rosmarinus officina/is, but its reduction could influence the flowering pattern
of the species.

Introduction plants showed that although growth was unaffected

Enhanced UV-B radiation is generally characterized
as harmful for plants and its effects are species spe-
cific (Terramura & Sullivan 1993; Tevini & Teramura
1989). Furthermore, some researchers claim that even
ambient UV-B radiation may be adverse for growth
(Rousseaux eta!. 1998) or alter plant-consumer inter-
actions (Rousseaux et a!. 1998; Searles et al. 1999).
Also, there are indications that the natural fluctuations
of UV-B radiation could be an important regulatory
factor for plants (Manetas 1999; Rozema et a!. 1997)
and in some cases specific UV-B photoreceptors ap-
pear to be involved (Balian:~ et a!. 1995). A common
way to get information on the role of ambient UV-B
radiation has been the exclusion or drastic reduction of
natural UV-B radiation, using various combinations of
UV-B transmitting and absorbing plastic filters under
field conditions (Day et al. 1999; Deckmyn & Impens
1995; Deckmyn & Impens 1998; Krizek ct a!. 1997;
Krizek eta!. 1998; Rousseaux eta!. 1998; Tosserams
et a!. 1996). We report here such an experiment with
the Mediterranean plant Rosmarinus officina/is. Field
UV-B enhancement experiments with Mediterranean
(see Manetas 1999, and the literature therein), some
reproductive parameters could be considerably influ-
enced (Grammatikopoulos et al. 1998; Stephanou and
Manetas 1998; Stephanou et a!. 2000). Therefore, in
the present experiment we examined the effects of
natural (sub-ambient) UV-B radiation on growth and
flower demography.
Materials and methods
Plant material and growth conditions
Seedlings of Rosmarinus officina/is L. were raised
in 20 em clay pots containing local soil in an open
nursery under natural solar radiation. At the age of
I year (early May 1998), 36 similar seedlings (based
on plant height and leaf number) were selected and
randomly assigned into two treatments (high and low
UV-B), with three experimental plots per treatment
(i.e., 6 plants per plot, 18 plants per treatment). The
plots were located in an open unshaded area within
the Patras University campus. On each plot, a metal
framework (80 x 150 em) provided the support for
3 mm, UV-B transmitting Plexiglass sheets (Rohm
120
2458). In three of the plots, the UV-B transm1ttmg
Plexiglass was covered with UV-B absorbing 0.1 mm
Mylar film (Dupont Co., DE, USA). The plants were
located in the central part of the plot and rotated within
the plot weekly to avoid site effects. The obtained re-
duction in ambient UV-B radiation reaching the plants
is presented in Table l. As shown, 'high' and 'low'
UV-B treatments corresponded to near-ambient (22%
reduction) and considerably reduced (94-97% reduc-
tion) UV-B radiation doses. Differences in UV-A and
PAR were negligible. The plants were watered with
200 ml H20 every other day during the hot/dry period
of the experiment (May to October) and twice a week
from November to the end of the experiment (early
February 1999). The experiment lasted for 9 months.
Measurements
Solar spectral irradiance in the UV-B (280-320 nm)
region of the spectrum was measured in situ with
an OL-752 Optronic (Orlando, FL, USA) spectrora-
diometer, calibrated against an OL-752-10 spectral
irradiance standard and an OL-752-150 module for
wavelength accuracy. For biologically effective UV-
B radiation (UV-BsE), the absolute spectral iuadiance
(Bjorn & Murphy 1985) was weighted with the gen-
eralized plant action spectrum normalized at 300 nm
(Caldwell 1971 ).
At final harvest, the plants were divided into
leaves, stems and roots and dried (80 °C, 24 h) for dry
mass determination. Dry leaves were powdered and
used for the determination of total phenolics (Folin-
Ciocalteu method as described by Waterman & Mole,
1994) and nitrogen (Kjeldahl method as described
by Allen 1989). The number of flowers was counted
each day during the flowering period (September 9 to
February 5).
Statistics
All statistical tests were performed with SPSS 9.0 sta-
tistical package, using Type Ill square sums. Since
individual plants were rotated only within but not
between plots, differences between treatments (near-
ambient UV-B, reduced UV-B) for dry mass (leaves,
stems, roots), total phenolics, total nitrogen as well
as plant height and total branch length, were analysed
using nested ANOVA. For flower demography, a
repeated measures (general linear model) statistical
analysis was used to test the etlects of UV-B, time and
their combination (sphericity assumed for calculation
of significances).
Results
Table 2 shows that biomass and its partition between
roots and shoots was not significantly affected by UV-
B radiation at the doses used in this investigation. The
same was true for leaf nitrogen and total phenolics
(Table 2) and for plant height and total branch length
(results not shown).
Flowering, under both treatments commenced in
early September, peaked in early November and was
again increasing during the last period of the experi-
ment (Figure 1). There was no UV-B x time interac-
tion nor a UV-B effect if data from the whole flowering
period were taken into account. However, the results
in Figure I indicate a consistent trend for flowering
superiority of the plants receiving near-ambient UV-
B during autumn. This trend was gradually reversed
during early winter. We arbitrarily chose the date of
December 26 to consider two sets of flowering data.
During this date the modeled UV-B radiation dose is
the lowest of the year for our site (Bjorn & Murphy
1985). This model takes into account both zenith angle
and normal seasonal variation in the ozone column.
Accordingly, our experimental period can be divided
into a descending and an ascending part, as far as the
daily UV-B dose is concerned. A repeated measures
analysis for the first set of data (September to De-
cember 26) showed that the observed trend for more
flower under near-ambient UV-B was not statistically
significant (p>0.05), while the same test for the rest of
data (December 26 to early February) showed a statis-
tically significant UV-B x time interaction (p=0.004),
expressed as a flowering preponderance under reduced
UV-B.
Discussion
The results of this investigation indicate that near-
ambient UV-B radiation does not suppress growth of
Rosmarinus officina/is. This is in contrast with some
previous findings showing that even present day UV-
B radiation levels are inhibitory for plant growth (Day
et al. 1999; Krizek et al. 1997, 1998). However, it
is in agreement with the results of Dcckmyn & 1m-
pens (1995) and Tosserams et al. (1996). In these
cases, reduction of ambient UV-B radiation had no
significant inhibitory effects on growth of Phaseo-
lus vulgaris and four species of the coastal grassland
vegetation. Thus, the previous assumption that am-
bient UV-B radiation effects on growth are species
specific is confirmed by the present study and it is
 121

Table I. Daily doses of absolute and hiologically effective UV-B irradiance (2S0-320 nm) outside the
frames (ambient). under the Plexiglass frames (high UV-B) and under the Plcxiglass + Mylar frames
(low UV-B) measured on a clear day !September 7. 1998).

Absolute irradiance 0(· of ambient Biologically effective 0 /c of ambient

(k.J m- 2 day- 1) irradiance (kJ m- 2 day- 1 1

Ambient 43.16 4.212

High UV-B :1364 77.9 3.253 77.2
LowUV-B 2.52 5.8 0.117 2.8

Table 2. Final plant dry mass (g plant-! ). leaf nitrogen and total phenolics under
near-ambient or reduced UV-B radiation, measured at plant harvest. Mean ± SD from
three experimental plots (with six plants per plot, sec Materials and Methods). Results
of nested A NOVA for comparison between treatments arc given in the last two columns.
Differences were not statistically significant.

Near-ambient Reduced C:+ Change F p

L:v-B UV-B

Dry mass (g)

Leaves 12.97 ± 2.44 1:1.63 ± 1.96 +5.0 0.13 >0.05
Stems 6.66 ± 0 95 6.93 ± 1.03 +4.1 0.10 >0.05
Roots 16.10±0.89 17.97 ± 0.97 +11.3 13.21 >0.05
Total phenol ies 40.81 ± 7.49 36.19 ± 2.92 -10.9 0.99 >(l.OS
(mg per g OM)
Total nitrogen 19.04 ± 0.68 20.08 ± 1.99 +5.5 0.50 >0.05
(mg per g OM)

20
~-near ambient UV-8

-~ 18
16
-- ·- · · redu:ed UV-8

0.. 14
r/l
;.... 12
~
s
4-<
10
8 I.j
].
0
;....
v 6

1
l : [(! /
4 l' l[[\111 ··jl
I' ·!I~
2
11u1roh, 1un1)J1
hl
0
Sep
Figure 1. Flowering number versus time in Rosmarinus officina/is, grown under near-ambient ( - - ) or reduced (- - - - -) UV-B radiation.
Values are means+ SD from three experimental plots (with six plants per plot, see Materials and methods).
122
also strengthened by the fact that even positive growth
responses to near-ambient UV-8 radiation have been
observed (Deckmyn & Tmpens 1998).
The differences in the UV-8 sensitivity between
the species in the above mentioned studies can not
be correlated to the ability of the plants to increase
their phenolic levels with increasing UV-8 radiation.
In most of the cases the phenolic levels were not af-
fected (Day et al. 1999; Deckmyn & Impens 1995,
1998; Krizek et al. 1997: Rousseaux et al. 1998; re-
sults of the present study), irrespective of any effects
on growth.
Although the results on growth were clear, the
flowering pattern was affected by UV-8 radiation in
a peculiar manner. The choice of 26 December to di-
vide the anthesis data into two sets was based on the
fact that close to this date we have the lowest possi-
ble UV-8 radiation dose of the year (27 December).
We may therefore advance the hypothesis that dur-
ing the descending period of ambient UV-8 radiation,
flowering is not affected by UV-8, while UV-8 seems
to suppress flowering during the period of ascending
ambient UV-8 radiation. Field UV-8 supplementation
studies have, in some cases, shown positive effects
on flower number (Grammatikopoulos et al. 1998)
and reproductive effort (Gwynn-Jones et al. 1997;
Stephanou & Manetas 1998; Stephanou et al. 2000).
Since flowering in many cases is photoperiodically
induced and UV-8 radiation has photomorphogenctic
effects (Balian; et al. 1995), we introduce the idea
that the seasonal changes in ambient UV-B radiation
may be considered as a photoperiodic signal. The sig-
nal could be the UV-B daylcngth itself. Alternatively,
conventional photoperiodic responses may be modi-
fied by changing UV-8 radiation. Apparently, more
work is needed to confirm these hypotheses under field
conditions.
Acknowledgement
We are grateful to the Commission of the European
Communities for financial support.
References
Allen, S. E. 1989. Chemical Analysis of Ecological Materials.
Blackwell, Oxford.
Ballare, C. L., Barnes, P. W. & Flint, S. D. 1995. Inhibition of
hypocotyl elongation by ultraviolet-B radiation in de-etiolating
tomato seedlings. I. The photoreceptor. Physiol. Plant. 93: 584-
592.
Bjorn. L. 0. & Murphy, T. M. 1985. Computer calculation of solar
ultraviolet radiation at ground level. Physiol. Veg. 23: 555-561.
Caldwell, M. M. 1971. Solar UV radiation and the growth and de-
velopment of higher plants. Pp. 131-177. In: Giese, A.C. (ed),
Photophysiology. Academic Press. New York.
Day, T. A., Ruhland, C. T., Grobe. C. W. & Xiong, F. 1999.
Growth and reproduction of Antarctic vascular plants in res ponce
to warming and UV radiation reductions in the field. Oecologia
119: 24-35.
Deckmyn, G. & Impens, I. 1995. UV-B increases the harvest index
of hean (Phaseo/us vulgaris L.). Plant Cell Environ. 18: 1426-
1433.
Deckmyn, G. & Impens. I. 1998. Effects of solar UV-B imtdia-
tion on vegetative and generative growth of Bromus catharticus.
Environ. Exp. Bot. 40: 179-185.
Grammatikopoulos, G., Karousou, R., Kokkini, S & Manetas, Y.
1998. Ditlerential etlects of enhanced UV-B radiation on repro-
ductive effort in two chemotypes of Mentha spica/a L. under field
conditions. Aust. J. Plant Physiol. 25: 345-351.
Gwynn-Jones, D., Lee, J. A. & Callaghan, T. Y. 1997. Effects of
enhanced UV-B radiation and elevated carbon dioxide concen-
trations on a sub-arctic forest heath ecosystem. Plant Ecol. I 28:
242-249.
Krizek, D. T., Mirecki, R. M. & Britz, S. J. 1997. Inhibitory effects
of ambient levels of solar UV-A and UV-B radiation on growth
of cucumber. Physiol. Plant. 100: 886-893.
Krizek, D. T., Britz, S. J. & Mirecki, R. M. 1998. Inhibitory effects
of ambient levels of solar UV-A and UV-B radiation on growth
of cv. New Red Fire lettuce. Physiol. Plant. 103: 1-7.
Manetas, Y. 1999. Is enhanced UV-B radiation really damaging for
plants 0 Some case studies with European Mediterranean species.
Pp. 250-263. In: Rozema, J. (ed.), Strutospheric Ozone De-
pletion; the Etlects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden.
Rousseaux, M. C., Balian,, C. L., Scope!, A. L., Searles, P. S. &
Caldwell, M. M. 1998. Solar ultrabiolet-B radiation affects plant-
insect interactions in a natural ecosystem of Tierra del Fuego
(southern Argentina). Oecologia 116: 528-535.
Rozema, J., Van de Staaij, J., Bjtirn, L. 0. &Caldwell, M. 1997. UV-
B as an environmental factor in plant life: stress and regulation.
Trends Ecol. Evol. 12: 22-28.
Searles, P. S., Flint, S. D., Diaz, S. B., Pousseaux, M. C., Ballare,
C. L. & Caldwell, M. M. 1999. Solar ultraviolet-B radiation in-
fluence on Sphargum bog and Carex fen ecosystems: first season
findings in Tierra del Fuego, Argentina. Global Change Bioi. 5:
223-228.
Stephanou, M. & Manetas. Y. 1998. Enhanced UV-B radiation in-
creases the reproductive effort in the Mediterranean shrub Cistus
creticus under field conditions. Plant Ecol. 134: 91-96.
Stephanou, M., Petropoulou, Y., Georgiou, 0. & Manetas, Y. 2000.
Enhanced UV-B radiation, flower attributes and pollinator behav-
iour in Cistus creticus: a Mediterranean field study. Plant Ecol.
147: 165-171.
Teramura, A. H. & Sullivan. J. H. 1993. Effects of UV-B radiation
on plant productivity. Pp. 37-44. In: Yamamoto, H. Y. & Smith,
C. M. (eds), Photosynthetic Responses to the Environment. Cur-
rent Topics in Plant Physiology. Vol. 8. American Society of
Plant Physiologists, Rockville.
Tevini, M. & Teramura, A. H. 1989. UV-B effects on terrestrial
plants. Photochem. Photobiol. 50: 479-487.
Tosserams, M., Pais de Sa, A. & Rosema, J. I 996. The effect of
solar UV radiation on four plant species occurring in a coastal
grassland vegetation in The Netherlands. Physiol. Plant. 97: 731-
739.
Waterman, P. G. & Mole, S. 1994. Analysis of Phenolic Plant
Metabolites. Blackwell, Oxford.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

A small UV-B and PAR sensor. developed for measurements wilhin grass canopies_
 Plant Ecology 154: 125·-133, 200 I.
125
© 200 I Kluwer Academic Publishers.

UV-B and PAR in single and mixed canopies grown under different
UV-B exclusions in the field

Gaby Deckmyn, Erwin Cayenberghs & Reinhart Ceulemans

Laboratory of Plant Ecology, University (){Antwerpen UIA, Universiteitsplein 1, 2610 Wilr(jk,
Belgium ( lc'-mail: gdeckmyn @uia.ua.ac.be)

Key words: Clover, Grass, Light penetration, Mixed canopies, UV-B, PAR

Abstract
The purpose of this study was to investigate whether differences in canopy architecture due to the investigated
species (planophile versus erectophile, single versus mixed canopies) or to UV-B effects on plant morphology, lead
to differences in UV-B and UV-B/PAR doses within canopies.
The development of a very small (I 0 mm diameter) UV-B and PAR sensor on a long 5 mm wide stick allowed
us to measure the penetration of UV-B and PAR in single and mixed canopies of the grass Dactyli.1· glome rata and
white clover, Trif'olium repens. The plants were grown in greenhouses covered with different thicknesses (3 and
5 mm) of UV-transmittant plexi ( 12 and 18% UV-B exclusion).
For clover, a planophile vegetation, radiation penetration was very low for both UV-B and PAR. UV-B penetra-
tion was much less than for PAR, resulting in low UV-B/PAR ratio's within the canopy. This is explained by the
low UV-B transmittance of the leaves ( <0.1 %) in combination with the planophile leaves.
In the grass species, both UV-B and PAR penetrated much deeper into the canopy due to the erectophile
structure. The difference between UV-B and PAR penetration was generally quite small except in very tall canopies.
The mixed species canopies showed results comparable to the clover canopies. Due to the strongly increased
grass growth in these plots, light penetration was generally much lower than in the single species cultures. The
increased growth of grass in these mixed plots could be linked to the lower UV-B/PAR dose they received.
In plots grown under the higher UV-B level there was a relative decrease in UV-B/PAR ratio within the canopy
for both species, compared to canopies from the lower UV- B greenhouses. This could not he explained hy changes
in leaf angle or biomass, but might be linked to the increase in leaf transmittance of PAR.

Introduction to know the UV-B/PAR ratio received by leaves at

UV-B induced effects on plant growth and physiology
have been intensively studied during the past decades
(for reviews, see Teramura & Sullivan 1994; Caldwell
eta!. 1998). Considerably less attention has been paid
to the study of the light environment to which the ex-
perimental plants are subjected. It has been shown that
UV-B damage is strongly related to the amount of PAR
and UV-A which the plants receive during and after
the UV-B treatment (Cen & Bornman 1990; Caldwell
et al. 1994; Teramura 1980). Apparently, the ratio UV-
B/PAR is highly correlated to the size of the effects
(Deckmyn et al. 1994 ). Therefore, it is interesting
the top and within the canopy. A number of models
and some experiments have been used to show that
UV-B and PAR behave differently in canopies (Allen
et al. 1975; Anisimov & Fukshansky 1993; Flint and
Caldwell 1998; Grant 1997). For example UV-B/PAR
ratios in forest gaps were increased in shaded. but re-
duced in sun-lit areas (Flint & Caldwell 1998). Lear
transmittance of UV-B is generally very low, much
lower than PAR transmittance (Day 1993). However,
penetration of UV-B can be relatively high in plant
canopies, depending on the leaf angles and solar el-
evation (Caldwell 1981; Dcckmyn & Tmpens 1998;
126
Grant 1991 ). So far, there has been no experimental
comparison of different types of canopies as regards
their transmittance of UV-B and PAR.
UV-B is also known to induce a number of changes
in plant morphology. These changes include reduced
leaf angles, reduced plant height, increased tillering
and many more (Barnes et al. 1996; Deckmyn &
Impens 1999; Rozema et al. 1997 ). Obviously such
changes can influence the penetration of both UV-B
and PAR in the canopy. Several of these effects have
been shown to be independent from damage to the
photosynthetic apparatus of the plants, and are inter-
preted as true photomorphogenic effects (Dumpert &
Knacker 1985; Rozema et al. 1997). Although the
pathway to these effects is still unclear, the existence
of specific UV-B photoreceptors and etiects on plant
hormones (IAA) are often postulated (Greenberg et al.
1996; Jenkins 1997). This might be of importance in
erectophile canopies such as of grass species where
the meristimatic zone is located at the bottom of the
canopy: apparently UV-B damage is not necessarily
restricted to photosynthetic tissues. In grass canopies,
where UV-B penetration is relatively high, a high UV-
B/PAR ratio can reach these mcristimatic zones and
might be responsible for photomorphogenic changes
(Dcckmyn & lmpens 1998). Clearly this possibility
deserves to be investigated.
Small changes in plant morphology can also have
important consequences when the plants are grown in
a mixed canopy (Barnes et al. 1988, 1990). Besides
the changes in competition for PAR (Barnes et al.
1996), changes of UV-B/PAR ratio might be impor-
tant in mixtures of planophile and erectophile species.
Many crcctophilc species, including the grasses, have
their mcristimatic zone at the bottom of the canopy,
while in other species it is located at the top or the
canopy. Changes in UV-B/PAR ratio might influence
the balance between such species.
The purpose of this study was to investigate
whether differences in canopy architecture due to
the investigated species (planophile versus erectophile
canopies) or to UV-B effects on plant morphology lead
to differences in UV-B and UV-8/PAR doses within
the canopy. Furthermore, mixed canopies were stud-
ied to investigate the behaviour of light within these
canopies and the possible consequences for competi-
tion of the species.
Dactylis r;lomerata was used as a model grass
because it is known to be very UV-sensitive: above-
ground biomass was reduced by 14.6% when com-
paring a 90% of ambient to a 80% of ambient UV-B
100
90
<f.
c· 80
0
·u;
(/) 70
E
(/)
c
60
f
50
40 ---iHI111iliHIIIIiilillltiitiHtlllll·llli:lll~·iHl~:11tttllltltlltHIIIIt:ltttttnttttHttt:tlltftHHttl·ltHtHIHftttHIIIIHiilii··HIIIIhltll·ltt:lttttttHI
280 300 320 340 360 380 400 420 440
Wavelength, nm
Figure /. UV-B transmittance of plexi-glass of 3 and 5 mm in the
UV-B and UV-A
treatment (Deckmyn & Tmpens 1999) and because it
has a very erectophile architecture. White clover was
used since it is a typical competitor in grasslands,
although no data on possible UV-B sensitivity were
found.
Materials and methods
Plant material and growth conditions
Seeds of Dactylis glomerata cv. Athos and Trifolium
Repens cv. Mervi (both are local commercial cultivars)
were sown in large trays (I x 0.7 m) in April 1999.
After emergence the seedlings were transplanted in
rectangular plastic boxes, 20 by 30 em, at 4-cm in-
tervals leading to 40 plants per box. For the mixed
canopies alternating species were planted every 4 em,
resulting in a perfectly even mixed canopy.
The boxes were placed in four ventilated, square
greenhouses ( 1.5 x 1.5 m), covered on all sides with
5 or 3 mm UV-B transmittant plexiglass, resulting in
UV-BsE (biologically effective UV-B, Caldwell 1971)
doses of 82% and 88% ( 18% and 12% exclusion,
respectively) compared to ambient levels (Figure I).
UV-B in the greenhouses was measured at the be-
ginning and the end of the growing season with an
Optronic OL 754 spectroradiometer. UV-B/PAR ra-
tio inside and outside the greenhouses was measured
throughout the growing season with the UV-BIPAR
sensor (see description below) several times a week.
The average reduction in UV-B and PAR was calcu-
lated from the latter, while the measurements with the
spectroradiometer were used to determine the spectral
distribution of the light in the greenhouses. Both UV-
8 and PAR transmission were slightly (2.4%) lower

at the end of the growing season. Two greenhouses of
each treatment were used. PAR was reduced by I 0.1%
in the greenhouses. Every greenhouse contained 20
boxes (6 to 8 of each kind). Due to the strong venti-
lation temperatures within the greenhouses (measured
with a thermocouple) never differed by more than 0.8°
from outside temperatures. The plants were watered
daily and cut once every 4 weeks. After each cut
fertiliser was added (N (8.33 gm- 2 ), P (6.25 gm- 2 )
and K (9.75 gm- 2 )). Ambient UV-B and PAR were
measured hourly with the Optronic OL 754 spcctrora-
diometer whenever weather permitted. Values at solar
noon on sunny days varied from 22.5 mW m- 2 to
43.3 mW m- 2 for UV-BBE while PAR varied from
1250 to 1532 {lmolm- 2 s- 1.
Measurements
For the measurements of UV-B and PAR a small,
portable UV-B/PAR ratio meter was constructed con-
sisting of a long (60 em), narrow (5 mm) stick with
aligned UV-B and quantum sensor (I 0 mm diameter)
and a reference UV-B and PAR measuring unit al-
lowing parallel measurements of radiation above and
within the canopy. The UV-B sensor was a broad-band
SiC-photodiode (Laser Components JEFC I I-DE),
with an integrated teflon diffuser (cosine corrected),
peak sensitivity at 285 nm and spectral response ac-
cording to erythema. By using a symmetric design and
a chopper-stabilised amplifier (MAX430CPA) a total
signal amplification of 4000 was reached, which al-
lowed measurements down to 0.1 °1< of ambient UV-B.
As quantumsensor we used a PAR corrected photodi-
ode EG&G VTB 8440. Measurements at ground level
under the canopies were made within the greenhouses
(so as not to disturb the canopies) at different moments
throughout the summer, under both sunny and overcast
weather conditions, at a distance of at least 20 em from
the edge of the canopy. All measurements were made
close to solar noon (between 12 h and 15 h) to avoid
differences due to solar elevation. Measurements of
extinction in function of depth in the canopy were
made once (on 21 July, at a plant age of 28 days) under
overcast sky conditions. Leaf angles (as degrees devi-
ating from horizontal) and plant height were measured
at the same time with a clinometer.
Leaf transmittance of PAR was measured once in
august on 16 random leaves from the top layer of the
canopies with the same quantum sensor. Leaf trans-
mittance of UV- B was always below the sensitivity of
127
the UV-B sensor(± 0.1% of UV-B at solar noon) and
could not be detected.
Leaf biomass (all biomass harvested) was deter-
mined every 4 weeks at harvest (on 24 June, 21
July and 18 August 1999), SLA (specific leaf area,
measured on small samples from each box) was de-
termined at the harvest in August.
Chlorophyll content was measured once in Au-
gust on 8 random, 2 em length leaf samples from
the top of the canopy of each greenhouse for grass
and on 8 random leaves from the top of the canopy
from each greenhouse for clover. Immediately after
determining the area of the leaf samples, the pig-
ment was extracted in DMF in the dark for 72 h
and spectrophotometrically determined according to
Moran ( 1982).
Data analvsis
The penetration data were analysed with split-plot
Anova (statistical programs+) to distinguish between
treatment effects (plants from the 12% versus the 18%
UV-B exclusion treatment) and differences between
the penetration of PAR and UV-B (wavelength effect).
Since UV-B and PAR penetration were always mea-
sured on the same plants, a split-plot design was used.
All other data were analysed with nested ANOVA.
Results
Transmittance through short stands (Table 1)
Short stands of Dactyl is glome rata (DG) stands grown
under 12% UV-B exclusion showed a significantly
higher penetration of both UV-B and PAR compared to
plants from the 18% UV-B exclusion treatment. UV-B
penetration was either lower or equal to PAR penetra-
tion in all DG canopies. At a canopy age of 13 days,
the interaction between UV-B and wavelength effect
(= difference between UV-B and PAR penetration)
was significant: UV-B penetration of the plants from
the 129'<) UV- B exclusion treatment was low, compared
to PAR penetration.
Trij(J/ium repens (TR) stands grown under 12%
UV-B exclusion showed a significantly lower penetra-
tion of both UV-B and PAR. UV-B penetration was
significantly lower than PAR penetration in all TR
canopies. No interaction between these effects was
found.
Canopy transmittance in TF was generally low
compared to penetration through DG stands.
128
Table I. Influence of UV-B exclusion ( 12% versus 18o/c of ambient) on canopy transmittance of UV-B and
PAR radiation through short, single and mixed, stands of Trifolium repens (TR) and Dact\·lis glomemta (DG ).
Means of I0 to 20 measurements ± SE are given. All measurments were made under clear sky conditions (PAR
= 1076-1320 11mol m- 2s- 1). The effects of the UV-B treatment (82% versus 88%) and of the difference
between UV-B and PAR penetration (A) were analysed using a split-plot ANOVA (using s+ ). Significant
effects (p<0.05) are indicated with an asterisk.

Date J\gc Species UV-B Transmittance, % Significance Interaction

days UV-B PAR UV-B )"

5 July 9 DG 18% 37.6 ± 4.6 41.8±5.4 N.S.

12% 51.9 ±4.3 60.5 ± 4.2
TR 18% 32.6 ± 4.1 42.5 ± 4.3 N.S.
12% 25.5 ± 2.2 29.2 ± 3.3
9 July 13 DG 18% 34.4 ± 3.7 34.8 ± 4.6 N.S.
12% 33.6 ± 2.9 41.0±5.2
TR 18% 16.0 ± 4.2 24.7 ± 1.8 N.S.
12% 7.2 ± 3.8 19.3 ± 2.9
Mixed 18% 3.8 ± 3.7 8.3 ± 2.7 N.S.
12'!( 7.3 ± 4.2 13.1 ±4.6

In mixed stands canopy penetration of both PAR UV-BIPAR ratios

and UV-B was very low. It was significantly reduced
in canopies from the lower UV-B (18% exclusion)
treatment.
Transmission through high stands (Table 2)
In high stands, UV-B penetration was significantly
reduced compared to PAR penetration at all heights
in the canopy for both DG and TR. DG plants from
the 12% UV-B exclusion treatment had a lower UV-
B penetration at 15 em above the ground, but not at
25 em above the ground compared to plants from the
18% UV-B exclusion treatment. There was significant
interaction between the treatment and the wavelength
(UV-B versus PAR) effect. Penetration of UV-B in the
TF canopy was so low it could not be detected (<I%).
Effect (if cloud cover (Table 3)
Cloud cover significantly reduced the penetration of
both UV-B and PAR in DG and TR canopies of dif-
ferent ages. UV-B penetration was always reduced
compared to PAR penetration, under overcast as well
as under sunny conditions. There was no significant
interaction between the effect of cloud cover and the
wavelength (both UV-B and PAR penetration were
reduced to the same extent under overcast sky).
Relative UV-B/PAR penetration ratios under the
canopies (from % penetration data) declined as the
plants grew, for both DG and TF (Figures 2 and 3).
The UV-B/PAR penetration ratio was higher in plants
grown under the lower UV-B dose. The profile of the
UV-B/PAR ratio in function of the height above the
soil in DG (Figure 4) shows that the ratio declined with
depth in the canopy.
Leaf and plant characteristics
TF plants from the 12% UV-B exclusion treatment
showed a significant increase in total biomass (Ta-
ble 6) in June and August. Biomass of DG was not
significantly affected by the UV-B level. There was
no significant change in SLA of either species due to
UV-B (Table 6). There was no significant effect of
UV-B treatment on leaf angles of either DG or TF
(Table 5). DG height was decreased under the higher
UV-B level, while TF height increased. TF had a more
planophile canopy (leaf angles from 23° to 39°) while
DG had an erectophile structure (leaf angles from 64°
to 85°). Clorophyll content was significantly reduced
in DG and TF plants from the 88% UV-B treatment,
while leaf transmittance of PAR was increased on
these plants.
Total biomass of the mixed canopies was inter-
mediate between the values of the DG and the TF
canopies. Grass growth was increased in the mixed
 129
Table 2. Influence of UV-B exclusion ( 12'/f versus 18'7c reduction from ambient) on penetration of UV-B
and PAR radiation at ditTerent heights above the ground through high stands of Trifolium repens and Dactvlis
glomerata. Means of 10 to 20 measurements ± SE are given. All rneasurrnent~ were made under overcast
sky conditions (PAR = 250-520 /I mol m- 2 s- 1) on 21/7 (canopy age 2S Jays). The effects of the UV-B
treatment (82% versus 88%) and of the JitTcrence between UV-B and PAR penetration within each treat-
ment (!c) were analysed using a split-plot AN OVA (statistical programs+). Significant effects (p<l).(J5) arc
indicated with an asterisk.

Species UV-B Depth. Penetration, % Significance Interaction

exclusion ern UV-B PAR UV-B ),

DG 18'/r () 4.2 ± 2.4 N.S. N.S.

15 4.6 ± 3.1 18.5 ± 2.9 N.S.
25 31.8 ± 2.7 49 ± 4.S N.S. :-.I.S.
12% 5 0 6.5 ± 2.4
15 0 19.6 ± 3.1
25 36.9 ± 3.7 52.7 ± 5.3
Clover 18o/r. 'i () 0.4 ± 0.6 'J.S. N.S. N.S.
15 0 7.7 ± 2.5 :-.I.S. N.S. N.S.
l2°k 5 () 0.5 ± 1.6
IS () 4.6 ± 1.8

compared to the single canopies. while clover growth
was reduced. The higher UV-B level did not affect
DG biomass, but increased TF biomass in the mixed
canopies. Leaf angles were lower compared to values
in monocultures. No significant effects of UV-B on
the clorophyll content and leaf transmittance on leaves
from the mixed canopies were detected.
Discussion
Effect (!f plant species
As expected (Jones 1992) there were important dif-
ferences in light penetration between the erectophile
(grass) and the planophile (clover) canopy. These are
partially explained by the differences in leaf angle,
though there were other differences as well (biomass,
leaf transmittance). Although the leaf angles of clover
were considerably more planophile than the grass leaf
angles, they were still quite high (25-30°), which ex-
plains why the data do not follow the predicted results
of a perfect planophile canopy.
As expected, transmittance through a planophile
canopy is low. As soon as the canopy closes, transmit-
tance is comparable to leaf transmittance, since there
arc few sunflccks (Jones 1992). As leaf transmittance
of UV-B was very low, UV-B penetration in the clover
canopy was very low too. Therefore the ratio UV-
B/PAR at the bottom of the canopy decreased rapidly
as the plants grew. In a planophile vegetation such as
clover, therefore, UV-B damage and effects arc likely
limited to the uppermost leaves of the canopy.
In an erectophile canopy a portion of the sunlight
is able to penetrate through the canopy without hit-
ting leaves even at relatively high LAI (Grant 1996;
Jones 1992). Therefore, light penetration depends not
only on leaf transmittance but also on the angle of the
incoming radiation compared to the leaf angle. This
explains why the difference between the UV-B and
the PAR penetration was smaller in our grass plots,
resulting in relatively higher UV-B/PAR doses in the
canopy compared to the clover canopies.
UV-B versus PAR
Average UV-B penetration was always lower than PAR
penetration, as has been described in several studies
before (Grant 1996; Flint & Caldwell 1998). How-
ever, in less dense canopies, it has been shown that
UV- B penetration sometimes exceeds PAR penetration
(Deckmyn & Impens 1998; Grant 1996), and in our
study under the youngest stands, the ratio UV~B/PAR
penetration was >I (though UV-B penetration was
lower). This apparent discrepancy is probably caused
hy differences in variability of UV-B and PAR in the
canopy (Grant 1996; Flint & Caldwell 1998).
Since diffuse radiation clearly penetrated less in
both the clover and the grass stands, and UV-B radi-
ation is known to be relatively more diffuse (Grant
130

Table 3. Influence of cloudiness on penetration of UV-B and PAR radiation through high stands of Trifolium repens (TR) and Dactyli.\·
glome rata (DG) grown under diiTerenl UV-B exclusion levels ( 12% ve"us 18% reduction from ambient). Measurements were made on days
when sunny and overcast conditions followed each other. Ambient PAR is also given. Means of I0 to 20 measurements ± SE are given. The
difference between UV-B and PAR (A) and of the cloud cover (sunny versus overcast) were analysed using a split plot AN OVA (statistical
programs+). Significant effects (p<0.05) are indicated with an asterisk.

Date Age, Species UV-B Penetration, % PAR, significance Tnteraction

days exclusion UV-B PAR 11mol m ""'S !c cloud cover

15 June 21 DG 12% 11.2 ± 1.7 21.2 ± 3.1 1208 N.S.

1.4 ± 0.6 6.7 ± 1.1 429
TR 18% 53.3 ± 4.2 63.2 ± 2.3 1252 N.S.
50.9 ± 8.8 71.5 ± 2.4 520
22 June 26 DG 12% 4.1 ±2.6 12.4 ± 2.5 872 N.S.
1.2 ±0.8 9.2± 1.4 259
2 August 14 DG 18o/c 42.2 ± 0.9 53.4 ± 3.6 923 N.S.
30.5 ± 2.3 42.3 ± 4.1 295
TR 18% 9.5 ± 1.2 15.2 ± 3.1 1081 N.S.
2.1 ±0.7 11.7 ± 2.2 277

Table 4. Influence ofUV-B exclusion (18% versus 12%) on plant height and av-
erage leaf angle of Trifolium repens (TR) and Dactylis glomerata (DG) grown
in single and mixed stands. Means of 10 to 20 measurements ± SE arc given.
Significantly different means (p<0.05, nested ANOVA, statistical programs+)
are followed by different letters.

Date Age, Species UV-B Height, Leaf angle,

days exclusion em

15 June 21 DG 12% 16.9 ± 2.1 84.6 ± 1.9

TR 18% 10.4 ± 1.8 31.2 ± 2.4
22 June 26 DG 12'11 34.9 ± 2.8 73.2 ± 2.7
5 July 9 DG 18% 17.6 ± 1.8 a 80.7 ± 1.2 a
12% 14.1 ± 1.2 b 80.4 ± 1.1 a
TR 18% 12.3 ± 1.6 a 23.7 ± 2.3 a
12% 14.8 ± 2.3 a 25.2 ± 3.4 a
9 July 13 DG 18% 25.1 ± 1.6a 73.4 ± 1.4 a
12% 20.9 ± 1.1 b 78.6 ± 2.1 a
TR 18% 14.0 ± 2.9 a 35.8 ± 4.2 a
12% 17.2 ± 1.3 a 31.7 ± 4.6 a
Mixed DG 18% 31.2 ± 3.2 a 67.9 ± 1.9 a
12°/c 24.8 ± 2.1 b 67.7 ± 2.7 a
Mixed TR 18% 16.8 ± 1.4 a 41.6 ± 2.8 a
12% 18.9 ± 1.8 a 44.9 ± 2.3 a
21 July 28 DG 18% 36.5 ± 1.2 a 78.9 ± 1.8 a
12'11 39.0 ± 2.4 a 79.7 ± 1.3 a
TR 18% 19.9 ± 1.5 a 27.7 ± 1.4 a
12% 24.4 ± 2.2 b 29.2 ± 2.3 a
2 August DG 18% 24.9 ± 1.7 74.6 ± 2.1
TR 18'11 15.8 ± 1.2 39.6 ± 2.9
 131
Table 5. Lear tran,mittancc of PAR and total chlorophyll content or
leaves of Trifolium repem (TR) and Dae1ylis glomera/a (DG) from uni-
form and mixed stands grown under 82 and 88% of ambient UV-B.
Means of 16 measurements arc given. Data were analysed with nested
ANOYA (statistical programs+) and significant differences (p<0.05J
arc indicated by different letters following the data.

Species UV-B Stand Lear transmillancc. Chlorophyll,

flg 111 '
~

DG 820( Uniform 8.6 ± 0.6 a 41.1 ± 2.4 a

88'/, Uniform l1.7±1.4h 37.8 ± 2.8 h
82'1< Mixed 9.2±0.7a 43.0 ± 4.7 a
88°/r Mixed 9.0 ± 0.6 a 46.9 ± 3.8 a
TR 82'X Uniform 4.5 ± 0.2 a 45.7 ± 1.3 a
88'k Uniform 8.3 ± 0.7 b 42.1 ± 1.5 b
82'k Mixed 6 ..1 ± 0.5 ab 44.9 ± 2.3 a
88r!, Mixed 7.2±0.7h 44.7 ±0.9 a

Table 6. Aboveground biomass and SLA of Triji1lium repms ITR) and Dactylis glomeratu (DG)
from uniform and mixed stands grown under 18% and l2'k exclusion of ambient UV-B. Means of
16 measurements arc given. Data were analysed with nested ANOVA (statistical programs+) and
significant differences ( p <0.05) are indicated by ditlerent letters following the data.

Species UV-B June July August

exclusion DW,g DW. g DW.g SLA. cm 2 g~l

DG 18"/r 9.91 ± 0.8a 17.1 ±0.9a 8.2 ± 1.2a 395 ± 14a

12'/c 11.3 ± 0.7a 15.5 ± 0.7a 6.2 ± 0.4a 386±21a
TR 189( 5.4 ± 0.8a 21.4±1.2a 15.6 ± 0.9a 337 ± l7a
12'/r X.6 ± 0.5b 23.X ± 1.5a 17.4±0.7b 339±lla
DG mixed 18'/c ll.6±0.7a
12Cf, 12.3 ± 0.9a
TR mixed 189( 6.0 ± 0.7a
12'/r 8.3 ± 0.9b

1996; Deckmyn & Impcns 1998), this increased
the difference between UV-B and PAR penetration
through the canopy (next to the important difference
in leaf transmittance).
Therefore, even in the gras:;, stand the UV-B/PAR
ratio decreased as the stand grew (Figure 2) or with
increasing depth in the canopy (Figure 3), although
to a lesser extent than in clover. However, in young
stands some leaves received higher UV-B/PAR doses
than ambient solar radiation.
The UV-B levels reaching the meristematic zones
in the grass canopy could be high enough to induce
damage in young stands, and both in young and in
older stands the possibility of photomorphogenic ef-
fects cannot be dismissed, since there are no data on
critical levels of UV-B for such effects. Furthermore,
the exact location of a putative UV-B photoreceptor is
unknown.
Mixed canopies
Although at the harvest, total biomass of the mixed
canopies was intermediate between the values of DG
and TR, at the early stages of growth, the vigourous
DG growth probably resulted in high LAI and thus
reduced canopy transmittance. Leaf angles were lower
in the mixed canopy, which explains why the UV-
B/PAR ratio within the canopy was low, as in a
planophile canopy.
This can be important for plant growth. The meris-
tematic zone of grass is at the bottom of the canopy.
In the single stands, this zone receives relatively high
132

1.2 0.7
[ l 18% 12% T
0.6 n 1s% 12% *
0
,g 0.5
~ 0.8 ~
a:: a:: 0.4
0: 0.6 <(
Q_
CD CD 0.3
:>
::J
0.4 :>::J 0.2
0.2 1 0.1

0 0 +----==----__,__JL.._
5d 9d 14d 28d 5 15 25
Canopy age, days Height in canopy, em
Figure 2. Ratio of UV-B/PAR penetration (in % of incoming ra- Figure 4. Ratio of UV-B/PAR penetration at different heights (in
diation at ground level. SE indicated) in Trifolium repens stands of % of incoming radiation at ground level. SE indicated) in Dactvlis
different ages, grown under 12 and 18% exclusion of ambient UY-B. !;lomerata standsgrown under 12 and 18% exclusion from ambi-
Significantly different ratios (nested ANOYA, pdl.05, statistical ent UY-B. Significantly different ratios (nested ANOYA,p < 0.05,
program s+) are indicated with different asterisks. statistical programs+) arc indicated with different asterisks.

1.4 was lower, though not significantly so. This clearly

1.2 explains the increased canopy transmittance through
0
:p the canopies of the 12% UV-8 exclusion treatment.
~
Clover growth was increased by UV-B. This ex-
~ 0.8 plains the higher absorption of the TR plots from the
Q_

~ 0.6 higher UV-8 treatment. Presumably, therefore, dur-

> ing the experiment there was an increasing difference
::::> 0.4
0.2
0
5d 9d 14d
r28d
Canopy age, days
Figure 3. Ratio of UV-B/PAR penetration (in % of incoming radi-
ation at ground level, SE indicated) in Dactylis glomera/a stands
of different ages, grown under 12 and 18% exclusion from ambi-
ent UY-B. Significantly different ratios (nested ANOVA, p < O.OS,
statistical program s+) are indicated with different asterisks.
UV-B doses during the first weeks of regrowth (see
above). In the mixed stand, the clover leaves quickly
shade the lowest canopy layer. This might partially
explain the increased growth of grass in the mixed
canopies. Since a ll plants received optimal N monthly,
the N- fixation of clover should not have influenced
grass growth significantly.
UV-B induced changes
Although in a previous study, leaf angles of Dactylis
glomerata were influenced by UV-B (unpublished
data), in this study no significant differences were de-
tected. The DG plants grown under the higher UV-8
level were always shorter, and biomass at the harvest
in leaf area between the treatments, hut this was not
measured.
The increase in leaf PAR transmittance was linked
to a significant decrease in chlorophyll content in both
species. Although increased leaf thickness is often
found in response to UV-B (Flint et al. 1985), no effect
was found in this experiment. Although it is difficult
to compare the results from the different treatments,
since growth was influenced as well, it can be seen
that the relative UV-8/PAR ratio tended to be lower in
plants grown under higher UV-B (Figures 2-4). Since
this ratio decreased with growth, the reverse would
be expected in the smaller plants from the 12% UV-
B exclusion stands. This change in UV-8/PAR ratio
can be explained partially by the increased leaf trans-
mittance of PAR. However, UV-8 penetration appears
to be relatively low as well. This could be the result of
changes in canopy architecture such as tillering, leaf
area distribution or others that were not analysed here.
To investigate this, however, modelling of the light cli-
mate in the canopy would be necessary, using a model
that encompasses morphological parameters (Sinoquet
et al. 2000).
The small change in UV-B/PAR ratio found in this
experiment might indicate a protective response in the

plants: by small changes in canopy architecture pen-
etration of UV-B could be reduced, relative to PAR.
Whether this really is the case cannot be proved from
these results and needs further investigation.
Acknowledgements
First of all we wish to thank our marvellous tech-
nical wizard, Fred Kockelbergh, who developed the
UV/PAR sensor on which this research was based. G.
Deckmyn is also indebted to the FWO-Vlaanderen, for
her position as postdoctoral researcher.
References
Allen, L. H., Gausman, H. W. & Allen, W. A. 1975. Solar ultraviolet
radiation in terrestrial plant communities. J. Environ. Qual. 4:
285-294.
Anisimov, 0. & Fukshansky, L. 1993. Light-vegetation interac-
tion: a new stochastic approach for description and classification.
Agric. Forest Meteorol. 66: 93-110.
Barnes, P. W., Balian~. C. L. & Caldwell, M. M. 1996. Photomor-
phogenic effects of UV-B radiation on plants: consequences for
light competition. J. Plant Physiol. 148: 15-20.
Caldwell, M. M. 197l.Solar ultraviolet radiation and the growth and
development of higher plants. Pp. 131-177. In: Giese. A. C. (ed.),
Photophysiology, Vol. 6, Academic Press, New York.
Caldwell, M. M. 1981. Plant response to solar ultraviolet-B radi-
ation. Pp. 170-186. In: Lange. 0. L., Nobel, P. S., Osmond,
S. B. & Ziegler, H. (eds). Physiological Plant Ecology 12A.
Springer-Verlag, Berlin.
Caldwell, M. M., Flint, S.D. & Searles, P. S. 1994. Spectral balance
and UV-B sensitivity of soybean: a field experiment. Plant, Cell
Environ. 17: 267-276.
Caldwell, M. M. 1998. Effects of increased solar ultraviolet ra-
diation on terrestrial ecosystems. J. Photochem. Photobiol. 46:
40-52.
Cen, Y P. & Bornman, J. F. 1990. The response of bean plants to
UV-B radiation under different irradiances of background visible
light. J. Exp. Bot. 41: 1489-1495.
Day, T. A. 1993. Relating UV-B screening effectiveness to
absorbing-compound concentration and anatomical characteris-
tics in a diverse group of plants. Oecologia 95: 542-550.
Deckmyn G. & Impens I. 1998. UV-B and PAR in a grass (Latium
perenne L.) canopy. Plant Ecol. 137: 13-19.
133
Deckmyn G. & Impens I. 1999. Seasonal responses of six poaceae
to differential levels of solar UV-B radiation. Environ. Exp. Bot.
41: 177-184.
Deckmyn, G., Martens, M. & Impens I. 1994. The importance of the
ratio UV-B photosynthetic active radiation (PAR) during leaf de-
velopment as determining factor of plant sensitivity to increased
UV-B irradiance: effects on growth. gas-exchange and pigmenta-
tion of bean plants (Phaseolus vulgaris L. ). Plant, Cell Environ.
17: 295-301.
Dumpert. K. & Knacker, T. 1985. A comparison of the effects of
enhanced UV-B radiation on crop plants exposed to greenhouse
and field conditions. Biochemy Physiol. Pflanzen 180: 599-612.
Flint, S. D. & Caldwell, M. M. 1998. Solar UV-B and visible radia-
tion in tropical forest gaps: measurements partitioning direct and
diffuse radiation. Global Change Bioi. 4: 863-870.
Flint. S. D., Jordan. P. W. & Caldwell, M. M. 1985. Plant protective
response to enhanced UV-B radiation under field conditions: leaf
optical properties and photosynthesis. Photochem. Photobiol. 41:
95-99.
Grant, R. H. 1991. Agroclimatology and modeling. Ultraviolet and
photosynthetically active bands: plane surface irradiance at corn
canopy base. Agron. J. 83: 391-396.
Grant, R. H. 1997. Partitioning of biologically active radiation in
plant canopies. Int. J. Biometeorol. 40: 26-40.
Greenberg, B. M .. Wilson, M. 1., Gerhardt, K. E. & Wilson, K. E.
1996. Morphological and physiological responses of Brassica
napus to ultraviolet-B radiation: photomodification of ribulose-
1.5-bisphosphate carboxylase/oxygenase and potential acclima-
tion processes. J. Plant Physiol. 148: 78-85.
Jenkins, G. I. 1997. UV-B and blue light signal transduction in
Arabidopsis. Plant Cell Environ. 20: 733-778.
Jones, H. G. 1992. Plants and Microclimate. Cambridge University
Press, Cambridge.
Moran, R. 1982. Formulae for the determination of chlorophyllous
pigments extracted with N,N-dimethylformamide. Plant Physiol.
69: 413--416.
Rozema, J., Van de Staaij, J., Bjorn. L. 0. & Caldwell. M.
1997. UV-B as an environmental factor in plant life: stress and
regulation. Trees 12: 22-28.
Sinoquet, H., Rakocevic M. & Varlet-Grancher C. 2000, Com-
parison of models for daily light partitioning in multispecies
canopies. Agric. Forest Meteorol. 2774: 1-13.
Teramura, A. H. 1980. Effects ofultraviolet-B radiation on soybean.
I. Importance of photosynthetically active radiation in evaluating
ultraviolet-B irradiance effects on soybean and wheat growth.
Physiol. Plant. 49: 333-339.
Teramura, A. H. & Sullivan, J. H. 1994. Effects of UV-B radiation
on photosynthesis and growth of terrestrial plants. Photosynth.
Res. 39: 463--473.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

M orphological changes in response to enhanced UV- B radiation under low ('PAR' ) and near ambient (' >> PAR ' ) PAR
.
levels. (Photograph hy B . B Meijkamp)
 Plant Ecology 154: 137-146,2001. 137
© 2001 Kluwn Academic Publishers.

The response of Viciafaba to enhanced UV-B radiation under low and

near ambient PAR levels

B. B. Meijkamp, G. Doodeman & J. Rozema

Institute of Ecological Science, Department of Systems Ecology, De Boelelaan 1087, 1081 HV Ams/erdwn,
The Netherlands

Key words: Faba bean, Light quality, Morphology, Photomorphogenic effects, UV-B-absorbing compounds

Abstract
The effects of enhanced UV-B are often overestimated in greenhouse studies due to low levels of photosynthetically
active radiation (PAR). For this reason, we studied effects of enhanced UV-B ( 12 kJ m- 2 d- 1) at low and near
ambient PAR levels on young vegetative plants of Vicia faba, in the greenhouse. It was hypothesized that near
ambient PAR levels could reduce the negative UV-B effects on growth, due to higher amounts of UV-B absorbing
compounds in the leaves and to morphological changes attenuating UV-B damage.
We found that effects of enhanced UV-B on the growth were not negative. We found an increase in biomass in
response to enhanced UV-B at low and near ambient PAR levels. The increase in biomass was related to increased
branching, which leads to a higher interception of PAR. Enhanced irradiance of both PAR and UV-B had similar
photomorphogenic effects: thicker and smaller leaves and reduced plant height and internode length. Moreover,
the concentration of UV- B absorbing compounds was increased. We conclude that in this study effects of enhanced
UV-B were mainly photomorphogenic effects, which were also induced by radiation in the PAR region.

Abbreviations: PAR: photosynthetic active radiation, RGR: relative growth rate, SLA: specific leaf area, LWR: leaf
weight ratio, LAR: leaf area ratio, NAR: net assimilation rate, IAA: indole-3-acetic acid

Introduction damage by the formation of DNA dimers (Taylor et al.

During the last three decades, a decline in
stratospheric ozone amounts has occurred. This
decrease is ascribed to anthropogenically emitted
CFC's (chlorofluorocarbons) and other ozone deplet-
ing chemicals reaching the stratosphere (Herman et al.
1996; Madronich et al. 199R). Also greenhouse gases
which cause cooling of the stratospheric ozone layer
above the arctic, appear to be an indirect factor lead-
ing to ozone depletion (Shindell et al. 1998). As a
result of this decline in stratospheric ozone concen-
tration, plants receive increasing solar UV-B radiation
levels (Caldwell & Flint 1994; Herman et al. 1996;
Madronich et al. 1998).
Enhanced UV-B fluence rates can cause damaging
effects in plants (Dumpert & Knacker 1985; Runeck-
les & Krupa 1994; Kim et al. 1998), for instance DNA
1997). On the plant level, a reduced biomass pro-
duction may occur. The growth reduction can be the
result of a changed allocation of biomass, increasing
amounts of secondary compounds or morphological
alterations which lead to lower photosynthetic produc-
tivity (Teramura et al. 1980; Caldwell et al. 19R9;
Fiscus & Booker 1995; Allen et al. 1998). Responses
to UV-B include morphological alterations such as re-
duced leaf size, thicker leaves (Adamse & Britz 1992),
reduced hypocoty I length (Kim et al. 1998) and curl-
ing and bronzing of leaves (Teramura et al. 1980;
Visser et al. 1997a; Allen et al. 1998). These effects
are more pronounced at lower PAR levels (Teramura
et al. 1980; Warner & Caldwell 1983; Mirecki &
Teramura 1984). Morphological UV-B effects could
either be interpreted as damaging effects when they
are caused by photodcstructivc processes or as pho-
138

tomorphogenic responses mediated via photoreceptors
(Barnes et al. 1996; Kim et al. 1998).
Photomorphogenic UV-B effects are observed at
low UV-B doses, causing no damage (Tevini & Ter-
amura 1989; Kim et al. 1998). Another UV-B re-
sponse, mediated by a photoreceptor is the increase
of UV-B absorbing compounds, such as flavonoids
in the leaves, particularly in the epidermis (McClure
1975; Caldwell et al. 1989; Runeckles & Krupa 1994;
Meijkamp et al. 1999 and references therein).
Some photomorphogenic effects and the produc-
tion of flavonoids give mesophyll cells protection
against UV-B radiation and thus have a role in adap-
tation to UV-B radiation (Teramura 1986; Balian~
et al. 1992; Barnes et al. 1996). When leaves be-
come thicker, UV-B as well as PAR are absorbed in
higher amounts in the leaves implying that leaf tissue
is exposed to reduced levels of both UV-B and PAR
(Adamse & Britz 1992; Balian~ et al. 1992). Also the
increased amounts of flavonoids which are produced
in response to UV-B may be favourable in a UV-B
irradiated environment. Flavonoids absorb specifically
in the UV region and not in the PAR region (e.g.,
McClure 1975; Balian~ et al. 1992). At higher PAR
levels, the interaction between UV-B and PAR effects
may lead to compensation of negative UV-B effects
(Warner & Caldwell 1983; Cen & Bornman 1990;
Adamse & Britz 1992; Balian~ et al. 1992). Firstly,
radiation with a wavelength range between 300 and
500 nm is required for the activity of the enzyme DNA
photolyase, repairing DNA dimers induced by UV-B
(Jordan 1993; Taylor et al. 1997). Secondly, some UV-
B effects such as reduced plant height, thicker leaves
and enhanced concentrations of phenolics, which have
a protective function against UV-B, are also observed
in response to enhanced PAR levels (Teramura 1980;
Cen & Bornman 1990; Balian~ et al. 1992). In most
cases, PAR levels in the greenhouse and in climate
chambers are lower than outside. Also, the light spec-
trum inside differs from the spectral composition of
the light outside. Thus, when results from greenhouse
experiments are extrapolated to the field situation, this
may lead to an overestimation of UV-B effects on
growth in the field (Kramer et al. 1992; Barnes et al.
1996; Rozema et al. 1997; Caldwell et al. 1998).
In the greenhouse and the climate room where
environmental conditions can be standardized, we in-
vestigated the UV-B effects of Vicia faba with low
PAR (250 11mol m- 2 s- 1) and near ambient PAR lev-
els (600 11mol m- 2 s- 1). We investigated whether
greenhouse experiments overestimate the growth re-
duction by enhanced UV-B radiation due to low PAR
levels. We measured growth, morphology and ac-
cumulation of UV-B absorbing compounds. It was
hypothesized that enhanced UV-B radiation, in com-
bination with near ambient PAR levels, should lead to
a less pronounced growth reduction, due to enhanced
flavonoid production and an altered morphology (cf.,
Flint et al. 1985; Barnes et al. 1990; Visser et al.1997a,
b; Tosserams et al. 2000).
Material and methods
Growth conditions
The experiment was conducted in a ventilated green-
house compartment with a 14 h photoperiod and
day/night temperature regime of 22-26 °C I 14-
16 oc. Relative humidity varied between 50% and
95% depending on temperature. The experiment was
conducted between September 23, and October 14,
1996. Daily global irradiance at Schiphol Airport, lo-
cated about 10 km from the greenhouse, is shown in
Figure 1.
Experimental setup and light treatments
Two Vicia faba L. (cv Minica) seeds were sown per
pot (2.6 I) filled with a mixture of commercial potting
soil (Jongkind BV, Aalsmeer, NL), 0.1 1of potting soil
inoculated with Rhizobium bacteria, and 3 g I- 1 of a
controlled release fertilizer (Osmocote 13:13:13:3:2,
N:P:K:Mg:Fe; Grace Sierra Int., Heerlen, NL). Ten
days after sowing, thinning to one plant per pot took
place.
Four treatments were used: minus (-UV-B) and
plus UV-B (+UV-B) (Biologically Effective dose, UV-
BsE: 0 and 12 kJ m- 2 d- 1, respectively) in combi-
nation with low (at least 250 11mol m- 2 s- 1) (LL)
and near ambient (at least 600 11mol m- 2 s- 1) (AL)
additional PAR, indicated as -UV-B LL, -UV-B AL,
+UV-B LL and +UV-B AL, respectively. Spectra of
the PAR and UV-B treatments were measured with
a double-monochromatorspectroradiometer (Optronic
Model OL 752) at noon in the greenhouse (Figure 2).
Plants were divided at random over the treatments.
The treatments started 14 days after sowing. Plants
were rotated twice a week within the treatment plot.
The treatment plots were rotated within the green-
house once a week to avoid site effects. Per treatment,
one experimental plot with 12 plants was used. Twenty
 139

this purpose, a calibration curve of the UV-X me-

ter and the biologically active UV-B dose (UY-BaE)
n was constructed. The UY-BaE dose was calculated by
~~
multiplying UV-B spectra measured with a spectrora-
2
diometer and weighting factors from the generalized
~ 800

i>, 600
plant action spectrum (Caldwell 1971 ), normalized at
300 nm. The UV-BHE dose of 12 kJ m- 2 d- 1 sim-
il
ulated 35% ozone reduction at clear sky on 21 June
"a-g 400
in Amsterdam according to the model of Green et a!.
200
(1980).

9/23/96 9/30/96 10/7/96 10/14/96 Growth analvsis, mo11Jiw/og_v and UV-B absorbing
Date
compounds
Figure/. Glohal daily irradiance at Schiphol Airport. located ahoul
10 km from the greenhouse, during the experiment. Plants were harvested after twenty days of the UV-
B/ PAR treatment. The number of adventitious shoots
and leaves per shoot were counted, and shoot length
days after starting the treatments, the plants were har-
and leaf area (Licor 3100 area meter, Li-Cor Inc.,
vested for growth and analysis of UV-B absorbing
Lincoln, USA) per shoot were measured. For deter-
compounds. _0
mining biomass accumulation, fresh weight of leaves
The average daily global irradiance was 736 J em ~
and stems of main and adventitious shoots were mea-
d- 1 (Figure I). The near ambient PAR treatment had
sured separately. Dry weight of all stems and leaves
a minimal flux of 600 f.1mol m- 2 s- 1 during 14 h per
were measured (48 h, 70 °C). With the fresh weight to
day (intensity of the lamps plus the minimal natural
dry weight ratio, dry weight of leaves and stems per
light intensity). This led to a daily irradiance dose
shoot was calculated. Dry weight of roots of the plants
of 673 J cm- 2 d- 1. We converted the PAR flux to
was determined after rinsing with water and drying at
daily irradiance using a PAR spectrum measured in
70 oc for 48 h.
the greenhouse (Figure 2). For the low PAR level
Internode length was calculated by dividing shoot
(250 J.Lmol m- 2 s- 1) we calculated a daily dose of
length by number of leaves. Leaf area per leaf was
280 J cm- 2 d- 1.
calculated by dividing leaf area of the whole shoot by
The PAR and UV-B doses were realized at canopy
number of leaves. Specific leaf area (SLA) per shoot
height and adjusted twice a week by adapting lamp
is the ratio between leaf area and dry weight of the
levels above the canopy. The PAR dose was measured
leaves per shoot. Leaf weight ratio (LWR) is the ratio
with a Li-185b quantum sensor (LI-COR Inc .. Lin-
of biomass between leaf and whole plant.
coln, NA, USA). Additional PAR was supplied 14 h
For the analysis of UV-B absorbing compounds,
a day by 400 W Philips HPI-T lamps. For the low
two leaf discs ( 1 cm 2 ) of young, just unfolded leaves
PAR (LL) treatment, one lamp was used per plot.
of the main shoot were sampled. After measuring fresh
For the near ambient PAR CAL) treatment, two HPT-1
weight of the samples, they were frozen in liquid nitro-
lamps were used per plot of which the lamp hold-
gen and stored at -20 oc for 14 days. UV-B absorbing
ers were covered inside with aluminum foiL Plants
pigments were extracted with 5 ml of a mixture of
were exposed to UV-B by irradiation with two Philips
methanol: water: hydrochloric acid (79:20: 1) at 90 oc
40W/12 lamps per plot, switched on from 10.00-
for 90 min. The absorption spectrum of the extract was
16.00 h, which were wrapped in cellulose acetate foil
measured between 280 to 320 nm. Integration of this
(0. 1 mm, Tam boer & Co. Chemie B.V., Haarlem,
spectrum in the UV-B region was used as a relative
NL). This foil transmits the radiation above 290 nm
concentration of UV-B ahsorhing compounds in the
(Figure 2). For the control UV-B treatment (minus
extracts.
UV-B), UV lamps were wrapped in polyester foil (my-
lar, 0.13 mm) which excludes UV-B radiation below
Statistics
313 nm (Figure 2). Mylar foil was renewed once a
week and cellulose acetate foil twice a week. The UV- Data were statistically tested with SPSS software
B dose was adjusted with a portable UV-X radiometer (SPSS Inc. version 8.0). Normality was tested with
with a UV-X 31 sensor (San Gabriel. CA, USA). For Shapiro-Wilk and homogeneity of variance was tested
140

6 .-------------------------------------------- --------------.
0.07 , - - - - - - - - - - - - ,
8: UV-B treatments
A: PAR treatments
0.06
5 ~ E 0.05
2-0.04
Q)
(.)
~0.03
4 '0
~0.02
~
E 0.01
3
0.00 1---'----~...;_--~----~
Q)
3
280 290 300 310 320 330 340
(.)
c
ca wavelength (nm)
'0
~
2

320 400 480 560 640 720 800

wavelength (nm)

Figure 2. Examples of spectra taken around noon, measured with a spectroradiometer. (A) Spectra of the low PAR (LL) (250 11mol m- 2 s- 1,
equivalent to 280 J cm-2 day- 1 (dotted line) and near ambient PAR (AL) (600 11mol m-2 s- 1 equivalent to 673 J cm- 2 day- 1) (solid line).
(B) Spectra of minus UV-B (-UV-B) (dotted line) and plus UV-B, 12 kJ UV-BsE m- 2 day- 1 (+UV-B) (solid line).

with Levene's test. Data of dry weight of the main
shoot were transformed to their natural logarithm,
to obtain homogeneity of variance. UV-B and PAR
effects and interaction of UV-B * PAR was tested
with two-way ANOVA followed by a LSD post-hoc
test on the four treatments. Data of the number of
shoots and number of leaves were not normally distrib-
uted and were tested non-parametrically by multiple
comparisons with the Kruskal Wallis test (Zar 1984 ).
Results
Biomass accumulation
of the main shoot was found, whereas at near am-
bient PAR levels biomass accumulation of the main
shoot decreased (p<0.001). Biomass accumulation of
the first adventitious shoot increased in response to
enhanced UV-B (p<0.001). A second and third ad-
ventitious shoot was developed in plants exposed to
enhanced UV-B (Figure 3, Table 1).
We found an increased allocation of biomass to the
roots in response to enhanced UV-B (p<O.OOI), re-
flected as a decrease in shoot to root ratio (Table 2).
The leaf weight ratio (LWR) increased in response to
near ambient PAR levels (p = 0.002) but decreased
in response to enhanced UV-B radiation (p <0.00 I)
(Table 2).

The total dry biomass of plants exposed to enhanced Plant architecture

UV-B was higher than that of plants without expo-
sure to UV-B (p<0.001 ), whereas there was no effect
of PAR (Figure 3). A similar response was observed
for the biomass of the roots and total above-ground
dry weight. No UV-B effect on biomass accumulation
More branching occurred in response to UV-B, while
there was no PAR effect on branching (Table I). The
number of leaves per shoot can be seen as a measure
for the developmental stage of the shoot. No differ-
 141
Table I. Number of shoots and number of leaves of main and first adventitious shoot of Viciu j(tba.
in response to UV-B radiation under low and near ambient PAR levels. Data are means ± SE with
11 = 12. Different letters in columns indicate significant difference between treatments ( p <().05. LSD
test). Treatments are minus UV-B (-UV-BJ and pit" UV-B: 12 kJ m- 2 J 1 t+ UV-B) in combination
with low PAR. 250 l'molm 2 s -I tLLJ or near ambient PAR. 600 I' mol m- 2 s· 1 (ALJ.

Leaf number of first

Treatment Number of shoots Leaf number of main shoot adventitious shoot

- UV-B LL 1.50±0.19 a 14.3±0.26 a 1.83±0.87 a

- UV-B AL 1.92±0.08 a 14.8±0.25 a 5.75±0.93 ab
+ UV-B LL 3.08±0.23 b 14.6±0.36 a 8.33±0.58 b
+ UV-13 AL H2±0.19b 14.7±0.33 a 8.42±0.57 b

Table 2. Shoot to root ratio (S/R) and Leaf weight

ratio (LWR) of the total plant of Vici" .fitba. in re-
sponse to UV-B radiation under low and near am-
bient PAR levels. Treatments according to Table I.
Data are means ± SE with n = 12. Different let-
ters in columns indicate significant difference between
treatments (p<0.05, LSD test).
S/R LWR
- UV-B LL 3.0±0.2 a 0.46±0.02 a
- UV-B AL 3.1±0.1 a 050±0.01 b
+ UV-B LL 2.4±0.3 b 0.42±0.01 c
+ UV-B AL 2.2±0.1 b 044±0.01 ac
ence in the number of leaves of the main shoot was
found. Thus, there were no differences in developmen-
tal stage of the main shoot. The number of leaves of
the first adventitious shoot increased in response to en-
hanced UV-B, which was comparable to the increased
branching.
The differences in length of the adventitious shoots
could be caused by different developmental stages
(less leaves) or by less elongation (shorter internodes).
To estimate elongation of the internodes we calculated
internode length. Internode length of the main shoot
was reduced by enhanced UV-B radiation and PAR
(p<O.OO I) (Figure 4). There was an interaction effect
of the UV-B and PAR treatments on the main shoot
(p = 0.003) and a significant UV-B effect for the
internode length of the first adventitious shoot (p =
0.034). Plant height also decreased by high levels of
PAR and UV-B radiation (data not shown).
Leaf morphology
Leaf size of the main shoot became smaller in response
to near ambient PAR (p<O.OOI) and UV-B radiation
(p = 0.007) (Figure 5). The combination of near am-
bient PAR and enhanced UV-B was not additive. Leaf
area of the first adventitious shoot was lower because
of the younger developmental stage of the shoot. There
was no treatment etlect (UV-B or PAR) on the leaf size
of the first adventitious shoot.
The specific leaf area (SLA) per shoot was calcu-
lated to estimate changes in leaf thickness. The SLA
decreased in response to UV-B (p<O.OOl) and near
ambient PAR ( p<O.OO I). Thus, leaves became thicker
(Figure 6). The combination of UV-B and near am-
bient PAR was not additive. There were no effects of
UV-B and PAR on the SLA of the first adventitious
shoot.
Pigments
Higher amounts of UV-B absorbing compounds were
synthesized in leaves in response to UV-B (p<O.OO 1)
and PAR (p<O.OO 1) (Figure 7). The accumulation of
UV-B absorbing compounds in response to ncar am-
bient PAR and enhanced UV-B radiation was additive
(Figure 7).
Discussion
Growth
In contrast to our expectations, growth, measured
as biomass accumulation, increased in response to
enhanced UV-B radiation at both PAR levels (Fig-
ure 3 ). The increased biomass accumulation of the
142

.l!l IS::SJ third adventitious shoot

0
0
.c IZ2J second adventitious shoot
"'3
&S.S.l first adventitious shoot

2
~ mainshoot

~ DWroots

- UV-B LL - UV-B AL + UV-B LL + UV-B AL

Figure 3. Dry weight of the roots and shoots of Vicia faba in response to UV-B radiation at low and near ambient PAR levels. Treatments
according to Table 1. Data arc means ± SE per plant part (n = 12). Different letters above and within bars indicate significant difference
between treatments of dry weight of total plant and main shoot. respectively (p<0.05, LSD test).

whole plant was caused by increased growth of adven- ~main shoot

a
titious shoots (Figure 3) bearing increased numbers of ~ first adventitious shoot

leaves (Table I) so that more PAR could be captured b

and used for growth (Schmitt 1997). Furthermore, in-
creased branching has led to relatively more young
leaves with a higher photosynthetic rate. Some other
experiments with Vicia faba showed no significant
difference in biomass accumulation in response to
UV-B, although morphology was altered (e.g. Barnes
et al. 1990; Visser et al. 1997b). However, Tosser-
ams et al. (200 I) found a growth reduction of the
same cultivar (Minica) of Vicia faba in response to
enhanced UV-B, at a PAR level of250 flmol m- 2 s- 1
(day 24, 380 fLmol C02 mol-l, 0 and 10.6 kJ UV-
BsE m- 2 day- 1).
An explanation for these contrasting results may
be differences in PAR intensity at the plant level at
the time of the year in the greenhouse. The aver-
age global irradiance level outside during the UV-B
treatment period of Tosserams et a!. (2001) (end of
December 1993-January 1994) was 131 J cm- 2 day- 1
(KNMI, 1993, 1994). During our experimental pe-
riod (end of September/ October 1996), this dose was
736 J cm- 2 day- 1 (KNMI, 1996), As the additional
PAR levels were equal in both experiments, the to-
tal PAR level during the experiment of Tosserams
c
d c d
d
d
· UV·B LL · UV·B AL + UV·B LL + UV·B AL
Figure 4. Internode length of Vicia faba in response to UV-B radi-
ation at low and near ambient PAR levels. Treatments according to
Table l. Data are means ± SE with n = 12. Different letters above
bars indicate significant difference between treatments of similar
shoot type (p<0.05, LSD test).
eta!. (2001) was much lower than during our exper-
iment. The phenomenon that at lower PAR levels the
UV-B growth responses were negative, which is not
the case for higher PAR levels or outside situations,
was also found in soybean, pea and many other crop
species (Dumpert & Knacker 1985; Teramura & Sul-
livan 1987; Kramer eta!. 1992; Deckmyn eta!. 1994;
 143
50 -,----
I ~ 40

.
~main shoot N

a cz:z:zJ first adventitious shoot ~ 35

I
40 ~ ~

~
N~
I
1
~
30

25
~ 30 8

.
rn
c
~ 20
~

20 ~
m 15
>
~ ::l
0
c 10
10
il
~ 5

- UV-B LL - UV-8 AL + UV-8 LL + UV-B AL - UV-B LL - UV-8 AL + UV-B LL + UV-B AL

Figure 5. Leaf size of Vicio .filhu in response to UV-B radiation at
low and near ambient PAR levels. Treatments according to Table I.
Data are means ± SE with 11 = 12. Ditferent letters above bars
indicate significant dirrerence between treatments or similar shoot
type (pdJ.OS, LSD test).
50~----------------------~==~m-a~in-s~ho~ot______
Figure 7. Relative amounts ofUV-B ahsorbing compounds of Vicio
.f!tbo in response to UV-B radiation at low and near ambient PAR
levels. Relative amounts arc measured between 280 and J20 nrn
calculated on a leaf area base. Treatments according to Table I. Data
arc means ± SE with n = 12. Different letters above bars indicate
signilicanl difference between treatments (p<O.OS, LSD lest).

~ first adventitious shoot

d
a
Deckmyn et al. ( 1994) ascribed the disappearance
of negative UV-B effects at higher PAR levels to a
rn
-~ 30
higher PAR to UV-B ratio. However, it is unlikely that
trn the ratio between UV-B and the whole PAR range is
important because there is no a general PAR recep-
C\J~ 20
:5 tor. Enhanced flavonoid concentration and photomor-
<f)

phogenic responses such as internode length, leaf size

10
and leaf thickness, are responses induced by red, far
red, UV~A/blue and UV~B light. It is conceivable that
the balance between red, far red, blue/UV-A and UV-
- UV-B LL - UV-B AL + UV-8 LL + UV-8 AL
B is important for the final growth UV-B response,
Figure 6. Specific leaf area (SLA) of Vicio .fi1ha in response to
UY-B radiation at low and near ambient PAR levels. Treatments
influenced by morphology and chemical content of the
according to Table l. Data are means ± SL with 11 = 12. Different plants (Baraldi et al. 1998; Bornman 1999; Meijkamp
letters above bars indicate significant difference between treatments et al. 1999 and references therein; Sullivan & Rozema
of similar shoot type (p<O.OS. LSD test). 1999).

Antonelli et a!. 1997; Nogues et a!. 1998; Allen et al. Photomorphogenic effects: morphology
1999).
Increased branching of UV-B treated plants was re-
Besides differences in PAR doses, spectral dif-
lated to increased biomass (Table I). The internode
ferences may help to explain contrasting growth re-
length and leaf size of the main shoot were decreased
sponses to UV-B in our experiment and those of
and leaf thickness, measured as SLA, was increased
Tosserams et a!. (20()] ). In addition, the plant density
by enhanced UV-B and PAR (Figures 4 and 5). Plant
was higher in Tosserams eta!. (200 I) resulting in more
height and leaf size arc partly determined hy internode
shaded spaces with higher UV-B to PAR ratio's in the
and leaf elongation, respectively. These photornor-
canopy (Grant 1997; Flint & Caldwell1998; Deekmyn
phogenic characteristics are common characteristics
& Impens 1998). In a more dense canopy, also the red
of UV~B responses in Vi cia faba and many other crop
to far red ratio would be decreased and there would be
species (Dum pert & Knacker 1985; Cen & Bornman
a steeper drop in fluence rate of blue, red and far-red
1990; Barnes et al. 1996; Tosserams et al. 2000).
light (Ballare et al. 1992). Altered light quality might
Many of these photomorphogenic UV-B etlects are
also affect the morphology of plants (see below).
also initiated or modulated by other light qualities
such as red, far red, blue and UV-A radiation and
144
high PAR (Chabot et al. 1979; Teramura 1980; Cen
& Bornman 1990; Balian~ et al. 1992; Adamse et al.
1994). Red and far red are perceived via phytochrome
and blue/UV-A light is primarily sensed by cryp-
tochromes (Balian~ et al. 1992; Kraepiel & Miginiac
1997; Jenkins 1997). A possible explanation for the
similarities and interactions between UV-B and red to
far red ratio on photomorphogenic effects is that re-
sponses are mediated at least partly by phytochrome.
Red, UV-A/blue as well as UV-B can transform the
phytochrome activity (Young et al. 1992; Middleton
& Teramura 1994; Jenkins 1997).
Based on this knowledge, we suggest that the pho-
tomorphogenic changes, which we found in response
to UV-B and PAR, are possibly the result of activity
of cryptochrome, phytochrome, and other receptors.
These receptors are regulated via UV-B, UV-A/Blue
and I or red and far red light (Lingakumar & Ku-
landaivelu 1993; Barnes et al. 1996; Jenkins 1997;
Kobzar et al. 1998). It seems that the photomor-
phogenic effects in response to UV-B, arc not directly
damaging UV-B effects (Balian~ et al. 1992; Barnes
et al. 1996).
Increased branching and reduced plant height are
characteristics of loss of apical dominance, which
could be caused by reduction of indole-3-acetic acid
(IAA) activity or concentration of this auxin (Schmitt
1997; Rozema et al. 2001). IAA, is also involved in
the light induced inhibition of plant cell elongation
(Kraepiel & Miginiac 1997). It is suggested that IAA
can be destructed directly by UV-B or that IAA ac-
tivity can be reduced by interaction with quercetin
flavonoids which occur in the UV-B irradiated Vi-
cia faba leaves in enhanced amounts (Meijkamp
et al. 1999 and references therein). Ros & Tevini
(1995) found that specific IAA photoproducts inhibit
hypocotyl elongation in sunflower seedlings. All these
data considered together, IAA might be involved in
the UV-B transduction pathway leading to photomor-
phogenic responses. However, the role of IAA is
still not clear in the light regulation of morphology
(Kraepiel & Miginiac 1997).
UV-B-absorbing compounds
Enhanced intensity of both PAR and UV-B led to
higher concentrations of UV-B absorbing compounds.
The effect of both radiation treatments is additive, thus
enhanced PAR radiation gives additional UV-B protec-
tion (Figure 7). It is often found that the concentration
of UV-B absorbing compounds is enhanced in leaves
exposed to UV-B and enhanced PAR levels (Warner
& Caldwell 1983; Cen & Bornman 1990; Meijkamp
et al. 1999).
For the induction of flavonoids, it seems that differ-
ent light qualities (UV-B, UV-A/blue and red/far-red)
and thus different photoreceptors are working together
(Balian~ et al. 1992; Beggs & Wellman 1994; Barnes
et al. 1996; Jenkins 1997).
Damaging UV-B effects mediated by PAR
In addition to photomorphogenic effects, damage such
as bronzing and curling of leaves has been found in
Faba bean at a UY-BsE dose of 10.6 kJ m- 2 day- 1
(Visser et al. 1997a) and cucumber (Krizek et al.
1993). However, we did not find any visible damage
on the leaves at a dose of 12 kJ m- 2 day- 1. It seems
that the PAR quality and quantity prevented leaves
from damage by inducing UV-B protective responses.
Also Adamse & Britz (1992), who used a UY-BsE
dose of 18 kJ m- 2 day- I in combination with a PAR
dose of 1000 pmol m- 2 s- 1 did not find visible dam-
age. Reduction of leaf size and internode length can
also be considered UV-B damage, when this occurred
by disturbance of cell division and elongation (Ter-
amura et al. 1980; Mirecki & Teramura 1984 ). The
UV-B fluence rate is determining whether damage or
photomorphogenic effects take place (Teramura et al.
1980; Kim et al. 1998). Maybe the threshold for oc-
currence of UV-B damage is dependent also on other
light qualities than just UV-B.
The question was if near ambient PAR (as com-
pared to lower PAR levels) could reduce UV-B dam-
age. As discussed above, PAR and UV-B can have the
same photomorphogenic effects, which can be favor-
able to protect the mesophyll cells against deleterious
UV-B radiation. However, for the direct growth and
damaging effects, the effects of PAR and UV-B are not
comparable. It is still incompletely understood which
light quality and which mechanisms cause that PAR
may ameliorate these damaging UV-B effects.
We conclude that, in the greenhouse, the effects of
UV-B on growth of young vegetative Vi cia faba plants
are not always negative, even at 'low' PAR levels.
Photomorphogenic effects and an increase in UV-B
absorbing compounds (Figure 7), are at least partly
responsible for avoiding UV-B damage.

Acknowledgements
We would like to thank Prof. Dr W. H. 0. Ernst and
Prof. Dr R. Aerts for helpful comments on the manu-
script. The authors thank H. Verschoor of Nickerson-
Zwaan (Barendrecht) for providing seeds of cultivar
Minica.
References
Adamse. P. & Britz. S.J. 1992. Amelioration ol" UV-B damage
under high irradiance. l. Role of photosynthesis. Photochem.
Photobiol. 56: 645-650.
Adamse, P., Britz, S.J. & Caldwell. C.R. 1994. Amelioration
of UV-B damage under high irradiance. 2. Role of blue light
photoreceptors. Photochem. Photobiol. 60: 110-115.
Allen, D.J., Nogues, S. & Baker, N.R. 1998 Ozone depletion and in-
creased UV-B radiation: is there a real threat to photosynthesis"'-
!. Exp. Bot. 49: 1775-1788.
Allen, D.J., Nogues, S .. Morison. J.I.L., Greenslade. P.O .. McLeod.
A.R. & Baker, N.R. 1999. A thirty percent increase in UV-B has
no impact on photosynthesis in well-watered and droughted pea
plants in the field. Global Change Bioi. 5: 235-244.
Antonelli, F., Grifoni, D., Sabatini, F.& Zipoli, G. 1997. Morpho-
logical and physiological responses or bean plants to supplemen-
tal UV radiation in a mediterranean climate. Plant Ecol. 128:
127-136.
Ballare, C.L., Scope!, A.L., Sanchez, R.A. & Radosevich. S.R.
1992. Photomorphogenic processes in the agricultural environ-
ment. Photochem. Photobiol. 56: 777-788.
Barnes, P.W., Balian~, C.L. & Caldwell, M.M. 19'!6. Photomor-
phogenic etfects of UV-B radiation on plants: Consequences for
light competition. J. Plant Phys. 148: 15-20.
Barnes, P.W., Flint. S.D. & Caldwell M.M. 1990. Morphological
responses of crop and weed species of different growth forms to
ultraviolet-B radiation. Am. J. Bot. 77: 1354-1360.
Beggs, C.J. & Wellmann, E. 1994. Photocontrol of flavonoid
biosynthesis. pp. 733-75 l. In: Kendrick, R.E.& Kronenberg,
G.H.M. (eds), Photomorphogencsis in Plants. Kluwcr Academic
Publishers, Dordrecht.
Bornman, J.F. 1999. Localisation and functional significance of
flavonoids and related compounds. pp. 59-69. In: Rozema. J.
(ed.), Stratospheric Ozone Depletion: the Effects of Enhanced
UV-B Radiation on Terrestrial Ecosystems. Backhuys Publishers
Leiden, The Netherlands.
Caldwell M.M. 1971. Solar UV irradiation and the growth and de-
velopment of higher plants. pp. 131-177. In: Giese. A.C. (ed.).
Photophysiology, Vol. 6. Academic Press. New York.
Caldwell, M.M. Tcramura, A. H. & Tevini. M. ( 19891. The chang-
ing solar ultraviolet climate and the ecological consequences for
higher plants. TREE 4: 363-367.
Caldwell, M.M. & Flint, S.D. 1994. Stratospheric ozone reduction,
solar UV-B radiation and terrestrial ecosystems. Clim. Change
28: 4 375-394.
Caldwell, M.M., Bjorn, L.O .. Bornman. J.F.. Flint. S.D .. Ku-
landaivelu, G., Teramura, A.H. & Tevini. M. 1998. Effects of"
increased solar ultraviolet radiation on terrestrial ecosystems. J.
Photochem. Photobiol. B: Bioi. 46: 40--152.
Cen, Y. & Bornman. J.F. 1990. The response of bean plants to UV-B
radiation under different irradiances of background visible light.
J. Exp. Bot. 41: 1489-1495.
145
Chabot. B.F., Jurik. T. W. & Chabot, J.F. 1979. Influence of instan-
taneous and integrated light-flux density on leaf anatomy and
photosynthesis. Am. J. Bot. 66: 940-945.
Deckmyn, G .. Martens, C. & Impens, I. 1994. The importance of
the ratio CV-R/Photosynthctic Active Radiation (PAR) during
leaf development as determining factor of plant sensitivity to
increased UV~B irradiance - EtTects on growth, gas exchange
and pigmentation of Bean plants (Phaseolus Vulgaris CV Label).
Plant Cell Environ. 17: 295-30 I.
Deckmyn. G .. & 1m pens, I. 1998. UV-B and PAR in a grass (Loliwn
perenne L) canopy. Plant Ecol. 137: 13-19.
Dumpert. K. & Knacker. T. 1985. A comparison of the etfccts
of enhanced UV-B radiation on some crop plants exposed to
greenhouse and field conditions. Biochem. Physiol. Ptlanz. 180:
599-612.
Fiscus. E.L.& Booker. F.L. 1995. ls increased UV-13 a threat to crop
photosynthesis and productivity? Photosynth. Res. 43: X1-92.
Flint, S.D., Jordan P.W. & Caldwell. M.M. 19X5. Plant protective
response to enhanced UV-B radiation under field conditions: leaf
optical properties and photosynthesis. Photochem. Photobiul. 41:
95-99.
Flint, S.D. & Caldwell. M.M. 1998. Solar UV-B and visible radia-
tion in tropical forest gaps: measurements partitioning direct and
diffuse radiation. Global Change Bioi. 4: 863-X70.
Grant. R.H. 1997. Partitioning of biologically active radiation in
plant canopies. Int. J. Riometcorol. 40: 26--40.
c;rcen. A.E. S .. Cross, K.R. & Smith. L.A. 1980. Improved analytic
characterization of ultraviolet skylight. Photochcm. Photobiol.
31: 59-65.
Herman, J.R .. Bhartia, P.K .. Ziemke. J .. Ahmad. Z. & Larko. D.
1996. UV-B increases ( 1979-1992) from decreases in total ozone.
Geogr. Res. Lett. 23: 2117-2120.
Jenkins. Ci.I. 1997. UV and blue light signal transduction in .1\ru-
bidopsis. Plant Cell Environ. 20: 773-778.
Jordan. B.R. 1993. The molecular biology or plants exposed to
ultraviolct-B radiation and the interaction with other stresses.
pp. 153-170. In: Jackson M.B. & Black C.R. (cds). Interacting
Stresses on Plants. NATO ASI series. Springer-Verlag, Berlin.
Vol. 16.
Kim. B.C., Tennessen. D.J. & Last, R.L. 1998. UV-B-induced pho-
tomorphogenesis in Arabidopsis thaliww. Plant J. 15:667-674.
KNMI. 1993. 1994.1996. Annual whcather reports KNMI 1993.
1994. 1996, respectively Bilthoven. the Netherlands.
Kobzar, E.F., Kreslavski. V.D. & Muzafarov. E.N. 1998. Photomor-
phogenetic responses to UV radiation and short-term red light in
lettuce seedlings. Plant Growth Reg. 26: 73-76.
Kraepiel. Y. & Miginiac. E. 1997. Photomorphogenesis and phyto-
hormones. Plant Cell Environ. 20: 807--812.
Kramer. G.F.. Krizek. D.T. & Mirecki, R.M. 1992. Influence of
photosynthetically active radiation and spectral quality on UV-
B-induced polyamine accumulation in soybean. Phytochemistry
31:1119-1125.
Krizek. D.T.. Kramer, G.F.. lipadhyaya. A. & Mirecki. R.M.
1993. LV-B response of cucumber seedlinas grown under hi<>h
pressure sodium/deluxe lamps. Physiol. Pla~t. S8: 350-358. b
Lingakumar. K. & Kulandaivelu. G. 1993. Regulatory role of
phytochrome on ultraviolet-B (280-315 nm) induced changes
in growth and photosynthetic activities of Vi!Jna sinmsis L.
Photosynthetica 29: 341-351.
Madronich, S., McKenzie. R.L.. Bjorn. L.O. & Caldwell, M.M.
1998. Changes in biologically active ultraviolet radiation reach-
ing the earth's surface. J. Photochem. Photobiol. B: Riol. 46:
5 19.
146
McClure, J.W. 1975. Physiology and functions of ftavonoids.
pp. 970-1055. In: Harborne J.B .. Mabry, T.B. & Mabry, H. (eds).
The Flavonoids. Chapman and Hall London, UK.
Meijkamp, B.. Aerts. R.. Van de Staaij, J.. Tosserams. M ..
Ernst. W.H.O .. Rozema, J. 1999. Effects of UV-B on sec-
ondary metabolites in plants. pp. 71-99. In: Rozema, J. (ed.),
Stratospheric Ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers
Leiden. The Netherlands.
Middleton. E.M. & Teramura, A.H. I 994. Understanding photo-
synthesis, pigment and growth responses induced by UV-B and
UV-A irradiances. Photochem. Photobiol. 60: 38-45.
Mirecki. R.M. & Teramura, A.H. I 984. Effects of ultraviolet-B
irradiance on soybean. V. The dependence of plant sensitivity
on the photosynthetic photon ftux density during and after leaf
expansion. Plant Physiol. 74: 475-480.
Nogues, S., Allen, D.J., Morison. J.I.L. & Baker, N.R. I998.
Ultraviolet-B radiation effects on water relations, leaf develop-
ment, and photosynthesis in droughted pea plants. Plant Physiol.
I 17: 173-181.
Ros, J. & Tevini, M. 1995. Interaction of UV-radiation and IAA
during growth of seedlings and hypocotyl segments of sunflower.
J. Plant Phys. 146: 295-302.
Rozema, J., Van de Staaij, .J., Bjorn, L.O. & Caldwell, M.M.
1997. UV-B as an environmental factor in plant life: stress and
regulation. Trends Ecol. Eva!. 12: 22-28.
Rozema, J., Brockman, R., Lud, D., Huiskes, A., Moerdijk, T.,
de Bakker, N., Meijkamp. B. & Van Beem, A. 2001. Con-
sequences of depletion of stratospheric ozone for terrestrial
antarctic ecosystems: the response of Deschampsia antarctica
to enhanced UV-B radiation. Plant Ecol. 154: 101-115 (this
volume).
Runeckles. V.C. & Krupa S. V. 1994. The impact of UV-B radiation
and ozone on terrestrial vegetation. Environ. Pollut. 83: 191-213.
Schmitt, J. 1997. Is photomorphogenic shade avoidance adaptive"
Perspectives from population biology. Plant Cell Environ. 20:
826-830.
Shindell, D.T., Rind, D. & Lonergan, P. 1998. Increased polar
stratospheric ozone losses and delayed eventual recovery ow-
ing to increasing greenhouse-gas concentration. Nature 392:
589-592.
Sullivan, 1.H. & Rozema J. 1999. UV-B effects on terrestrial plant
growth and photosynthesis. pp. 39-57. In: Rozema, 1. (ed.),
Stratospheric ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers
Leiden, The Netherlands.
Taylor, R.M., Tobin, A.K. & Bray, C.M. 1997. DNA damage
and repair in plants. pp. 53-76. In: Lumsden, P.L. (ed.), Plants
and UV-B: Responses to Environmental Change. Cambridge
University Press, Cambridge.
Tcramura, A.H. 1980. Effects of ultraviolet-B irradiances on soy-
bean I. importance of photosynthetically active radiation in
evaluating ultraviolet-B irradiance effects on soybean and wheat
growth. Physiol. Plant. 48: 333-339.
Teramura, A.H. Biggs, R.H. & Kossuth, S. 1980. Effects of
ultraviolet-B irradiances on soybean 2. Interaction between
ultraviolct-B and photosynthetically active radiation on net pho-
tosynthesis, dark respiration and transpiration. Plant Physiol. 65:
483-488.
Teramura, A.H. 1986 Interactions between UV-B and radiation
and other stresses in plants. pp. 327-343. In: Worrest R.C.
& Caldwell M.M. (eds), Stratospheric ozone Reduction, Solar
Ultraviolet Radiation and Plant Life. Springer-Verlag. Berlin.
Teramura, A.H. & Sullivan .J.H. \987. Soybean growth responses
to enhanced levels of ultraviolet-B radiation under greenhouse
conditions. Am. 1. Bot. 74: 975-979.
Tevini. M. & Teramura, A.H. 1989. UV-B effects on terrestrial
plants. Photochem. Photobiol. 50: 479-487.
Tosserams, M., Visser, A., Groen, M., Kalis, G., Magendans, E.
& Rozema, 1. 200 I. Combined effects of C02 concentration and
enhanced UV-B radiation on faba bean. Plant Ecol. 154: 167-1 Hl
(this volume).
Visser, A.1., Tosserams, M., Groen, M.W., Kalis. G., Kwant, R.,
Magendans. G.W.H. & Rozema, J. l997a. The combined effects
of C02 and solar UV-B radiation on faba bean. III. Leaf optical
properties, pigments, stomatal index and epidermal cell density.
Plant Ecol. 128: 208-222.
Visser, A.1., Tosserams, M., Groen, M.W., Magendans, G.W.H. &
Rozema. J. l997b. The combined effects of C02 and solar UV-B
radiation on t'aba bean grown in open top chambers. Plant Cell
Environ. 20: 189-199.
Warner, C.W. & Caldwell, M.M. 1983 Influence of photon flux den-
sity in the 400-700 nm waveband on inhibition of photosynthesis
by UV-B (280-320 nm) irradiation in soybean leaves: separa-
tion of indirect and immediate effects. Photochem. Photobiol. 38:
341-346.
Young, J.C., Liscum, E., Hangarter & R.P. 1992. Spectral-
dependence of light-inhibited hypocotyl elongation in photo-
morphogenic mutants of Arabidopsis: evidence for a UV-A
photosensor. Planta 188: 106-114.
Zar, J.H. 1984. Biostatistical analysis, second edition. Prentice-Hall,
Englewood Cliffs, N.J.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

Experimental set-up for high-light exposure of leaf discs of UV-B-treated and control plants.
(Photograph hy D. Visser)
 Plant Ecologv 154: 149-156, 200 I.
149
© 200 I Kluwer Academic Publishers.

Growth under UV-B radiation increases tolerance to high-light stress in

pea and bean plants

Esther M. Bolink, lise van Schalkwijk, Freek Posthumus & Philip R. van Hasselt
Department of Plant Biolog_v. Universitv of Gmningen. PO. Box 14, 9750 AA Haren, The Netherlands
(E-mail: e.m.bolink@ bioi. rug.nl)

Key words: Fv/ Fm. Glutathione, Oxygen evolution, Photoinhibition, UV-B, Xanthophyll cycle

Abstract
Pea (Pisum sativum L.) and bean (Phaseolus vulgaris L.) plants were exposed to enhanced levels ofUV-B radiation
in a growth chamber. Leaf discs of UV-B treated and control plants were exposed to high-light (HL) stress (PAR:
1200 [LillO I m- 2 s- 1) to study whether pre-treatment with UV-B affected the photoprotective mechanisms of the
plants against photoinhibition. At regular time intervals leaf discs were taken to perform chlorophyll a fluorescence
and oxygen evolution measurements to assess damage to the photosystems. Also, after I h of HL treatment the
concentration of xanthophyll cycle pigments was determined. A significantly slower decline of maximum quantum
efficiency of PSTT (Fv/ F111 ), together with a slower decline of oxygen evolution during HL stress was observed in
leaf discs of UV-B treated plants compared to controls in both plant species. This indicated an increased tolerance
to HL stress in UV-B treated plants. The total pool of xanthophyll cycle pigments was increased in UV-B treated
pea plants compared to controls, but in bean no significant differences were found between treatments. However.
in bean plants thiol concentrations were significantly enhanced by UV-B treatment, and UV-absorbing compounds
increased in both species, indicating a higher antioxidant capacity. An increased leaf thickness, together with
increases in antioxidant capacity could have contributed to the higher protection against photoinhibition in UV-B
treated plants.

Abbreviations: Fv/ F111 : maximum quantum efficiency of PSII; HL: high-light; PAR: photosynthetically active
radiation (400-700 nm); PSII: photosystem II; UV-A: ultraviolet-A (320-400 nm); UV-B: ultraviolet-B (280-
320 nm); UV-BsE: biologically active UV-B radiation.

Introduction rescence, it can drop to the excited triplet state. From

In spite of the importance of sunlight as primary en-
ergy source for life on Earth through absorption by
photosynthetic organisms, excess light can lead to se-
rious injury in plants. In high light (HL) situations
(full sunlight) plants absorb more light than can he
used for photosynthesis, resulting in over-excitation
of the photochemical reaction centers in the chloro-
plast, which can lead to serious damage (Barber &
Andersson 1992; Owens 1996; Eskling et al. 1997 ).
When a chlorophyll molecule in the singlet excited
state can not transfer its energy to the photochemical
reaction centers or release the energy as heat or fluo-
this state it may return to the ground state by interact-
ing with oxygen, giving rise to reactive singlet oxygen
which can cause photo-oxidative damage and break-
clown of the reaction center D !-protein of photosystem
II (PSII) (Richter et al. 1990a, b; Barber & Anders-
son 1992). This results in photoinhihition: repressed
electron transport and decreased photosynthesis.
Excess light absorption may occur as a result of a
decreased rate of photosynthesis clue to environmen-
tal stress (Owens 1996), for example by an increase
in UV-B radiation. The UV-B (280-320 nm) frac-
tion of solar light reaching the Earth's surface has
increased steadily over the last few decade,, clue to
!50
thinning of the ozone layer (Blumthaler & Ambach
1990; Herman et al. 1996). UV-B radiation can have
negative effects on plant growth, photosynthesis and
pigmentation (for reviews: Tevini & Teramura 1989;
Teramura & Sullivan 1994; Jansen et al. 1998). How-
ever, due to damage repair and stimulation of UV-B
protective mechanisms, like increased reflectance and
UV absorbance by flavonoids, many plants are able
to mitigate UV-B damage effectively, especially in
field situations (Fiscus & Booker 1995; Rozema et al.
1997). In sensitive plants, UV-B has been shown to
inhibit photosynthetic electron transport, with PSII
as the major site of damage (Bornman 1989). UV-B
treated plants would therefore have a lower threshold
for light-induced damage.
In HL situations carotenoids play an important
role in protecting the photosynthetic apparatus against
oxidative damage. Carotenoids stabilize and protect
the lipid phase of the thylakoid membranes (Havaux
1998), and they are quenchers of the excited triplet
state of chlorophyll and singlet oxygen (Siefermann-
Harms 1987). In addition, the xanthophyll cycle pig-
ment zeaxanthin quenches the singlet excited state
of chlorophyll (Demmig-Adams 1990). In excessive
light zeaxanthin is rapidly formed by de-epoxidation
of violaxanthin, via the intermediate antheraxanthin, a
reaction reversed in the dark.
The concentrations of the xanthophyll cycle pig-
ments have been found to increase by some environ-
mental stresses, due to a higher need of energy dis-
sipation (Demmig-Adams & Adams 1992). However,
Pfiindel et al. ( 1992) found that the concentrations
of xanthophyll cycle pigments were not increased in
pea plants treated with enhanced UV-B. Instead, the
activity of the enzyme violaxanthin de-epoxidase was
inhibited by 50%, resulting in a decrease of zeaxanthin
availability in HL situations. Therefore, one would ex-
pect that UV-B treated plants would be more sensitive
to HL conditions than control plants.
In the experiments of Pfiindel et al. ( 1992), plants
were exposed to very high UV-B irradiances, in which
I h of UV-B treatment corresponded to the daily UV-
BsE at a latitude of 40° at 1500 m elevation, in short
periods of time ( 12-64 h), lacking diurnal rhythms.
This could have led to an overestimation of the dam-
age by UV-B. The goal of the present study was to
investigate whether or not prolonged pre-treatment of
pea and bean plants with enhanced UV-B, using nat-
ural diurnal rhythms, would also lead to reductions in
zeaxanthin availability during HL treatment. Although
the UV-B/PAR ratio was still too high compared to
natural situations in this laboratory experiment, the
mechanisms of the effects of pre-treatment with UV-
B on photoinhibition could be analyzed in detail in
this study. To assess the sensitivity of UV-B treated
and control plants to HL conditions, maximum quan-
tum efficiency of PSII (Fv/ Fm) and photosynthetic
performance were measured as an indication of pho-
toinhibition.
Materials and methods
Plant material and growth conditions
Seeds of the pea mutant Argenteum (Pisum sativum
L. mutant Argenteum) were obtained from the Plant
Genetic Resources Unit (US, DA/ARS, NYS Agricul-
tural Experimental Station, Geneva, NY, USA). Seeds
of bean (Phaseolus vulgaris L. cv Saxa) were obtained
from Fleurmerc (Wormerveer, NL). Seeds were ger-
minated in a growth chamber on wet vermiculite. The
day/night temperature was ±20/16 °C. A photosyn-
thetic photon flux density of 190 J-Lmol m- 2 s- 1 was
provided by a 1:1 combination of Osram L58 W/21
and W/31 (Luminax, Germany) lamps during a 12-h
photoperiod. Before the emergence of the first leaf,
seedlings were transferred to potting soil in 0.5 litre
pots and treatment with UV irradiation was started in
the same growth chamber.
UV-B radiation was provided by UV-B fluorescent
tubes type TL40W/12 (Philips, Eindhoven, NL) for
6 h per day in the middle of the photoperiod. Differ-
ent treatment sectors were created by placing 2 UV
translucent Plexiglas (PMMA) plates (3 mm poly-
methyl methacrylate, Vink, Didam, NL) at a distance
of 10 em underneath the UV-B tubes. One of the UV
translucent PMMA plates was covered with a 0.1 mm
cellulose diacetate foil (CA, transmittance >290 nm;
Tamboer & Co., Haarlem, NL) to create a sector in
which the plants received both UV-A and UV-B, the
'UV-B' treatment. The other UV translucent plate was
covered with a 0.1 mm polyester foil (Mylar type D,
transmittance> 313 nm; DuPont de Nemours, Lux-
embourg) to create a control treatment. Control plants
received a similar portion of UV-A, but no UV-B. The
treatment sectors were exchangeable by altering the
sides of the growth room at which the CA and My-
lar foils were placed. The treatments were separated
in the growth chamber by non-UV translucent 4 mm
PMMA (Vink, Didam, NL) to prevent UV-B radiation
from reaching control treatment. Since the transmis-

sion spectra of CA and Mylar are atlected by UV light
(Strid eta!. 1990) the foils were replaced once a week.
The spectral irradiances were measured with a
MACAM SR9910 double monochromator scanning
spectroradiometer (Macam Photometries). According
to the generalized plant action spectrum (Caldwell
1971) normalized at 300 nm, the daily biologically ac-
tive UV-B (UV-BsE) radiation in the UV-B treatment
was 11.3 kJ m- 2 day-i. Plants were watered daily
and rotated within their treatment area every other
day to assure an equal light distribution. A k strength
Hoagland's solution was provided once a week to
avoid nutrient deficiencies.
HL treatments, chlorophyll a .fluorescence and gas
exchange
Leaf discs, with a diameter of I em, of control and
UV-B-treated plants were placed in petri dishes on
demineralized water. The discs were then subjected
to HL (1200 Jlmolm- 2 s-i), provided by two 400W
HPL lamps (type IP22, Philips, Eindhoven, NL), at
20 oc in an experimental chamber. After various times
(0.25-5 h) some of the petri dishes were removed
from the HL chamber and the leaf discs were either
frozen in liquid nitrogen and stored at -80 oc (xantho-
phyll determination), placed in the dark (fluorescence
measurements), or used immediately for 02 evolution
measurements. The leaf discs used for the HL treat-
ment were cut from the first leaves of bean plants and
from the 5th and 6th leaves of pea plants, which were
the youngest fully developed leaves in all experiments.
After cutting the discs, leaves were placed under a
photocopier to exactly determine their leaf area.
Chlorophyll a fluorescence (at 20 °C) was mea-
sured using a modulated fluorometer (PAM-I 01/-103,
Heinz Walz GmbH, Effeltrich, Germany). Two Schott
lamps (KL 1500, Mainz, Germany) provided satu-
rating flashes and actinic illumination. The minimum
fluorescence level Fo (dark adapted leaves) was mea-
sured at a light intensity of about 0.1 Jlmolm- 2 s-i.
The maximum fluorescence level F111 (dark-adapted
leaf discs) was determined using a saturating light
flash (I s) with an intensity of 3500 Jlmol m- 2 s-i.
The maximum quantum efficiency of photosystem II
(PSII) was estimated from F,,j F111 values, measured
after 15 min of dark adaptation.
Photosynthetic performance (at 20 oq of high-
light treated leaf discs was measured by placing the
discs in a cuvette with a Clark oxygen electrode con-
nected to an 02 electrode control box (Hansatech,
151
King's Lynn, Norfolk, UK). 02 evolution was de-
termined with a gas mixture of 2% 02 and 4.5%
C02 in nitrogen at a non-saturated light intensity of
I00 flmol m- 2 s-i.
Pigment and thiol-pool determination
To determine the concentration of xanthophyll cycle
pigments, lutein and chlorophyll, bean and pea plants
were treated with high-light after growing under UV
conditions for 14-21 days. Leaf discs were treated for
I hour with HL and stored at -80 oc. The discs were
then powdered in liquid N2 with some solid Na 2 co 3 .
Pigments were extracted in I ml ice-cold I 00% ace-
tone and separated by HPLC according to Venema
et al. ( 1999). A Waters Delta Pak reversed-phase col-
umn (5 Jlm, Cis, 100 A, 3.9 x 150 mm, 17% carbon
load, fully end capped) and a Water Nova-Pak guard
column (5 Jim, Cis) were used. Column tempera-
ture was maintained at 15 oc by a column thermostat
(model BF0-0415, W.O. Electronics). The binary sol-
vent system consisted of acetonitrolc:methanol: Tris-
HCI (0.2 M, pH 8.0) (60:20:20 v/v/v; solvent A) and
acetonitrile: ethanol: ethyl acetate (50:30:20 v/v/v;
solvent B). Pigments were identified as based upon
their retention times relative to known standards and
by their absorption spectra, measured with a Cary 3E
spectrophotometer. Concentrations were calculated as
based on standard-curves, created with known stan-
dards. Xanthophyll-cycle pool size was calculated as
the sum of V + A + Z, were V, A, and Z correspond
to the contents of violaxanthin, antheraxanthin, and
zeaxanthin, respectively.
The amount of UV-absorbing compounds (mostly
ftavonoids) was determined spectrophotometrically af-
ter extracting leaf discs in 4 ml ethanol: acetic acid
solution (99: I v/v), according to Tosserams et al.
( 1996). Absorption was measured at 300 nm.
Water soluble non-protein thiol components were
extracted according to Stuiver et al. (1992) and the
content of the DTNB-reactive compounds of the
30 000 g supernatant was measured as described by
De Kok et al. ( 1988). The first leaves of bean plants
and whole shoots of pea plants were used for this
determination, since a minimum of I g FW was re-
quired. Thiols were measured after 12 to 14 days of
UV treatment.
Sampling and statistics
All HL experiments were performed several times for
both plant species, using various leaves, with very
152
Tah/e I. Leaf area (cm 2 ) and specific leaf area (SLA, cm 2 g- 1) of the youngest fully
developed leaf of pea (leaves 5 and 6) and bean plants (leaf l), after respectively 30 and 12
days of UV treatment. Values presented are means of 4-7 replicates± SD. Values marked
with different letters are significantly different according to Students t- test (p < 0.05).
Pea
Control
Leaf area 11.2 ± !.52 a
SLA 51.0 ± 1.65 a
similar results. To be able to make comparisations be-
tween experiments, only the data acquired from leaves
5 and 6 of pea and leaf I of bean plants are presented in
the current paper. In pea, plants were randomly chosen
from a group of plants which were all of a similar
physiological age, in which the 5th and 6th leaves
were the youngest fully-developed ones. Because of
small ditl'erences in the size of the root at the time
the UV treatment started, the physiological age of the
plants varied within the groups of both treatments later
in development. For this reason, the time at which a
number of plants of both treatments reached the phys-
iological age in which the 5th and 6th leaves were the
youngest fully developed ones varied between 21 to
30 days. The authors have chosen to compare plants
used in different experiments based on a physiological
age rather then on the time under UV treatment, since
senescence of leaves would bring about changes in
pigment composition. In bean plants much less vari-
ation in physiological age occurred, and plants were
chosen randomly for each experiment when the first
leaves were fully developed. All experiments were
performed with plants treated for 12-14 days with or
without UV-B.
UV-B treatment effects were tested statistically
with the GraphPad Prism Package (GraphPad Soft-
ware, Inc. 1994-1995, San Diego, CA, USA). Differ-
ence between means was tested with Students t-test
(p < 0.05). Differences between treatments in re-
sponse to photoinhibition in time were tested with a
2-way Analysis of Variance (ANOVA).
Results
Treatment with enhanced UV-B radiation significantly
reduced leaf area in both pea and bean plants, com-
pared to controls. In Table I, leaf area and specific
leaf area (SLA) of the leaves which were used in the
Bean
UV-B Control UV-B
8.05 ± 0.89 b 238 ± II. I a 96.5 ± 30.0 b
44.4 ± 2.10 b 59.6 ± 1.68 a 50.0 ± 3.65 b
photoinhibition experiments arc represented. UV-B re-
duced leaf area by 28% in pea and 59% in bean plants.
Furthermore, SLA decreased significantly by UV-B
treatment, indicating increased leaf thickness.
To determine whether treatment with UV-B radia-
tion had changed the concentration or the conversion
rate of the xanthophyll cycle pigments, discs of fully
developed pea and bean leaves were taken and exposed
to high-light. Concentrations of xanthophyll cycle pig-
ments after 1 h of HL treatment are represented in
Table 2. It appeared that the total VAZ pool was sig-
nificantly higher in leaf discs of UV-B treated pea
plants compared to controls. In bean plants there were
no differences in total VAZ pool. There were no dif-
ferences between treatments in the conversion state
(de-epoxidation of violaxanthin to antheraxanthin and
zeaxanthin) in both pea and bean plants. In both
species the neoxanthin and lutein concentrations were
higher in the leaf discs of UV-B treated plants, but
only significantly in pea. There were no differences
between treatments in the concentration of chlorophyll
a + h in pea, but a significantly higher concentration
in leaf discs of UV-B treated bean plants.
The decline of maximum quantum efficiency of
PSTT (Fv/ Fm) by HL stress versus time was measured
with leaf discs of pea and bean plants after respectively
30 and 14 days of pre-treatment with or without UV-
B (Figures 1A and 1B). In both species the decline
of Fv/ Fm due to HL stress was significantly lower
in the UV-B treated leaf discs (p < 0.000 I; 2-way
AN OVA). Recovery of Fv/ Fm after 4 h of HL under
low light (45 11mol m- 2 s- 1) was also significantly
faster in discs of UV-B treated bean plants.
To determine whether the slower decline of Fv j F111
would be accompanied by a better photosynthetic
performance during HL stress, 02 evolution was mea-
sured in pea and bean leaf discs directly after exposure
to HL (Figures 2A and 2B). The decline of 02 evo-
lution, like Fv/ Fm, was also significantly slower in
 153
1(tble 2. Pigment concentrations after high-light treatment I I hour) in discs of leaves S and 6 of
pea plants and leaf I of bean plants. after 21 (pea) and 14 (bean) days of CV treatment. chi"+ h:
total chlorophyll lit mol m- 2 ). V: violaxanthin: A: antheraxanthin; Z: zeaxanthin; A+ZIV +A+Z :
conversion state. V+A+Z. neoxanthin and lutein arc expressed in mmol moi- 1 chi"+ h. Values
presented arc means of 6 replicates ± SO. Values marked with different letters are significantly
different according to Students 1- test (JI < 0.05).

Pea Bean
I Control UV-B I Control UV-B

chi"+ h 275 ± 14.5 282 ± 16.2 279 ± 30.0 a 338 ± 31.4 b

V+A+Z fl9.\ ± 5.49 a 79.4 ± 3.69 h 38.2 ± 8.04 37.9 ± 9.7X
A+ZIVAZ 0.70 ± 0.02 0.72 ± 0.02 0.73 ± 0.04 0.69 ± 0.04
Neoxanthin 36.8 ± 0.93 a 38.4 ± 1.32 b 32.6 ± 3.97 37.6 ± 6.15
Lutein 212 ± 5.94 a 228±5.19b 214 + 20.0 239 ± 33.7

Table 3. Concentration of thiols (flmol g- 1 FW) and amount of UV-absorbing com-

pounts, expressed as absorbance at 300 nm IA3oo: cm 2 ml- 1 J in pea and bean plants.
Thiol concentrations were measured after 14 (pea. whole shoot) and 12 (bean. leaf I)
days of treatment. A 300 was measured after 30 (pea, leaf 5) and II (bean. leaf I) days
of treatment. Values presented are means of 4-7 replicates ±SO. Values marked with
different letters are significantly different according to Students 1- test lp < 0.05).

Pea Bean
Control UV-B Control L:v-B

Thiols 0.41 ± 0.06 0.42 ± 0.02 0.32 ± 0.07 a 0.47 ± 0.04 b

A3oo 0.22 ± 0.01 a 0.27 ± 0.03 b 0.13±0.0\a 0.17 ± 0.03 b

leaf discs of UV-B treated plants compared to controls. Discussion

This difference was found both on chlorophyll (Fig-
ure 2) and on area basis. Before HL treatment (t = 0),
02 evolution (chi based) was significantly lower in
UV-B than in control plants in both plant species.
At saturated light intensity (500 11 mol m- 2 s- 1) 0 2
evolution as well as dark respiration (chi based) did
not differ between treatments in bean plants (data not
shown; not determined in pea plants).
The thiol concentration in leaves of UV-B treated
bean plants was significantly higher than in the leaves
of controls (Table 3). In pea plants no ditl'erences
in thiol concentration of the shoots were found. The
pea plants used for thiol extraction were of a younger
physiological age then in the other experiments shown
in this paper. Still, also in these plants the decline of
F,,j F111 by HL stress was significantly slower in leaf
discs of the youngest fully developed leaves (leaves 3
and 4) of UV-B-treated plants (data not shown). UV
absorbing capacity, measured at 300 nm, was signif-
icantly higher in leaf discs of UV-B-treated plants in
both species (Table 3).
Our data show that pre-treatment with UV-B radiation
can harden the plants against photoinhibition by HL.
The decline of Fpj f~ 11 by HL stress was significantly
slower in leaf discs of UV-B-treated than of control
plants (Figure I). This slower decline of F.,j F, 11 was
accompanied by a better photosynthetic performance
after HL treatment (Figure 2). Clear correlations be-
tween the F,,j f; 11 ratio and 02 evolution during HL
treatment have been found before, indicating that this
ratio can be well used for monitoring photoinhibition
(Bjorkman & Demmig 1987).
Although some authors have found indications
that xanthophyll cycle pigments have a photoprotec-
tive role in HL situations (Demmig-Adams & Adams
1996; Thiele et al. 1996; Eskling et al. 1997), the
present study does not show such a clear correla-
tion. When pea plants were treated with UV-B total
VAZ pool, neoxanthin and lutein concentrations were
significantly increased in leaf discs after 21 days (Ta-
ble 2), which could explain the higher photoprotec-
tion. However, there was no correlation found between
154

A
0.8 125 A
,-._
0

0.6 ,_lL 100

=
r...
>
0
~
0.4 ~
,_.,
c 75
.9
0.2 .a0
50
:>
(I)
0.0 N
0 2 3 4 5 6 0
25
HL LL B
0.8
0
~!c !

!
0.6
= y B
!
~
~
,..--._

- 100
~
0.4 0
II
,_
0.2 0
2 ~ 75

0.0 +---r-----r--r---r---,-----,r--.-..--i
0 2 3 4 5 6 7 8 23 24 25
Time (hours)
Figure I. Decline of maximum quantum efficiency of PSII
(Fv/Fml in time under HL (1200 /[mol m- 2 s- 1) after 30 (pea)
and 14 (bean) days of UV treatment. (A) Pea, leaves 5 and 6.
(B) Bean, leaf I, decline of Fv/ Fm under HL and recovery under
low-light (45 /[mol m- 2 s- 1 ; LL). Values presented are means of
5-6 replicates± SO. Open circles: control; closed circles: UV-B.
xanthophyll cycle pigments and photoprotection in
bean plants.
In the leaves of UV-B treated bean plants the thiol
content was strongly increased compared to control
plants (Table 3). In pea plants no increase in thio11ev-
e1s was observed, but since whole shoots have been
analyzed instead of leaves, like in the HL experi-
ments, the results might not be representative. Thiols,
in pea and bean mainly present as (homo-)glutathione,
can serve as antioxidants (Teramura & Ziska 1996).
Glutathione (GSH) together with ascorbate can de-
crease the decline of Fv/ Fm during photoinhibition in
spinach thylakoids (Richter eta!. 1990b). In pea plants
UV-B treatment has been found to increase levels of
glutathione reductase (GR), the enzyme which con-
verts GSH back to the reduced form in the process of
detoxification of reactive oxygen species (Strid 1993).
Therefore, increased levels of GSH, and possibly also
GR, may have contributed to the better protection
against HL stress in leaves of UV-B treated plants.
-
'-'
c
.9
.a0 50
(>
N
0 25
0
0 2 3 4 5
Figure 2. Photosynthetic Oz evolution under 2% Oz and 4.5% COz
in nitrogen at a low-light intensity of 100 Jimol m- 2 s- 1, after HL
treatment of 0 to 5 h. (A) Pea, leaves 5 and 6 (30 days of UV
treatment). (B) Bean, leaf I (12 days of UV treatment). Data are
expressed as percentage of t = 0, measured as nmol Oz mg- 1
chi s- 1. Values presented are means of 4 replicates ± SO. Open
circles: control; closed circles: UV-B.
The concentration of UV absorbing compounds,
mainly ftavonoids, was significantly increased by UV-
B, both in bean and in pea plants (Table 3). Flavonoids
have effective radical-scavenging capabilities (Bors
et a!. 1990), and could have contributed directly to
the enhanced photoprotection of UV-B treated plants
during HL stress. The primary site of HL induced
free radical formation is the chloroplast and although
ftavonoids are mainly localized in the vacuoles of
the epidermis (Weissenbock et a!. 1986), chloroplasts
of Fabaceae do contain ftavonoids (Saunders & Mc-
Clure 1976), which can serve as antioxidants in the
chloroplast (Takahama 1982).

Next to increases in antioxidant capacity, an impor-
tant reason for the better protection of UV-B treated
pea and bean plants may originate in structural and
chemical changes in the leaf induced by UV-B, caus-
ing changes in leaf reflection, absorption and scatter-
ing of light. Increases in leaf thickness (lower SLA)
and pigmentation (UV absorbing compounds), both
measured in this study as a response to UV-B treat-
ment in pea and bean plants, may cause a decrease
in penetration of PAR in the leaf (Bornman 1989:
Bornman & Vogelmann 1991 ). An indication for this
can be found in the results of the 02 evolution ex-
periments with bean, measured before HL treatment
was started. 02 evolution (chlorophyll based) was
lower in leaf discs of UV- B treated bean plants at
low light intensities (100 Mmol m- 2 s- 1 ), but sim-
ilar between treatments at saturated light intensities
(500 Mmol m- 2 s- 1). During the low light intensity
less PAR may have reached the chloroplasts of UV-B
treated plants, causing a lower 02 production than in
control plants (in which PAR penetrated better into the
leaf). This difference would not be detectable at satu-
rated light intensities, because enough PAR reached
the chloroplast to evoke maximum 02 evolution. The
attenuation of PAR could have been caused by the
increase in leaf thickness and possibly by movement
of chlorophyll to deeper parts of the leaf, as has been
found earlier as a response to enhanced UV-B (Alenius
et al. 1995; Day & Vogel mann 1995). Because of the
longer route to deeper layers in the leaf the amount
of PAR reaching the chloroplast can be reduced. It is
thought that the UV-B induced increase in chlorophyll
content, found in many studies and in our experiments
in bean plants, is triggered by this chlorophyll move-
ment in an attempt to compensate for the decreased
irradiance (Alenius et al. 1995). Penetration of PAR
may also decrease by increases in retlectivity of the
leaves, as Cen & Bornman ( 1990) found in UV- B
treated bean plants.
From this study with low PAR levels in the growth
chamber and unbalanced UV-B/UV-A/PAR levels, it
is hard to predict whether similar protection against
HL situations would occur outside, when plants would
receive enhanced natural UV-B radiation. Neverthe-
less, this experiment shows that the mechanism of
this effect is based on the general. often found re-
sponses of plant species to enhanced UV-B. Potential
beneficial effects of UV-B against photoinhibitory or
other kinds of stress have also been reported in field
experiments. Carnivorous plants exposed to enhanced
UV-B in the field have been found to be less suscep-
155
tible to photoinhibitory (low temperature/high-light)
treatments (Mendez et al. 1999). Like in the present
study, a correlation was found between protection
against photoinhihition and increases in pigments, in
this case anthocyanins. Furthermore, Manetas and co-
writers ( 1997) have found improvements of needle
water relations in seedlings of Pinus pinea, exposed
to enhanced UV-B radiation and water stress, in which
cuticle thickness was increased almost two-fold com-
pared to water stressed plants which received no UV-
B. Other hardening effects of UV-B radiation, studied
in greenhouse or controlled chamber experiments, are
a reduction in oxidative damage during HL stress
in UV-B treated oilseed rape (Bornman & Sundby-
Emanuelsson 1995 ), and an increased heat resistance
in UV-B treated cucumber (Caldwell 1994).
In conclusion, growth under UV-B radiation in-
creased photoprotection in HL situations in both pea
and bean plants. The higher tolerance to HL was cor-
related with significant increases in the total pool of
xanthophyll cycle pigments and lutein, hut only in
pea plants. No reductions were found in the conver-
sion from violaxanthin to antheraxanthin and zeax-
anthin in either plant species. Increased antioxidant
capacity, like the increased glutathione content in
bean. and increased levels of UV absorbing com-
pounds (flavonoids) in both species, could also have
provided protection against photo-oxidative damage.
Furthermore. structural changes caused by UV-B, like
increased leaf thickness, movement of chloroplasts to
deeper layers in the leaf and a higher reflection, re-
ducing penetration of PAR into the leaf, may explain
the increased tolerance to HL stress in UV- B treated
plants.
Acknowledgements
We would like to thank Dr J. H. Venema for useful
discussions during the preparation of this manuscript,
L. Villerius and A. Eggert for technical assistance on
HPLC pigment analyses and L. Hess, E. Hesse and H.
Kortrijk for laboratory assistance.
References
Alenius. C. M., Vogelmann, T. C. & Bornman, J. F. 1995. A three-
dimensional representation of the relationship between penetra-
tion of UV-B radiation and UY- screening pigments in leaves ol"
Brussica napus. New. Phytol. 131: 297-302.
Barber. J. & Andersson. B. 1992. Too much of a good thing: light
can be bad l"or photosynthesis. TIBS 17: 61-66.
156
Bjorkman, 0. & Demmig, B. 1987. Photon yield of 02 evolu-
tion and chlorophyll fluorescence characteristics at 77 K among
vascular plants of diverse origin. Planta 170: 489-504.
Blumthaler, M. & Ambach, W. 1990. Indication of increasing solar
ultraviolet-B radiation flux in alpine areas. Science 248: 206-
208.
Bornman, J. F. 1989. Target sites ofUV-B radiation in photosynthe-
sis of higher plants. J. Photochem. Photohiol. 4: 145-158.
Bornman, J. F. & Vogelmann, T. C. 1991. Effect ofUY-B radiation
on leaf optical properties measured with fibre optics. J. Exp. Bot.
42: 547-554.
Bornman, J. F. & Sundby-Emanuelsson, C. 1995. Response of
plants to UV-B radiation: some biochemical and physiologi-
cal effects. Pp. 245-262. In: Smirnoff. N. (ed.), Environment
and Plant Metabolism: Flexibility and Acclimation. Oxford Bios
Scientific Publishers, Oxford.
Bars, W., Heller, W., Michel, C. & Saran. M. 1990. Flavonoids
as antioxidants: determination of radical-scavenging efficiencies.
Methods Enzymol. 186: 343-355.
Caldwell, C. R. 1994. Modification of the cellular heat sensitivity of
cucumber by growth under supplemental ultraviolet-B radiation.
Plant Physiol. 104: 395-399.
Caldwell. M. M. 1971. Solar UV irradiation and the growth and
development of higher plants. Pp. 131-177.ln: Giese A. C. (ed.),
Photophysiology, Vol. 6. Academic Press, New York.
Cen, Y. P. & Bornman, J. F. 1990. The response of bean plants to
UV-B radiation under different irradiances of background visible
light. J. Exp. Bot. 41: 1489-1495.
Day, T. A. & Vogelmann, T. C. 1995. Alterations in photosyn-
thesis and pigment distributions in pea leaves following UV-B
exposure. Physiol. Plant. 94: 433-440.
De Kok. L. J., Buwalda, F. & Bosma, W. 1988. Determination
of cysteine and its accumulation in spinach leaf tissue upon
exposure to excess sulfur. J. Plant Physiol. 133:502-505.
Demmig-Adams, B. 1990. Carotenoids and photoprotection in
plants: a role for the xanthophyll zeaxanthin. Biochim. Biophys.
!\eta I 020: 1-24.
Demmig-Adarns, B. & Adams, W. W. III. 1992. Photoprotection
and other responses of plants to high light stress. Ann. Rev. Plant
Physiol. Plant Mol. Bioi. 43: 599-626.
Dcmmig-Adams, B. & Adams, W. W. III. 1996. Xanthophyll cy-
cle and light stress in nature: uniform response to excess direct
sunlight among higher plant species. Planta 198: 460-470.
Eskling, M., Arvidsson, P. 0. & Akerlund, H. E. 1997. The xan-
thophyll cycle, its regulation and components. Phys. Plant. 100:
806-816.
Fiscus, E. L. & Booker, F. L. 1995. Is increased UV-B a threat to
crop photosynthesis and productivity? Photos. Res. 43: 81-92.
Havaux, M. 1998. Carotenoids as membrane stabilizers in chloro-
plasts. Trends Plant Science 3: 147-151.
Herman, J. R., Bhartia, P. K., Ziemke, J., Ahmad, Z. & Larko, D.
1996. UV-B increases ( 1979-1992) from decreases in total oLone.
Gcophys. Res. Lett. 23: 2117-2120.
Jansen, M.A. K., Gaba, V. & Greenberg, B. M. 199R. Higher plants
and UV-B radiation: balancing damage, repair and acclimation.
Trends Plant Sci. 3: 131-135.
Manctas, Y., Petropoulou, Y., Stamatakis, K., Nikolopoulos, D.,
Livizou, E., Psaras, G. & Karabourniotis, G. 1997. Beneficial
effects of enhanced UV-B radiation under field conditions: im-
provement of needle water relations and survival capacity of
Pinus pinea L. seedlings during the dry Mediterranean summer.
Plant Eeol. 128: 100-108.
Mendez, M., Gwynn Jones, D. & Manetas, Y. 1999. Enhanced
UV-B radiation under field conditions increases anthocyanin and
reduces the risk of photoinhibition but does not affect growth
in the carnivorous plant Pinguicula vulgaris. New Phytol. 144:
275-282.
Owens, T. G. 1996. Processing of excitation energy hy antenna
pigments. pp. 1-23. In: Baker, N. (ed.), Photosynthesis and the
Environment. Kluwer Academic Publishers, Dordrecht.
Pfiindel, E. E., Pan, R. S. & Dilley, R. A. 1992. Inhibition of vi-
olaxanthin deepoxidation by ultraviolet-B radiation in isolated
chloroplasts and intact leaves. Plant Physiol. 98: 1372-1380.
Richter, M.. RUhle. W. & Wild, A. 1990 a. Studies on the
mechanism of photosystem II photoinhibition. I. A two-step
degradation of D !-protein. Photos. Res. 24: 229-235.
Richter, M., RUhle, W. & Wild, A. 1990 b. Studies on the mecha-
nism of photosystem II photoinhihition. II. The involvement of
toxic oxygen species. Photos. Res. 24: 237-243.
Rozema, J., Van de Staaij, J., Bjorn, L. 0. & Caldwell, M. 1997. UV-
B as an environmental factor in plant life: stress and regulation.
Trends Ecol. Evol. 12: 22-28.
Saunders, J. A. & McClure, J. W. 1976. The distribution of
ftavonoids in chloroplasts of twenty five species of vascular
plants. Phytochemistry 15: 809-810.
Siefermann-Harms. D. 1987. The light harvesting and protective
functions of carotenoids in photosynthetic membranes. Physiol.
Plant. 69: 561-568.
Strict, A. 1993. Alteration in expression of defense genes in Pisum
sativum after exposure to supplementary ultraviolet-B radiation.
Plant Cell Physiol. 34: 949-953.
Strid, A., Chow, W. S. & Anderson. J. M. 1990. Effects of sup-
plementary ultraviolet-B radiation on photosynthesis in Pisum
sativum. Biochim. Biophys. Acta I 020: 260-26R.
Stuiver.C. E. E, De Kok. L. J. & Kuiper. P. J. C. 1992. Freezing
tolerance and biochemical changes in wheat shoots as affected
by H2S fumigation. Plant Physiol. Biochem. 30: 47-55.
Takahama, U. 1982. Suppression of carotenoid photobleaching by
kaempfcrol in isolated chloroplasts. Plant Cell Physiol. 23: 859-
864.
Teramura. A. H. & Sullivan. J. H. 1994. Effects of UV-B radiation
on photosynthesis and growth of tetTestrial plants. Photos. Res.
39: 463-473.
Tcramura, A. H. & Ziska, L. H. 1996. Ultraviolet radiation and pho-
tosynthesis. Pp. 435-450. In: Baker. N. (ed.), Photosynthesis and
the Environment. Kluwer Academic Publishers, Dordrecht.
Tevini, M. & Teramura, A. H. 1989. UV-B effects on teiTestrial
plants. Photochem. Photobiol. 50: 479-487.
Thiele, A., Schirwitz, K., Winter K. & Krause, G. H. 1996. In-
creased xanthophyll cycle activity and reduced D I protein inacti-
vation related to photoinhibition in two plant systems acclimated
to excess light. Plant Sci. 115: 237-250.
Tosserams, M. Pais de Sit, A. & Rozema, J. 1996. The effect of
solar UV radiation on four plant species occurring in a coastal
grassland vegetation in the Netherlands. Physiol. Plant. 97: 731-
739.
Venema, J. H., Posthumus, F., De Vries, M. & Van Hassel!, P. R.
1999. Differential response of domestic and wild Lycopersican
species to chilling under low light: growth. carbohydrate content,
photosynthesis and the xanthophyll cycle. Physiol. Plant. 105:
81-88.
Weisscnbiick, G., Hedrich, R. & Sachs, G. 1986. Secondary pheno-
lic products in isolated guard cell. epidermal cell and mesophyll
cell protoplasts from pea (Pisum sativurn L.) leaves: distribution
and determination. Protoplasma 134: 141-148.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

A dune grassland ecosystem. with Planwgo lanceolaw. At the background UV-B fi ltration and UY-B
lamp systems. (Photograph by J. Rozema)
 Plant Ecologv 154: 159-168. 2001.
159
© 2001 Kluwer Academic Publishers.

Nutrient availability influences UV-B sensitivity of Plantago lanceolata

Marcel Tosserams*, Jaqueline Smet, Erwin Magendans & Jelte Rozema

Department of Systems Ecology, Faculty (~fBiologv, Vrije Universiteit, De Boe/elaan 1087, 1081 HV Amsterdam.
The Netherlands(* Address for correspondence: Montferland 8, 8245 BM Lelvstad, The Netherlands,
E-mail: m.tosserams@ riza. rws.minvenw.nl)

Key words: Carbohydrates, Dune grassland ecosystem, Nutrients, Ozone depletion, Plantago lanceolata, UV
absorbing pigments, UV-B radiation

Abstract
Seeds of Plantago lanceolata were collected in a dune grassland ecosystem in the Netherlands. Plants were grown
in a greenhouse for 61 days under either low or high nutrient conditions and were exposed to four different
levels of biologically effective UV-B radiation. The highest UV-B exposure level simulated 30% reduction of
the stratospheric ozone layer during summertime in the Netherlands. Total biomass production of plants at low
nutrient supply was 50% lower compared to plants grown at high nutrient supply, while net photosynthesis was
decreased by only 12%. Increased levels of UV-B reduced biomass production under non-limiting nutrient condi-
tions only. Biomass production of plants grown at limited nutrient supply was not affected by UV-B. This response
was correlated to increased accumulation of carbohydrates under nutrient limitation, which agrees well with the
carbon/nutrient balance hypothesis. It is concluded that the increased accumulation of carbon in nutrient-stressed
plants, may lead to a reduction of UV-B induced damage because of increased foliar UV- B absorbance by enhanced
accumulation of phenolic compounds and leaf thickening.

Introduction the UV-B response of plant species may be modified

Predictions indicate that the continued reduction in the
stratospheric ozone layer will have reached its max-
imum around the year 2000, with a slow recovery
over the subsequent 50 years (World Meteorological
Organization 1994). Relative to 1960 the maximum
ozone reductions expected at northern mid-latitudes
are 13% in winter and spring and 7% in summer and
fall (Madronich et al. 1995). As a result, the solar UV-
B fluence at the earth's surface increases (Blumthaler
& Ambach 1990; World Meteorological Organization
1994). This may have implications for plant life, since
plant development and physiology may already he af-
fected by present-day solar UV-B levels (Tosserams
et al. 1996; Visser et al. 1997).
Sensitivity to enhanced UV-B radiation, however,
differs considerably among agricultural and natural
plant taxa (Biggs et al. 1981; Krupa & Kickert 1989;
Rozema et al. 1997; Tosserams ct al. 1997). In addi-
tion, results of several studies have demonstrated that
by other environmental factors like photosynthetically
active radiation (PAR; Cen & Bornman 1990; Adamse
& Britz 1992; Kramer et al. 1992), atmospheric C02
concentration (Van de Staaij et al. 1993; Sullivan
1997; Visser et al. 1997), water availability (Tevini
et al. 1983; Murali & Teramura 1986; Balakumar
et al. 1993) and nutrient availability (Bogenrieder &
Doute 1982; Murali & Teramura 1985, 1987; Musil
& Wand 1994; Deckmyn & Impens 1997; Ernst et al.
1997 ). Studies on the combined effects of UV- B and
other environmental factors on plant performance are,
however, scanty. These studies are of importance for
the accurate assessment of potential consequences of
stratospheric ozone reduction on plant species of nat-
ural ecosystems. In their natural environment plants
are seldomly affected by a single stress factor only but
rather by a combination of stresses.
Nutrient availability may alter the plant's response
to enhanced UV-B levels. Phosphorus-deficient soy-
bean plants were less sensitive to UV-B than plants
160
grown at optimum phosphorus levels, which was re-
lated to the accumulation of secondary plant com-
pounds and leaf thickening (Murali & Teramura
1985),
An increased production of secondary plant com-
pounds at low nutrient availability as observed in
the study by Murali & Teramura ( 1985), agrees well
with the carbon/nutrient balance hypothesis (Bryant
ct al. 1983). This hypothesis proposes that the rel-
ative availability of carbon and nutrient resources in
the local environment greatly influences the amount
of carbon-based secondary plant compounds found in
plant tissues by mediating plants' carbohydrate stores.
One important prediction that results from the car-
bon/nutrient balance theory is as follows (Fajer et a!.
1992): under low-nutrient conditions the reduction in
plant growth is generally greater than the reduction in
photosynthesis. This leads to the accumulation of car-
bohydrate concentrations in excess of those needed for
plant growth, which will drive the additional synthesis
of carbon-based secondary compounds. Thus plants
grown under low-nutrient conditions will have higher
concentrations of carbon-based secondary compounds
in their tissues than plants grown under high-nutrient
conditions (Bryant et al. 1983).
Support for this carbon/nutrient balance theory
came from experiments where plants were grown at
low availability of nutrients or water. These plants
show increased concentrations of condensed tannins,
lignin, total phenols and/or phenol glycosides (Lam-
hers 1993, and references therein).
Both the accumulation of carbon-based secondary
compounds as well as the increase in leaf thickness
that might occur as a result of nutrient limitation could
have important consequences for the UV-B sensitivity
of plants. Apart from their ecological functions such
as allelopathy, deterrence of herbivores, attraction of
pollinators and of organisms that feed on herbivores
(Lambers 1993), secondary carbon-based plant com-
pounds, such as flavonoids and related phenolics are
well known for their UV-B absorbing (Caldwell et al.
1983) and antioxidant properties (Larson 1988).
In response to increased UV-B radiation epider-
mal concentrations of flavonoids and related phenolics
may increase (Teramura & Sullivan 1994). As a con-
sequence, transmittance of UV-B radiation can be
reduced to a large extent (Robberecht & Caldwell
1978) thus protecting underlying leaf structures from
potential damage. The protective function of these
secondary compounds was illustrated by experiments
in which Arabidopsis thaliana mutants deficient in
flavonoid biosynthesis appeared to be much more sen-
sitive to UV-B than wild-type plants (Li eta!. 1993;
Lois & Buchanan 1994; Landry eta!. 1995).
In addition to the protective function of UV-B
absorbing compounds, leaf thickening may reduce
UV-B damage too. Upper leaf tissue layers might
act as anatomical screens or filters to decrease UV-B
transmittance to lower cell layers (Teramura 1983).
In recent experiments on the effects of UV-B radi-
ation on species of a Dutch dune grassland vegetation
(Tosserams et al. 1997), greenhouse-grown as well
as field-grown Plantago lanceolata proved to be sen-
sitive to elevated doses of UV-B radiation. In these
experiments plants were grown under conditions of
sufficient water and nutrient availability. In view of the
above mentioned carbon/nutrient balance hypothesis,
however, the effects of enhanced UV-B on P lanceo-
lata growth and physiology might be less pronounced
at low nutrient conditions. It is the objective of this
experiment to test this hypothesis by studying the
combined effects of nutrient availability and UV-B
radiation on growth and physiology of P lanceolata.
Material and methods
Growth conditions
Seeds of Plantago lanceolata L. were collected in a
dune grassland near Heemskerk (NL). A random sam-
ple of the collected seeds was sown on plastic trays
(45 x 30 x 8 em), containing commercial potting soil
(Jongkind, Aalsmeer, NL) and cultivated in a green-
house. After germination individual seedlings were
transplanted to pots (2.6 dm 3 ), containing a mixture
of commercial potting soil and sand ( 1:1 ). In 50% of
the pots (80), seedlings were supplied with 0.5 g dm- 3
soil (low nutrient treatment) Osmocote controlled re-
lease fertiliser (N:P:K:Mg:Fe:,l3:13:13:3:2:, Grace
Sierra Int., Heerlen, NL), while in the other pots
4 g dm- 3 soil (high-nutrient treatment) was added.
Three days later the experiment was started. The aver-
age temperature during the experiment varied between
15.3 oc (night) and 26.rc (day). Relative humidity
varied between 50% (day) and 98% (night). During
the experiment plants were watered regularly with
demineralised water.
Experimental set-up
Plants were grown at four UV-B (280-320 nm) lev-
els. Solar UV-B and most of the UV-A (320-400 nm)

was totally absorbed by the greenhouse cover. UV ra-
diation was artificially supplied by Philips 40 W/12
fluorescent lamps. UV-B tubes were attached at both
sides of a 400 W Philips HPI/T lamp that provided
250 {Lmol m- 2 s- 1 of photosynthetically active ra-
diation (PAR) 16 h daily. Maximal PAR received
by the plants at clear sky days was approximately
1000 {Lmol m- 2 s- 1.
Both nutrient treatments were divided into four
groups of 20 plants. One group received no UV-B
radiation (control), while the other three groups re-
ceived different UV-B levels. For these three UV-B
treatments, the radiation emitted by the 40 W/12 flu-
orescent tubes was filtered with cellulose diacetate
(0.1 0 mm thick; Tam boer & Co. Chemic, Haarlem,
NL), which absorbs radiation below 290 nm (UV-B-
treated plants). For the control treatment, polyester foil
(Mylar 0.13 mm thick), which absorbs all radiation
below 313 nm, was used. Because of photodegrada-
tion the cellulose diacetate was replaced twice a week
and the Mylar polyester foil once a week. All UV-B
treatments were carried out in duplo and plants were
rotated between duplicate treatments twice a week
to minimise positional effects within the greenhouse
compartment.
The spectral irradiance of the UV-B lamps was
measured with a double-monochromator spectrora-
diometer (Optronic Model OL 752, Orlando, FL,
USA). The calibration procedure of the spectrora-
diometer was similar to the one described in Tosse-
rams et al. ( 1997).
The generalised plant action spectrum (Caldwell
1971 ), normalised at 300 nm, was used to deter-
mine the biologically effective UV-B dose (UV-BBE).
Weighted daily UV-BBE dose was 0 for control plants
and 4.6, 7.6 and 10.6 kJ m- 2 day- 1 for UV-B treated
plants. Plants were irradiated in a square-wave fashion
from I 0.00 to 16.00 daily. Different UV-B treatments
were obtained by adjusting the height of the UY-B
lamps above the top of the plants. As plants grew, the
height of the tubes was adjusted in such a way that
UV-8 levels remained the same during the course of
the experiment. This was checked daily with a portable
UYX radiometer equipped with a UVX 31 sensor (San
Gabriel, CA, USA).
According to the empirical model of Green et al.
( 1980), the UV-BBE dose of the UV treated plants sim-
ulate a 0 (approximately ambient), 15 and 30% ozone
reduction, respectively, during clear sky conditions
on 21 June in Amsterdam (52 °N latitude ).The long-
term (1971-1993) mean stratospheric ozone thickness
161
measured in June at the KMI in Ukkel (Belgium,
51 oN latitude) and the RIVM in Bilthoven (NL; 52 oN
latitude), was used for the model calculations. The ab-
solute UV-A radiation emitted by the 400 W HPI/T
lamp and the UV-B tubes (excluding solar UV-A ra-
diation) was 1.6 for control plants and 1.2, 1.6 and
1.9 W m- 2 for UV-B treated plants respectively.
Growth measurements
After 61 days plants were harvested and separated into
leaves, roots and reproductive parts. Dry weights of
plant parts were measured after drying for at least
48 h at 70 oc. The number of additional shoots, leaves
and inflorescences per plant, as well as maximal
leaf length were determined. Leaf area was measured
with a Licor 3100 area meter (Li-Cor. Inc., Lincoln,
Nebraska, USA).
Photosynthesis
Net photosynthetic rates of the youngest fully devel-
oped leaves were measured 53 days after the start of
the experiment, using a portable infrared gas analyser
(LCA3 and broad-leaf 'Parkinson leaf chamber', An-
alytical Development Co., Hoddesdon, Herts, UK).
All measurements were performed inside a climatised
room at saturating PAR (1200 ±50 ttmol m- 2 s- 1).
Room temperature was maintained at 24.3 ± 0.2 oc.
Gas-exchange parameters were calculated according
to von Caemmcrer & Farquhar ( 1981 ).
Determination of carbohydrates, carbon and nitrogen
Soluble carbohydrate content and starch content of the
total dry leaf material per plant, was determined using
the anthrone method (Morris 1948; Yemm & Willis
1954).
Carbon and nitrogen percentages of dried leaf and
root material were measured by gas chromatography
(Kirsten 1979), using a Perkin-Elmer 2400 series II
CHNS/0 analyser.
Pigment analysis
After 32 and 49 days of treatment, UV-B ab-
sorbing pigments of the youngest fully developed
leaf were extracted from fresh leaf material with
a MeOH:H20:HCl (79:20: I v/v) solution. Samples
were heated in a waterbath (90 °C) for one hour. The
absorbance at 300 nm was recorded using a Perkin-
Elmer Lambda 15 UV /VIS scanning spectrophotome-
ter.
162
Table I. The effect of nutrient availability and UV-B radiation on total plant dry
weight and shoot-to-root ratio (SRR) of Plantago lanceo/ata after 61 days of treat-
ment. Values represent means of 16-20 replicates. Values in parenthesis indicate the
relative increase or decrease as a result of UV-B treatment when compared to the
control treatment (0 kJ m~ 2 day~ I UV-BBEl; ns =not signiticanl (P>0.05).

Nutrients UV-BsE Dry weight (g) SRR

(kJ m~ 2 day~l) Shoot Root Total

Low 0 2.19(100) 1.02 (100) 3.21 (100) 2.31

4.6 2.36 ( 108) 1.12(110) 3.48 (108) 2.17
7.6 2.09 (95) 1.18 (115) 3.26 (102) 1.90
10.6 2.30 ( 105) 1.04 (102) 3.34 ( 104) 2.38

High () 5.69 (100) 1.2H (100) 6.98 ( 100) 4.72

4.6 6.35 (115) 1.28 (99) 7.62 ( 109) 5.42
7.6 5.71 (100) 120 (94) 6.92 (99) 4.99
10.6 4.59 (81) 0.84 (65) 5.43 (78) 5.68

Statistics

Nutrient <0.001 ns <0.001 <0.001

UV-B 0.002 0.006 0.002 0.030
Nutrient x UV-B 0.002 0.045 0.008 ns

After 60 days of treatment, samples of the
youngest fully expanded leaves were taken for deter-
mination of chlorophyll content. Samples for chloro-
phyll determination were weighed, frozen in liquid
nitrogen and stored at -20 °C. Chlorophyll content
was estimated using the method of Arnon ( 1949).
Statistics
Most data were analysed by a two-way analysis of
variance (ANOVA; Sokal & Rohlf 1981 ), which tested
for main effects of nutrient supply, UV-B radiation and
their interaction. Dry weight and leaf area data were
transformed to their natural logarithms. To analyse the
data concerning the absorbance of methanolic leaf ex-
tracts a three-way ANOVA was used, which tested for
main etfects of nutrient supply, UVB radiation, time
and their interaction.
Results
After 61 days of treatment, the effect of nutrient
availability on the aboveground biomass production
(including reproductive material) of Plantago lance-
olata was evident. Shoot biomass of plants receiving
the low nutrient treatment was 50 to 62% lower as
compared to plants grown at the high nutrient treat-
ment (Table 1). Biomass of roots, however, was not
significantly affected by nutrient availability. As a re-
sult, shoot to root ratio of plants grown at low nutrient
level was lower than that of plants grown at the high
nutrient level.
UV-B radiation affected biomass production of
plants grown at high nutrient supply only at
10.6 kJ m~ 2 day~ I UV-BBE· Shoot biomass of plants
at low nutrient supply was not influenced by UV-8,
which explains the observed nutrient and UV-B inter-
action. The effect of UV-B on root biomass of plants
grown at high nutrient supply was more pronounced
than the effect on the shoot (maximum decrease of
35% and 19% respectively). Total biomass production
(Table 1) of plants grown at low nutrient availability
was not affected by enhanced UV-B levels, while total
biomass of plants grown at high nutrient supply was
reduced by 22% when exposed to 10.6 kJ m~ 2 day~ I
UV-BBE·
Leaf area per plant of low nutrient plants was sig-
nificantly less than the leaf area per plant of high
nutrient plants (Table 2). This was primarily caused
by a reduced production of leaves per plant but the
leaf area per leaf was also significantly smaller (P =
0.004) at low nutrient availability. A significant nutri-
ent and UV-B interaction was observed for total leaf
area (Table 2), as enhanced UV-B only reduced leaf
area of high nutrient treatment plants. The SLA was
affected by nutrient treatment only (Table 2). Plants of
 163
Table 2. The effect of nutrient availability and UV-B radiation on morphological characteristics and the specific leaf area
(SLA) of Plantago lanceolata after 61 days of treatment. Values represent means of 16-20 replicates: ns =not significant
IP > 0.05).

Nutrients UV-BsE Leaf area SLA Total number of Maximal leaf length
(kJ m- 2 day- I) (cm 2 ) (m2kg-11 Leaves Shoots lnH.orcsccnccs (em)

Low 0 425 21.1 2() 1.9 2.5 29.7

4.() 446 19.8 30 2.3 2.4 28.6
7.6 410 20.4 26 2.0 1.1 27.4
10.6 449 20.8 26 2.8 1.9 31.9

High 0 1345 24.9 72 6.5 8.2 35.8

4.6 1272 23.() M 5.9 12.8 35.4
7.6 1268 23.9 67 7.1 7.2 32.7
10.6 1083 24.8 64 7.3 7.3 33.8

Statistics

Nutrient <0001 <0.001 <1).001 <IJ.OOI <1).001 <0.001

UV-B ns ns ns ns ns 0.027
Nutrient x UV-B 0.011 ns ns ns ns ns

the high nutrient treatment had a significantly higher
SLA compared to plants receiving the low nutrient
treatment.
Of the morphological parameters studied only
maximal leaf length was significantly affected by both
nutrient availability and UV-B (Table 2). At high nutri-
ent availability leaf length was increased, while at both
nutrient treatments maximal leaf length was slightly
reduced by enhanced UV-B levels. The number of in-
florescences of low nutrient treatment plants was lower
compared to plants receiving sufficient nutrients. UV-
B however did not affect the number of inflorescences.
Timing of flowering was neither affected by nutrient
availability nor UV-B radiation (data not shown).
Net photosynthetic rate was only affected by nutri-
ent availability (Table 3). Plants grown at high nutrient
availability exhibited a stimulated photosynthetic rate
when compared to plants grown at low nutrient avail-
ability. When expressed on a fresh weight basis, total
chlorophyll content of low nutrient plants was higher
compared to plants grown at high nutrient supply. To-
tal chlorophyll content of low nutrient plants increased
with UV-BBE enhancement. In contrast to low nutrient
plants, chlorophyll content of high nutrient plants was
decreased at 10.6 kJ m- 2 day- 1 UV-BBE· Etfects of
nutrients and UV-B were similar for both chlorophyll
a and chlorophyll b (data not shown).
The C/N ratio of leaf material of low nutrient plants
was about twice as high compared to high nutrient
plants (Table 4 ). Compared to plants at high nutri-
ent supply, plants grown at low nutrient supply had a
higher carbon content. which in combination with the
lower nitrogen content resulted in an increase of the
C/N ratio. UV-B radiation slightly increased carbon
content but did not significantly affect nitrogen content
and C/N ratio of leaves.
Similar to the leaves, C/N ratio of roots grown
at low nutrient supply was higher compared to roots
grown at high nutrient supply (Table 4). As for leaves,
this was due to an increased carbon content in com-
bination with a strongly reduced nitrogen content.
Compared to low nutrient plants, the nitrogen content
of roots of plants grown at high nutrient supply was
increased at the highest UV-BsE level.
P lanceolala plants grown at low nutrient supply
accumulated between I Rand 35% more carbohydrates
in their leaves compared to plants grown at high nu-
trient supply (Figure I). The amount of both soluble
sugars and starch was increased under conditions of
low nutrient supply. Soluble sugars were increased by
28 to 49%, while starch content was increased by II
to 29%. UV-B did not influence the accumulation of
carbohydrates.
After 32 days of treatment, the absorbance of
methanolic leaf extracts at 300 nm was significantly
affected by nutrient supply (Figure 2). Except for the
highest UV-B exposure level, where no effect was
observed, plants grown at low nutrient availability ex-
164
Table 3. Net photosynthesis and total chlorophyll content of Plantago lance-
olata as affected by nutrient availability and UV-8 radiation. Measurements
were conducted 53 (photosynthesis) and 60 (chlorophyll) days after the start
of the experiment. Values represent means of 6 replicates; ns = not significant
(P>0.05).

Nutrients UV-BBE Net photosynthesis Total chlorophyll

(kJ m- 2 day- I) (limo! C02 m -2 ,-1) (mgg- 1 FW)

Low 0 12.8 2.39

4.6 12.2 2.40
7.6 12.4 2.77
10.6 12.1 3.18

High () 12.6 2.35

4.6 13.6 2.57
7.6 16.2 2.67
10.6 14.1 2.25

Statistics

Nutrient 0.001 0.015

UY-B ns 0.020
Nutrient x UV-B ns 0.001

hibited an increased UV-B absorbance compared to
plants grown at high nutrient supply. UV-B also tended
to increase absorbance at 300 nm (P = 0.056). After
49 days of treatment the absorbance of leaf extracts
from both nutrient treatments had increased compared
to the absorbance after 32 days. The difference in UV-
8 absorbance between both nutrient levels was still
present. After 49 days the stimulation of absorbance
due to nutrient limitation varied between 22 and 44%
(0 and 7.6 kJ m- 2 day- I UV-8sE respectively).
Discussion
Increased levels of UV-B decreased total biomass pro-
duction of P. lanceo/ata only under conditions where
nutrients were non-limiting for growth. Total biomass
production of plants grown under conditions of low
nutrient availability was not affected by enhanced lev-
els of UV-8 radiation. The observed UV-8-induced
reduction of biomass production under high nutrient
availability agrees well with results of earlier find-
ings on P. /anceolata, which were collected at the
same location as the plants used in the present study
(Tosserams eta!. 1997).
Murali & Teramura ( 1985, 1987) found similar re-
sults for soybean plants, which were grown at various
P nutrition levels. Although both UV-8 radiation and
low P supply produced deleterious etfects on plant
biomass, the effects were non-additive. Total plant
biomass at low P levels was not affected by UV-
8 enhancement while total biomass of plants grown
at high P levels was reduced by UV-8. They con-
cluded that plants experiencing P deficiency are less
sensitive to UV-8 than plants grown at optimum P
levels. In contrast Musil & Wand ( 1994) observed that
UV-8 inhibited above-ground biomass production of
the winter ephemeral Dimorphotheca pluvial is during
early developmental stages but only under conditions
of nutrient limitation. This interaction between nutri-
ent availability and UV-8 was, however, no longer
present in mature plants. Eventually growth of these
low nutrient plants was even stimulated by increased
UV-8 radiation. In this study of Musil & Wand (1994 ),
however, no evidence was found that carbohydrate ac-
cumulation had taken place and foliar organic nitrogen
content was not affected by the nutrient treatment. 8o-
genrieder & Doute ( 1982) also observed that the effec-
tiveness ofUV-8 radiation to limit biomass production
of Lactuca sativa and Rumex a/pinus diminished as
nutrient concentrations increased.
The decrease in maximal leaf length of P. lance-
o/ata in response to increasing UV-8 levels is in
agreement with earlier findings on this plant species
(Tosserams et a!. 1997). Reduction of leaf length or
shoot height in response to enhanced levels of UV-B is
 165
Table 4. Carbon and nitrogen content of leaves and roots of Plantago lanceolatu as affected
by nutrient availability and UV-B radiation after 61 days of treatment. A sample of the total
leaf material per plant was used for the determination. Values represent means of 6 replicates:
ns =not significant ( P >0.05 ).

Nutrients UV-BsE Leaf Root

(k.l rn- 2 day- 1 1 Ctfi N c;,. C!N c 'I<· N'lr C!N

Low 0 40.9 2.45 14.6 41.6 0.89 3.9

4.6 41.4 2.93 14.3 42.1 0.~4 5.9
7.6 41.6 3.04 13.8 42.5 0.73 3.7
10.6 40.6 2.84 14.5 42.8 0.90 4.0

High 0 35.4 7.34 5.0 41.1 2.13 2.2

4.6 37.5 5.71 6.8 41.3 1.88 2.5
7.6 37.7 5.66 6.9 41.4 2.24 2.3
10.6 37.0 5.79 6.6 40.9 2.93 1.2

Statistics

Nutrient <0001 <0.001 <Cl.OOl 0.042 <0.001 <0.001

UV-B ().()46 ns ns ns 0.014 ns
Nutrient x UV-B ns ns ns ns 0.037 ns

one of the most frequently reported UV-B effects for
plants. However, reductions in leaf length and shoot
height are not necessarily accompanied by reductions
of total plant biomass (Barnes et a!. 1990; Tosse-
rams et a!. 1997). Instead reduced leaf length may
reflect a specific photomorphogenic response of plants
to enhanced UV-B mediated by UV-B photoreceptors
(Balian~ et a!. 1995) or may be caused by reduction or
destruction of auxin (Ros & Tevini 1995).
The observed increase of UV-B absorbance for
P. lanceolata grown under conditions of nutrient lim-
itation is in agreement with the predictions resulting
from the carbon/nutrient balance hypothesis (Bryant
et a!. 1983; Fajer et al. 1992). Indeed the reduc-
tion in biomass accumulation of plants grown under
low-nutrient conditions was greater (50%) than the
reduction of net photosynthesis ( 12% ). As a result, ac-
cumulation of non-structural carbohydrates occurred
in leaves of low nutrient treatment plants. This was
reflected in the carbon content of the leaves. Foliar car-
bon content of low nutrient treatment plants was about
4% higher than in high nutrient plants. In addition.
carbon accumulation was reflected in an increased
accumulation of secondary compounds as indicated
by the increased UV-absorbance of methanolic leaf
extracts.
Lambers ( 1993) discussed two possible hypotheses
that may explain the accumulation of secondary com-
pounds when nutrient availability is limited. The first
hypothesis suggests that the synthesis of secondary
compounds is enhanced by sucrose levels in excess of
those required for protein synthesis, thus functioning
as a form of 'energy overflow· for plants in nutrient
limited environments. According to the second hy-
pothesis, it is the demand for amino acids for protein
synthesis which determines the incorporation of car-
bon into secondary compounds. The excess carbon
was at least partly used for the additional synthesis of
UV-B absorbing compounds since the UV-absorbancc
of mcthanolic extracts increased in response to nutri-
ent limitation.
UV-B enhancement by itself may also stimulate
the biosynthesis of UV absorbing compounds (Strid
et a!. 1994: Teramura & Sullivan 1994 ). In the present
experiment exposure to enhanced UV-B levels did
not affect UV-B absorbance of leaf extracts. This
is in accordance with earlier findings for P. lanceo-
lata obtained from greenhouse and field experiments
(Tosserams et a!. 1997 ).
A decrease of the SLAin response to UV-B radi-
ation has been frequently reported (Bornman & Tera-
mura 1993 ). In the present study however, UV-13 did
not affect the SLA. Instead, the accumulation of car-
bohydrates in leaves of low nutrient treatment plants
may have lead to the observed decrease of the SLA for
these plants, since reductions in SLA are often accom-
166
0.5
A D low nutrients
IZI high nutrients
0.4

120
~ D low nutrients
0.3
~
.... 121 high nutrients
'c.o 100 0.2
c.o
! 80
"'
~
"C
. 60
. 0.1

.~
;..
..CI ,.Q
Q
0
'f 40 Q
B
5 "'
..CI
<
:E 20 0.4
-§
VJ
0 0.3

100
0.2
~
80
....~ 0.1
c.o
c.o 60
!
..CI 0

s
<>

VJ
40 0 4.6 7.6 10.6

20
Figure 2. Effects of nutrient availability and UV-B on UV-B ab-
0 sorbance of methanolic leaf extracts of Plantago lanceolata after
32 (A) and 49 (B) days of treatment. Samples were taken from
~ 175 the youngest fully developed leaf. Absorbance was measured at
~
.... 300 nm and recalculated to the absorbance of I mg fresh leaf em- 3
0.0 150 extraction medium. Values are means of 5-6 replicates ± SEM.
c.o
! 125
Time and nutrient effects as well as their interaction were signifi-
cant (P <0.00 I for time and nutrient effects an P = 0.009 for their
"'
~
"C
100 interaction) when tested by a three-way analysis of variance. Effects
of UV-B and other interactions were not significant (P >0.05).
.S
.
75
Q
,.Q
01 50

s panied by increases in total carbohydrates (Farrar &

<>
25 Williams 1991 ).
Q
.....
0 The SLA gives an approximation of leaf thickness
0 4.6 7.6 10.6 and denotes leaf compactness (density) as welL Al-
UVBBE (kJ m-2 day-1) though preferably fresh weight data should be used
because the ratio between dry weight and fresh weight
Figure 1. Effects of nutrient availability and UV-B on soluble car-
bohydrate content, starch content and total carbohydrate content of leaves may vary (Dijkstra 1990), the reduced SLA
of Plantago lanceolata after 61 days of treatment. A sample of might indicate an increased leaf thickness/density for
the total leaf material per plant was used for the determination. low nutrient plants. Teramura ( 1983) suggested that an
Bars represent means of 5 replicates ± SEM. Only the nutrient
increased leaf thickness may act as a protective mech-
treatment caused significant effects on carbohydrate content, in all
cases the ANOVA P values for two-way analysis of variance were anism for plants to avoid potential damage caused
P :;:: 0.001. Effects ofUV-B or UV-B x nutrient were not significant by increased levels of UV-B. Upper leaf tissue layers
(P > 0.05). might act as anatomical screens or filters to decrease
UV-B transmittance to lower cell layers. Murali et al.
(1988) found for two cultivars of soybean that dif-
ferences in SLA correlated with differences in sensi-

tivity between both species. Bornman & Vogelmann
(1991) reported increases in leaf thickness of 45% in
Brassica campestris in response to UV-B exposure.
They concluded that leaves may compensate for UV
stress by increasing leaf thickness but they may con-
trol simultaneously the internal light gradient through
modifications of pigment content and light scattering
properties. However, decreases of SLA are not al-
ways found to correlate with UV-B radiation tolerance
(Biggs et al. 1981 ).
It is concluded that the effects of reduced nutri-
ent supply and increased UV-B radiation on growth
of P. lanceolata are non-additive. In contrast to plants
grown at high nutrient supply, growth of plants un-
der conditions of low nutrient supply was not af-
fected by increased levels of UV-B radiation. This
non-responsiveness of low nutrient supply plants to el-
evated UV-B radiation may be related to the enhanced
accumulation of phenolic compounds in response to
nutrient limitation and possibly also to an increase
of leaf thickness. These processes are related to the
accumulation of non-structural carbohydrates under
conditions of low nutrient supply.
Similar responses were also obtained in experi-
ments where UV-B enhancement was combined with
water stress. Balakumar et al. (1993) observed that
Vigna unguiculata plants were less sensitive to UV-B
enhancement under water stress due to accumulation
of phenolic compounds and leaf thickening.
Murali & Teramura (1986) found comparable re-
sults for field-grown soybean. Increased levels of UV-
B reduced leaf area, total plant biomass and net photo-
synthesis under well-watered conditions hut no signif-
icant UV-B effects were found in plants subjected to
water stress. The present and above mentioned experi-
ments indicate that nutrient or water stress may reduce
the potential damage caused by enhanced UV-B ra-
diation levels through anatomical and/or biochemical
changes.
The present experiment clearly illustrates that al-
though plants may exhibit UV-B sensitivity under op-
timal growth conditions this does not necessarily mean
that the same response can be expected in their natural
environment were plants usually have to respond to
several stress factors acting in concert.
Acknowledgements
We would like to thank Prof. Dr W. H. 0. Ernst for
helpful comments on the manuscript. This work was
167
financially supported by the Dutch National Research
Programme on Global Air Pollution and Climate
Change (NRP; Project: 850022).
References
/\damse, P. & Britz. S. 1. 1992. Amelioration of UV-B damage
under high iiTadiance. l: Role of photosynthesis. Photochem.
Photobiol. 56: 645-650.
Arnon. D. I. 1949. Copper enzymes in isolated chloroplasts.
Polyphenoxidase in Beta rulgarii. Plant Physiol. 24: 1-15.
Balakumar, T.. Hani Babu Vincent. V & Paliwal. K. 1993. On the
interaction of UV-B radiation (280-315 nm) with water stress in
crop plants. Physiol. Plant. 87: 217-222.
Balian;. C. L.. Barnes, P. W & Flint, S. D. 1995. Inhibition of
hypocotyl elongation by ultraviolet-B radiation in de-etiolating
tomato seedling. l The photoreceptor. Physiol. Plant. 93: 584-
592.
Barnes. P W.. Flint, S.D. & Caldwell. M. M. 1990. Morphological
responses of crop and weed species of different growth forms to
ultraviolct-B radiation. Am. 1. Bot. 77: 1354-1360.
Biggs. R. H., Kossuth. S. Y. & Teramura, A. H. 19X I. Response
of 19 cultivars of soybeans to ultraviolet-B irradiance. Physiol.
Plant. 53: 19-26.
Blumthaler, M. & Ambach, W. 1990. Indication of increasing solar
ultraviolet-B radiation flux in alpine regions. Science 248: 206.
Bogcnricder, A. & Doute, Y. 1982. The effect of UV on photosyn-
thesis and growth in dependence of mineral nutrition (l,actuca
satim L. and Rumex a/pinus L. ). Pp. 164-168. In: Bauer. H. eta!.
(cds), Biological Effect of UV-B Radiation. Munich, Germany.
Bornman, 1. F. & Teramura, A. H. 1993. Effects of ultraviolct-B
radiation on tetTestrial plants. Pp. 427-471. In: Young, A. R.
eta!. (eds). Environmental UV Photobiology. Plenum Press, New
York.
Bornman, 1. F. & Vogelmann, T C. 1991. Effect ofUV-B radiation
on leaf optical propet1ies measured with fibre optics. 1. Exp. Bot.
42: 547-554.
Bryant. J.P., Chapin. F. S. & Klein, D. R. 1983. Carbon/nutrient
balance of boreal plants in relation to vertebrate herbivory. Oikos
40: 357-368.
Caldwell. M. M. 1971. Solar UV iiTadiation and the growth and
development of higher plants. Pp. 131-177 In: Giese. A. C. (ed. ).
Photophysiology, Vol. 6. Academic Press, New York.
Caldwell, M. M. 1983. Internal filters: Prospects for CV-acclimation
in higher plants. Physiol. Plant. 58: 445-450.
Cen, Y-P. & Bornman, 1. F. 1990. The response of bean plants to
UV-B radiation under ditlcrent iiTadianccs of background visible
light. 1. Exp. Bot. 41: 1489-1495.
Dcekmyn. G. & lmpens. I. 1997. Combined etlects of enhanced UV-
B radiation and nitrogen deficiency on the growth. composition
and photosynthesis of rye (Scca/c cereci/e). Plant Ecol. 128: 235-
240.
Dijkstra, P. 1990. Cause and effect of ditlerences in specific leaf
area. Pp. 125-140. In: Lambers, H. et a!. (eds), Causes and
Consequences of Variation in Growth Rate and Productivity
of Higher Plants. SPB Academic Publishing. The Hague. the
Netherlands.
Ernst. W. H. 0 .. Van de Staaij, 1. W. M. & Nelissen, H. 1. M. 1997.
Reaction of savanna plants from Botswana on UV-B radiation.
Plant Ecol. 128: 162-170.
F'uer. E. D., Bowers, M. D. & Bazzaz, F. A. 1992. The effect of nu-
trients and enriched C0 2 environments on production of carbon-
168
based allelochemicals in Plantago: a test of the carbon/nutrient
balance hypothesis. Am. Nat. 140: 707-723.
Farrar, J. F. & Williams, M. L. 1991. The effects of increased
atmospheric carbon dioxide and temperature on carbon partition-
ing, source-sink relations and respiration. Plant Cell Environ. 14:
819-830.
Green. A E. S., Cross, K. R. & Smith, L. A. 1980. Improved
analytic characterization of ultraviolet skylight. Photochem. Pho-
tobiol. 31: 59-65.
Kirsten, W. G. 1979. Automatic methods for the simultaneous de-
termination of carbon, hydrogen, nitrogen and sulfur, and for
sulfur alone in organic and inorganic materials. Anal. Chern. 51:
1173-1197.
Kramer, G. F., Krizek, D. T. & Mirccki, R. M. 1992. Influence of
photosynthetically active radiation and spectral quality on UV-
R-induced polyamine accumulation in soybean. Phytochem. 31:
1119-1125.
Krupa, S. Y. & Kickert, R. N. 1989. The greenhouse effect: The
impacts ofultraviolet-B (UV-B) radiation, carbon dioxide (C02)
and ozone (03) on vegetation. Environ. Pollut. 61: 263-293.
Lambcrs, H. 1993. Rising C02. secondary plant metabolism, plant-
herbivore interactions and litter decomposition: Theoretical con-
siderations. Vegetatio 104/105: 263-271.
Landry, L. G., Chapple, C. C. S. & Last, R. L. 1995. Ara-
bidopsis mutants lacking phenolic sunscreens exhibit enhanced
ultraviolct-B injury and oxidative damage. Plant Physiol. 109:
1159-1166.
Larson, R. A. 1988. The antioxidants of higher plants. Phytochem.
27: 969-978.
Li, J .. Ou-Lec, T., Raba, R., Amundson, R. G. & Last, R. L.
1993. Arabidopsis flavonoid mutants arc hypersensitive to UV-B
irradiation. Plant Cell 5: 171-179.
Lois, R. & Buchanan, B. B. 1994. Severe sensitivity to ultraviolet
radiation in an Arabidopsis mutant deficient in flavonoid accu-
mulation. II Mechanisms ofUY-rcsistance in Arahidopsis. Planta
194: 504-509.
Madronich, S., McKenzie, R. L.. Caldwell, M. M. & Bjorn,
L. 0. 1995. Changes in ultraviolet radiation reaching the earth's
surface. Ambia 24: 143-152.
Morris, D. L. 1948. Quantitative determination of carbohydrates
with Dreywood's anthrone reagent. Science 107: 254-255.
Murali, N. S & Teramura, A. H. 1985. Effects of UV-B inadiance
on soybean. VI. Influence of phosphorus nutrition on growth and
flavonoid content. Physiol. Plant. 63: 413-416.
Murali, N. S. & Teramura, A. H. 1986. Effectiveness of UV-B ra-
diation on the growth and physiology of field-grown soybean
modified by water stress. Photochem. Photobiol. 44: 215-219.
Murali, N. S. & Teramura, A. H. 1987. Insensitivity of soy-
bean photosynthesis to ultraviolet-B radiation under phosphorus
deficiency. J. Plant Nutr. 10:501-515.
Murali, N. S., Teramura, A. H. & RandalL S. K. 1988 Response
differences between two soybean cultivars with contrasting UV-
B radiation sensitivities. Photochem. Photobiol. 44: 1-5.
Musil, C. F. & Wand, J. E. 1994. Ditferential stimulation of
an arid-environment winter ephemeral IJimorphoteca pluvialis
(L.) Moench by ultraviolet-B radiation under nutrient limitation.
Plant Cell Environ. 17: 245-255.
Robberecht, R. & Caldwell, M. M. 1978. Leaf epidermal trans-
mittance of ultraviolet radiation and its implications for plant
sensitivity to ultraviolet-radiation induced injury. Oecologia 32:
277-287.
Ros, J. & Tevini, M. 1995. Interaction of UV-radiation and IAA
during growth of seedlings and hypocotyl segments of sunflower.
J. Plant Physiol. 146: 295-302.
Rozema, J., Van de Staaij, J. W. M. & Tosserams. M. 1997. Etlects
of UV-B radiation on plants from agro- and natural ecosys-
tems. Pp. 213-232 In: Lumsden, P. L. (ed), Plants and UV-8.
Cambridge University Press, Cambridge.
Sakal, R. R. & Rohlf. F. J. 1981. Biometry. Freeman Press, San
Fransisco, California, USA.
Strid, A., Chow, W. S. & Anderson, J. M. 1994. UV-B damage and
protection at the molecular level in plants. Photosynth. Res. 39:
475-489.
Sullivan, J. H. 1997. Etfccts of increasing UV-B radiation and at-
mospheric C02 on photosynthesis and growth: Implications for
tenestrial ecosystems. Plant Ecol. 128: 194-206.
Sullivan, J. H. & Tcramura, A. H. 1994. The effects of UV-B ra-
diation on loblolly pine. 3. Interaction with C02 enhancement.
Plant Cell Environ. 17:311-317.
Teramura, A. H. 1983. Effects of ultraviolet-R radiation on the
growth and yield of crop plants. Physiol. Plant. 58:415-427.
Teramura, A H. & Sullivan, J. H. 1994. Effects of UY-B radiation
on photosynthesis and growth of tcncstrial plants. Photosynth.
Res. 39: 463-473.
Tcvini, M., Iwanzik, W. & Tcramura, A. H. 1983. Etlects of UV-B
radiation on plants during mild water stress II. Etlects on growth,
protein and flavonoid content. Z. Pflanzenphysiol. 110:459-467.
Tosserams, M., Pais de Sa, A. & Rozema, J. 1996. The effect of
solar UV radiation on four plant species occurring in a coastal
grassland vegetation in The Netherlands. Physiol. Plant. 97:731-
739.
Tosscrams, M., Magendans, G. W. H. & Rozema, J. 1997. Ditferen-
tial effects of elevated ultraviolet-B radiation on plant species of
a dune grassland ecosystem. Plant Ecol. 128: 266-281.
Van de Staaij, J. W. M., Lenssen. G. M., Stroetenga, M. & Rozema,
1. 1993. The combined e!Tects of elevated C02 and UV-B ra-
diation on growth characteristics of Elymus athericus. Vcgetatio
104/105: 433-439.
Visser, A. J., Tosserams, M., Groen, M. W., Magendans, G. W. H.
& Rozema. J. 1997. The combined effects of C02 concentra-
tion and solar UV-B radiation on faba bean grown in open-top
chambers. Plant Cell Environ.20: 189-199.
Yon Caemmerer, S. & Farquhar, G. D. 1981. Some relationships be-
tween the biochemistry of photosynthesis and the gas exchange
of leaves. Planta 160: 320-329.
Yemm, E. W. & Willis, A. J. 1954. The estimation of carbohydrates
in plant extracts by anthrone. Biochem. J. 57: 508-514.
World Meteorological Organization 1994. Scientific assessment of
ozone depletion. Executive summary of WMO Ozone report
No. 37, pp. 1-12.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

External hyphae of arbuscular mycorrhizal fungi: (AMF) entwining roots or Co/omagrostis epigeios or the dune
grassland. (Photograph by J. Van de Staaij)
 Plont Ecoiogv 154: 171-177, 200 I.
171
© 2001 Kluwer Academic Publishers.

Increased solar UV-B radiation may reduce infection by arbuscular

mycorrhizal fungi (AMF) in dune grassland plants: evidence from five
years of field exposure

J. van de Staaij, J. Rozema, A. van Beem & R. Aerts

Department of Systems Ecology, Faculty of' Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HVt Amsterdam,
The Netherlands

Key words: AMF, Arbuscules, Arbuscular mycorrhizal fungi, Biodiversity, Calamagrostis epigeios, Carex
arenaria, Flavonoids, Grasslands, Myconhiza, Root exudates, UV-B radiation, YAM

Abstract
An area of coastal dune grassland, dominated by the gramineous species Calamagrostis epigeios and Co rex are-
naria, was exposed to enhanced levels of UV-B radiation during a five year period. These species showed reduced
AM-fungal infection percentages in their roots. In C. epigeios AM infection was reduced by 18%, C. arenaria
showed a reduction by 20%. The major effect of enhanced UV-B on AM associations was a reduction of the
number of arbuscules. This indicates a reduction in the exchange of nutrients between the symbionts. Since the
effect of UV-B on AM associations may result from altered flavonoid levels in the root exudates of the host plants,
flavonoid levels in the roots were investigated. No detectable flavonoid concentrations were found in the roots of
C. epigeios and C. arena ria . Less effective AM associations can have pronounced negative effects on biodiversity
and nutrient dynamics of the dune grassland ecosystem. The possible mechanisms causing these indirect effects of
elevated UV-B on below ground AM associations are discussed. We conclude that UV-B induced changes in plant
hormone levels are more likely to be the mechanism reducing AMF infection than UV-B induced alterations in
flavonoid concentrations in the root exudates of the host plant.

Abbreviations: AMF- arbuscular mycorrhizal fungus (fungi, fungal)

Introduction mass production are predicted (Rozema et al. 1997;

Since the mid-seventies it is known that human activ-
ity is causing a breakdown of the stratospheric ozone
layer (Molina & Rowland 1974, Russell et al. 1996).
This knowledge triggered research on the possible
effects of the resulting increase in ultra violet-B radi-
ation (280-315 nm) exposure (Biumthaler & Ambach
1990) of organisms. Present knowledge on the effects
of increasing UV-B radiation fluxes on plants grow-
ing in their natural habitat indicates that the direct
effects on individual plants will, for most species, be
restricted to alterations in their morphology (Corlett
et al. 1997) and in their secondary metabolism (Van
de Staaij et al. 1995 ). No major reductions in bio-
Caldwell et al. 1998; Rozema et al. 1999).
Changes in the secondary metabolism are centred
around the phenylpropanoid pathway. UV-B induces
an increased synthesis of phenylalanine ammonia
lyase (PAL) and chalcone synthase (CHS). Increased
levels of these enzymes may result in an enhanced
metabolic activity of the phenylpropanoid pathway
(for a review see Meijkamp et al. 1999). Flavonoids
and related (poly)phenolic compounds resulting from
this metabolic route act as an UV-B absorbing screen
in the epidermis, protecting plant tissues from UV-B
radiation damage (Van de Staaij et a!. 1995; Reuber
et al. 1996).
In addition to their function in UV-B absorp-
tion, many of these phenolics are multi-functional and
172
can also act as messengers in plant-environment in-
teractions (Shirley 1996). Therefore, UV-B induced
changes in the secondary metabolism may affect in-
teractions between plants and their associated (mi-
cro)organisms. Recent research suggests an impor-
tant impact of (above ground) solar UV-B on below-
ground biological processes. In a greenhouse exper-
iment (vesicular)-arbuscular mycorrhiza (AM) asso-
ciations of Acer saccharum (sugar maple) showed
reduced numbers of arbuscules when the host plants
were grown under increased UV-B fluxes (Kiironomos
& Allen 1995). These plant root-soil fungus interac-
tions are mutualistic in nature and their main impact is
a stimulation of plant growth by increasing the uptake
of various nutrients, phosphorus in particular. Plants
grown under elevated UV-B, showed AM associations
with a reduced number of arbuscules. Since these
organs are the interface in which nutrient exchange
between plant and fungus takes place a reduction in
numbers indicates a less active association.
As discussed above UV-B radiation induces an in-
creased synthesis of flavonoids in many plant species.
Flavonoids excreted by the plant roots influence AM
fungal spore germination and hypha! growth (Siqueira
et al. 1991; Chabot et al. 1992). Whether the ob-
served reductions in AM associations can be attributed
to alterations in the secondary metabolism of the host
plant remains uncertain at present (Van de Staaij et al.
1999).
AM fungi form mutualistic associations with the
roots of about 80% of all terrestrial plant species and
are abundant in the soils of most ecosystems (Ozinga
et al. 1997). AM associations are especially important
in grassland species growing on mineral soils (Allen
1993). The composition of the AM-fungal populations
may determine the biodiversity and productivity of
ecosystems (Van der Heijden et al. 1998a, b). Further-
more infection with AM fungi may offer protection
against pathogenic fungi (Newsham et al. 1995) and
against herbivorous insects (Gange & West 1994 ).
Therefore it is possible that elevated UV-B induced
changes in AM associations could affect ecosystem
functioning.
UV-B radiation is known to affect other plant-
fungus interactions as well. The colonisation of Vi'Ic-
cinium spp. and Quercus robur litter by saprotrophic
fungi is reduced by UV-B (Gehrke et al. 1995; New-
sham eta!. 1997a). Furthermore UV-B can reduce the
frequency of phylloplane fungi on plants (Newsham
et al. 1997b ). Infection with fungi can modify the
effect UV-B has on plants. When infected with the
leaf endophytic fungus Neotyphodium lolii the fertil-
ity of Lolium perenne is reduced under elevated UV-B
(Newsham et al. 1998).
Individual plants grown in pots under controlled
environmental conditions may show different re-
sponses to experimental manipulation compared to
plants grown under field conditions. Therefore, must
be recognised that both for studies on UV-B effects
(Rozema et al. 1997) and for studies on AMF (New-
sham 1995), results obtained in greenhouse experi-
ments cannot always be directly extrapolated to a field
situation.
In this paper we report on the observed changes in
AM associations in plants from a long-term field ex-
periment. A semi-natural coastal dune grassland was
radiated with UV-B, simulating a 15% ozone reduc-
tion, over a 5-year period. At the end of this period the
two dominant plant species, the monocots C. epigeios
and C. arenaria, were screened on the presence and
composition of AM associations in their roots. To as-
sess whether UV-B might influence the composition
of root exudates involved in the formation and mainte-
nance of AM associations, we determined the levels of
flavonoids in the roots of C. epigeios and C. arenaria.
Materials and methods
Experimental site
The experimental site is located at the inland site
of a coastal dune complex within the nature reserve
'Het Noordhollands Duinreservaat' near Heemskerk
(52°30' N, 4°40' E), NL. UV-B lamps, installed in
racks, were placed above the vegetation in a semi-
natural grassland. Two elevated and two ambient
UV-B radiated plots, measuring I .2 m x 2 m, were
created. To assure homogeneity of radiation only the
central 0.9 m x 1.6 m were used for experiments.
The plots have been radiated for a 5-year period
(1992-I 997).
Since I960 the site is mechanically mown once a
year in autumn or winter (Rozema et al. 1999). During
the experimental period the area has not been mown.
The soil consists of sand low in nutrients (Ernst et al.
I 984 ). The top soil layer contains the bulk of the plant
roots and organic matter is approximately 5 em thick.
UV-B radiation
Philips TL 12/40 fluorescent tubes were used to gener-
ate UV-B radiation. At an ambient background level of
 173

5 kJ m- 2 day- 1 UV-BBE (June 21 , cloudless sky), el- 100

evated UV-B plots received a suppleme ntal UV-B dose

of 2.5 kJ m- 2 day- 1 UV-BBE, calculated using the 80 ,.......
Generalised Plant Action Spectrum (Caldwell 1971 ), c
0 )_ *
normalised at 300nm. This simulates a stratospheric ".::l
<.)
60
~
ozone depletion of 15% (Green et al. 1980). Radiation
·=
from the lamps above the elevated UV-B plots was
filtered using cellulose diacetate foil (0.1 mm) to elim- ~
40 ~
~

J
inate radiation below 290 nm. Lamps above control 20
plots were filtered using Mylar foil (0. 13 mm) elimi-
nating radiation below 31 3 nm. The combined use of 0
cellulose diacete and Mylar foils ensured that UV-A June Apnl June October

fluxes in the ambient and elevated plots were the same.
Foils were replaced weekly to avoid etfects of photo
degradation on radiation levels.
AMF infection percentages
Root samples were collected in the field in 1997, dur-
ing spring (April 10), summer (June 14) and autumn
(October 6). On each date one vegetation-soil sample
(30 em x 30 em x 10 em) per plot, was taken. The
samples were taken to the laboratory where the soil
was washed from the plant roots.
After washing roots of Ca /amagrostis epigeos and
Carex arenaria were selected. Only roots attached to
shoots were used to make species identification pos-
sible. To obtain enough root material for both deter-
mination of AMF infection percentages and flavonoid
extractions, the roots of the elevated UV-B plots were
pooled. Roots from ambient plots were pooled as well.
Of the collected roots for each treatment -species com-
bination 4 microscope samples were prepared. For
each sample arbuscules, vesicles, spores and hyphae
were scored. A minimum of seventy five root sec-
tions per microscope sample was examined. Infection
percentages were calculated for each microscope sam-
ple. Infection percentages of samples from elevated
plots were tested against infection percentages from
ambient plots.
After clearing the selected roots with a I 0% KOH
solution (45 minutes at 90 °C), roots were stained us-
ing chlorazol black E in a lactoglycerol solution ( 120
minutes at 90 °C) (Brundrett et al. 1984 ).
AMP infection percentages were determined
by microscopical inspection of root-sections, one
microscope-field (0.19 mm) wide, on the occurrence
of external hyphae, internal hyphae, spores, vesicles
and/r arbuscules. Roots used measured between l 0
and 20 J.Lm in diameter.
C. arenaria C. epigeios
Fi!!.urc I. AMF infection perce ntages (with s.e. m. ) for the spec ies
Care.r orenarht and Ca famagrostis epigeios. Plants were grown un-
der ambient (white bars) or elevated (dark burs) leve ls of UV-B.
Infection pcrce ntoges were determi ned in June for C. arenario and
in Apri l, Ju ne and October lor C. epigeios. Statistically signiticam
difference between treatments is indicated w ith an asterisk.
Since all fungal structures (non AMF included)
are coloured using chlorazol black E, infection was
only considered being AM if internal hyphae were
accompanied by vesicles and-or arbuscul es.
Flavonoid extraction and sample preparation
Methods used for fl avonoid extraction and analyses
are described in Van de Staaij et al. 1995 .
Statistics
All data were stati sticall y analysed using one way
analysis of variance (ANOVA) to test significance
(p < 0.05) of UV-B treatment (Sokal & Rolf 1995).
Prior to analysis data were tested on homogeneity of
vari ances.
Results
AMF infection
UV-B radiation affected AMF infection of Ca/ama-
g rostis epigeios in the field. In June 1997, C. epigeios
showed a statistical significant reduction in AMF in-
fection percentages when grown under the elevated
UV-B regime (Figure l ).
The reduction in AMF infection percentages in C.
epigeios in June was mainly the result of a reduction
in the number of arbusculcs (Table I) . The number of
vesicles showed no UV-B induced reduction (Table I).
174
Table I. Average percentages (SD in parenthesis) of root sections containing hyphae
(no distinction between AMF and non-AMF hyphae is made), arbuscules, vesicels or
spores of the species Ca/amagrostis epigeios and Carex arenaria. Plants were grown
under ambient (-) or elevated (+) UV-B radiation. C. epigeios was harvested in
April, June and October. C. arenaria was harvested in June. Statistically significant
differences ( P < 0.05) between treatments are indicated with•.

Species UV-B %hyphae % arbuscules %vesicles %spores

treatment

C. epigeios
April 96 (3) 20 (8.6) 23 (4.8) 5 (0.9)"
+ 97 (2) 12 (0.1) 35 (6.8) I (O.l)a
June 95 (3) 57 (9.5)a 17(13.0) 8 (3.9)
+ 93 (4) 37(1.4)a 15 (3.7) 6 (4.2)
October 99 (3) 27 (8.0) 32 (5.8) 5 (1.0)
+ 100 18(10.5) 22 (10.0) 6 (5.6)

C. arenaria
June 94 (3) 64 (19.7) 22 (22.5) 6 (1.7)
+ 98 (4) 35 (27.1) 38 (5.7) 14 (13.6)

For C. epigeios infection percentages were also
scored for April and October, for these periods no
significant UV-B induced reductions were observed
(Figure I, Table I). The percentage of root-sections
containing spores was significantly depressed in C.
epigeios in April only (Table I).
The percentage of root-sections containing fungal
hyphae was very high in all C. epigeios and Carex
arenaria samples (Table 1). Since chlorazol black E
stains all fungal structures, it has to be noticed that
no distinction between AMF hyphae and other fungal
hyphae could be made.
For C. arenaria the reduction in AMF infection
percentages was a trend only and not statistically
significant (Figure I) (p=0.07).
In C. arenaria in June the number of vesicles nor
the number of arbuscules was affected by enhanced
UV-B radiation.
Flavonoids
Analysis of the root extracts made from roots collected
in June, using HPLC techniques, did not show de-
tectable levels of flavonoids in C. epigeios roots nor
in C. arenaria roots.
Discussion
The present experiment shows that UV-B can nega-
tively affect mutualistic vesicular arbuscular mycor-
rhizal fungi-plant associations in monocots grown in a
field situation. This is in accordance with UV-B effects
found in AM associations in the greenhouse-grown
dicot Acer saccharum (Klironomos & Allen 1995).
The absence of UV-B effects on AMF infection in
April and October may be due to the low metabolic
activity of in the plants and the fungi. A reduced ex-
change of nutrients results when plants and fungi are
not growing.
The main effect of high levels of UV-B radiation
on AMF infection is a decreased number of arbus-
cules in the host plant roots. Since arbuscules are the
structures in which the exchange of nutrients between
the partners takes place, a reduction in their number,
as found in the present experiment, indicates a re-
striction in the nutrient flow. The uptake of nitrogen
(Tobar et al. 1994), phosphorus (Scheiner et al. 1997;
Dickson et al. 1999) and water (Rozema et al. 1986;
Subramanian et al. 1995) from the soil is improved
by AM associations. At the experimental site the soil
is poor in nutrients (Ernst et al. 1984 ). Furthermore,
the water holding capacity of the dune sand is limited.
This causes water stress in the vegetation during the
dry summer period (Rozema et al. 1999).
An indication of the importance of AM associa-
tions for this dune grassland ecosystem may be the
high infection rates found in the host plants. Carex
arenaria showed an infection of 80% in this ecosys-
tem, which is a very high level of infection for species
from the genus Carex. Carex fiacca growing in an

alpine calcareous grassland had infection percentages
between 2 and 10% (Van der Heijden, pers. com-
mun.). For North America 16 out of 23 species of
the genus Carex showed AMF infection, percentages
varied between 5 and 30% (Miller et al. 1999).
For grassland ecosystems it is clearly shown that
the presence of well developed AM associations is
of the great importance in maintaining biodiversity
and ecosystem productivity (Watkinson & Freckleton
1997; Vander Heijden eta!. 1998a, b). Therefore it
seems reasonable to expect changes in the vegetation
of Dutch coastal dune grasslands under elevated UV-8
fluxes resulting from less effective AM associations.
For dune grasslands it has been been reported that
after one year of exposure to enhanced UV-8 radiation
C. epigeios showed reduced biomass accumulation
and reduced tiller density (Oudejans et al. 2001 ). Since
no data on AMF infection are available for this ex-
periment, it is impossible to determine to what extent
possible reductions in AMF infection are causing this
growth depression.
It is not very likely that the reduction in the number
of fungal spores found in C. epigeios roots in April
will have a noticeable influence on AMF infection
percentages in dune grasslands. The naturally occur-
ring infection is so well developed that it is doubtful
whether fungal dispersion by spores in this ecosys-
tem is of great importance. The reported effects of
UV-8 on phylloplane (Newsham et al. 1997a; 1998)
and saprotrophic fungi (Gehrke et al. 1995; Newsham
et al. 1997b) can, at least in part, result from direct
effects of UV-8 on the fungus. For both phylloplane
and saprotrophic fungi direct UV-8 effects on spore
germination, spore production and mycelial extension
rates have been reported (Moody et al. 1999) UV-8
radiation does not penetrate into the soil. Therefore the
effect of UV-8 on AM associations must be indirect,
via the above ground parts of the host plant.
Two possible mechanisms causing this indirect
UV-8 effect were proposed by Van de Staaij et a!.
( 1999). The first mechanism, explains changes in
AM under elevated levels of UV-8 radiation as a re-
sult of the induced changes in the phenylpropanoid
pathway in the host plant. This results in increases
in the flavonoid and related phenolics concentrations
(Meijkamp eta!. 1999). These phenolics when exuded
by the roots, influence spore germination and hypha!
growth of AM fungi (Siqueira et al. 1991; Chabot et al.
1992).
Although flavonoids are present in the shoot of
C. epigeios and C. arenaria (pers.comm. Rozema),
175
chemical analysis of the roots of C. epigeios and
C. arenaria revealed an absence of detectable lev-
els of flavonoids in the root tissue (this paper).
Furthermore, the levels of UV-8 absorbing com-
pounds (flavonoids and related phenolics) in green-
house grown C. epigeios plants did not show an
increase under elevated UV-B radiation (Tosserams
& Rozema 1995; Tosserams et al. 1997). Combin-
ing these facts it seems unlikely that enhanced UV-B
influences AM associations via differences in UV-
8 absorbing flavonoids in above and below ground
host plant tissues. More generally, UV-8 induced en-
hanced synthesis of difficult to digest polyphenolics
(e.g .. tannins and lignin) could reduce the develop-
ment of micro-organisms including fungi. However
hypha! infection was not affected by enhanced UV-8.
The second mechanism involves changes in the
plant hormone levels. Fungal development in the host
plant may be partly regulated by phytohormone levels
(8eyrle 1995). Auxins absorb strongly in the UV-8
part of the spectrum, and UV- 8 radiation can reduce
the levels of these phytohormones in plants (Ros &
Tevini 1995 ). Therefore elevated levels of UV-8 may
indirectly affect AM associations through effects on
phytohormone balance in the host plant.
The experimental work done so far supports this
hypothesis. The main etlect of UV-8 on AM is a re-
duction in the number of arbuscules present. Arbuscle
formation takes place in the host plant after fun-
gal hyphae have penetrated. Internal fungal develop-
ment seems to be partly regulated by phytohormones
(8eyrle 1995) and since UV-8 influences auxin levels,
this mechanism seems to give a likely explanation for
the effects observed.
The enhanced UV-8 induced decreases in AMF
percentages in the field are intriguing, but much needs
to be done to assess possible impacts on clune grass-
lands. Therefore our present research is focused on
two important remaining questions. The first is what
the effects of decreases in AMF on individual plant
growth and nutrient fluxes within the grassland system
are. The second is whether changes in phytohormones
are really involved in decreases in AMF infection
percentages.
Acknowledgements
The authors are indebted to Drs 8.8. Meijkamp
and R.A. Brockman for developing suitable flavonoid
analyses techniques. We thank Dr M. Van der Heij-
176
den for useful comments which helped to improve the
manuscript. We acknowledge NV PWN Waterleiding
Bedrijf Noord-Holland (De Vries, MSc., Mr Hidde
Posthuma and the late Mr E. Koet) for permission
and support to work in the Dune Reserve. This re-
search has been partially financed by the EC under the
project numbers ENV4-CT96-0208 and ENV4-CT97-
0580 (Environmental and Climate Programme) which
is acknowledged.
References
Allen, M. F. 1993. The ecology of mycorrhizae, Cambridge Univer-
sity Press. Cambridge.
Beyrle. H. 1995. The role of phytohormones in the function and
biology of mycorrhizas. Pp. 365-390. In Varma. A. & Hock. B.
(eds), Mycorrhiza Structure, Function, Molecular Biology and
Biotechnology. Springer-Verlag. Berlin.
Blumthaler. M. and Ambach. W. 1990. Indications of increasing
solar ultraviolet-B radiation flux in alpine regions. Science 248:
206-208.
Brundrett. M. C.. Piche, Y. & Peterson, R. L. 1984. A new
method for observing the morphology of vesicular-arbuscular
mycorrhiza. Can. J. Bot. 62: 2128-2134.
Caldwell, M. M. 1971. Solar UV radiation and the growth and de-
velopment of higher plants. Pp. 131-268. In Giese, A. C. (ed.),
Photophysiology, Vol. 6. Academic Press. New York.
Caldwell, M. M., Bjorn, L. 0., Bornman, J. F., Flint, S.D., Ku-
landaivelu, G., Teramura, A. H. & Tevini. M. 1998. Effects of
increased solar ultraviolet radiation on terrestrial ecosystems. J.
Photochem. Photobiol. B46: 40-52.
Chabot, S., Bel-Rhlid, R .. Chenevert, R. & Piche, Y. 1992. Hypha!
growth promotion in vitro of the VA mycorrhizal fungus, Gi-
gmpora margarita Becker & Hall, by the activity of structural
specific flavonoid compounds under C02-enriched conditions.
New Phytol. 122:461-467.
Corlett, J. E., Stephen. J., Jones, G. H., Woodfin, R., Mepsted, R.
& Paul, N.D. 1997. Assessing the impact of UV-B radiation on
the growth and yield of field corps. Pp. 195-211. In Lumsden,
P. J. (ed.), Plants and UV-B. Society for Experimental Biology,
Seminar Series 64. Cambridge University Press, Cambridge.
Dickson, S., Smith, S. E. & Smith, F. A. 1999. Characterisation
of two arbuscular mycorrhizal fungi in symbiosis with Allium
porrun: colonisation, plant growth ami phosphate uptake. New
Phytol. 144: 163-172.
Ernst, W. H. 0., Van Duin, W. E. & Oolbekking, G. T. 1984.
Vesicular-arbuscular mycorrhiza in dune vegetation. Acta Bot.
Neerl. 33: 151-160.
Gange, A. C. and West, H. M. 1994. Interactions between arbus-
cular myconhizal fungi and foliar feeding insects in Plantago
lanceulata L. New Phytol. 128: 79-87.
Gehrke, C., Johanson, U .. Callaghan, T. Y., Chadwick, D. &
Robinson, C. H. 1995. The impact of enhanced ultraviolet-
B radiation on litter quality and decomposition processes in
Vaccinium leaves from the subartic. Oikos 72: 213-222.
Graves, J. D., Watkins, K. N., Fitter, A. H .. Robinson, D. &
Scrimgcour, C. 1997. Intraspecific transfer of carbon between
plants linked by a common mycorrhizal network. Plant Soil 192:
153-159.
Green, A. E. S., Cross, K. R. & Smith, L. A. 1980. Improved
analytic characterization of ultraviolet skylight. Photochem. Pho-
tobiol. 31: 59-65.
Kape, R., Wex, K., Parniske, M., Gorge, E., Wetzel, A. &
Werner, D. 1992. Legume root metabolites and VA-mycorrhiza
development. J. Plant Physiol. 141: 54-60.
Klironomos, J. N. & Allen, F. N. 1995. UV-B mediated changes
on below-ground communities associated with the roots of Acer
saccharum. Funct. Ecol. 9: 923-930.
Meijkamp, B. B., Aerts, R., Van de Staaij, J., Tosserams.
M., Ernst, W. & Rozema. J. 1999. Effects of UV-B on sec-
ondary metabolisms in plants. Pp. 71-99. In: Rozema, J. (ed.),
Stratospheric ozone Sepletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden, The Netherlands.
Miller, M. R., Smith, C. 1., .Jastrow, .f. D. & Bever, J. D. 1999.
Mycorrhizal status of the genus Carex (Cyperaceae). Am. J. Bot.
86: 547-553.
Molina, M. J. & Rowland, F. S. 1974. Stratospheric sink for chlo-
roftuoromethanes: chlorine atom-catalyzed destruction of ozone.
Nature 249: 810-812.
Moody, S. A., Newsham, K. K., Ayres, P. C. & Paul, N.D. 1999.
Variation in the responses of litter and phylloplane fungi to UV-B
radiation (290-315 nrn). Mycol. Res. 103: 1469-1477.
Newsham, K. K., Fitter, A. H. & Watkinson, A. R. 1995a. Arbus-
cular mycorrhiza protect an annual grass from root pathogenic
fungi in the field. J. Ecol. X3: 991-1 000.
Newsham, K. K., Fitter, A. H. & Watkinson, A. R. 1995b. Multi-
functionality and biodiversity in arbuscular mycorrhizas. Trends
Ecol. Evol. 10:407-411.
Newsham, K. K.. Lewis, G. C .. Greenslade, P. D. & McLeod, A. R.
1998. Neotyphodiurn lolii, a fungal leaf endophyte, reduces lertl-
ity of Lolium perenne exposed to elevated UV-B radiation. Ann.
Bot. 81: 397-403.
Newsham, K. K., !.ow, M. N. R., McLeod, A. R., Greenslade,
P. D.& Emmet, B. A. 1997a. UV-B radiation influences the abun-
dance and distribution of phylloplane fungi on pedunculate oak
(Quercus Robur). New Phytol. 136: 287-297.
Newsham. K. K., McLeod, A. R., Roberts, J.D .. Greenslade, P. D.&
Emmet, B. A. 1997b. Direct effects of elevated UV-B on the
decomposition of Quercus Rohur leaf litter. Oikos 79: 592-602.
Oudejans, A. M. C., Nijssen, A., Huls, J. S. & Rozema, .J. 200 I.
The reduction of aboveground Calamagrostis epigeios mass and
tiller numer by enhanced UV-B in a dune grassland ecosystem.
Plant Ecol. 154: 37-48 (this volume).
Ozinga, W. A., Van Andel, J. & McDonnell-Alexander, M.P. 1997.
Nutritional soil heterogeneity and rnycorrhiza as determinants of
plant species diversity. Acta Bot. Neerl.46: 237-254.
Reuber, S., Bornman, J. F. & Weissenbock, G. 1996. Phenyl-
propanoid compounds in primary leaf tissues of rye (Secrile
cereale). Light response of their metabolism and the possible role
in UV-B protection. Physiol. Plant. 97: 160-168.
Ros, J. & Tevini, M. 1995. Interaction of UV-radiation and IAA
during growth of seedlings and hypocotyl segments of sunflower.
J. Plant Physiol. 146: 295-302.
Rozema, J. 1999. UV-B radiation and terrestrial ecosystems:
processes, structure and feedback loops. Pp. 101-114. In:
Rozema J. (ed.). Stratospheric Ozone Depletion: the Effects of
Enhanced UV-B Radiation on Terrestrial Ecosystems. Backhuys
Publishers, Leiden, The Netherlands.
Rozema, J., Arp, W., Van Diggelen, J., Esbroek, M., Brockman, R.
& Punte, H. 1986. Occurence and significance of vesicular arhus-
cular mycorrhiza in the salt marsh environment. Acta Bot.Neerl.
35: 457-467.

Rozema. L Arp. W., Van Esbroek, M .. Brockman. R.. Puntc.
H. & Schat, H. 1986. Vesicular arbuscular mycorrhiza in salt
marsh plants in response to soil salinity and flooding and the
significance of water relations. In: Mycorrhizae: physiology and
genetics - Les mycchorites: physiologic et gcnctiquc. INRA,
Paris.
Rozema, 1., Oudejans, /\., Hooter, N .. Schoonheim, H .. Walraven.
I.. Van 't Klooster, C., Van de Staaij. J.. To"erarrb, M .. De
Bakker, N., Van Beem, A., Stroetenga, M .. Broekman. R., Van
Heerwaarden, L., Nclisscn, H. & Aerts. R. 1999. Responses of
plants from a dune grassland ecosystem in the Netherlands to
solar liV-B: UV-B filtration and supplementation experiments.
Pp. 203-225. In: Rozema J. (ed.). Stratospheric Owne Deple-
tion: the Effects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden. The Netherlands.
Rozema, J., Van de Staaij, J., Bji:irn, L. 0. & Caldwell. M. 1997. UV-
ll as an environmental factor in plant life: stress and regulation.
Trends Ecol. Evol. 12: 22-2H.
Russell, J. M .. Luo, M., Cicerone, R. 1. & Deaver. L. E. 1996.
Satellite conformation of the dominance of chloroftuoroearbones
in the global stratospheric chlorine budget. Nature 379: 526-529.
Schreiner, R. P., Mihara, K. L., McDaniel. H. & Bethlcnfalvay. G. 1.
1997. Mycorrhizal fungi influence plant and soil functions and
interactions. Plant Soil 188, 199-209.
Shirley, B. W. 1996. Flavonoid biosynthesis: new functions for an
old pathway. Trends Plant Sci. II: 377-382.
Siqueira, J. 0., Safir, G. R. & Nair. M.G. 1991. Stimulation of
vesicular-arbuscular mycorrhiza formation and growth of white
clover by flavonoid compounds. New Phythol. 118: 87-93.
Sakal, R. R. & Rohlf, F. J. 1995. Biometry (3rd edn.). heeman and
Company, New York.
Stafford, H. A. 1997. Roles of tlavonoids in symbiotic and defence
functions in legume roots. Bot. Rev. 63: 27-39.
Subramanian, K. S., Charest, C., Dwyer. L. M. & Hamilton. R. I.
1995. Arbuscular mycorrhizas and water relations in maize under
drought stress at tasseling. New Phytol. 129: 643-650.
177
Tobar. R .. Azc6n, R. & Barca, J. M. 1994. Improved nitrogen up-
take and transport from I SN-Iabellcd nitrate by external hyphae
of arbuscular mycorrhiza under water-stressed conditions. New
Phytol. 126: 199-122.
Tosserams. M. & Rozema. 1995. Effects of ultraviolet-Fl radia-
tion on growth and physiology of the dune grassland species
Colomogrostis epigeios. Environ. Poll. 89: 209-214.
Tosserams. M .. Magendans, E. & Ro£ema. J. 1997. Differential
effects of elevated ullraviolet-B radiation on plant species of a
dune grassland ecosystem. Plant Ecol. 128: 266-281.
Van de Staaij, J. W. M., Rozema, J. & Aerts. R. 1999. The impact
of UV-I3 radiation on mutualistic plant/micro-organism interac-
tions at the soil-root interface. Pp. I59-171. In: Rozema. J. (ed. ).
Stratospheric Ozone Depletion: the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystems. Backhuys Publishers,
Leiden. The Netherlands.
Van de Staaij. J. W. M .. Ernst, W. H. 0 .. Hakvoort, H. W. J.
& Rozema. J. 1995. Ultraviolet-B (280-320 nm) ahsorhing
pigments in the leaves of Silene vulgaris: their role in UV-B
tolerance. J. Plant Physiol. 147: 75-80.
Van der Heijden. G. A .. Boller. T., Wiemken. A. & Sanders, I. R.
1998a. Different arbuscular mycorrhizal fungal species are po-
tential deterrnents of plant community structure. Ecology 79:
2082-2091.
Vander Heijden. G. A .. Klironomos. J. N .. Ursie. M., Moutoglis.
P.. Streitwolf-Engcl, R .. Boller. T.. Wienrken, A. & Sanders,
L R. 1998b. Mycorrhizal fungal diversity determines plant bio-
diversity. ecosystem variability and productivity. Nature 396:
69-72.
Watkinson, A. R. & Freckleton, R. P. 1997. Quantifying the impact
of arbuscular mycorrhiza on plant competition. J. Ecol. 85: 541-
545.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

Phlomis fruticosa (L.) in the field: the Mediterranean drought semi-deciduous shrub used in the UV-B x
Wounding experiment.
 Plant Ecologr 154: 181-186, 2001.
181
© 200 I Kluvcer Academic Puh/ishers.

Combined effects of enhanced UV-B radiation and additional nutrients on

growth of two Mediterranean plant species

E. Levizou & Y. Manetas

Laboratory of Plant Physiology, Department of Biolog_v, Universit.v of Patras, 26500 Patras, Greece
(E-mail: levizou@hiology. upatras.gr)

Key words: Ceratonia siliqua, Mediterranean, Nutrients, Phlomisfruticosa, UV-B

Abstract
Seedlings of two Mediterranean plants, the slow-growing, evergreen sclerophyll Ceratonia siliqua L. and the fast
growing drought semi-deciduous Phlomisfruticosa L., were grown for one year in the field at ambient or ambient
plus supplemental UV-B radiation (equivalent to a 15% ozone depletion) and two levels of applied fertilizers
(NPK). The effects on growth, morphological, anatomical and physiological parameters were measured at final
plant harvest. Additional nutrients increased leaf nitrogen, improved growth and reduced the root/shoot ratio in
both plants, yet these effects were more pronounced in the fast growing P. fruticosa. A nutrient-induced increase
in chlorophyll content was also observed in this plant. The growth responses to UV-B radiation were different for
the two species. Growth in C. siliqua was not affected by UV-B radiation at both nutrient levels and the same was
true for P. fruticosa at low nutrients. However, at the high nutrient level, supplemental UV-B radiation improved
growth in P.fruticosa, indicating a strong interaction between the treatments. Photosystem II (PSII) photochemical
efficiency, methanol-extractable UV-B absorbing capacity, total phenolics and tannins were not affected by either
treatment in both plants. It is concluded that nutrient levels can strongly modify the UV-B radiation effects on
growth of P. fruticosa. We presume that this may be correlated to the fast growing habit of this species.

Introduction UV-B radiation on growth (Drilias et al. 1997; Mane-

Measured trends in stratospheric ozone concentration
(Stolarski et al. 1992) and in UV-B reaching the sur-
face of the earth (Zerefos et al. 1995) indicate that
this radiation is increasing even at mid latitudes. Ear-
lier studies have shown that UV-B radiation may be
detrimental to some plants (see Tevini 1993 and the
literature therein). However, the effects of enhanced
UV-B radiation on plants can be modified by other
co-occurring stresses or by simply changing environ-
mental parameters like atmospheric C02 (Bjorn et al.
1997), temperature (Mark & Tevini 1996), water avail-
ability (Manetas et al. 1997) or visible light intensity
(Cen & Bornman 1990). The extent of modification or
even the direction of changes are species-specific. For
example, additional watering during the dry summer
period abolished the detrimental (Nerium oleander)
and beneficial (Pinus pinea) effects of supplemental
las eta!. 1997). In order to investigate the possible rea-
sons for these modifications, one may consider that the
levels of UV-B absorbing phenolic compounds (which
protect plant tissues against UV-B radiation damage,
Caldwell et a!. 1983) can be changed in response
to a variety of environmental factors (Gershenzon
1984 ), including UV-B radiation (Beggs & Wellman
1994). Therefore, any environmental factor that in-
duces biosynthesis of phenolics may afford protection
against UV-B radiation. Manipulation of internal phe-
nolic concentration can be accomplished by changing
nutrient availability. According to the carbon/nutrient
balance hypothesis (Bryant et a!. 1983 ), nutrient de-
ficiency atlects growth more than photosynthesis. As
a result, assimilated surplus carbon is diverted to
the production of carbon-based secondary metabo-
lites (i.e., phenolics and terpenoids), rendering the
nutrient-deficient plants less vulnerable to both gener-
182
alist herbivores and UV-B radiation damage. Another
prediction of the same hypothesis is that inherently
slow growing plants invest more on carbon-based sec-
ondary metabolites. Based on the above, it seems of
interest to investigate the combined effects of nutri-
ents and UV-B radiation on growth and physiology of
plants.
Such studies have been performed only under
glasshouse (Murali & Teramura 1985; Musil & Wand
1994; Hunt & McNeil 1998) or growth chamber
(Deckmyn & Tmpens 1997) conditions and, in all
cases, with fast growing, mostly cultivated plants.
In the present field study, two Mediterranean species
with contrasting growth strategies (the slow-growing
evergreen sclcrophyll Ceratonia siliqua and the fast-
growing drought semi-deciduous Phlomis fruticosa)
were used. These plants are known to have different
leaf phenolic levels in their leaves (Nikolopoulos eta!.
1995; Grammatikopoulos eta!. 1998).
Materials and methods
Plant material and growth conditions
Seedlings of C. siliqua and P. fruticosa were raised
from seeds in small clay pots with local, low fer-
tility soil (Manetas et al. 1997), under natural con-
ditions in the field. When the plants reached the
age of five months (mid-March 1998) 64 apparently
similar seedlings from each species (based on leaf
number, total leaf area and plant height) were se-
lected and randomly assigned to 8 plots (4 controls,
4 UV-8, 8 plants per plot). Half of the plants in each
plot were watered with 600 ml of tap water per pot
and week, while the rest received the same amount
of water enriched with solid fertilizer equivalent to
10 mg N, 1 mg P and 1.3 mg K (given as NH4N03
and KH2P04, respectively) per pot and week, result-
ing into four groups: control, low nutrients (CON-L);
UV-8, low nutrients (UV-8-L); control, high nutri-
ents (CON-H); UV-B, high nutrients (UV-B-H). UV-B
radiation was provided by Q-Panel UV-B 313 fluores-
cent tubes (Cleveland, OH), wrapped in 'pre-burnt'
0.1 mm cellulose acetate film (Courtlauds Chemicals,
Derby, UK) and suspended 170 em above plant apex.
This distance was kept constant throughout the ex-
periment. The tubes were mounted in metal frames
with minimum shading (ca. 10% reduction in PAR)
and cellulose acetate was replaced every 20 h of lamp
operation. In control frames, plastic tube effigies of
the same diameter replaced the Q-panel tubes. The
six lamps per plot were turned on and off in groups
of three. Accordingly, the supplemental irradiance
was increased (and decreased) in two steps centered
at solar noon and the lamp duration was modified
monthly, in order to follow the natural course of
ambient UV-B radiation. Lamps were controlled by
timers. To calculate the duration the lamps should be
on each day, the absolute spectral irradiance measured
at night (Optronic OL 752, Orlando, FL) and the gen-
eralized plant action spectrum normalized at 300 nm
(Caldwell 1971) were used in conjunction with the
computer program of Bjorn & Murphy ( 1985). Ac-
cording to this program ambient UV-BsE radiation
doses are 0.85 kJ m- 2 day- 1 and 8.55 kJ m- 2 day- 1
for the winter minimum and summer maximum, re-
spectively. Supplemental irradiation was based on a
15% ozone depletion scenario. More details of the UV-
B irradiation system can be found in Manetas et al.
(1997).
Sampling and measurements
Chlorophyll fluorescence induction was followed for
4 s from the upper leaf surface (10 leaves per plant)
with a Hansatech Plant Efficiency Analyser. Exciting
red light at 1500 fimol m- 2 s- 1 was routinely used.
All fluorescence measurements were performed about
one hour after sunset. Chlorophyll content (I 0 leaves
per plant) was measured with a portable chlorophyll
meter (Minolta, SPAD-502). The SPAD values were
transformed into actual chlorophyll concentrations by
using a reference curve. For this purpose, leaves from
plants not used in the experiment and having various
SPAD values, were extracted with 100% methanol
and chlorophylls were estimated using the equations
of Lichtenthaler & Well burn ( 1983). For estimation of
UV-B absorbing capacity, two leaf discs were removed
from each plant and UV-B absorbing compounds were
extracted in boiling methanol:H20:HCI (90: I: I) (v:v)
according to Day ( 1993) and the absorbance at 300 nm
was measured with a Simadzu UV-160-A recording
spectrophotometer. Leaf area (all leaves) was mea-
sured with a portable LICOR LI-3000 leaf area meter
and leaf thickness (middle of the leaf avoiding the
middle vein) with a friction-stop caliper (Mitutoyo,
Japan). Due to its pubescence, leaf thickness was not
measured in P.fruticosa. After plant harvest, the plants
were divided into leaves, stems and roots. The dry
mass of plant parts was measured after drying at 80 °C
for 24 h. Specific leaf mass (SLM) was calculated
from mass and area measurements. All dried leaves
 183
Tahle I. Results of two-way ANOYA testing the differences between treatments and their in-
teractions in C. siliqua. In those cases where significant differences were observed, the arrow
denotes the direction of change ( t increase. t decrease) and the number in parenthesis the percent
change.

Additional nutrients Enhanced UV-B Interaction

(nutrients x UV-B)

Plant height 0.008 t (23) 0.542 0.666

Total leaf number 0.002 t (26) 0.894 0.149
Total leaf area 0.002 t (49) 0.872 0.304
Stem length 0.03 t (17) 0.520 0.338
Total length of branches <0.001 t (60) 0.322 0.701
Number of Branches 0.006j(16) 0.501 0.337
Total leaf OW 0.014 t (38) 0642 0.304
Total above ground OW 0.012 t (37) 0.521 0.664
Root OW 0.444 0.793 0.878
Root/shoot <0.00 I ~ (33 I (J.358 0.266
Leaf thickness 0.098 0.665 0.140
Adaxial cuticle thickness 0.001 ~ (16) < 0.001 t (21 I 0.254
Abaxial cuticle thickness 0.158 0.397 0.532
N(%0W) <0.00 I t (27) 0.786 0.786
Fv/f;" 0.816 0.781 0.875
Chlorophylls/cm 2 0.750 0.120 0.690
A300 I cm 2 0.072 0.727 0.718
Total Phenolics 0.509 0.400 0.076
(mg TA/g OW!
Tannins 0.304 0.494 0.292
(mgTA/gOW)
N ffotal phenolics <0.001 t (3\) 0.301 0.153
N I Tannins 0.001 t (36) 0.716 0.891

from each plant were ground into powder and used
for estimation of total phenolics, tannins and nitro-
gen content. For total phenolics and tannins 50 mg of
the dry powder was extracted for 3 h in 3 ml, 50%
methanol at 40 oc (Waterman & Mole 1994 ). Total
phenolics were measured using the Folin-Ciocalteu
method as described by Waterman & Mole ( 1994) and
tannins following the blue-dye labeled bovine serum
albumin (BSA) method of Asquith & Butler ( 1985).
Tannins could not be detected in P. fruticosa. Toes-
timate the total nitrogen content of the leaves, the
Kjeldahl method was used (Allen 1989).
Cuticle thickness was assessed microscopically
from hand-cut transverse sections of fresh material
viewed immediately after staining with Sudan IV (sat-
urated in 90% ethanol) under an Axioplan (Zeiss)
microscope. Two mature leaves per plant were used
and the sections were taken from the middle of the
leaves in order to avoid variability due to a possible
ditlerential cuticle thickness along the leaf. In each
section, cuticle thickness was measured at 5 ditler-
ent points. Sudan IV stains with a deep red color the
cuticle components (Jensen 1962). A discrimination
between the cuticle proper and the possible occurrence
of a thin layer of cutin in the outer cell wall of the
epidermis (Fahn 1974) was not made as the entire
cutinized complex serves an antitraspirant function.
Due to its extremely low thickness, cuticle was not
measured in P. fruticosa.
Statistics
As described under 'sampling and measurements', all
parameters were measured on each individual plant
and a mean value characteristic for the plot (replica-
tion) was calculated. Thus, the results are expressed as
means± SO from four replicates (plots) per treatment.
Homogeniety of variances was checked and the fol-
lowing transformations were performed: artan for total
leaf number, root/shoot, N/phenolics data of C.siliqua
and log!O for total leaf number of P. fruticosa. Dif-
ferences between treatments were assessed through a
184
Table 2. Results of two-way ANOVA testing the differences between treatments and
their interactions in P fruticosa. In those cases where significant differences were ob-
served, the arrow denotes the direction of change (t increase, t decrease). For percent
changes see Table 3.

Additional Enhanced UV - B Interaction

nutrients (nutrients x UV-fl)

Plant height 0.078 0.716 0.788

Total leaf number <0.001 t 0.070 0.050 t
Total leaf area <0.001 t 0.140 0.048 t
Petiole length 0.050 t 0.013 t 0.022 t
Total leaf OW <0.001 t 0.107 0.080 t
Total above ground DW <0.001 t 0.036 t 0.085
Root OW <0.001 t 0.002 t <0.001 t
Root/shoot <0.001 t 0.127 0.003 t
N(%DW) <0.001 t 0.105 0.186
F,.fF,, 0.754 0.735 0.878
Chlorophylls/cm 2 <0.001 t 0.833 0.053
A300/cm 2 0.685 0.383 0.814
Total phenolics 0.501 0.323 0.761
(mgTA/gDW)
N I Total Phenolics <0.001 t 0.653 0.515

Table 3. Effects of UV-B radiation and nutrients on growth parameters of P fruticosa. For statistics, see Table 2.

UV-B-H CON-H 9( UV-B- L CON -L 9(

change change

Height 16.41 ± 4.31 I 5.48 ± 4.07 6.0 13.35 ± 3.50 13.21 ± 3.47 1.1
Leaf number 169.92 ± 24.16 129.71 ± 18.44 31.0 95.46 ± 13.57 96.41±13.71 -1.0
Total leaf area 774.71 ± 197.34 553.37 ± 140.96 40.0 333.94 ± 85.06 350.62 ± 89.31 -4.8
Total petiole length 72.49 ± 26.73 38.56 ± 14.22 88.0 35.71 ± 13.17 34.28 ± 12.64 4.2
Leaf dry mass 9.18 ± 2.3 7.01 ± 1.76 31.0 4.05 ± 1.13 4.'i9± 1.15 -2.0
Total above ground dry mass 15.20 ± 3.46 11.43 ± 2.60 33.0 8.00± 1.82 7.60± 1.73 5.3
Root dry mass 21.09 ± 3.09 13.96 ± 2.05 51.1 12.48 ± 1.83 13.72 ± 2.01 -9.0
Total dry mass 45.47 32.40 40.3 24.98 25.91 -3.6

two-way ANOVA statistical test (SPSS 9.0.1, SPSS
Inc.)
Results
Ceratonia siliqua
Table I presents the results of a two-way ANOVA
testing for differences between treatments and their
interactions. As shown, there were no interactions be-
tween nutrient and UV-B radiation levels and there
was almost no response of the plants to supplemen-
tal UV-B radiation. The only significant difference
was found in the thickness of the adaxial leaf cuticle,
which was increased by 21% under UV-B supplemen-
tation. It is worth-noting that the cuticle thickness was
also responsive to nutrient levels, showing a 16% re-
duction under high nutrients. As expected, additional
nutrients resulted in a significant increase in leaf nitro-
gen concentrations and a growth improvement, which,
however, was limited to the above ground part of the
plant. As a result, the root/shoot ratio was consider-
ably reduced. In addition, the increase in leaf nitrogen
and the absence of any effect on leaf phenolics, led
to a considerable improvement of leaf palatability, as
assessed from the ratio of nitrogen to total phenolics
or tannins.
Phlomis fruticosa
The results were quite different for P. jruticosa. As
shown in Table 2, not only nutrients, but UV-B radia-

tion as well had a positive effect on growth parameters
of this plant. Thus UV-B radiation had a significant
positive effect on petiole length, root and total above
ground biomass, while the positive effects on leaf
number, area and mass were not significant. In addi-
tion, a strong positive interaction was also observed
between high nutrients and enhanced UV-B radiation
leading to significant improvements in the growth of
leaves and roots, while the effects on the other growth
parameters were marginally significant (Table 2). The
nature of this interaction can be better understood by
giving the absolute values of growth parameters for
each treatment. As shown on Table 3, UV-B radiation
had no effect on growth of P. fruticosa at low nutri-
ent levels. At high nutrients, however, UV-B radiation
improved growth considerably and as a result, plants
grown under the combination of high nutrients and en-
hanced UV-B radiation were heavier, wider, taller and
had more leaves compared to all other treatments.
Among the effects on other parameters, we may
note the increase in chi content and N, as well as the
reduction in the root/shoot ratio under high nutrients
(Table 2 and 3). It is worth noting that phenolic levels
were not affected either by nutrient or UV-B radiation
treatments
Discussion
The initial prediction, based on the carbon/nutrient
balance hypothesis, that growth promotion by addi-
tional nutrients would result in less investment into
phenolic compounds and accordingly, into plants more
vulnerable to UV-B radiation damage, was not con-
firmed. Although growth was promoted in both plants,
additional nutrients had no effect on phenolic com-
pounds measured either chemically or through their
UV-B absorbing capacity. The reasons for this unre-
sponsiveness can not be assessed from the results of
the present investigation. However, similar lack of re-
sponse to additional nutrients is not uncommon (see
Haukioja et a!. 1998 and the literature therein). In
some cases, additional nutrients even increased the
phenolic levels (Musil & Wand 1994; Hunt & McNeil
1998). In our case, phenolic compounds did not re-
spond to enhanced UV-B radiation as well. In addition,
no indication of higher vulnerability to UV-B radiation
damage in P. .fi-uticosa was obtained, in spite of the
fact that the UV-B absorbing capacity in the leaves of
this plant is considerably lower compared to the corre-
sponding capacity of C. siliqua leaves (Nikolopoulos
eta!. 1995, Grammatikopoulos et a!. 1998). Accord-
185
ingly, the growth responses to UV-B radiation ob-
served in the present study (promotion in P. fruticosa,
no effect in C. siliqua) can not be ascribed to the dif-
ferent levels of UV-B absorbing compounds. However,
the more responsive (not only to UV-B radiation but
to high nutrients as well) P. fruticosa is a fast growing
plant. On the contrary, the effects ofUV-B radiation on
the slow growing C. siliqua were negligible and those
of high nutrients were less pronounced. Therefore the
species-specific differences may in fact be related to
the physiological growth forms, as suggested recently
by Gwynn-Jones et a!. ( 1999). Perhaps. longer expo-
sure times are needed for the slow growing species
in order to get significant UV-B radiation effects. The
induction of a thicker cuticle, for example, may attenu-
ate UV-B radiation. In addition, it may be beneficial to
the plant in the long term, reducing cuticular transpira-
tion and improving water relations under extreme dry
episodes. Thicker cuticles, under UV-B supplemen-
tation have also been observed in the Mediterranean
sclerophylls Pinus pinec1 (Manetas et a!. 1997) and
Nerium oleander (Drilias eta!. 1997).
Concerning growth, it is clear that the exploitation
of the surplus soil nutrients by P. fruticosa is con-
siderably improved under enhanced UV-B radiation.
We have no clues from our measurements to explain
this strong growth promotion. Our results partly agree
with those of Musil & Wand ( 1994) who found sig-
nificant, UV-B induced increases in vegetative growth
of Dimorplwtheca pluvia/is grown at low nutrients
and increasing trends at high nutrients. In the same
plant, the UV-B promoting effects on the growth of
reproductive organs were significant at both nutrient
levels. The above results are in contrast to those of Mu-
rali & Teramura (1985 ), Hunt & McNeil ( 1998) and
Tosserams et a!. (2001 ). These investigators, work-
ing with Glycine max, Cucumis sativus and Plantago
lanceo/ata, respectively, found that nutrient deficiency
hardens the plant against UV-B radiation damage. As
a result, UV-B radiation reduced growth at high nu-
trients but was ineffective at low nutrients. The above
contradictions could be due to the ditlerent test plants,
irradiation protocols or growth conditions. Por ex-
ample, all previous studies have been conducted in
greenhouses. However, the results of Murali & Ter-
amura ( 1985) and Tosserams et al. (200 I) could be
correlated to the phenolic contents of the leaves, since
in their cases phenolics were increased by nutrient
deficiency.
In conclusion, the results of this field study showed
that the effects of UV- B radiation on growth of some
186

plants can be strongly modified by other, co-occuring Timmermann, B. N .. Steelink. C. & Loewus, F. A., (eds.),
stresses. Thus, in P. fruticosa, supplemental UV-B ra- Recent Advances in Phytochemistry, Vol. 18. Phytochemical
Adaptations to Stress. Plenum Press, New York.
diation reduces growth under water stress (Nikolopou-
Grammatikopoulos, G., Kyparissis. A., Drilias, P., Petropoulou,
los et al. 1995), has no effect on well-watered plants Y. & Manetas, Y. 1998. Effects of UV-B radiation on cuticle
and promotes growth under high water and nutrients thickness and nutritional value of leaves in two Mediterranean
supply (results of the present investigation). evergreen sclerophylls. J. Plant Physiol. 153: 506-512.
Gwynn-Jones, D., Lee, J. A., Johanson, U., Phoenix, G. K.,
Callaghan, T. Y. & Sonesson, M. 1999. The responses of plant
Acknowledgement functional types to enhanced UV-B radiation. Pp. 173-185. In:
Rozema, J. (ed.). Stratospheric Ozone Depletion: the Effects of
We are grateful to the Commission of the European Enhanced UV-B Radiation on Terrestrial Ecosystems. Baekhuys
Publishers, Leiden.
Communities for financial support. Haukioja, E., Ossipov, Y., Koricheva, J., Honkanen, T., Larsson,
S. & Lempa, K. 1998. Biosynthetic origin of carbon-based sec-
References ondary compounds: cause of variable responses of woody plants
to fertilization'' Chemoecology 8: 133-139.
Hunt, .1. E. & McNeil, D. L. 1998. Nitrogen status affects UV-B
Allen. S. E. 1989. Chemical Analysis of Ecological Materials.
sensitivity of cucumber. Aust. .1. Plant Physiol. 25: 79-86.
Blackwell Oxford UK.
Jensen, A. 1962. Botanical Histochemistry. Freeman and Company.
Asquith, T. N. & Butler. L. G. 1985. Use of dye-labelled protein as a
San Fransisco.
spectrophotometric assay for protein precipitants such as tannin.
Lichlenthaler, H. K. & Well burn A. R. 1983. Determinations of total
J. Chern. Ecol. 11 : 1535-1543.
carotenoids and chlorophylls a and b of leaf extracts in different
Beggs, C. J. & Wellman, E. 1994. Photocontrol of flavonoid
solvents. Biochem. Soc. Trans. II: 591-592.
biosynthesis. Pp. 733-751. In: Kendrick, P. E. & Kronenberg,
Manetas, Y, Petropoulou, Y., Stamatakis, K., Nikolopoulos, D.,
G. H. (eds). Photomorphogenesis in Plants. Kluwer Academic
Levizou, E., Psaras, G. & Karaboumiotis, G. 1997. Beneficial
Publishers, Dordrecht.
effects of enhanced UV-B radiation under field conditions: im-
Bjorn, L. 0. & Murphy, T. M. 1985. Computer calculation of solar
provement of needle water relations and survival capacity in
ultraviolet radiation at ground level. Physiol. Veg. 23: 555-561.
Pinus pinea L. seedlings during the dry Mediterranean summer.
Bjorn, L. 0., Callaghan, T. V., Johnsen, 1., Lee, J. A., Manetas,
Plant Ecol. 128: 100-108.
Y., PauL N. D., Sonesson, M., Wellburn, A. R., Coop, D.,
Mark. U. & Tevini, M. 1996. Combination Effects of UV-B radi-
He ide-Jorgensen, H. S., Gehrke, C., Gwynn-Jones, D., Johanson,
ation and temperature on sunflower (Helianthus annus L., cv.
U .. Kyparissis, A .. Levizou, E .. Nikolopoulos, D., Petropoulou.
Polstar) and maize (Zea mays L. cv. Zenit 2000) seedlings. J.
Y., & Stephanou, M. 1997. The effects of UV-B radiation on
Plant Physiol. 148: 49-56. ~
European heathland. Plant Ecol. 128: 252-264.
Murali, N. S. & Teramura, A. H. 1985. Etfects of ultraviolet-B
Bryant, J. P., Chapin 111, F. S. & Klein, D. R. 1983. Carbon I nutrient
radiation on soybean. VI. Influence of phosphorus nutrition on
balance of boreal plants in relation to vertebrate herbivory. Oikos
growth and flavonoid content. Physiol. Plant. 63:413-416.
40: 357-368.
Musil, C. F. & Wand, S. J. E. 1994. Differential stimulation of
Caldwell. M. M. 1971. Solar UV radiation and the growth and de-
an arid-environment winter ephemeral Dimorphotheca pluvial is
velopment of higher plants. Pp. 131-177. In: Giese, A. C. (ed.),
(L.) Moench by ultraviolet-B radiation under nutrient limitation.
Photophysiology. Academic Press. New York.
Plant Cell Environ. 17: 245-255.
Caldwell, M. M., Robberecht, R. & Flint, S. D. 1983. Internal filters,
Nikolopoulos, D., Petropoulou, Y., Kyparissis, A. & Manctas
prospects for UV-B acclimation in higher plants. Physiol. Plant
Y. 1995. Effects of enhanced UV-B radiation on the drought
58 : 445-450.
semi-desiduous Mediterranean shrub Phlomis fruticosa under
Cen, Y. P. & Bornman, .1. F 1990. The response of bean plants to
field conditions are season-specific. Aust. J. Plant Physiol. 22:
UV-R radiation under different irradiance of background visible
737-745.
light. J. Exp. Bot. 41: 1489-1495.
Stolarski, R., Bojkof, R., Bishop, L., Zerefos, C. S., Staehelin, J.
Day, T. A. 1993. Relating radiation screening effectiveness of
& Zawodny, J. 1992. Measured trends in stratospheric ozone.
foliage to absorbing-compound concentration and anatomical
Science 256: 342-349.
characteristics in a diverse group of plants. Oeeologia 95: 542-
Tevini, M. 1993. Effects of UV-B radiation on terrestrial plants.
550.
Pp. 125-153. In: Tevini M. (ed.), UV-B Radiation and Ozone
Deckmyn, G. & Impens, I. 1997. Combined effects of enhanced UV-
Depletion: Effects on Humans, Animals, Plants, Microorganisms
B radiation and nitrogen deficiency on the growth, composition
and Materials. Lewis Publishers, London.
and photosynthesis of rye (Seca!e cereale). Plant Ecol. 128: 235-
Tosserams, M .. Smet, J., Magendans, E. & Rozema, J. 2001.
240.
Nutrient availability influences UV-B sensitivity of Plantago
Drilias, P., Karabourniotis, G., Levizou, E., Nikolopoulos, D.,
lanceolata. Plant Ecol. !54: I 57-168 (this volume).
Petropoulou, Y. & Manetas, Y. 1997. The effects of enhanced
Waterman, P. G. & Mole S. 1994. Analysis of Phenolic Plant
UV-B radiation on the mediterranean evergreen sclerophyll Ner-
Metabolites. Blackwell, Oxford. UK.
ium oleander depend on the extent of summer precipitation.
Zerefos, C. S., Meleti, C., Bais, A. F. & Lambros, A. 1995. The
Aust. J. Plant. Physiol. 24: 301-306.
recent UV-B variability over southeastern Europe. J. Photochem.
Fahn, A. 1974. Plant Anatomy. Pergamon Press, Oxford, UK.
Photobiol. 31: 15-19.
Gershneson, J. 1984. Changes in the levels of plant secondary
metabolites under water and nutrient stress. Pp. 273-320. In:
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

A general view of the field experimental site.

 Plant Ecology 154: 189-193, 200L
189
© 200 I Kluwer Academic Publishers.

Effects of UV-B radiation and additional irrigation on the Mediterranean

evergreen sclerophyll Ceratonia siliqua L. under field conditions

Aris Kyparissis, Periklis Drilias, Yiola Petropoulou, George Grammatikopoulos &

Yiannis Manetas
Laboratory of Plant Physiology, Department of Biology, University of Patras, GR-26500 Patras, Greece
(E-mail: y.manetas@ upatras.gr)

Key words: Ceratonia siliqua, Field experiment, UV-B, Water availability

Abstract
Seedlings of Ceratonia siliqua L were grown for I year in the field under ambient or ambient plus supplemental
UV-B radiation (corresponding to 15% ozone depletion over Patras) and received two levels of additional irrigation
during the summer dry period. The experiment was started during February 1998 and two major samplings were
performed, the first at the end of the dry period (September 1998) and the second at the end of the experiment
(January 1999). Plants receiving additional irrigation showed significantly higher leaf number, plant height and
chlorophyll content at the end of the summer, but these differences were abolished at the final harvest. Plants
growing under enhanced UV-B radiation had significantly fewer leaves and less nitrogen content at the end of
the dry period, but these effects were also abolished at the final harvest, during which significant UV-B induced
increases in stem dry mass were observed. None of the other measured parameters (mean leaf area, leaf dry mass,
leaf thickness, UV-B absorbing compounds, phenolics, tannins and photochemical efficiency of PSII) were affected
by either treatment. Combined UV-B I water etfects were not significant We may conclude that although some
minor responses to enhanced UV-B radiation were evident, C. siliqua is resistant against UV-B radiation damage
at the level applied.

Introduction feets are species specific and depend on interactions

Earlier studies investigating the effects of UV-B radi-
ation on plants were usually performed in controlled
chambers and greenhouses with artificial light and the
experimental protocols were rather unrealistic with
regard to the absolute doses of UV-B and the spec-
tral distribution of radiation (UV-8/UV-A/PAR ratio)
(Allen et al. 1998; Caldwell et al. 1994 ). Quite often,
the UV-B doses were extremely high, whereas UV-A
and PAR were low, compared to natural sunlight. It
has now been shown, that a natural balance of UV-
B/UV-A/PAR is necessary for the adequate function
ofUV-B protection mechanisms (Rozema et al. 1997).
Recent studies under semi-natural field conditions re-
vealed that UV-B radiation is not as detrimental for
plant growth and physiology, as previously believed
(Bjorn et al. 1997). Furthermore, UV-B radiation ef-
with other environmental parameters (i.e. C02 con-
centration, water and nutrient availability) (Drilias
et al. 1997; Gwynn-Jones et al. 1997; Manetas et al.
1997; Sullivan & Teramura 1990).
Among the environmental parameters that are ex-
pected to change in the next decades is the precip-
itation pattern (Rambal & Debussche 1995). This
may be especially important for Mediterranean plants,
since the absence of precipitation during the summer
months (Mooney 1987) is considered as the major lim-
iting factor for their growth and development. Taking
all the above into consideration, we conducted a field
study with the evergreen sclerophyll Ceratonia sili-
qua, in order to examine the possible etlects of UV-B
radiation and increased water availability during the
summer.
190
Materials and methods
Plant material and growth conditions
Seedlings of Ceratonia siliqua were raised from seeds
in small plastic pots with local soil, under natural
conditions in the field (Patras University Campus,
38.3° N, 29.1 o E; for details see Manetas et a!. 1997).
The soil was slightly alkaline (pH 7.78) and consisted
of sand, silt and clay (55.4/28.2116.4) with negligible
organic material. When the plants reached the age of
five months (late February 1998) 128 apparently sim-
ilar seedlings (based on leaf number, total leaf area
and plant height) were selected and randomly assigned
to 16 plots (8 controls, 8 UV-B, 8 plants per plot).
Half of these plots (4 controls, 4 UV-B) were well
watered while the rest were water stressed during the
summer, resulting into four treatments: control, well
watered (CON+), UV-B, well watered (UV + ), con-
trol, water stressed (CON-), UV-B, water stressed
(UV- ). Prior to burying the plastic pots in the ground,
their bottoms were removed to allow root growth. De-
tails of the UV-B irradiation system can be found in
Manetas et al. (1997). In brief, UV-B radiation was
provided by Q-Panel UV-B 313 fluorescent tubes (The
Q-Panel Company, Cleveland, OH, USA), wrapped
in 'pre-burnt' 0.1 mm cellulose acetate film (Court-
lauds Chemicals, Derby, UK) and suspended 170 em
above plant apex. This distance was kept constant dur-
ing the experiment. The tubes were mounted in metal
frames with minimum shading and cellulose acetate
was replaced regularly. In control frames, the Q-panel
tubes were replaced by plastic tube effigies of the same
diameter. The lamps were on and off in two steps cen-
tered at solar noon and the lamp duration was modified
monthly, in order to follow the natural march of am-
bient UV-B radiation. To calculate the duration the
lamps should be on each day, the absolute spectral
irradiance measured at night (Optronic OL 752, Or-
lando, FL) and the generalized plant action spectrum
normalized at 300 nm (Caldwell 1971) were used in
conjunction with the computer program of Bjorn and
Murphy ( 1985). Supplemental irradiation was based
on a 15% ozone depletion scenario.
In preliminary trials it was shown that young
C. siliqua seedlings suffered a high mortality rate dur-
ing the summer dry period, unless they had received
a minimum amount of water. Therefore, from May to
October 'well watered' and water stressed plants were
receiving 200 and 7 5 ml of water per pot twice a week,
respectively.
Sampling and measurements
Non-destructive measurements (plant height, leaf
number, leaf area and thickness, photochemical ef-
ficiency of PSII and chlorophyll content) were per-
formed on all plants at the beginning of the experiment
(February 24, 1998), at the start and the end of the
dry period (July I and September 24, respectively)
and at the end of the experiment (January 20, 1999).
For destructive measurements (dry mass, cuticle thick-
ness, total phenolics and tannins, UV-B absorbing
compounds and total nitrogen content) two samplings
were performed. At the end of the dry season, 4 plants
per plot (i.e. 16 plants per treatment) were randomly
selected, while the remaining ones were used at final
plant harvest.
Chlorophyll fluorescence induction was followed
for 4 s from the upper leaf surface with a Hansat-
ech Plant Efficiency Analyser. Exciting red light at
1500 f1 mol m- 2 s- 1 was routinely used. All fluores-
cence measurements were made about one h after sun-
set. Chlorophyll content was measured with a portable
chlorophyll meter (Minolta, SPAD-502). The SPAD
values were transformed into chlorophyll concentra-
tion by using a calibration curve. For this purpose,
leaves from plants not used in the experiment and hav-
ing various SPAD values, were extracted with 100%
methanol and chlorophylls were estimated by using
the equations of Lichtenthaler & Well burn ( 1983). For
estimation of UV-B absorbing capacity two leaf discs
were removed from each plant, UV-B absorbing com-
pounds were extracted in boiling methanol according
to Day (1993) and the absorbance at 300 nm, was
measured with a Shimadzu UV-160-A recording spec-
trophotometer. Leaf area was measured with a portable
LICOR LI-3000 leaf area meter and leaf thickness
with a friction-stop caliper (Mitutoyo, Japan). Af-
ter plant harvest, the plants were divided into leaves,
stems and roots. The dry mass of plant parts was mea-
sured after drying at 80 °C for 24 h. Specific leaf mass
(SLM) was calculated from mass and area measure-
ments. All dried leaves from each plant were ground
into powder and used for estimation of total pheno-
lics, tannins and nitrogen content. For total phenolics
and tannins 50 mg of the dry powder were extracted
for 3 h in 3 ml, 50% methanol at 40 °C (Waterman
& Mole 1994). Total phenolics were measured with
the Folin-Ciocalteu method as described by Waterman
& Mole ( 1994) and tannins following the blue-dye la-
beled bovine serum albumin (BSA) method of Asquith
 191
Table I. Effects of supplemental UV-B radiation on C. siliqua. Only the parameters with a significant effect in at least one
sampling date are presented. + or - indicate a positive or negative effect. respectively.

Parameter Date CON+ CON- UV+ UV- %change p

Number of Sep. 24 24.72 ± 2.02 20.71 ± 2.07 19.59 ± 4.93 16.19 ± 2.19 -21.2 0.040
leaves Jan. 20 42.74 ± 3.81 35.15 ± 2.84 37.85 ± 7.38 31.31 ± 5.37 -11.2 0.217

StemDW,g Sep. 24 0.23 ± 0.02 0.22 ± 0.06 0.22 ± 0.06 0.22 ± 0,03 -2.3 0.386
Jan. 20 0.80 ± 0.10 0.63 ± 0.16 1.34 ± 0.28 1.02 ± 0.22 +65.0 0.002

Leaf nitrogen Sep. 24 20.07 ± 1.48 19.20 ± 0.42 18.62 ± 0.75 17.80 ± 0.82 -7.3 0.023
content, mg g DW-l Jan. 20 13.13 ± 0.90 13.46 ± 1.38 13.17 ± 0.55 11.94 ± 0.66 -5.6 0.195

Table 2. Effects of additional watering during the summer on C. siliqua. Only the parameters with a significant effect in at
least one sampling date are presented. + or- indicate a positive or negative effect, respectively.

Parameter Date CON+ CON- UV+ UV- %change p

Number of Sep. 24 24.72 ± 2.02 20.71 ± 2.07 19.59 ± 4.93 16.19 ± 2.19 +20.1 0.049
leaves Jan. 20 42.74 ± 3.81 35.15 ± 2.84 37.85 ± 7.38 31.31 ± 5.37 +21.3 0.035

Plant height, Sep. 24 8.19 ± 0.23 6.84 ± 0.90 7.69 ± 0.81 6.48 ± 0.75 +19.2 0.010
em Jan. 20 16.44 ± 1.36 13.86 ± 2.45 14.53 ± 2.50 11.93 ± 1.80 +20.1 0.052

Chlorophyll Sep. 24 39.0 ± 2.3 33.5 ± 3.1 39.0 ± 1.3 35.8 ± 3.4 +12.6 0.014
content, 11g em -2 Jan. 20 26.3 ± 3.3 28.1 ± 6.1 29.4 ± 6.4 23.9 ± 2.5 +7.1 0.534

& Butler (1985). For total nitrogen estimation, the
Kjeldahl method was used (Allen 1989).
Statistics
For all measurements, the results were expressed as
means ± S.D. from four replicates (plots) per treat-
ment. For non-destructive measurements the values
arise from 8 plants per plot at all dates, with the ex-
ception of the final harvest in which the values arise
from 4 plants per plot. For destructive measurements,
the values arise from 4 plants per plot. Differences
between treatments were assessed through a two-way
ANOVA statistical test.
Results and discussion
Statistical analysis showed that there were no signif-
icant interactive effects between supplemental UV-
B radiation and additional watering on all sampling
dates. This is in contrast to previous field experi-
ments with other Mediterranean plants, showing that
additional watering during the summer abolished the
negative (Drilias et a!. 1997) or positive (Manetas
et a!. 1997) UV-B irradiation effects on growth of
Nerium oleander and Pinus pinea, respectively. Con-
cerning the effects of UV-B radiation per se, these
were season specific, as shown on Table I. Thus,
leaf number and nitrogen content were significantly
lower in UV-B plants at the end of the dry season
(September) but the differences were abolished at fi-
nal harvest (January). On the contrary, the substantial
positive effect on stem dry mass was observed only at
final harvest. None of the other measured parameters
(leaf area, dry mass and thickness, UV-B absorbing
compounds, total phenolics, tannins, chlorophylls and
PSII photochemical efficiency) were affected by UV-
B supplementation (data not shown). The increase in
stem dry mass has not been reported elsewhere. Both
increases (Murali & Teramura 1985; Wand et al. 1996)
and decreases (Dai et a!. 1992; He et al. 1993; Ku-
landaivelu & Nedunchezhian 1993; Vu et al. 1982)
of leaf nitrogen content due to increased UV-B radi-
ation have been reported, while in other cases UV-B
radiation was ineffective (N aidu et a!. 1993; Vu et al.
1982; Wand et al. 1996). A decrease in nitrogen in-
dicates a lower nutritional value making the leaves
less prone to herbivore attack or, alternatively, may
192
lead to over-consumption to compensate for low food
nitrogen levels (Schoonhoven et al. 1998).
Concerning irrigation, the effects were as ex-
pected, with well-watered plants being taller and
having more leaves compared to water stressed ones
(Table 2). These effects were sustained throughout the
experiment. Additionally, well-watered plants had sig-
nificantly higher chlorophyll contents during the dry
period (Table 2). In fact, this was due to chlorophyll
loss in water-stressed plants, which was abolished
with additional watering. This type of response is con-
sidered a common photoprotective adaptation under
photooxidative conditions (Kyparissis et al. 1995) and
has also been found under water stress situations for
several Mediterranean semi-decidual and sclerophyl-
lous species (Faria et al. 1998; Nufiez-Oiivera et al.
1996; Stephanou and Manetas 1998). In all other mea-
sured parameters, the effects of additional irrigation
were negligible and only non-significant trends for in-
creased total leaf area and above ground dry mass were
observed (data not shown).
We may assume from the above that the growth of
the evergreen sclerophyll, slow-growing plant C. sili-
qua is not much affected by both UV-B radiation
and additional watering, at least under the conditions
used in this experiment. However, the subtle, mostly
season-specific effects observed on some parameters
could have a long-term impact on the fitness of this
plant.
Acknowledgement
This work was funded by the Commission of Euro-
pean Communities (project code: ENV4-CT96-0208).
References
Allen, D. J., Nogues, S. & Baker, N. R. 1998. Ozone depletion and
increased UV-B radiation: is there a real threat to photosynthesis?
J. Exp. Bot. 49: 1775-1788.
Allen, S. E. 1989. Chemical Analysis of Ecological Materials.
Blackwell, Oxford.
Asquith, T. N. & Butler, L. G. 1985. Use of dye-labelled protein as a
spectrophotometric assay for protein precipitants such as tannin.
J. Chern. Ecol. II: 1535-1543.
Bjorn, L. 0. & Murphy, T. M. 1985. Computer calculation of solar
ultraviolet radiation at ground level. Physiol. Veg. 23: 555-561.
Bjorn, L. 0., Callaghan, T.V., Johnsen, 1., Lee, J. A., Manetas, Y.,
Paul, N. D., Sonesson, M., Wellburn, A. R., Coop, D., Heide-
Jorgensen H. S., Gehrke, C., Gwynn-Jones, D., Johanson, U.,
Kyparissis, A., Levizou, E., Nikolopoulos, D., Petropoulou, Y. &
Stephanou, M. 1997. The effects ofUV-B radiation on European
heathland. Plant Ecol. 128: 252-264.
Caldwell, M. M. 1971. Solar UV radiation and the growth and de-
velopment of higher plants. Pp. 131-177. In: Giese, A. C. (ed.),
Photophysiology. Academic Press, New York.
Caldwell, M. M., Flint, S.D. & Searles, P. S. 1994. Spectral balance
and UV-B sensitivity of soybean: a field experiment. Plant Cell
Environ. 17: 267-276.
Dai, Q., Coronel, V. P., Vergana, B.S., Barnes, P. W. & Quintos A. T.
1992. Ultraviolet-B radiation effects on growth and physiology
of four rice cultivars. Crop Sci. 32: 1269-1274.
Day, T. A. 1993. Relating radiation screening effectiveness of
foliage to absorbing-compound concentration and anatomical
characteristics in a diverse group of plants. Oecologia 95: 542-
550.
Drilias, P., Karaboumiotis, G., Levizou, E., Nikolopoulos, D.,
Petropoulou, Y. & Man etas, Y. 1997. The effects of enhanced
UV-B radiation on the mediterranean evergreen sclerophyll Ner-
ium oleander depend on the extent of summer precipitation.
Aust. J. Plant. Physiol. 24: 301-306.
Faria, T., Silverio, D., Breia, E., Cabral, R., Abadia, A., Abadia,
G., Pereira, J. S. & Chaves, M. M. 1998. Differences in the
response of carbon assimilation to summer stress (water deficits,
high light and temperature) in four Mediterranean tree species.
Physiol. Plantarum 102:419-428.
Gwynn-Jones, D., Lee, J. A. & Callaghan T. V. 1997. Effects of
enhanced UV-B radiation and elevated carbon dioxide concen-
trations on a sub-arctic forest heath ecosystem. Plant Ecol. 128:
242-249.
He, J., Huang, L-K., Chow, W. S., Whitecross, M. I. & Anderson,
J. M. 1993. Effects of supplementary ultraviolet-B radiation on
rice and pea plants. Ausl. J. Plant. Physiol. 20: 129-142.
Kulandaivelu, G. & Nedunchezhian N. 1993. Synergistic effects of
ultraviolet-B radiation and growth temperature on ribulose I ,5-
bisphosphate carboxylase and 14 co 2 fixation in Vigna sinensis
L. Photosynthetica 29: 377-383.
Kyparissis, A., Petropoulou, Y. & Manetas, Y. 1995. Summer
survival of leaves in a soft-leaved plant (Phlomis fruticosa L.,
Labiatae) under Mediterranean field conditions: avoidance of
photoinhibitory damage through decreased chlorophyll contents.
J. Exp. Bot. 46: 1825-1831.
Lichtenthaler, H. K. & Wellburn, A. R. 1983. Determinations of
total carotenoids and chlorophylls a and b of leaf extracts in
different solvents. Biochem. Soc. Trans. II: 591-592.
Manetas, Y., Petropoulou, Y., Stamatakis, K., Nikolopoulos, D.,
Levizou, E., Psaras, G. & Karaboumiotis G. 1997: Beneficial
effects of enhanced UV-B radiation under field conditions: im-
provement of needle water relations and survival capacity in
Pinus pinea L. seedlings during the dry Mediterranean summer.
Plant Ecol. 128: I00-108.
Mooney, H. A. 1987. The impact of environmental stress on plant
performance in Mediterranean climate ecosystems. Pp. 661-668.
In: Tenhunen, J. D., Catarina, F. M., Lange, 0. L. & Oechel,
W. C. (eds), Plant Response to Stress-functional Analysis in
Mediterranean Ecosystems. Springer-Verlag, Berlin.
Murali, N. S. & Teramura A. H. 1985. Effects of ultraviolet-B irra-
diance on soybean VII. Biomass and concentration and uptake of
nutrients at varying P supply. J. Plant Nutr. 8: 177-192.
Naidu, S. L., Sullivan, J. H., Teramura, A. H. & DeLucia E. H.
1993. The effects of ultraviolet-B radiation on photosynthesis of
different aged needles in field-grown loblolly pine. Tree Physiol.
12: 151-162.
Nufiez-Olivera, E., Martinez-Abaigar, J. & Escudero, J. C. 1996.
Adaptability of leaves of Cistus /ada/lifer to widely varying
environmental conditions. Fun ct. Ecol. I0: 636-646.

Rambal, S. & Debussche, G. 1995. Water balance of Mediterranean
ecosystems under changing climate. Pp. 386-407. In: Monero
J. M. & Oeehel W. C. (eds), Global Change and Mediterranean
Type Ecosystems. Ecological studies. Vol. 117. Springer-Verlag,
New York.
Rozema, J., van de Staaij, J., Bjorn. L. 0. & CaldwelL M. M.
1997. UV-B as an environmental factor in plant life: strc>S and
regulation. Trends Ecol. Evol. 12: 22-28.
Schoonhoven L. M., Jerrny. T & Van Loon, J. J. A. 1998. Insect-
plant Biology. Chapman & Hall, London.
Stephanou. M. & Manetas, Y. 1998. Enhanced UY-B radiation in-
creases the reproductive effort in the Mediterranean shrub Cistus
creticus under field conditions. Plant Ecol. 134: 91-96.
Sullivan, J. H. & Teramura. A. H. 1990. Field study of the
interaction between solar ultraviolet-B radiation and drought on
193
photosynthesis and growth in soybean. Plant Physiol. 92: 141-
146.
Yu, C. V.. Allen. Jr. L. H., & Garrard L.A. 1982. Effects of supple-
mental UV-B radiation on primary photosynthetic carboxylating
enzymes and soluble protcines in leaves of c3 and c4 crop
plants. Physiol. Plant. 55: 11-16.
Wand. S. J. E., Midgley. G. F. & Musil C. F. 1996. Physiological
and growth rcsponces of two African ~pe~.:ies, Acacia korroo
and Themeda triandra. to combined increases in C02 and UY-B
radiation. Physiol. Plant. 98: 882-890.
Waterman, P. G. & Mole. S. 1994. Analysis of Phenolic Plant
Metabolites. Blackwell, Oxford.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

The effect of enhanced UV-B radiation on the growth of faba bean. (Photograph by .1. Rozema)
 Plant EcologY 154: 197-210,2001.
197
© 2001 Kluwer Acudemic Publishers.

Combined effects of C0 2 concentration and enhanced UV-B radiation

on faba bean

Marcel Tosserams*, Andries Visser, Mark Groen, Guido Kalis, Erwin Magendans &
Jelte Rozema
Department of' Systems Ecology, Faculty of' Biology, Vrije Universiteit, De Boelelaan I 087, I 081 HV Amsterdam,
the Netherlands(* Addressji1r correspondence: Montf'erland 8, 8245 BM Lelyslad, The Netherlands,
E-mail: m. tosserams@ riza. rws.minvenw. nl)

Key words: Apical dominance, Biomass allocation, Biomass production, Carbohydrates, C02 enrichment, Faba
bean, Photosynthetic acclimation, UV-B radiation

Abstract
Due to anthropogenic influences, both solar UV-B irradiance at the earth's surface and atmospheric [C02] are
increasing. To determine whether effects of C02 enrichment on faba bean (cv. Minica) growth are modified by
UV-B radiation, the effects of enhanced [C02] on growth and photosynthetic characteristics, were studied at four
UV-B levels. Faba bean was sensitive to enhanced UV-B radiation as indicated by decreases in total biomass
production. Growth stimulation by C02 enrichment was greatly reduced at the highest UV-B level. [C02] by
UV-B interactions on biomass accumulation were related to loss of apical dominance. Both [C02] and UV-B
radiation affected biomass partitioning, UV-B effects being most pronounced. Effects of [C02] and UV-B on
faba bean growth were time-dependent, indicating differential sensitivity of developmental stages. [C02] and UV-
B effects on photosynthetic characteristics were rather small and restricted to the third week of treatment. C02
enrichment induced photosynthetic acclimation, while UV-B radiation decreased light-saturated photosynthetic
rate. It is concluded that the reduction in biomass production cannot be explained by UV-B-induced effects on
photosynthesis.

Introduction the year 2000 (Madronich et a!. 1995 ), with a slow

Due to the combustion of fossil fuels and defor-
estation, atmospheric C02 concentration ([C02]) in-
creases at a rate of 1.5 Jimol mol- 1 year-!. Current
projections suggest that by the middle of the next cen-
tury atmospheric [C02] will have doubled compared
to pre-industrial levels (Watson et al. 1990). Concur-
rently with the increasing [C02], stratospheric ozone
concentration is being reduced as a result of anthro-
pogenic emissions of chlorofluorocarbons (CFCs) and
other trace gasses (Molina & Rowland 1974). Due
to this depletion of ozone. the solar UV-B fluence at
the earth's surface is increasing (Biumthaler & Am-
bach 1990; World Meteorological Organization 1994 ).
Under the current CFC phase-out schedules, peak re-
ductions of stratospheric ozone are expected around
recovery over the subsequent 50 years (World Me-
teorological Organization 1994 ). As a consequence,
in the next century plant life will encounter both
an increased atmospheric [C02] and enhanced UV-B
radiation environment.
The effects of C02 enrichment (see Bazzaz 1990;
Rogers & Dahlman 1993, for reviews) and UV-B
radiation (see Caldwell & Flint 1994; Teramura &
Sullivan 1994; Rozema et al. 1997a, for reviews) on
plants are well documented. The limited experimen-
tal data on the combined effects of [C02] and UV-B
(Sullivan 1997), suggests that enhanced solar UV-B
radiation may counteract the stimulation of growth in-
duced by C02 enrichment. However, considerable in-
terspecific and intraspecific variability in responses are
observed. In general, growth responses to a combina-
198
tion of [C02] and UV-B were found to be independent
(Rozema et al. 1997b; Sullivan 1997) and interac-
tions were only reported in a limited number of studies
(Ziska & Teramura 1992; Sullivan & Teramura 1994 ).
The mechanisms responsible for the observed interac-
tions still have to be unravelled, although results of
some studies suggest that UV-B radiation may restrict
photosynthetic capacity at elevated [C02] (Teramura
et al. 1990; Ziska & Teramura 1992).
The present study describes a greenhouse exper-
iment in which the response of faba bean to C02 en-
richment was studied at four levels of UV-B. The main
objective of this study was to determine, whether, how
and to what extent, effects of C02 enrichment on
growth and biomass allocation of faba bean are modi-
fied by UV-B radiation during faba bean development.
Materials and methods
Growth conditions and experimental design
Seeds of faba bean (Vicia faba L. cv. Minica) were
sown in pots (4 dm 3 ) filled with commercial potting
soil (Jongkind, Aalsmeer, NL) and cultivated in a
greenhouse. Pots contained 3.5 g dm- 3 osmocote con-
trolled release fertiliser (13:13:13:3:2, N:P:K:Mg:Fe;
Grace Sierra Int., Heerlen, NL). Mean air temperature
during the experiment was 13.0 ± 0.5°C (night) and
22.5 ± 0.7°C (day). Relative humidity was 82 ± 2%
(night) and 62 ± 2% (day).
Twelve days after germination, seedlings were
thinned to single pots. One day later, the experi-
ment was initiated (day 0). Plants were either trans-
ferred to a greenhouse compartment with a [C02] of
380 11mol mol- 1, or to a compartment with a [C02]
of 750 11mol mol- 1. In each compartment four UV-B
treatments were applied. Forty plants per UV-B treat-
ment were studied (320 plants in total). As all UV-B
treatments were duplicated in both greenhouse com-
partments, these plants were divided into two groups
of 20 plants. Plants within each greenhouse compart-
ment were rotated and randomized between duplicate
UV-B treatments twice a week, to minimise positional
effects.
For each UV-B treatment, radiation was supplied
by two Philips 40W/12 tubes. The tubes were attached
at both sides of a 400 W Philips HPI/T lamp that
operated 14 hours daily, providing a minimal photo-
synthetic photon flux density (PPFD) of 12.6 mol m- 2
day- 1• For the UV-B treatments, radiation emitted by
the Philips 40W/12 tubes was filtered with 0.10 mm
thick cellulose acetate foil (Tamboer and Co. Chemic
B.V., Haarlem, NL), which absorbs all radiation
<290 nm. For the control treatment, the UV-B tubes
were filtered with 0.13 mm thick polyester foil (Mylar,
Dupont Industries, USA), which absorbs all radiation
<313 nm. Cellulose acetate was replaced twice a week
and the polyester foil was replaced once a week.
The spectral irradiance of the UV-B lamps was
measured with a double-monochromator spectrora-
diometer (Optronic Model OL 752, Orlando, FL,
USA). The spectroradiometer was calibrated for ab-
solute responsivity against a 200 W tungsten-halogen
standard lamp (Optronic Model OL 752-10, Orlando,
FL, USA). Before the measurements, wavelength ac-
curacy was checked with a dual calibration and gain
check source module (Optronic Model OL 752-150,
Orlando, FL, USA), by scanning a low-pressure mer-
cury vapour lamp with a known peak emission at
312.9 nm.
The generalised plant action spectrum (Caldwell
1971 ), normalised at 300 nm, was used to deter-
mine the biologically effective UV-B dose (UV-BsE).
Weighted daily UV-BsE dose was 0 for control plants
and 4.6, 7.6 and 10.6 kJ m- 2 dar' for UV-B treated
plants. Plants were irradiated from 10:00 to 16:00 h
daily. The different UV-B treatments were obtained by
adjusting the height of the UV-B lamps above the top
of the plants. As plants grew, the height of the tubes
was adjusted in such a way that UV-B levels at the
top of the plants remained constant. This was checked
daily using a portable UV-X radiometer equipped with
a UV-X 31 sensor (San Gabriel, CA, USA).
Absolute UV-A radiation emitted by the 400 W
Philips HPT/T lamps and the 40 W/12 UV-B tubes was
1.56 for control plants and 1.21, 1.56 and 1.91 W m- 2
for the UV-B-treated plants.
Growth measurements
During the experiment three harvests were conducted
(day I 0, 24 and 41 ). Ten randomly selected plants per
treatment were divided into leaves, stems, roots and
flowers. After determination of the fresh weight, all
plant parts were oven-dried (70°C, 48 h) after which
dry weight was determined. Total leaf area per plant
was measured with a Li-31 00 leaf area meter (LI-COR
Inc., Lincoln, NE, USA). In addition, the develop-
mental stage of the plants was determined at each
harvest using the Plastochron Index (PI) according to
the equation of Erickson & Michelini ( 1957):

PI= n + (ln(L 11 ) -ln(Lrct)/ln(L 11 ) -ln(L[n+IJ)),
where n is the number of leaves longer than the ref-
erence length (Lref), L 11 is the length of leaf n, and
Lrn+ 11 is the length of leaf n + I. Measurements of the
leaflets nearest to the stem were used for the latter two
parameters.
Photosynthesis
Photosynthesis was measured using a portable infrared
gas analyser (LCA3 and broad-leaf 'Parkinson leaf
chamber', Analytical Development Co., Hoddesdon,
Herts, UK). All measurements were conducted on the
youngest fully developed leaflet in a climatised room
at a chamber temperature of 25 ± 0.2 °C. Prior to
the measurements, the IRGA was calibrated against
a known [C02]. For all measurements, the air leaving
the cuvette was maintained at a relative humidity of
70%-80% by diverting part of the inlet air through a
column filled with FeS04 · 7H20. Assuming complete
mixing within the cuvette, the relative humidity inside
the chamber was estimated between 70% and 80%.
During the experiment three types of photosyn-
thetic measurements were conducted. At day 5, 17,
and 31 the light-saturated photosynthesis ( 1250 ±
20 ~tmol m- 2 s- 1 PPFD) was measured at an exter-
nal [C02] (Ca) of 380 ~tmol mol- 1. At day 7, 20 and
33 A/ Ci curves were obtained by decreasing theCa in
8 steps from 1500 ~tmol mol- 1 to 75 ~tmol mol- 1 at
saturating light intensity (1250 ~tmol m- 2 s- 1 PPFD).
The A/ Ci curves were fitted to the non-linear model:
A = A00 [l - e(-k(C;-Xo))], where A is the net pho-
tosynthesis, Aoo is the predicted rate of assimilation
at saturating [C02], k the rate of increase in A with
increasing [C02] and Xo the C0 2 compensation point
(Potvin et al. 1990). Plants grown at the highest UV-
BsE level were measured at day 7 only, because later
on in the experiment leaves were damaged to such an
extent by UV- B that photosynthesis could no longer be
measured.
At day 14 and 35 light-response curves were made
by decreasing PPFD in 9 steps from 1250 ~tmol m-2
s- 1 to 50 {lmol m- 2 s- 1, with 5 measuring points
in the range between 0 and 200 r<mol m- 2 s- 1. The
Ca during the measurements was 350 {lmol moi- 1.
Light-response curves were fitted with the non-linear
model: A = A max[ 1- e-(Qapp * 1I AmaJ]- RJ, where A
is net photosynthesis, I is PPFD, A max the maximum
gross photosynthetic rate, Qapp the 'apparent' light ef-
ficiency and Rd the dark respiration rate (Goudriaan
1982). As with the A/ Ci curves, leaves receiving the
199
highest UV-BsE level were only used at the first mea-
surement (day 14). Gas-exchange parameters were
calculated according to von Caemmerer & Farquhar
( 1981).
Determination (!f carbohydrates and statistical
analysis
Soluble and insoluble carbohydrate content of the
youngest fully developed leaflet were determined us-
ing the anthrone method (Yemm & Willis 1954 ). Only
plants harvested between 12:00 and 15:00 hours were
analysed.
All data were analysed by a three-way analysis of
variance (ANOVA, Sokal & Rohlf 1981), testing for
[C02], UV-B, time effects and their interactions. Data
were also subjected to a two-way AN OVA for the sep-
arate harvests. The ?-values of the latter analysis are
presented in the text when relevant. Before analysis,
plant dry weight and leaf area data were transformed
to their natural logarithms to obtain homogeneity of
variance. In addition, leaf weight ratio (LWR, leaf
dry weight/total dry weight), root weight ratio (RWR,
root dry weight/total dry weight), stem weight ratio
(StWR, stem and petiole dry weight/total dry weight)
and flower weight ratio (FWR flower dry weight/total
dry weight) were arcsin JX transformed.
A/ C curves and light-response curves were fitted
using the SYSTAT non-linear regression procedure.
Obtained parameters were analysed by two-way or
three-way AN OVA (Sokal & Rohlf 1981 ).
Results
At all harvests, C02 enrichment significantly in-
creased total plant dry weight ( P <0.00 I; Table I).
At the first harvest, in the absence of UV-B radiation.
stimulation of biomass accumulation by C0 2 enrich-
ment was 9%. At the final harvest the increase was
37%. The stimulation of biomass production by C0 2
enrichment varied with increasing UV-B levels. At
the final harvest stimulation of biomass accumulation
was largest at 7.6 kJ m- 2 day- 1 UV-BsE and small-
est at I 0.6 kJ m- 2 day- 1 UV-BsE (62% and 19%.
respective! y).
In contrast, UV-B enhancement resulted in a re-
duction of biomass production (?<0.001; Table I).
At 380 f1mol mol- 1 C02, a UV-B dose-response re-
lationship was observed at all harvests with a maximal
reduction of 48% at l 0.6 kJ m- 2 day- 1 UV-BHE at
200

Table I. The combined effects of COz concentration and UV-B radiation on biomass partitioning of Vicia faha plants. DLWR. dead
leaf weight ratio; FWR. flower weight ratio; LAR, leaf area ratio; LWR. leaf weight ratio; RWR, root weight ratio; SLA, specific
leaf area; SRR, shoot to root ratio; StWR, stem weight ratio. Values represent means of 10 replicates. In the final row the ANOVA
P-values for three-way analysis of variance are presented; ns =not significant (P > 0.05).

COz UV-BsE Total OW LWR DLWR StWR RWR FWR SRR

(fLmol mol- 1) (kJ m- 2 day- 1) (g)

Day 10
380 0 0.91 0.40 0.23 0.37 1.71 40.9 14.7
380 4.6 0.92 0.40 0.21 0.39 1.61 39.5 14.0
380 7.6 0.88 0.42 0.21 0.37 1.73 36.8 13.8
380 10.6 0.78 0.41 0.22 0.36 1.77 34.6 12.1

750 0 0.98 0.41 0.23 0.36 1.80 40.2 14.5

750 4.6 0.99 0.42 0.22 0.37 1.74 37.9 13.5
750 7.6 0.92 0.42 0.22 0.35 1.83 37.2 13.7
750 10.6 0.81 0.40 0.23 0.37 1.76 32.3 11.3

Day 24
380 0 2.10 0.44 0.28 0.26 0.020 2.94 39.6 15.2
380 4.6 2.01 0.44 0.26 0.27 0.026 2.74 37.8 14.7
380 7.6 2.01 0.46 0.25 0.27 0.027 2. 77 36.7 14.6
380 10.6 1.45 0.47 0.27 0.23 0.037 3.38 31.3 12.7

750 0 2.76 0.42 0.30 0.26 0.022 2.87 42.6 15.7

750 4.6 2.63 0.43 0.28 0.27 0.026 2.79 37.8 14.7
750 7.6 2.78 0.45 0.27 0.26 0.029 2.93 36.1 14.4
750 10.6 1.78 0.45 0.27 0.24 0.040 3.18 29.9 11.6

Day 41
380 0 4.45 0.34 0.05 0.33 0.22 0.063 3.54 39.9 12.4
380 4.6 4.27 0.36 0.05 0.30 0.21 0.076 3.89 36.9 12.3
380 7.6 3.62 0.35 0.08 0.29 0.19 0.087 4.22 35.2 11.4
380 10.6 2.32 0.26 0.15 0.30 0.19 0.104 4.41 31.9 7.6

750 0 6.08 0.32 0.05 0.35 0.22 0.069 3.54 38.2 11.4
750 4.6 5.34 0.32 0.05 0.32 0.23 0.084 3.38 36.6 10.9
750 7.6 5.87 0.35 0.05 0.30 0.21 0.094 3.81 35.3 11.3
750 10.6 2.76 0.28 0.12 0.33 0.17 0.105 4.91 37.3 9.5

Statistics
C02 <0.001 0.040 0.031 <0.00 1 ns ns ns ns ns
UV-B <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Time <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.009 <0.001
C02 x UV-B 0.029 ns ns ns ns ns ns ns ns
UV-B x Time <0.001 <0.001 <0.001 <0.001 0.002 <0.001 <0.001 ns
C02 x UV-B x Time ns 0.002 ns 0.012 ns <fl.OOI «UJOI <0.001
 201
Table 2. The combined effects of C02 concentration and UV-B radiation on morphological character-
istics and development of Vicia.fizlm plants. Values represent means of I0 replicates. In the final row the
AN OVA ?-values for three-way analysis of variance are presented. ns = not significant ( P > 0.05):
nm = not measured.

C02 UV-B13E No. leaves No. additional No. flowers Plant height
(fimol mol-l) (kJ Ill -2day-IJ shoots (em)

Day 10
380 0 4.2 0.3 21.2
380 4.6 4.3 0.6 19.4
380 7.6 4.2 0.5 17.7
380 10.6 3.9 0.1 18.2

750 0 4.1 0.1 21.6

750 4.6 4.2 0.5 19.6
750 7.6 4.0 0.3 19.7
750 10.6 4.2 0.6 18.7

Day 24
380 0 8.6 0.2 nm 40.4
380 4.6 9.1 0.7 nm 35.6
380 7.6 10.7 1.2 nm 31.9
380 10.6 9.0 0.1 nm 32.6

750 0 8.9 0.5 nm 44.7

750 4.6 9.3 0.6 nm 38.6
750 7.6 10.7 1.2 nm 38.1
750 10.6 9.8 0.6 nm 36.1

Day41
380 () 16.4 0.3 7.2 60.7
380 4.6 17.1 0.8 7.7 54.6
380 7.6 18.8 l.2 8.1 49.0
380 10.6 \8.3 0.5 6.8 48.6

750 0 \8.7 0.8 9.1 68.8

750 4.6 18.5 1.4 10.0 60.0
750 7.6 20.9 1.2 10.6 56.0
750 10.6 17.5 0.5 7.8 56.3

Statistics
C02 0.042 ns ns <0.001
UV-B 0.001 <0.001 ns <0.001
Time <0.001 0.001 <0.001
C02 x Time ns ns <0.001
UB-B x Time ns ns ns <0.00\
Other ns ns ns ns

the final harvest. At increased [C02], a dose-response
relationship was only observed at the first harvest. At
the final harvest maximal reduction was 55% at I 0.6
kJ m- 2 dar 1 UV-BsE but at 7.6 k.T m- 2 day- 1 UV-
BsE only a slight reduction of biomass was found (4%
compared to 19% at ambient [C02]). This explains the
observed [C02] by UV-B interaction (P = 0.029).
This interaction was present in both the shoot and
root compartment (P = 0.018 and P = 0.005,
respectively).
202
800
Biomass allocation was influenced by both in-
creased [C02] and enhanced UV-B radiation (Table I). 700 380 J.lmol mol·l COz
The proportion of biomass invested in leaf material 600
(leaf weight ratio, LWR + dead leaf weight ratio,
DLWR) was reduced by C02 enrichment in favour of 500

stem biomass (StWR). The leaf area per leaf weight 400
(SLA) was reduced by elevated UV-B radiation at both
300
[C02]. In general, a clear UV-B dose-response rela-

s
tionship was observed at each harvest, although the 200

magnitude of decrease varied in time. A similar trend ~ 100

was observed for the total amount of leaf area per ~
t~ 800
total plant weight (LAR). After 24 and 41 days of
treatment, the proportion of biomass invested in the l.... 700 750 J.lffiOI mol· 1 COz

root system was reduced by enhanced UV-B levels. 600

This resulted in an increase of the shoot to root ratio
500
(SRR). LWR and StWR were also affected by UV-B
enhancement, although the response varied in time. At 400

the first harvest LWR and StWR remained unaffected 300

by UV-B, whereas after 24 days the proportion of bio-
mass invested in the leaves was increased by higher
UV-B levels at the expense of stem and root biomass.
At the final harvest the same trend was still present,
except for plants receiving the highest UV-B level,
where a reduction of the LWR was observed. This
was mainly due to an increase in the percentage of
dead leaves (DLWR). In spite of a decrease in absolute
flower dry weight by enhanced UV-B (P<O.OOI; data
not shown), flower biomass to total plant biomass
(FWR) was increased by UV-B enhancement at both
the second and final harvest. C02 enrichment in-
creased plant height (P <0.001; Table 2) at all UV-B
treatments. At the final harvest this increase was 13%
in the absence of UV-B. Elevated UV-B treatments re-
duced plant height at both ambient and elevated [COz]
( P < 0.00 I; Table 2). Both C02 enrichment and UV-
B enhancement resulted in increased number of leaves
(Table 2), the effect of enhanced UV-B being most
pronounced.
The increase of the number of leaves due to en-
hanced UV-B is related to the promotion of additional
shoot development by UV-B radiation (Table 2). The
number of additional shoots gradually increased with
increasing UV-B levels but was reduced again by the
highest UV-B treatment. After 24 days, the number
of shoots was largest for plants that were grown at
7.6 kJ m- 2 day- 1 UV-BsE for both [COz]. At this
UV-B level, growth of the main shoot was reduced
and a new additional shoot developed at the base of the
main shoot. By the end of the experiment, the length of
this newly developed shoot was almost the same as the
length of the main shoot. The number of flowers (only
200
100
0
5 10 15 20 25 30 35 40 45
Time (days)
Figure 1. The effect of C02 and UV-B on total leaf area produced
by Vicia faba plants during the course of the experiment. 380 JLmol
mol-l C02 (closed symbols); 750 )Lmol mol- 1 C02 (open sym-
bols). 0 kJ m- 2 day- 1 UV·BBE (control plants; 0); 4.6 kJ m- 2
day-! UV-BBE (o); 7.6 kJ m- 2 day-I UV-BsE (D) and 10.6
kJ m- 2 day- 1 UV-BBE (L1). Values represent means of 10 replicate
plants± SE.
measured at the final harvest) was not influenced by
[COz] and UV-B radiation (Table 2).
Total leaf area was increased by C02 enrichment
(P <0.00 I; Figure I), while increased UV-B radiation
levels resulted in decreased leaf area (P<O.OOI; Fig-
ure 1). At the final harvest, the maximal increase of
leaf area due to C02 enrichment was observed at 7.6
kJ m- 2 day- 1 UV-BsE (60%). The largest decrease
due to enhanced UV-B was observed at the highest
UV-B level for both [COz] (62%).
Since the area of newly formed leaf material de-
pends on the developmental stage of the plant, leaf
area is presented as a function of the PI (Figure 2).
When comparing the single leaf area of plants at
the same developmental stage (identical PI), [COz]
significantly increased the leaf area per leaf ( P <
0.001), while enhanced UV-B levels significantly re-
duced this parameter (P < 0.001). At a [COz] of
380 ttmol mol- 1, leaf area per leaf of plants at 7.6 and
 203
60
significantly at day 20. None of these effects were
380 11mol mol· 1 COz observed at day 7 and 33.
50 Parameters derived from light-response curves are
presented in Table 5. Only at day 14 effects were

~
40 observed. Qapp and Rd significantly decreased un-
der elevated [C02], whereas at the same time a trend
30 ( P = 0.090) towards a decreased A max was present.
UV-B radiation significantly decreased the Amax·

s
~
~

20 ~ Averaged over all UV-B treatments, C02 enrich-

ment increased the total carbohydrate content of the
~

.... youngest fully developed leaflet (Table 6), with a

10
"'
..2!
750 11mol mol· 1 C02
"'"'...
< 50
40
30
20
10
2 5 11 14 17 20
Plastochron index
Figure 2. The effect of C0 2 and UV-H on leaf area per leaf or
Vicia .faha plants during the course or the experiment in relation
to the plastoehron index. 380 tJ.mol mol- 1 C02 (closed symbols):
750 fL.mol mol- 1 C0 2 (open symbols). 0 kJ m- 2 day- I UV-BBE
(control plants: 0): 4.6 kJ m- 2 day- I UV-BBE (o): 7.6 kJ m- 2
day- I UV-BBE (D) and 10.6 kJ m 2 day 1 UV-BBE (.6). Values
represent means of 10 replicate plants ± SE.
10.6 kJ m- 2 day- 1 UV-BBE did not increase during
development.
In contrast, at 750 f.i.mol mol- 1lCOz] and l 0.6
kJ m- 2 day- 1 UV-BaE. leaf area per leaf did increase
as plants developed. C02 enrichment increased the PI
(P < 0.001; Figure 2), while UV-B enhancement
reduced the PI of faba bean (P = 0.00 I; Figure 2).
C0 2 enrichment did not affect light-saturated pho-
tosynthesis, stomatal conductance and C; when mea-
sured at the same Ca (Table 3). In contrast, light-
saturated photosynthesis and stomatal conductance
were decreased by UV-B radiation, although this was
only observed at day 17. This decrease could be fully
ascribed to the l 0.6 kJ m- 2 day- 1UV-BaE treatment.
Results obtained from A I C, curves are shown in
Table 4. At day 20, a decrease in A 00 at elevated
[C02] was observed, indicating that regeneration of
ribulose bisphosphate (RuBP) was limited. UV-B ra-
diation had the opposite effect since the A 00 increased
higher increase at day 20 compared to day 47 (II%
and 4%, respectively). This increase was caused by
an increase in soluble sugars alone. UV-B radiation
increased the total carbohydrate content averaged over
both [C02] by 28% and 26% at day 20 and 47, re-
spectively. On day 20. this increase was caused by
an increase in soluble sugars alone, whereas at day
47 both soluble sugars and starch were increased at
the 10.6 kJ m- 2 day- 1UV-BaE treatment. In general,
effects of UV-B appeared to be larger than effects of
lC02].
Discussion
In the absence of UV-B radiation, increased lCOz]
stimulated biomass production of the C3 species Vi-
cia faba by 62% at the final harvest (Table I). This is
consistent with previous studies on this species (Mori-
son & Gifford 1984; Dijkstra et al. 1993; Visser et al.
1997a).
As already suggested by Visser et al. ( 1997a), this
faba bean cultivar proved to be sensitive to UV-B radi-
ation. Results of previous combined lC02l and UV-B
experiments suggest that high levels of UV- B radi-
ation may reduce or fully eliminate the stimulatory
effect of C02 enrichment on biomass production (Sul-
livan 1997), which is in agreement with our findings
(Table 1).
The UV-B induced reduction in biomass accumu-
lation increased with time. This was accompanied by
visible damage to leaves. At the highest UV-B level,
curling, scorching and bronzing of leaves (Visser et al.
1997b) as well as premature leaf-loss were observed.
Similar UV- B effects were also found for other suscep-
tible crop species such as soybean, pea and cucumber
(Teramura 1983). It should, however, be noted that the
average PAR level during the course of the present ex-
periment was relatively low. Although the daily sum
of PAR was comparable to the average daily sum
204

Table 3. The effect or C02 concentration and UV-B radiation on net photosynthesis. stomatal conductance
and internal C02 concentration (Ci) or the youngest fully developed Vicia.filbaleallet after 5. 17 and 31 days
of treatment. Measurements were conducted at an external C02 concentration of 380 fimol mol- 1. Values
represent means of 4 replicate plants. In the final row at each day, the AN OVA ?-values for two-way analysis
of variance for main effects and interactions are presented: ns =not significant (P > 0.05).

C02 UV-BsE Net photosynthesis Stomatal conductance ci

(fimol mol-l) (kJ m- 2 day- I) (11mol C02 m -2 ,-1) (mmolH20m -2 ,-I) (!nnol mol- 1)

Day 5
380 0 9.9 187 250
380 4.6 10.4 224 258
380 7.6 12.2 295 267
380 10.6 11.8 274 261

750 0 10.5 247 266

750 4.6 11.6 259 264
750 7.6 11.0 249 270
750 10.6 11.4 277 272
col ns ns ns
UV-B ns ns ns
C02 X UV-B ns ns ns

Day 17
380 () 12.1 383 280
380 4.6 13.2 348 277
380 7.6 13.6 426 285
380 10.6 9.2 212 277

750 0 13.6 489 291

750 4.6 11.9 300 280
750 7.6 12.4 305 275
750 10.6 8.5 227 290

col ns ns ns
UV-B <0.001 0.022 ns
C02 x UV-B ns ns ns

Day 31
380 0 13.6 485 274
380 4.6 14.3 332 258
380 7.6 14.3 432 279

750 0 16.0 470 280

750 4.6 13.3 351 273
750 7.6 15.2 415 273

C02 ns ns ns
UV-B ns ns ns
C02 X UV-B ns ns ns
 205
Tohlc 4. The effect of C02 concentration and UV-8 radiation on A/C; curves of the
youngest fully developed Viciofaba leaflet. A/ C; curves were obtained 7, 20 and 33 days af-
ter the start of the treatment. Curves were fitted with the model A = A ex; [I - e 1-k(C; -Xo)l]
(Potvin et a!. 1990). Ax = C02-saturated net photosynthesis. k = time constant,
Xo = C02 compensation point. In all cases R2 was >0.99. Values represent means of 4
replicate plants. In the tina! row m each day. the ANOVA ?-values for two way analysis of
variance for main effects and interactions are presented: ns = not significant ( P > 0.05 ).

C02 UV-8sE A"' k Xo

(pmol mol-l 1 (kJ m- 2 day- 11 (pmo1 C02 m -2 ,-I) lrnno1 C02 I

Day7
380 0 24.2 3.20 57.1
380 4.6 21.5 3.17 70.2
380 10.6 24.3 2.77 70.5

750 () 21.0 .HIS 49.1

750 4.6 20.4 3.12 48.4
750 ]()() 23.6 2.72 47.9

C02 n~ ns <0.001
UV-8 ns ns ns
C02 X UV-B ns ns ns

Day 20
380 0 23.8 2.93 68.5
380 4.() 28.3 1.66 81.9
380 7.6 28.7 2.42 77.4

750 0 21.6 3.21 76.8

750 4.6 26.6 2.15 78.0
750 7.6 23.3 2.42 80.7

C02 0.042 ns ns
UV-8 0.037 ns ns
C02 x UV-8 ns ns ns

nay 33
380 0 26.2 2.69 62.1
380 4.6 25.5 1.91 61.8
380 7.6 30.1 2.27 60.0

750 0 25.9 3.70 71.0

750 4.6 27.5 2.53 65.2
750 7.6 28.5 3.00 65.5

C02 ns ns ns
CV-B ns ns ns
C02 X UV-8 ns ns ns

of PAR during an open-top chamber study with the
same species (Visser et aL 1997a), UV-B8E:PAR ra-
tio was relatively high. This most probably promoted
the effects of UV-B, as UV-B effects are known to be
more pronounced at high UV-B8E:PAR ratios (Cen &
Bornman 1990; Caldwell & Flint 1994).
Both the effects of l C02] and UV-B varied in time
(Table 1), suggesting differential UV-B sensitivity at
different developmental stages. Previous studies (Ter-
amura et al. 1990; Visser et aL 1997a) already men-
tioned the importance of sequential harvesting. These
examples indicate that conclusions drawn from studies
206
Table 5. The effect of COz concentration and UV-B radiation on maximum gross photosynthesis (A max), 'apparent' light
efficiency (Qapp) and dark respiration (RJ). Light-response curves were obtained 14 and 35 days after the start of the
treatments. Curves were fitted with the model A = A max [I - e -(Qapp * 1I Amax) J - Rd (Goudriaan 1982) where A is the
net photosynthesis and I the light intensity. In all cases R 2 was >0.99. Values represent means of 4 replicate plants. In the
final row at each day, the ?-values for two-way analysis of variance for main effects and interactions are presented; ns =
not significant ( P > 0.05).

C02 UV-BsE Amax Qapp Rd

(11mol mol- 1) (kJ m- 2 day- 1) (11mol C02 m- 2 s- 1) (nmol C02 11mol- 1 PAR) (11mol C02 m- 2 ,-I)

Day 14
380 0 10.7 45.0 1.10
380 4.6 9.5 40.4 0.87
380 10.6 8.9 43.6 1.22

750 0 9.0 36.8 0.55

750 4.6 9.9 38.6 0.78
750 !0.6 7.7 33.4 0.62

C02 ns ns 0.040
UV-B 0.026 ns ns
C02 x UV-B ns ns ns

Day 35
380 0 12.2 41.0 0.68
380 4.6 10.4 44.6 0.83
380 7.6 12.3 52.4 1.39

750 0 15.6 42.4 0.73

750 4.6 12.5 39.5 0.72
750 7.6 12.9 44.2 0.69

C0 2 ns ns ns
UV-B ns ns ns
C02 x UV-B ns ns ns

that use single point harvests may be influenced by
timing of sampling (Sullivan 1997).
[C02] and UV-B effects on total biomass accumu-
lation are generally found to be independent (Rozema
et al. 1997b ). However, in a study with three sequential
harvests, Teramura et al. (1990) showed significant
[COz] by UV-B interactions for rice and soybean
plants. Rice plants only showed significant interac-
tions after 5 and I 0 weeks but not at the final harvest.
In contrast, for soybean a significant [COz] by UV-
B interaction on total biomass production was only
apparent at the final harvest, when UV-B decreased
biomass production at ambient [C02] but increased
biomass production at elevated [COz]. Similar results
were obtained in the present experiment. When tested
per harvest, a [COz] by UV-B interaction on total bio-
mass accumulation, shoot biomass and root biomass
was only observed at the final harvest. The UV-B dose-
response relationship as observed at 380 11mol mol- 1
[COz] was not present at 750 11mol mol-l [COz]. The
development of new shoots at 7.6 kJ m- 2 day- I UV-
BBE at both [COz], caused this interaction, starting at
the second harvest. At the final harvest the develop-
ment of shoots at 380 11mol mol-l [COz] had already
been significantly affected by UV-8 irradiance, while
this was not yet the case at 750 {lmol mol-l [COz].
This result suggests that interactions between UV-B
and [COz] on biomass accumulation may only occur
at a specific combination of both factors and at specific
time periods during plant development.
It should be noted that due to the increase in plant
height, UV-B tubes have to be raised from time to
time, as the distance between the top of the plants and
the UV-B tubes has to be maintained constant through-
out the experiment. As a consequence, UV-B levels at
the base of the main shoot decrease during the ex peri-
 207
Table 6. The effect of C02 concentration and UV-B radiation on carbohydrate content of the youngest fuJiy developed
Viciafaba leaf. Leaves were harvested between 12:00 and 15:00 hours. Values represent means of 5 replicate plants. In
the final row the ?-values for three-way analysis of variance for main cffecb and interactions are presented: ns = not
significant (P > 0.05).

C02 UV-BBE Soluble carbohydrates Starch Total carbohydrates

(~tmol mol- 1 ) (kJ m- 2 day- 1 ) (mgg- 1 DW) (mgg- 1 DW) (mgg- 1 DW)
Day 20 47 20 47 20 47

380 0 40.1 53.6 67.4 75.4 107.5 129.0

380 4.6 44.2 55.6 68.9 61.7 113.1 117.3
380 7.6 49.7 53.1 68.0 55.9 117.7 109.0
380 10.6 60.2 76.5 63.9 89.2 124.1 165.7

750 0 45.2 62.3 67.2 69.0 112.3 131.2

750 4.6 49.4 62.1 61.2 72.5 110.6 134.6
750 7.6 58.8 60.9 71.3 54.6 130.1 115.5
750 10.6 83.2 82.8 72.1 80.2 158.0 161.4

Statistics
C02 <0.001 ns 0.048
lJV-B <0.001 0.005 <0.001
Time <0.001 ns 0.007
UV-13 x Time ns ns ns
Other ns ns ns

ment. Therefore, a newly developing shoot at the base
of the main shoot can grow relatively rapidly. Because
of the steep UV-B gradient, the newly developing
shoots of ambient [C02] plants, being smaller than
plants grown at increased [C02], are earlier exposed
to high levels of UV-B than elevated [C02l plants.
Both [C02] and UV-B can affect biomass alloca-
tion, although interspecific variability is large (Tera-
mura 1983; Cure & Acock 1986; Stu len & Den Hertog
1993; Caldwell & Flint 1994). In the present study,
biomass partitioning was predominantly affected by
increased UV-B radiation rather than by an increase of
the [C02]. Notwithstanding the absolute decrease of
leaf area, SRR was increased by enhanced UV-B ra-
diation. This was the result of an increased allocation
to the leaves and less allocation of biomass into sterns
and roots, which has also been observed for other sus-
ceptible crop species such as soybean, bean, cucumber
and pea (Teramura 1983). ln these species the increase
in LWR was accompanied by decreased SLA values.
Similarly, in the present experiment UV-B enhance-
ment also reduced the SLA (fresh weight basis) of the
youngest fully developed leaf, indicating leaf thicken-
ing. As a consequence, leaf optical properties of faba
bean are affected (Visser et al. 1997b ).
Morphological characteristics of plants are also
influenced by both C02 enrichment and enhanced
UV-B radiation (Tevini & Terarnura 1989; Rogers &
Dahlman 1993). In general, C02 enrichment increases
leaf area (Bazzaz 1990). Often such increases are due
to increased leaf number (Allen 1990). ln the present
experiment, an increase in the number of leaves (Ta-
ble 2) as well as an increase of the area per leaf
(Figure 2) was observed. The increase in the number
of leaves is most probably the result of an increased
developmental rate (Figure 2).
Jn contrast to C02 enrichment, UV-B radiation de-
creased plant height (Table 2), which is in agreement
with the results obtained in an open-top chamber ex-
periment using the same cultivar (Visser et al. 1997a).
Reduction of plant height may reflect specific photo-
morphogenic responses of plants to UV-B, mediated
by a UV-B photoreceptor (Lercari ct al. 1990; Bal-
Ian~ et al. 1995). Another potential mechanism for
UV-B induced growth inhibition may be a reduction
or destruction of auxin and the formation of growth
inhibiting IAA photoproducts (Ros & Tevini 1995).
Possibly these mechanisms are also involved in the
loss of apical dominance and the development of new
additional shoots in faba bean. Except for a significant
l C02] by UV-B interaction on leaf area per leaf, C02
208
and UV-B etlects on morphology were independent.
This is in agreement with the available literature on
the combined effects of both factors.
C02 enrichment stimulated photosynthesis through-
out the entire experiment. This is in accordance with
findings of Dijkstra et al. ( 1993), who observed no
significant shifts in the stimulation of canopy photo-
synthesis throughout the growing season for the same
cultivar. Even in the third week of treatment, when
photosynthetic acclimation (e.g., lower photosynthetic
rate measured at the same C;) was observed, photo-
synthesis was still stimulated by 50% as calculated
from A j C; curves (data not shown). The decreased
C02-saturated photosynthesis (Table 4) is an indica-
tion that RuBP regeneration was limited in the third
week of treatment.
In most studies C02 enrichment results in in-
creased levels of soluble carbohydrates and starch
(Stitt 1991; Long & Drake 1992), suggesting a change
in the source-sink balance of the plant. Increased car-
bohydrate concentrations may result in decreased ex-
pression of photosynthetic nuclear genes (Van Oosten
et al. 1994; Krapp & Stitt 1995) and a subsequent
down-regulation of photosynthesis. In the present ex-
periment changes in photosynthesis in response to
C02 enrichment were reflected in carbohydrate con-
tent of leaves (Table 6). At the time of acclimation,
total carbohydrate content of the youngest fully de-
veloped leaflet was increased by 11% whereas this in-
crease was only 4% when photosynthetic acclimation
had disappeared.
Although C02 enrichment effects on Rd show
large variation, a decrease seems to be the rule
(Amthor 1990; Poorter et al. 1992). In the present
experiment, Rd decreased in elevated l C02! grown
plants when measured at identical Ca (Table 3). A
possible cause for the decreased Rd may be found
in the decreased nitrogen content that was observed
at the same time (data not shown). A decreased pro-
tein content may result in a reduction of maintenance
respiration (Ryan 1991 ).
Light-saturated photosynthesis was decreased by
UV-B radiation 17 days after the start of the treat-
ment (Table 3). This occurred in spite of an increased
chlorophyll content (Visser et al. 1997b). The reduced
stomatal conductance found in the present experiment
has been reported by other investigators as well (Ne-
gash & Bjorn 1986). This, however, does not explain
the decreased photosynthesis since C; was not af-
fected. When these data were tested excluding the I 0.6
kJ m- 2 day- 1UV-BsE treatment, no UV-B effect on
light-saturated photosynthesis was found. The leaves
in the 10.6 kJ m- 2 day- 1UV-BsE treatment showed
bronzing and were curled (Visser et al. 1997b ). Most
likely, the observed decrease in photosynthesis was
caused by structural damage.
Both [C02] and UV-B effects on photosynthetic
characteristics were limited to a short time period.
The development of new sinks, like stems and flow-
ers, may explain the disappearance of photosynthetic
acclimation. The disappearance of UV-B effects on
photosynthesis may be the result of increased foliar
UV-B absorbing capacity (Visser et al. 1997b). Fur-
thermore, since all measurements were performed on
the youngest fully developed leaflet, we do not know if
effects measured in the third week of treatment were
still present in that specific leaflet later on in the ex-
periment. No interactions between [C02] and UV-B
radiation on photosynthetic parameters were found,
which is in agreement with the general notion that
effects of l C02] and UV-B are independent (Rozema
et al. 1997a; Sullivan 1997).
Summarising, [C02J by UV-B interactions on bio-
mass accumulation and leaf area per leaf, were appar-
ent and were related to the development of a newly
formed shoot at the base of the main shoot. [C0 2]
by UV-B interactions on biomass allocation and mor-
phology were not observed. In addition, at the highest
level of UV-B radiation the stimulatory effect of C02
enrichment was reduced considerably. UV-B effects
on photosynthetic parameters were small considering
the high dose of UV-BsE applied. The reductions in
biomass production are not the result of effects on
photosynthesis hut may rather be caused by structural
cellular and/or molecular damage.
Acknowledgements
We would like to thank Professor Dr W. H. 0. Ernst
for his constructive comments on the manuscript. This
work was supported by the Dutch National Research
Programme on Global Air Pollution and Climate
Change (NRP), projects 850020 and 850022.
References
Allen, L. H. Jr. 1990. Plant responses to rising carbon dioxide and
potential interactions with air pollutants. J. Environ. Qual. 19:
15-34.
Arnthor, J. S. 1991. Respiration in a future, high C02 world. Plant
Cell Environ. 14: 13-20.

Balian;, C. L., Barnes, P W. & Flint, S. D. 1995. Inhibition of
hypocotyl elongation by ultraviolet-B radiation in de-etiolating
tomato seedlings. I. The photoreceptor. Physiol. Plant. 93: 584-
592.
Bazzaz, F. A. 1990. The response of natural ecosystems to the rising
global C02 levels. Annu. Rev. Ecol. Syst. 21: 167-196.
Blumthaler, M. & Ambach. W. 1990. Indication of increasing solar
ultraviolct-B radiation flux in alpine regions. Science 248: 206-
208.
Caldwell, M. M. 1971. Solar UV irradiation and the growth and
development of higher plants. Pp. 131-177. In C>icse, A. C. (ed.),
Photophysiology. Vol. 6. Academic Press, New York.
Caldwell, M. m & Flint, S. D. 1994. Stratospheric owne reduction,
solar UV-B radiation and terrestrial ecosystems. Clim. Change
28: 375-394.
Cen, Y-P. & Bornman, J. F. 1990. The response of bean plants to
UV-B radiation under different in·adiances of background visible
light. J. Exp. Bot. 41: 1489-1495.
Cure J. D. & Acock B. 1986. Crop responses to carbon dioxide
doubling: a literature survey. Agric. For. Mcteorol. 38: 127-145.
Dijkstra, P, Schapendonk, A. H. C. M. & Groenwold. K. 1993.
Effects of C02 enrichment on canopy photosynthesis. carbon
economy and productivity of wheal and faba bean under field
conditions. Pp. 23-4 L ln: Van de Geijn, S. C., Goudriaan,
J. & Berendse, F. (eels), Climate Change: Crops and Terres-
trial Ecosystems, Agrobiologica\ Themes 9. DLO Centre for
Agrobiological Research, Wageningcn, The Netherlands.
Erickson, R. 0. & Michelini. F. J. 1957. The plastochron index. Am.
.1. Bot. 44: 297-305.
Goudriaan, J. 1982. Potential production processes. Pp. 98-113.
In: F.W.T Penning de Vries, F. W. T & Van Laar, H. H.
(eels). Simulation of Plant Growth and Crop Production. Pudoc.
Wageningen.
Krapp, A. & Stitt, 'VI. 1995. An evaluation of direct and in-
direct mechanisms for the ·sink-regulation' of photosynthesis
in spinach: Changes in gas exchange, carbohydrates, metabo-
lites, enzyme activities and steady-slate transcript levels after
gold-girdling source leaves. Planla 195: 313-323.
Lercari, B., Sodi, F. & Lipucci di Paola, M. 1990. Photo-
morphogenic responses to UV radiation: Involvement of phy-
tochrome and UV photoreceplors in the control of hypocotyl
elongation in Lycnpersicon escu/entum. Physiol. Plant. 79: 668-
672.
Long, S. P. & Drake, B. G. 1992. Photosynthetic C02 assimila-
tion and rising atmospheric C02 concentrations. Pp. 69-95. In:
Baker, N. R. & Thomas, H. (eds), Crop Photosynthesis: Spa-
tial and Temporal Determinants. Elsevier Science Publishers,
Amsterdam.
Madronich, S., McKenzie, R. L., Caldwell. M. M. & Bjorn,
L. 0. 1995. Changes in ultraviolet radiation reaching the earth's
surface. Ambio 24: \43-152.
Molina, M. J. & Rowland, F. S. 1974. Stratospheric sink for chlo-
rofluoromethanes: Chlorine atorn-catalysed destruction of ozone.
Nature 249: 810-812.
Morison, J. 1. L. & Gillord. R. M. 1984. Plant growth and water
use with limited supply in high C02 concentrations. 11. Plant
dry weight, partitioning and water use efficiency. Aust. J. Plant
Physiol. II: 375-384.
Negash, L. & Bjiirn, L. 0. 1986. Stomatal closure hy ultraviolet
radiation. Physiol. Plant. 66: 360-364.
Poorler, H., Gifford, R. M., Kriedemann, P. E. & Wong, S.C. 1992.
A quantitative analysis of dark respiration and carbon content as
factors in the growth response of plants to elevated C02. Aust. J.
Bot. 40:501-513.
209
Potvin. C .. Lechowicz. M. J. & Tardif, S. 1990. The statistical
analysis of ecophysiological response curves obtained from ex-
periments involving repeated measurements. Ecology 71: 1389-
1400.
Rogers. H. H. & Dahlman, R. C. \993. Crop responses to C02
enrichment. Vegelalio 104/105: 117-131.
Ros, J. & Tevini, M. \995. Interaction of UV-radiation and IAA
during growth of seedlings and hypocotyl segments of sunflower.
J. Plant Physiol. 146: 295-302.
Rozema, J., Van de Staaij. J. W. M., Bjorn, L. 0. & Caldwell, M.
:\11. 1997a. UV-B as an environmental factor in plant life: Stress
and regulation. Trends Ecol. Evol.: 12: 22-28.
Rozema, J., Lenssen. G. M., Van de Staaij. J. W. M., Tosserams,
M., Visser, A. J. & Brockman, R. A. 1997b. E!Tecls of UY-B
radiation on terrestrial plants and ecosystems: interactions with
C02 enrichment. Plant Ecol. 128: 182-191.
Ryan, M. G. 1991. Effects of' climate change on plant respiration.
Ecol. 1\ppl. I: 157-167.
Sage, R. F.. Sharkey, T. D. & Seemann. J. R. 1989. Acclimation of
photosynthesis to elevated C02 in live c, species. Plant l'hysiol.
89: 590-596.
Sokal, R. R. & Rohlf, F. J. 1981. The Principles & Practice of
Statistics in Biological Research. Biometry. 2nd edition. W.H.
Freeman & Co .. San Fransico, CA.
Stitt, M. 1991. Rising C02 levels and their potential significance
for carbon flow in photosynthetic cells. Plant Cell Environ. 14:
741-762.
Stu! en, I. & Den Hertog, J. 1993. Root growth and functioning under
atmospheric C02 enrichment. Vegetatio 104/105: 99-115.
Sullivan, J. H. & Teramura, A. H. 1994. The effects of UV-B ra-
diation on loblolly pine. 3. Interaction with C02 enhancement.
Plant Cell Environ. 17: 311-317.
Sullivan. J. H. 1997. Effects of increasing UV-B radiation and at-
mospheric C02 on photosynthesis and growth: Implications f'or
terrestrial ecosystems. Plant Ecol. 128: 194-206.
Teramura, A. H. 1983. Effects of ultraviolet-B radiation on the
growth and yield of crop plants. Physiol. Plant. 58:415-427.
Teramura, A. H., Sullivan, J. H. & Ziska, L. H. 1990. Interaction
of elevated ultraviolct-B radiation and C02 on productivity and
photosynthetic characterictics in rice. wheat and soybean. Plant
Physiol. 94: 470-475.
Teramura, A. H. & Sullivan, J. H. 1994. Effects of UV-B radiation
on photosynthesis and growth of terrestrial plants. Pholosynth.
Res. 39: 463-473.
Tevini. M. & Teramura. A. H. 1989. UV-B effects on terrestrial
plants. Photochem. Photobiol. 50: 479-487.
van Oosten, J. J., Wilkins. D. & Besford, R. T. \994. Regulation of
the expression of photosynthetic nuclear genes hy high C02 is
mimicked hy carbohydrates: a mechanism for the acclimation of
photosynthesis to high C02° Plant Cell Environ. 17: 913-923.
Visser, A. J., Tosserams, M .. Groen. M. W., Kalis. G., Kwant,
R., Magendans. G. W. H. & Rozema, J. \997b. The combined
etlccts of C02 and enhanced UV-B radiation on faba bean. 3.
Leaf optical properties, pigments, stomatal index and epidermal
cell density. Plant Ecol. 128: 208-222.
Visser, A. J., Tosserams. :\!! .. Groen, M. W., Magendans, G. W. H.
& Rozema, J. 1997a. The combined effects of C02 concentra-
tion and solar UV-B radiation on faba bean grown in open top
chambers. Plant Cell Environ. 20: 189-199.
Yon Caemmerer. S. & Farquhar, G. D. 1981. Some relationships be-
tween the biochemistry of photosynthesis and the gas exchange
of leaves. Planta 160: 320-329.
Watson, R. T., Rohde, H., Oeschleger. H. & Siegenthaler. U. 1990.
Greenhouse gasses and aerosols. Pp. 1-40. In Houghton, J. T.,
210

Jenkins. G. J. & Ephraums. J. J. (eds), The !PCC Scientific Ziska, L. H. & Teramura, A. H. 1992. C0 2 enhancement of
Assessment. Cambridge University Press, Cambridge. growth and photosynthesis in rice (Oryza sativa): Modification
World Meteorological Organization 1994. Scientific assessment of by increased ultraviolet-B radiation. Plant Physiol. 99: 473--4X I.
ozone depletion. Pp. 1-12. Executive summary of the WMO
Ozone Report No. 37.
Yemm, E. W. & Willis, A. J. 1954. The estimation of carbohydrates
in plant extracts by Anthrone. Biochem. J. 57: 508-514.
Section 4: Interactions of UV-B Radiation with Other Factors of
Terrestrial Environments

UV-B suppleme ntation system used for the UY-B-additional nutrie nts experiment.
 Plant Eco/ogv 154: 213-217, 200 I.
213
© 200 I Kluwa Acudemic Puh/i,hers.

Enhanced UV-B radiation, artificial wounding and leaf chemical defensive

potential in Phlomis fruticosa L.

Efi Levizou & Yiannis Manetas

Laboratory of Plant Physiology, Department of Biolog); University of Patras, 26500 Patras, Greece
(E-mail: Y.Manetas@ upatras.gr)

Key words: Artificial wounding, Mediterranean, Phenolics, Phlomisfruticosa, UV-B

Abstract
The combined effects of additional UV-B radiation and artificial wounding on leaf phenolics were studied in a short
term field experiment with the drought semi-deciduous Mediterranean shrub Phlomis fruticosa L. The seedlings
were grown under ambient or ambient plus supplemental UV-B radiation (biologically equivalent to a 15% ozone
depletion over Patras, 38.3° N, 29.1° E) for 7 months before wounding. Unexpectedly, supplemental UV-B radia-
tion decreased leaf phenolics. Subsequently, wounding was effected by removing leaf discs from some of the plants,
while the rest remained intact and served as controls. Wounding significantly increased phenolics of the wounded
leaves and the increase was more pronounced under supplemental UV-B radiation. In addition, wounding had a
significant positive effect on the phenolics of the opposite, intact leaf, but only under additional UV-B radiation.
We conclude that UV-B radiation, wounding and their combination may affect the chemical defensive potential
of Phlomis fruticosa. In addition, increased levels of phenolics after herbivore attack under field conditions may
afford extra protection against enhanced UV- B radiation levels.

Introduction caused by injuries to plant tissues. All the related

Plant phenolics absorb strongly in the UV region of the
spectrum, they occur mainly in the epidermis (Rob-
berecht & Caldwell 1978; Grammatikopoulos et al.
1999) and its appendages (Wollenweber & Dietz 1980;
Karabourniotis et al. 1992) and their biosynthesis is
accelerated by UV-B radiation (Beggs & Wellman
1994). Accordingly, it has been proposed that they
may constitute the first line of defense against UV-B
radiation (Caldwell et al. 1983). However, pheno-
lics may have other ecologically important functions
as well. Among them, phenolics function as antifun-
gal agents and, due to their bitter taste and astrin-
gency, they are considered as potent feeding deterrents
(Bernays et al. 1989; Matern & Kneusel 1988). Not
surprisingly, therefore, phenolics and other defensive
secondary metabolites in some cases can be induced
by folivory or simply by wounding the plant (Bald-
win 1994; Neuvonen & Haukioja 1991). This change
in the concentration of secondary metabolites is the
main response, among a wide array of plant responses
phenomena are included in the general term "induced
responses· (Karban et a!. 1989). Based on the above,
we may hypothesize that induction of phenolics by a
single stress factor (i.e., herbivory) can afford protec-
tion against UV-B radiation and vice versa. Recent
work has shown that UV-B radiation increases the
transcripts of pathogen related genes as well as the
production of jasmonic acid and ethylene (Mackerness
ct al. 1999). Since these molecules are components of
the signaling pathways associated with wounding and
infection (Blechert et al. 1995; Ecker 1995), a correla-
tion between UV-B and wounding-induced responses
was inferred. Up to now, extensive literature exists
on the separate effects of UV-B radiation (Rozema
et al. 1997; Caldwell et al. 1998) and wounding on
plant phenolics (Haukioja 1990, Wratten et al. 1984 ).
However, studies on the combined effects of enhanced
UV-B radiation and wounding are still lacking. We
have attempted to fill this gap in a field study with the
Mediterranean plant Phlmnisfruticosa.
214

Materials and methods Experimental design

Plant material, growth conditions, wounding and intact plants intact plants
(to be wounded) Icontrols)
sampling

Seeds of Phlomisfruticosa L. (harvested during June

.
1997 from a single, mature, individual growing in the
vicinity of the university campus) were germinated in ['-
Petri dishes on moist filter paper in a growth room I
with temperature and lighUdark cycles of 15 oc and
12112 h, respectively. PAR at plant level was about r-~ wounded plants

~ II\ ~-
20 tLmolc m- 2 s- 1. After four weeks, healthy plantlcts
were planted into 6-1 plastic pots (one plant per pot)
containing local soil. Further growth was allowed for
two more months in the field under ambient condi-
o fu···
~ wounded intact ~
~l ~)'

tions. On June 1998, 48 fully established and visually
identical seedlings were equally distributed under 4
control and 4 UV-B field plots (6 plants per plot). Each
pot was watered thrice a week with 500 ml oftap water
plus the natural precipitation. Description of the UV-B
and control frames, measurement of spectral irradi-
ance and calculation of the time required for the U V-B
tubes to be on each day in order to obtain supplemen-
tal radiation biologically equivalent to a I 5% ozone
depletion, arc given in Man etas et al.1997. In brief,
Q-Panel UV-B 313 lamps, wrapped in cellulose diac-
ctate film (Courtaulds Chemicals, Derby, UK) were
used. The cellulose diacetate film was changed regu-
larly. Spectral irradiance was measured at night (OL
752 Optronic spectroradiometer, Orlando, FL, USA),
weighted with the generalized plant action spectrum
(Caldwell 1971) normalized at 300 nm and the com-
puter program of Bjorn & Murphy (1985) was used
to compute the required daily duration of the lamps.
In control frames, the UV-B tubes were replaced with
white plastic tube effigies of the same diameter, in
order to have the same shading as in UV-B frames.
With our irradiation system, UV-A radiation under
UV-B frames was slightly higher than ambient. This
slight (1.9%) increase existed as long as all the tubes
were on (around mid-day) and disappeared completely
when all the tubes were off (early morning and late
afternoon). Accordingly, the increase in total daily
percentage was very small. Much higher differences
are needed for UV-A radiation to modify the effects of
UV-B radiation (Caldwell et al. 1994). For more de-
tails of the light conditions see Man etas et al. ( 1997).
After 7 months of growth under the above condi-
tions (Figure 1) there was no effect of supplemental
UV-B radiation on plant growth as judged from height,
leaf area and specific leaf mass measurements. Subse-
l
rl leaves leaves
~ &;
wounding
effect
/ wound
signal
/ time
effect
Figure I. The experimental design followed for wounding treat-
ment. All plants were grown for 7 months under ambient or ambient
plus supplemental UV-B radiation. Wounding was pert!Jrmed on 21
January 1999. After I 0 days. leaf phenolics were measured. For
details see Materials and methods.
quently, on January 21, 1999, half of the plants in each
plot were wounded by removing leaf discs (0.5 cm 2 )
with a cork borer. In particular, 2 leaf discs per leaf
were removed from the same side along the middle
vein and 8 leaves per plant were wounded. Care was
taken to avoid wounding of opposite leaves. The discs
from each wounded plant were accumulated for analy-
sis of phenolics and the values obtained denote the
pre-wounding phenolic levels. After I0 days (Fig-
ure I), leaf discs for phenolic analysis were removed
from:
- wounded leaves (the other side along the mid
vein), for testing the wounding effects;
- intact leaves of wounded plants in order to detect a
possible systemic wound signal;
- control, intact plants for examining if there is a
time effect.
On the basis of preliminary wounding experiment on
individual plants not participating in the experiment, it
was shown that a ten days induction period was needed
 215

in order to get measurable responses of phenolic levels

to wounding.

Measurements

In UV-B oriented research. the crude absorbance of a

methanolic extract at 300 nm (A300) has been adopted
as a measure of the UV-B protective potential of a
plant organ. Although A300 may be a measure of
phenolics as well, it is evident that not all phenolics
absorb at 300 nm (Harbone 1977) nor all methanol
extractable UV-B absorbing compounds are pheno-
lics (Tcvini 1993). Accordingly, the estimation of
A300 may be misleading if one aims on the assess-
ment of leaf phenolics as anti-herbivore or anti-fungal
agents. Instead, a chemical determination of total phe-
nol groups is required (Waterman and Mole 1994).
Therefore, leaf discs were dried at 80 °C for 24 h and
extracted for I h in 50% methanol at 40 °C. Total phe- Figure 2. Effects of supplemental UV-13 radiation and wounding
nolics were measured with the Folin-Ciocalteu method on leaf phenolics. Data are means ± SO from 4 replications per
treatment. See Materials and methods for dewils. T he wounded
as described by Waterman & Mole ( 1994) by using a
leaves/plants tested on 3 1 Jan uary were wounded on 2 1 January.
tannic acid reference curve. Different letters denote statistically signilicant differences ( p<0.05)
between treatments.
Statistics

The values of phenolics from the individual plants of
each plot/treatment were calculated and subjected to
one-way ANOVA for the effects of UV-B radiation
(until the wounding date), and two-way ANOVA for
the effects of time and these of wounding, UV-B radi-
ation and their combination (SPSS 9.0.1 , SPSS Inc.).
In the later case, the percentage differences of the cor-
responding values before and after wo unding for the
wounded and intact plants were used. Homogeniety of
variances was checked in all cases.
Results
As shown in Figure 2, supplemental UV-B radiation
resulted in a small (ca. 13.5%) but statistically signif-
icant decrease in total leaf phenolics of un-wounded
plants, while a time effect (i.e., a change in total phe-
nolics between the two sampling dates, before and
after wounding), was not observed. Wounding caused
an increase in the phenolics of the wounded leaf in
both ambient and e nhanced UV-B treated plants. How-
ever, the increase was more pronounced under UV-B
supplementation. T hus, a 32% increase was evident
in the enhanced UV-B treated plants, while the cor-
responding inc rease in plants grown under ambient
levels of UV-B was 17%. In addition, the phenolic
content of intact leaves of wounded plants (i.e., the
phenolics of the leaf opposite to the wounded one)
was also increased. However, the increase under UV-B
supplementation (ca. 20%) was statistically significant
while that under ambient UV-B (ca. 7%) was not.
The ultimate resul t was that the phenolic content of
wounded plants was similar under both ambient and
enhanced UV-B I 0 days after wounding, in spite of
the initial (pre-wounding) differences in their phenolic
contents (see Figure 2). The statistical anal ysis of the
results revealed an initial negative effect of UV-B radi-
ation on intact plants' leaf phenolics, a positive effect
of wounding and a synergism between wounding and
UV-B radiation.
Discussion
Unexpectedly, supplemental UV-B radiation caused
a small but significant decrease in leaf phenolics of
Phlomis fruticosa . Hatcher and Pau I ( 1994) working
with pea and Grammatikopoulos et al. (1998) work-
ing with Laurus nobilis have found increases in total
phenolics, while Gehrke et a!. ( 1995) and Rozema
et al. ( 1997) reported that tannin was not affected by
UV-B radiation, although lignin was considerahly in-
creased in Vaccinium uliginosum and Calamagrostis
216
epigeios, respectively. To the best of our knowledge,
these are the only investigations on the effects of UV-
B radiation on leaf phenolics measured chemically. In
all other cases, either specific phenolics were mea-
sured, or it was assumed that the methanol-extractable
UV-B absorbing capacity (A300) was a rough mea-
sure of total phenolics. This assumption seems to be
valid, since there is a positive correlation between
leaf phenolics and A300 in all tested plants (Levizou
& Manetas, in prep.). When phenolics were assessed
through their UV-B absorbance, usually an increase in
A300 was found, especially under laboratory condi-
tions (Tevini 1993), while in the field UV-B seldom
had an effect on leaf UV-B absorbing capacity (Bjorn
et al. 1997). This may be due to other co-occurring
environmental stresses that may force the phenolic
producing capacity to its limits (Stephanou & Manetas
1997). However, UV-B induced decreases in A300 are
not completely absent from the literature. Petropoulou
et al.( 1995), working with P. fruticosa and Swanson &
Fahselt ( 1997), working with the lichen Umplicaria
americana, have found slight decreases. Fourtouni
et al. (1998), in an attempt to explain the complex
effects of UV-B radiation on A300 of the phytopatho-
genic fungus Alternaria solanii, speculated that a
UV-B induced acceleration of the phenylpropanoid
pathway and a concomitant UV-B induced decompo-
sition of phenolics may act in parallel, with apparently
different rate constants for the two reactions. Indeed,
it has been shown that in vitro UV-B radiation of a
film of phenolics taken from Cistus lantifolius results
in a considerable decomposition of these compounds
(Vogt & Gulz 1994).
The response to wounding was small, probably be-
cause phytochemical induction is better stimulated by
herbivore secretions than simple mechanical damage
(Hartley & Lawton 1991 ). It was clear, however, that
UV-B treated plants were more responsive not only
in the wounded leaves, but in the intact leaves of a
wounded plant, implicating the systemic spread of the
wound signal (Baldwin & Ohnmeiss 1993). Based on
the previous discussion we may speculate that wound-
ing modifies the balance between phenolic production
and breakdown differentially under ambient or en-
hanced UV-B radiation conditions, favouring higher
rates of phenolic production in UV-B treated plants.
We may argue from the above that wounding ren-
ders the plant not only less palatable/digestible to
herbivores, but enhances the UV-B screening ability of
the leaves. This may have various implications. From
a methodological point of view, repeated destructive
sampling in UV-B supplementation experiments mim-
ics herbivory and may result in artifactual increase of
phenolics in the more responsive UV-B treated plants
compared to controls. On the other hand, induced re-
sistance in the field is mediated by fungal (Isaac 1992)
or herbivore (Schoonhoven et al.1998) attack. How-
ever, phylloplane (Gunasekera et al.1997) and phy-
topathogenic (Fourtouni et al 1998) fungi are inhibited
by UV-B radiation. In addition, recent field experi-
ments have shown that some insects (phytophagous
thrips, Mazza et al. 1999; pollinating bees, Stephanou
et al. 2000) tend to avoid UV-B radiation. There-
fore, we may predict that increased UV-B radiation
could lead to less fungal and herbivore attack and, ac-
cordingly, to less natural wounding and less phenolic
elicitors. In this case, the lower levels of phenolics in
the UV-B plants of the present investigation could be
due to less herbivore or fungal attack. More work is
needed to elucidate the above speculation.
Acknowledgement
We are grateful to the Commission of the European
Communities for financial support.
References
Baldwin, I. T. & Ohnmeiss, T. E. 1993. Alkaloidal responses to
damage in Nicotiana native to North America. J. Chern. Ecol.
19: 1143-1153.
Baldwin, I. T. 1994. Chemical changes rapidly induced by l'olivory.
Pp. 1-23. In : Bernays, E. A. (ed.), Insect- Plant Interactions,
Vol. 5., CRC Press. Boca Raton , FL.
Beggs, C. J. & Wellman, E. 1994. Photocontrol of flavonoid
biosynthesis. Pp. 733-751. In Kendrick, P. E. & Kronenberg,
G. H. (eds), Photomorphogenesis in Plants. Kluwer Academic
Publishers, Dordrecht, the Netherlands.
Bernays, E. A.. Cooper-Driver. G. & Bilgener. M. 1989. Herbivores
and plant tannins. Adv. Ecol. Res. 19: 263-302.
Bjorn, L. 0. & Murphy, T. M. 1985. Computer calculation of solar
ultraviolet radiation at ground level. Physiol. Veg. 23: 555-561.
Bjorn, L. 0., Callaghan, T. V., Johnsen, 1., Lee, J. A., Manetas, Y..
Paul, N. D., Sonesson, M .. Wellburn, A. R., Coop. D.. Heide-
Jurgensen, H. S., Gehrke. C., Gwynn-Jones, D., Johanson. U.,
Kyparissis, A., Levizou, E., Nikolopoulos, D .. Petropoulou. Y. &
Stefanou, M. 1997. The effects of UV-B radiation on European
heathland. Plant Ecol. 128: 252-264.
Blcckcrt, S., Brodschclm, W., Holder, S., Kammerer, L., Kutchan,
T. M., Mueller, M. J., Xia, Z-Q. & Zenk, M. H. 1995. The octade-
canoic pathway: Signal molecules for the regulation of secondary
pathways. Proc. Nat!. Acad. Sci. USA 92: 4099-4105.
Caldwell, M. M. 1971. Solar UV radiation and the growth and
development of higher plants. Pp. 131-177. In Giese, A. (ed.),
Photophysiology, Academic Press, New York.

Caldwell, M. M., Bjorn, L 0., Bornman, l F, Flint, S. D., Ku-
landaivelu, G., Teramura, A, H, & Tevini, M. 1998. Effects of
increased solar ultraviolet radiation on terrestrial ecosystems. J.
Photochem. Photobiol. B: Biol. 46: 46, 40-52.
Caldwell, M. M., Flint, S.D. & Searles, P. S. 1994. Spectral balance
and UV-B sensitivity of soybean: a field experiment. Plant Cell
Environ. 17: 267-276.
Caldwell, M. M, Robberecht, R. & Flint, S.D. 1983. Internal filters:
prospects for UV-acclimation in higher plants. Physiol. Plant. 58:
445-450.
Ecker, .1. R. 1995. The ethylene signal transduction pathways in
plants. Science 268: 667-675.
Fourtouni, A., Manetas, Y. & Christias, C. 1998. Effects of UV-
B radiation on growth, pigmentation and spore production in
the phytopathogenic fungus Alternaria so/ani. Can. J. Bot. 76:
2093-2099.
Gerhke, C., Johanson, U., Callaghan, T. Y.. Chadwick. D. & Robin-
son, C. H. 1995. The impact of enhanced UV-B radiation on litter
quality and decomposition processes in Vacciniwn leaves from
the subarctic. Oikos 72: 213-222.
Grammatikopoulos. G., Kyparissis, A,, Drilias. P, Pctropoulou. Y.
& Manetas, Y. 1998. Effects of UV-B radiation on cuticle
thickness and nutritional value of leaves in two Mediterranean
evergreen sclerophylls. J. Plant Physiol. 153: 506-512.
Grammatikopoulos, G., Petropoulou, Y. & Manetas, Y. 1999.
Site-dependent differences in transmittance and UV-B-absorbing
capacity of isolated leaf epidermes and mesophyll in Urginea
maritima (L.) Baker. .1. Exp. Bot. 50: S 17-521.
Gunasekera, T. S., Paul, N. D. & Ayres, P. G. 1997. Responses of
phylloplane yeasts to UV-B (290-320 nm) radiation: interspecific
differences in sensitivity. Mycol. Res. 101: 779-785.
Harborne, J. 1977. Phytochemical Methods. Chapman and Hall,
London.
Hartley, S. E. & Lawton, J. H. 1991. Biochemical aspects and signif-
icance of the rapidly induced accumulation of phenolics in birch
foliage. Pp. 105-132. In: Tallamy, D. W. and Ranpp, M. J. (eds)
Phytochemical Induction by Herbivores. John Willey. New York.
Hatcher, P. E. & Paul, N. D. 1994. The etfect of elevated UV-B
radiation on herbivory of pea by Autogmpho gamma. Entomol.
Exp. Appl. 71: 227-233.
Haukioja, E. 1990. Induction of defences in trees. Annu. Rev.
Entomol. 36: 25-42.
Isaac. S. 1992. f'ungai-Piant Interactions. Chapman and Hall. Lon-
don.
Karban. R. & Meyers, .1. H. 1989. Induced plant responses to
herbivory. Annu. Rev. Ecol. System 20: 331-348.
Karabourniotis, G., Papadopoulos, K., Papamarkou, M. & Manetas,
Y. 1992. Ultraviolet radiation absorbing capacity of leaf hairs.
Physiol. Plant. 86: 414-418.
Mackerness. S. A.-H., Surplus, S. L.. John, C. F.. Buchanan-
Wollaston, V.. Jordan, B. R. & Thomas, B. Ultraviolet-B-induced
stress and changes in gene expression in Arabidopsis thaliana:
role of signalling pathways controlled by jasmonie acid, ethylene
and reactive oxygen species. Plant Cell Environ. 22: 1413-1423.
Manetas, Y., Petropoulou, Y., Starnatakis, K., Nikolopoulos, D.,
Levizou, E., Psaras, G. & Karabourniotis G. 1997. Beneficial
217
effects of enhanced UV-B radiation under field conditions: im-
provement of needle water relations and survival capacity in
Pinus pinea L. seedlings during the dry Mediterranean summer.
Plant Ecol. 128: 100-108.
Matern. U. & Kneusel, R. I. 1988. Phenolic compounds in plant
disease resistance. Phytoparasitica 16: 153-170.
Mazza. CA .. Zavala. J., Scope!. A. L. & Ballare C. L. 1999. Effects
of solar UV-B on phytophagous insects: Behavioral responses
and ecosystem implications. Proc. Natl. Acad. Sci .. USA 96:
980-985.
Ncuvonen, S. & Haukioja, E. 1991. The effects of inducible resis-
tance in host foliage on birch-feeding herbivores. Pp. 277-291.
In: Tallarny, D. W. & Raupp, M. J. (eds), Phytochemical
Induction by Herbivores. John Wiley. New York.
Petropoulou, Y., Kyparissis, A., Nikolopoulos, D. & Manetas, Y.
1995. Perturbations of the normal UV-B radiation environment
alter leaf growth rates in Ph/omisji-Uiicosa L. seedlings. Environ.
Exp. Bot. 35: 371-377.
Robberecht, R. & Caldwell. M. M. 1978. Leaf epidermal trans-
mittance of ultraviolet radiation and its implication for plant
sensitivity to ultraviolet radiation induced injury. Oecologia 32:
277-287.
Rozema. J., Tosserams, H. J., Nelissen, H. J. M., Van Heerwaar-
den, L., Brockman. R. A. & Flierman, N. 1997. Stratospheric
ozone reduction and ecosystem processes: enhanced UV-B radi-
ation affects chemical quality and decomposition of leaves of the
dune grassland species Calamagmstis epigeios. Plant Ecol. 12X:
284-294.
Schoonhoven, L. M., Jenny. T. & Van Loon, J. J. i\. 1998. Insect-
Plant Biology. Chapman and Hall. London.
Stcphanou, M. & Manetas, Y. 1997. The effects of seasons. expo-
sure. enhanced UV-B radiation and water stress on leaf epicutic-
ular and internal UV-B absorbing capacity of Cistus creticus: a
Mediterranean field study. J. E.xp. Bot. 48: 1977-1985.
Stephanou, M .. Petropoulou, Y., Georgiou. 0. & Manetas. Y. 2000.
Enhanced UV-B radiation. flower attributes and pollination be-
haviour in Cistus creticus: a Mediterranean field study. Plant
Ecol. 147: 165-171.
Swanson. A. & Fahselt. D. 1997. Etl'ects of ultraviolet on polyphe-
nolics of Umbilicaria americana . Can. J. Bot. 75: 284-289.
Tevini, M. 1993. Molecular biological effects of ultraviolet radia-
tion, pp. 1-15. In: Tcvini. M. (ed.), UV-B Radiation and Ozone
Depletion: Effects on Humans, Animals, Plants, Microorganisms
and Materials. Lewis Publishers, London.
Vogt, T. & Glilz, P. G. 1994. Accumulation of flavonoids during leaf
development in Cisllls /aurifiilius. Phytochem. 36: 591-597.
Waterman, P. G. & Mole. S. 1994. Analysis of Phenolic Plant
Metabolites. Blackwell, Oxford.
Wratten, S. D .. Edwards. P. J. & Dunn. I. 1984. Wound-induced
changes in palatability of Beiula pubescens and B. pendula.
Oecologia 61: 372-375.
Wollenweber, E. & Dietz. V. H. 1980. Occurrence and distribution
of free aglyeones in plants. Phytochemistry 20: 869-932.
Section 5: Aquatic Plants and Aquatic Ecosystems

Cyanobacleria in !he wafer of a rice field in India. (Phorograph by R.P. Sinha)

 Plant Ecology 154: 221-236, 200 I.
221
© 2001 Kluwer Academic Publishers.

Responses of aquatic algae and cyanobacteria to solar UV-B

Rajeshwar P. Sinha, Manfred Klisch, Almut Groniger & Donat-P. Hader*

Institut fur Botanik und Pharmaz.eutische Biologie, Friedrich-Alexander-Universitdt, Staudtstr. 5, D-91058
Erlangen, Germany(* Authorfor correspondence, E-mail: dphaeder@biologie.uni-erlangen.de)

Key words: Cyanobacteria, Macroalgae, Mycosporine-like amino acids (MAAs), Phytoplankton, Scytonemin,
Ultraviolet-B radiation

Abstract
Continuous depletion of the stratospheric ozone layer has resulted in an increase in solar ultraviolet-B (UV-B;
280-315 nm) radiation reaching the Earth's surface. The consequences for aquatic phototrophic organisms of this
small change in the solar spectrum are currently uncertain. UV radiation has been shown to adversely affect a
number of photochemical and photobiological processes in a wide variety of aquatic organisms, such as cyanobac-
teria, phytoplankton and macroalgae. However, a number of photosynthetic organisms counteract the damaging
effects of UV-B by synthesizing UV protective compounds such as mycosporine-like amino acids (MAAs) and the
cyanobacterial sheath pigment, scytonemin. The aim of this contribution is to discuss the responses of algae and
cyanobacteria to solar UV-B radiation and the role of photoprotective compounds in mitigating UV- B damage.

Introduction few centimeters in brown humic lakes and rivers (Kirk,

Continued depletion of the stratospheric ozone layer,
mainly due to anthropogenically released atmospheric
pollutants such as chlorofluorocarbons (CFCs) is re-
sponsible for the increase in solar ultraviolet-B (UV-B;
280-315 nm) radiation reaching the Earth's surface
(Biumthaler & Ambach 1990; Crutzen 1992; Kerr &
McElroy 1993; Lubin & Jensen 1995). In addition
to the Antarctic ozone hole, ozone depletion has also
been reported in the north polar region (Hoffman &
Deshler 1991 ). Current estimates predict that ozone
losses will continue throughout the next century if we
fail to adhere to the 1992 Copenhagen Amendment
(Slaper et al. 1996).
Biologically effective doses of UV radiation pen-
etrate deep into the water column (Smith & Baker
1979); they have been detected down to a depth of
70 m (Smith et al. 1992) and may thus affect the
aquatic ecosystems (Hader et al. 1998). The pen-
etration of water by UV-B depends on the optical
properties of the water column. The depth of water re-
quired to remove 90% of the solar radiation at 310 nm
varies from about 20 m in the clearest ocean water to a
1994).
All aquatic organisms appear to be susceptible to
UV-B, but to a highly variable extent. UV-B is a small
(less than I% of total energy) but highly active com-
ponent of the solar spectrum which has the potential
to cause wide ranging effects, including alteration in
the structure of proteins. DNA and other biologically
relevant molecules, chronic depression of key physio-
logical processes and acute physiological stress. As a
result, the productivity of ecosystems may be affected
(Karentz et al. 1991 a; Vincent & Roy 1993; Bothwell
et al. 1994; Williamson 1995; Sinha & Hader 1996a).
However, certain photosynthetic organisms which
are simultaneously exposed to visible and UV radi-
ation in their natural habitat, have developed mech-
anisms counteracting the damaging effects of UV.
Besides repair ofUV-induced damage of DNA by pho-
toreactivation and excision repair (Britt 1995; Kim
& Sancar 1995) and accumulation of carotenoids and
detoxifying enzymes or radical quenchers and antiox-
idants that provide protection by scavenging harmful
radicals or oxygen species (Mittler & Tel-Or 1991;
Middleton & Teramura 1993), an important mecha-
nism to prevent UV-induced photodamage is the syn-
222
thesis of photoprotective compounds (Garcia-Piche!
& Castenholz 1991; Karentz et al. 1991 c; Dunlap &
Shick 1998; Sinha et al. 1998a). This review deals
with the responses of algae and cyanobacteria to so-
lar UV-B radiation and the role of photoprotective
compounds in mitigating UV-B toxicity.
Effects of UV-B radiation on cyanobacteria
Cyanobacteria are the largest and most widely dis-
tributed group of photosynthetic prokaryotes on Earth.
Cyanobacterial populations occupy an important place
in both aquatic and terrestrial ecosystems ranging
from hot springs to the Antarctic and Arctic regions.
The role of cyanobacteria in improving the fertility
of rice paddy fields and other soils is well docu-
mented (Singh 1961; Roger & Kulasooriya 1980;
Venkataraman 1981; Watanabe 1984; Sinha & Hader
1996a; Sinha et a!. 1998b; Vaishampayan eta!. 1998).
Any substantial increase in the solar UV-B radiation
might be detrimental to the cyanobacterial commu-
nities, which in turn may affect the productivity of
higher plants (Sinha & Hader 1996a).
All aquatic photosynthetic organisms depend on
solar radiation as the primary source of energy in their
natural environment. Light is one of the most impor-
tant factors determining cyanobacterial growth in their
natural habitats since cyanobacteria are predominantly
photoautotrophic organisms. In addition, light is an
environmental factor controlling orientation and habi-
tat selection. The cyanobacterial populations in rice
paddy fields, particularly in the tropics, are often ex-
posed to high white light and UV-B irradiances (Sinha
& Hader 1996a, b; Sinha et al. 1999a). Considering
the vital role of cyanobacteria as a biofertilizer in rice
and other crop production, the fluence rate of UV-B
radiation impinging on the natural habitats, seems to
be of major concern since UV-B radiation has been
reported not only to impair motility and photoorien-
tation (Donkor & Hader 1991) but also to affect a
number of physiological and biochemical processes
such as growth, survival, pigmentation, and total pro-
tein profile (Sinha et al. 1995a). Growth and survival
of several rice-field cyanobacteria have been reported
to be severely affected following UV-B irradiation
for different durations. Growth ceases and survival is
affected within 120-180 min of UV- B irradiation, de-
pending upon the species. Strains such as Scytonema
sp. and Nostoc commune, the filaments of which are
embedded in mucilagenous sheath. have been reported
1.2
1.0
ci
Q. 0.8
<I>
u
c: 0.6
Ill
..c
0
"'
..c
<(
0.4
0.2
0.0 -t-----,--,---,----,--,-,----,--,----j
250 300 350 400 450 500 550 600 650 700
Wavelength [nm]
Figure 1. Effects of UV-B irradiation on the pigmentation of a
rice-field cyanobacterium Anahaena sp.
to be more tolerant than Anabaena sp. and Nostoc sp.,
the filaments of which do not possess such covering
(Sinha et al. 1995a). Many workers have suggested
that the cellular constituents absorbing radiation he-
tween 280-315 nm are destroyed by UV-B radiation,
which may further affect the cellular membrane per-
meability and protein damage eventually resulting in
the death of the cell (Vincent & Roy 1993; Sinha et al.
1995a, 1997; Sinha & Hader 1996a).
The studies on the effects ofUV-B on pigmentation
of various rice field cyanobacteria have revealed that
the accessory pigment phycocyanin ().max 620 nm)
was bleached more rapidly and drastically than any
other pigment such as Chi a (),max 437 and 672 nm) or
the carotenoids (A.max 485 nm) (Sinha et al. 1995a, b)
(Figure I). A decrease in the phycobiliprotein contents
and disassembly of phycobilisomal complexes follow-
ing UV-B irradiation have been reported in a number
of cyanobacteria (Sinha et al. 1995b, c, 1997). Flu-
orescence emission spectra of phycobiliproteins after
UV-B irradiation first show an increase followed by a
shift towards shorter wavelength and finally a decrease
in fluorescence (Figure 2), indicating impaired energy
transfer from the accessory pigments to the photosyn-
thetic reaction centers and subsequent bleaching of the
pigments (Sinha et al. 1995b, c, 1997).
Differentiation of vegetative cells into heterocysts
has been reported to be severely affected by UV-B ir-
radiation in a number of rice field cyanobacteria. Most
probably the C:N ratio is altered following UV-B ir-
radiation, which in turn affects the spacing pattern of
heterocysts in a filament (Sinha et al. 1996). In ad-
dition, major heterocyst polypeptides of around 26,
54 and 55 kDa have been shown to be decreased in
 223
550
ruption of their structural entity (Sinha et al. 1997;
~ 500
Sinha & Hader 1998). Ultraviolet induced inhibition
·;:
450
:I of 14 C0 2 uptake in various rice-field cyanobacteria
Q)
400
.2: has been reported which could be due to the effect
n; 350
on the photosynthetic apparatus leading to the reduc-
~ 300
tion in the supply of ATP and NADPH2 (Kumar et al.
Q)
0 250
!:
Q)
1996a; Sinha et al. 1996 ). A disruption of the cell
200
0 membrane and/or alteration in thylakoid integrity as
e0
(/)
150
a result of UV-8 irradiation may partly or wholly de-
:I 100
~ stroy the component required for photosynthesis and
50
may thus affect the rate of C02 fixation (Sinha et al.
0
1996, 1997). In addition, ultraviolet-induced opening
630 640 650 660 670 680 690 700
of the membrane-bound calcium channels has been
Wavelength [nm]
demonstrated in the cyanobacterium Anabaena sp.
Figure 2. Fluorescence emission spectra of phycocyanin from An- (Richter et al. 1999).
abaena sp. after increasing UV-B exposure time, when excited at
620 nm.
Cyanobacteria are common organisms in fresh-
water and terrestrial ecosystems of Antarctica and
also in the north polar regions (Quesada & Vincent
concentration following UV-8 irradiation, suggesting 1997). Cyanobacteria have colonized a wide vari-
that the multilayered thick wall of heterocysts may be ety of polar environments including soils, the surface
disrupted resulting in the inactivation of the nitrogen- and interior of rocks, lakes, ponds. streams. moss
fixing enzyme nitrogenase by the penetrating oxy- beds, melt pools on glaciers and ice shelves, and
gen. UV-B-induced membrane disruption leading to littoral marine sediments. In some of these habitats
changes in membrane permeability and release of 14 C- they dominate in terms of total biomass and produc-
labeled compounds have been observed in a number tivity. Cyanobacteria are also a conspicuous element
of rice-field cyanobacteria (Sinha et al. 1997). UV- of mature microbial communities across the surface
8-induced inactivation of the nitrogen fixing enzyme of Antarctic rock faces, as well as under and within
nitrogenase has been reported in many cyanobacteria translucent rocks (Vincent & Quesada 1994 ). Antarc-
(Kumar et al. 1996a; Sinha et al. 1996). Total pro- tic cyanobacteria achieve their greatest abundance in
tein profiles of several cyanobacteria show a linear aquatic and wetland habitats. It is the benthic envi-
decrease in protein content with increasing UV-8 ex- ronment of lakes, ponds and streams that supports the
posure time, indicating that cellular proteins are one largest standing stocks of cyanobacterial biomass in
of the main targets of UV-B. Ultraviolet radiation is Antarctica. Cyanobacterial communities form crusts,
known to damage proteins and enzymes, especially films and spectacular mats up to several centimeters
those rich in aromatic amino acids such as tryptophan, in thickness, often intensely colored by pigments. Mat
tyrosine, phenylalanine and histidine, all of which forming cyanobacteria arc especially wide spread in
show strong absorption in the UV range from 270 to high latitude ponds and streams. Tn these environments
290 nm (Kumar et al. 1996a; Sinha et al. 1996; Sinha the cyanobacteria often occupy shallow water habitats
& Hader 1998). that are exposed to full sunlight. Cyanobacterial popu-
The activity of the ribulose I ,5-bisphosphate car- lations must therefore be capable of surviving frequent
boxylase (Rubisco), the primary C02 fixing enzyme, exposure to bright photosynthetically active radia-
has been reported to be inhibited by UV-8 irradiation tion (PAR) as well as high levels of solar ultraviolet
in a number of rice-field cyanobacteria which may radiation (Quesada & Vincent 1997).
be due to protein destruction or enzyme inactivation Tt has been reported that even moderate levels of
(Sinha et al. 1997). The control of RuBISCO biosyn- ultraviolet-8 radiation can have a major physiologi-
thesis is strongly influenced by the prevailing light cal impact on Antarctic cyanobacteria, but there are
environment. During UV-8 irradiation, proteins may substantial differences between closely related species
undergo a number of modifications including pho- in their ability to escape the damaging effects of this
todegradation, increased aqueous solubility of mem- high energy waveband (Quesada & Vincent 1997). It
brane proteins, and fragmentation of the peptide chain, has been concluded that the marine phototrophic or-
leading to inactivation of proteins (enzymes) and dis- ganisms living in cold environments may be especially
224
prone to the damaging etlects of ultraviolet radiation.
These findings are especially relevant to the cold wa-
ters found in the north and south polar zones, where
stratospheric ozone depletion and the associated in-
crease in ambient UV-B radiation are proceeding most
rapidly (Roos & Vincent 1998). Thus, in natural habi-
tats avoidance of UV radiation seems to be of utmost
importance for cyanobacterial growth and nitrogen
fixation.
Photoprotective mechanisms in cyanobacteria
Since it is clear that microorganisms evolved and
microbial mats were well established early in the Pre-
cambrian era (Rambler & Margulis 1980; Dillon &
Castenholz 1999), some mechanism(s) must have been
functioning to protect these organisms from the dele-
terious effects of UV radiation. There are at least
five adaptation strategies by which cyanobacteria try
to avoid high white light and ultraviolet radiation
stress (Vincent & Roy 1993; Castenholz 1997; Que-
sada & Vincent 1997; Sinha et al. 1998a; Cockell &
Knowland 1999):
(l) Production of ultraviolet-absorbing substances
such as mycosporine-like amino acids (MAAs)
and scytonemin (Garcia-Piche! & Castenholz
1991; Garcia-Piche! et al. 1993; B tide! et al. 1997;
Sinha et al. 1998a; Dillon & Castenholz 1999).
(2) Escape from ultraviolet radiation by migration
into habitats with reduced light exposure. Such
strategies include phototactic, photokinetic and
photophobic responses (Hader 1987a, h), vertical
migration into deeper strata of mat communities
(Bebout & Garcia-Piche! 1995) and sinking and
floating behavior by a combination of gas vac-
uoles and ballast (Reynolds et al. 1987). This
allows them to change their position in the water
column as environmental conditions change and
thus to always ensure a nearly constant external
environment.
(3) Production of quenching agents such as carotcnoids
(Burton & Ingold 1984) or systems such as su-
peroxide dismutase that react with and thereby
neutralize the highly toxic reactive oxygen species
produced by ultraviolct-B radiation (Quesada &
Vincent 1997).
(4) Availability of a number of repair mechanisms
such as photoreactivation and light-independent
nucleotide excision repair of DNA (Britt 1995;
Kim & Sancar 1995) and UV-A/blue-light me-
diated repair of the photosynthetic apparatus
(Christopher & Mullet 1994 ).
(5) A number of cyanobacteria have the ability to
vary their phycobiliprotein composition (phyco-
cyanin/phycoerythrin ratio), which allows regula-
tion of the balance of wavelengths of light ab-
sorbed, a phenomenon known as chromatic adap-
tation (Tandeau de Marsac 1977).
Below we discuss the role of MAAs and scytonemin
in mitigating the harmful UV-B radiation etlects in
cyanobacteria in more detail.
Mycosporine-like amino acids (MAAs) in
cyanobacteria
In the 1970s various substances were isolated and
characterized which had a maximum absorption in
the UV range. These substances were related to the
mycosporines found in terrestrial fungi (Favre-Bonvin
et al. 1987) and named mycosporine-like amino acids
(MAAs). To date, a number of MAAs are documented
of which mycosporine-glycine, palythine, palythene,
palythinol, asterina-330, porphyra-334 and shinorine
(Figure 3) have been characterized and well docu-
mented (Ito & Hirata 1977; Takano et al. 1978a, b,
1979; Tsujino et a!. 1980; Dunlap & Chalker 1986;
Gleason 1993).
MAAs are water soluble substances characterized
by a cyclohexenone or cyclohexenimine chromophore
conjugated with the nitrogen substituent of an amino
acid or its imino alcohol, having absorption maxima
ranging from 310 to 360 nm (Figure 3) and an average
molecular weight of around 300 (Sinha et al. 1998a;
Cockell & Knowland 1999). A number of cyanobac-
teria isolated from freshwater, marine or terrestrial
habitats contain MAAs (Garcia-Piche! & Castenholz
1993; Garcia-Piche! et al. 1993; Sinha et al. l998a).
Presence of MAAs has also been reported in Antarctic
(Quesada & Vincent 1997) as well as in a community
of halophilic cyanobacteria (Oren 1997).
The occurrence of high concentrations of MAAs
in organisms exposed to high levels of solar radiation
has been described to provide protection as a UV-
absorbing pigment (Garcia-Piche! et al. 1993; Sinha
et al. 1998a), but there is no conclusive evidence for
the exclusive role of MAAs as sunscreen, and it is
possible that they play more than one role in the cel-
lular metabolism of all or some organisms (Vincent &
Roy 1993; Castenholz 1997; Oren 1997). It has been
reported that MAAs may act as antioxidants to prevent
 225

~OCH, OH
HOOH
~0;1 HO

Basic structure of MAAs Scytonemin

R
Name
II AIIIDX 8 Reference

Mycosporine-glycine 0 310 28100 Ito & Hirata 1977; Dunlap et al.

II 1986; Gleason 1993

Palythine NH 320 36200 Takano et al. 1978a; Dunlap et al.

II 1986; Gleason 1993

()
Asterina-330 330 43500 Gleason 1993

c;
Palythinol 332 43500 Dunlap et al. 1986; Takano et al.
1978b

Porphyra-334 334 42300

i)
COfi Takano et al. 1979

Shinorine 334 44700 Tsujino et al. 1980; Gleason 1993

()
COJI

Palythene 360
~ 50000 Tak:ano et al. 1978b

II

Scytonemin 386 Proteau et al. 1993

Figure 3. Molecular structures, absorption maxima 0-max) and extinction coefficients (t:) of some principal photoprotective compounds.
226
cellular damage resulting from UV-induced produc-
tion of active oxygen species (Dunlap & Yamamoto
1995). Studies with cyanobacteria have shown that
MAAs prevent 3 out of 10 photons from hitting cy-
toplasmic targets. Cells with high concentrations of
MAAs are approximately 25% more resistant to ul-
traviolet radiation centered at 320 nm than those with
no or low concentrations (Garcia-Piche! et al. 1993).
This protection is unlikely to be effective for thin, soli-
tary filaments, but may be especially important in mat
communities or large phytoplankton.
The MAAs in Nostoc commune have been reported
to be extracellular and linked to oligosaccharides in
the sheath (Bohm et al. 1995). These glycosylated
MAAs represent perhaps the only known example
of MAAs that are actively excreted and accumu-
lated extracellularly and therefore act as a true screen
(Ehling-Schulz et al. 1997). Experiments with a rice
paddy field cyanobacterium, Anabaena sp., revealed
the existence and induction by UV radiation of a sin-
gle MAA, shinorine, a bisubstituted MAA containing
both glycine and serine groups, with an absorption
maximum at 334 nm and a fluorescence emission at
436 nm (Sinha et al. 1999b). There may be physio-
logical limitations to the accumulation of osmotically
active compounds such as MAAs within the cell, and
it seems probable that the maximal specific content of
MAAs in the cell is regulated by osmotic mechanisms
which is reflected by the fact that field populations
of halotolerant cyanobacteria contain unusually high
concentration of MAAs (Oren 1997).
Scytonemin
Scytonemin is a yellow-brown, lipid soluble dimeric
pigment located in the extracellular polysaccharide
sheath of some cyanobacteria. Scytonemin, which has
an in vivo absorption maximum at 370 nm, has been
proposed to serve as a UV sunscreen (Garcia-Piche!
et al. 1992; Dillon & Castenholz 1999). It has a mole-
cular mass of 544 Da and a structure based on indolic
and phenolic subunits (Figure 3). Purified scytonemin
has an absorption maximum at 386 nm, but it also ab-
sorbs significantly at 252, 278 and 300 nm (Proteau
et al. 1993; Sinha et al. 1998a, 1999c ).
Strong evidence for the role of scytonemin as ul-
traviolet shielding compound has been presented in
several cyanobacterial isolates and collected materi-
als from various harsh habitats, mostly exposed to
high irradiances (Garcia-Piche! & Castenholz 1991 ).
Its role as a sunscreen was clearly demonstrated
in the terrestrial cyanobacterium Chlorogloeopsis sp.
(Garcia-Piche! et al. 1992). A desiccation-tolerant
cyanobacterium, Lyngbya sp., collected from the bark
of mango trees in India has been found to contain
the yellow-brown sheath pigment, scytonemin (Sinha
et al. 1999c). In cyanobacterial cultures, as much
as 5% of the cellular dry weight may be accumu-
lated as scytonemin. Naturally occurring cyanobacte-
ria may have an even higher specific content (Casten-
holz 1997). The correlation between UV protection
and scytonemin presence has been established under
solar irradiance in a naturally occurring monospe-
cific population of a cyanobacterium, Calothrix sp.;
it was shown that high scytonemin content is re-
quired for uninhibited photosynthesis under high UV
flux (Brenowitz & Castenholz 1997). Studies indicate
that the incident UV-A radiation entering the cells
may be reduced by around 90% due to the presence
of scytonemin in the cyanobacterial sheaths (Garcia-
Piche! & Castenholz 1991; Garcia-Piche! et al. 1992;
Brenowitz & Castenholz 1997). Once synthesized, it
remains highly stable and carries out its screening ac-
tivity without further metabolic investment from the
cell. Rapid photodegradation of scytonemin does not
occur, which is evidenced by its long persistence in
terrestrial cyanobacterial crusts or dried mats (Garcia-
Piche! et al. 1992; Brenowitz & Castenholz 1997;
Quesada et al. 1999). This strategy may be invaluable
to several scytonemin containing cyanobacteria inhab-
iting harsh habitats, such as intertidal marine mats or
terrestrial crusts, where they experience intermittent
physiological inactivity (e.g., desiccation or freezing).
During these metabolically inactive periods, other ul-
traviolet protective mechanisms such as active repair
or biosynthesis of damaged cellular components are
ineffective (Brenowitz and Castenholz 1997; Ehling-
Schulz et al. 1997; Quesada et al. 1999). It has been
postulated that scytonemin may have evolved during
the Precambrian and allowed colonization of exposed,
shallow-water and terrestrial habitats by cyanobacte-
ria or their oxygenic ancestors (Dillon & Castenholz
1999).
In addition to the screening pigments described
above, another UV protective agent with absorption
maxima at 312 and 330 nm has been reported from the
terrestrial cyanobacterium Nostoc commune, a species
that also produces scytonemin (Scherer et a!. 1988).
A brown Nostoc sp. that produces three UV-absorbing
compounds with absorption maxima at 256, 314 and
400 nm, has been reported to be resistant to high

visible and UV radiation (de Chazal & Smith 1994 ).
The shielding role against UV-8-induced damage of
certain cyanobacterial pigments (a brown-colored pig-
ment from Scytonema hofmanii and a pink extract
from Nostoc spongiaej(Jrme) has been demonstrated
(Kumar et a!. I 996b).
Effects of UV-B radiation on phytoplankton
Phytoplankton organisms are the basis of marine food
webs and thus indirectly contribute to human nutri-
tion. They also play an important role in the regu-
lation of global climate. It has been estimated that
about 50% of the total carbon fixation is attributed
to marine ecosystems with most of it due to phyto-
plankton organisms (Hader et al. 1998). In addition
to the contribution of phytoplankton to carbon fixa-
tion, there are specific effects of phytoplankton on
climate. Some phytoplankton genera produce volatile
substances, mainly dimethyl sulfide (OMS), providing
a cooling effect on the atmosphere, since they are pre-
cursors of cloud condensation nuclei (Watson & Liss
1998). The cumulative effect of marine biota in the
uptake of atmospheric C02 and emission of OMS has
been estimated to cool the atmosphere by up to 6 oc
(Watson & Liss 1998).
The distribution of phytoplankton is not uniform
in the oceans. The dependence on solar radiation as
the primary source of energy restrict' phytoplankton
to depths at which the penetration of light supports
photosynthesis. This range is defined as the euphotic
zone. The horizontal distribution of phytoplankton is
also highly variable. In large regions of the oceans
a stable boundary exists between nutrient rich deep
sea water and nutrient poor surface water. Thus, the
accumulation of phytoplankton biomass is limited by
nutrient availability. Regions near the coast and at
the higher latitudes contain more phytoplankton bio-
mass because of higher nutrient availability due to
upwelling of nutrient-rich water from the deep sea and
terrestrial influxes. Especially the Southern Ocean is
highly productive (Valiela 1995).
The life of phytoplankton depends on solar radia-
tion that provides energy used via the photosynthetic
process. Simultaneously, the excess UV-B radiation
damages diverse targets within the organism. Inhibi-
tion of photosynthesis and growth, DNA damage, and
finally cell death are among the common effects of
UV-B on phytoplankton. High levels of visible radi-
ation induce the formation of active oxygen species
227
that affect cell integrity. UV-B reduces the content of
photosynthetic pigments in phytoplankton and leads
to lower photosynthetic rates (Gerber & Hader 1995).
Apart from the photosynthetic pigments a major target
of UV damage is the electron transport chain of pho-
tosystem II (Bornman 1989). Functional relationships
between the wavelength of radiation and the photoin-
hibition of photosynthesis (biological weighing func-
tions, BWFs) have been determined experimentally
(Cullen eta!. 1992; Helbling eta!. 1992; Neale et al.
1998a; Ghetti et a!. 1999). The inhibitory effect of
UV radiation increases exponentially with decreasing
wavelength in the UV-A and the UV-B portion of the
spectrum. However, due to its higher proportion in the
solar spectrum, the inhibitory effect of UV-A radiation
has been estimated to be higher than the UV-B effect,
in some cases even for the conditions of ozone deple-
tion (Cullen eta!. 1992; Arrigo 1994). The shape of a
BWF may be strongly influenced by the physiological
state of the organisms, e.g. the induction of tolerance
mechanisms. For example, the inhibitory effect of UV
radiation in the wavelength range of MAA absorp-
tion can be greatly diminished if high concentrations
of these UV-absorbing compounds are present in the
organisms (Neale et al. 1998a).
The peak absorption of DNA lies in the UV-C
range that is absorbed in the upper layers of the
atmosphere and does not reach the ozone layer. How-
ever, the absorption of UY-B radiation by DNA is
sufficient to induce severe damage to the DNA of phy-
toplankton cells. Absorbed quanta of UV can induce
changes in the molecular structure of the DNA (Kar-
entz ct a!. 1991 a). The main effect of UV-B radiation is
the formation of dimcrs between two adjacent pyrimi-
dine bases, cis-syn cyclobutan dimers and pyrimidine
(6-4) pyrimidone photoproducts. These DNA lesions
interfere with DNA transcription and replication and
can lead to misreadings of the genetic code causing
mutations and death. There arc big differences in the
susceptibility of phytoplankton species to DNA dam-
age that seem to be correlated with cell size and shape.
Taking into account that DNA is among the main
lethal targets of UV-B radiation, the susceptibility of
a species to UV-B induced DNA damage is a good
indicator of overall UV- B sensitivity (Karentz et a!.
199la).
Investigations of the effects of ozone depletion
on phytoplankton productivity in the Southern ocean,
where the increase in UV-B radiation is most pro-
nounced, show large differences. Smith ct al. ( 1992)
estimated a reduction in primary production of 6-12%
228

in the marginal ice zone of the Bellinghausen Sea. An-
other study simulating the effect on aquatic primary
production using model-derived radiation conditions
and BWFs, only results in a reduction of less than
I% integrated over the Southern Ocean (Arrigo 1994).
The large difference between the estimates reflects the
complexity of large scale estimates on UV-B effects.
A differential impact of UV-B radiation may be par-
tially a result of interactions with other factors, such
as the extent of vertical mixing. By vertical mixing or-
ganisms from deeper levels are brought to the surface
where they become photoinhibited while inhibited or-
ganisms from near surface are transported to deeper
levels where availability of light becomes limiting.
Thus the overall decrease in photosynthesis becomes
larger (Neale eta!. 1998b). In addition, solar radia-
tion and UV-B radiation is known to inhibit the ability
of phytoplankton to move and orient within the water
column (Tirlapur eta!. 1993).
Photoprotective mechanisms in phytoplankton
The recent effects ofUV-B are not new phenomena but
have accompanied the whole evolutionary process of
phytoplankton. Therefore, phytoplankton organisms
have developed certain tolerance mechanisms to avoid
harmful UV radiation. These include (a) vertical mi-
gration within the water column to avoid exposure to
excessive doses of harmful radiation. Many species of
phytoplankton actively move up and down in the wa-
ter column, controlled by gravitactic and phototactic
orientation (Hader 1988). This mechanism allows the
organisms to adjust the impinging radiation to a level
that is suitable for photosynthesis and to avoid excess
doses of visible as well as UV radiation (Hader 1988;
Tirlapur eta!. 1993), (b) repair of DNA damage (Kar-
entz eta!. 199lb). There are three mechanisms known
that can repair damaged portions of DNA:
- Photoenzymatic repair (PER) that involves the en-
zyme DNA photo lyase that monomerizes cyclobu-
tane dimers in the presence of visible or UV-A
light,
- Nucleotide excision repair (NER) has a broader
spectrum of action and involves the recognition
of damaged DNA portions and the excision and
resynthesis of the damaged strand by DNA poly-
merase, and
- Recombinational repair (postreplication repair)
may resolve DNA damage that has been bypassed
by the replication machinery (Karentz eta!. 1991 a,
b).
The capacity and kinetics of DNA repair differ greatly
between phytoplankton species. This includes differ-
ent relative importance of either PER and NER. The
removal of cyclobutane dimers by PER in the presence
of PAR or UV-A radiation may enhance the not light-
dependent NER of other DNA lesions (Karentz et a!.
199la, b), and (c) the production of UV-absorbing
compounds, MAAs (Carreto et a!. 1990a, b; Dunlap
eta!. 1995; Helbling eta!. 1996; Vernet & Whitehead
1996; Xiong et a!. I 997).
MAAs in phytoplankton
Several phytoplankton organisms from different re-
gions and taxonomic groups have been found to con-
tain MAAs. Most of the research has focused on
marine phytoplankton, but there are a few reports on
the occurrence of MAAs in freshwater algae (Xiong
et al. 1999). So far MAAs have been reported to
occur predominantly in species of the Dinophyceae,
Bacillariophyceae and Haptophyceae. In addition to
the reports from experiments with phytoplankton in
culture, there are numerous reports on UV-absorbing
properties of ocean waters that are assumed to be
caused by the presence of MAAs (Helbling et a!.
1994; Vernet et al. 1989). A UV-absorbing com-
pound was found in a phytoplankton bloom in the
Icelandic Basin, which showed an absorption spec-
trum and chromatographic behavior similar but not
identical to scytonemin. However, this pigment has not
yet been chemically characterized, and therefore, there
is no evidence for the presence of scytonemin or simi-
lar pigments in phytoplankton organisms (Llewellyn
& Mantoura 1997). Besides MAAs, sporopollenin,
a biopolymer of variable chemical composition, has
been proposed to play a possible role in screening UV
radiation in some freshwater phytoplankton species
(Xiong et a!. I 997).
The assumption that MAAs act as protectants
against UV radiation has been derived from the fact
that the distribution of MAAs in marine organisms
often shows a correlation with depth and thus with
the dosage of UV or PAR radiation (Dunlap & Shick
1998). In some phytoplankton species the accumu-
lation of MAAs is induced by UV radiation (Car-
reto et a!. 1990a, b) which supports the theoretical
assumption that the presence of UV-absorbing com-
pounds provides screening to the constituents of a cell
(Garcia-Piche! 1996). Direct evidence for their protec-
tive function in phytoplankton has been demonstrated

where high amounts of intracellular MAAs diminish
the inhibitory effect of UV radiation on photosynthesis
(Lesser 1996; Neale ct al. 1998a). The accumulation
of MAAs may also protect microalgae from inhibition
of motility by UV-B radiation (our unpublished data).
The effectivity of screening by UY-absorbing com-
pounds largely depends on cell size. Due to the longer
optical path length internal sunscreens are more effec-
tive in large cells. However, smaller cells may increase
the effectivity of screening if they form dense popu-
lations that provide mutual shading from deleterious
UV-B radiation. However, in this c<tse it is doubtful
that the presence of UV-screcning subst<tnccs will pro-
vide a competitive advantage to the cells producing
them since other species may also benefit from the
screening of UV radiation (Garcia-Piche! 1996). The
same applies to cases in which the UV-absorbing sub-
stances are released to the surrounding medium like in
the dinofl agellate Lingulodinium polyedra (Vernet &
Whitehead 1996).
The synthesis of MAAs is strongly influenced by
radiation intensity as well as by the spectral com-
position. In the dinoflagellate Alexandrium excava-
tum isolated from the continental shelf near Buenos
Aires, the transfer from low (20 ILE m- 2 s- 1) to high
(200 f.l·E m- 2 s- 1) PAR led to a c hange in MAA
composition and an overall increase in UV absorption
within a time scale of a few hours (Carrero et al. 1989,
1990a). Accumulation of MAAs in this o rganism also
depends on the spectral composition of the light; blue
light is more effective than green and red light, and
UV-A radiation strongly enhances M AA accumulation
(Carreto et al. 1990a). Exposure to sunlight leads to
a strong increase of MAAs content in Alexandriwn
excavatum (Carreto et al. 1990b). In the dinoflagel-
late Prorocentrum micans, after 21 days of growth
in the presence of UV radiation the organisms con-
tained higher concentrations of MAAs in comparison
to those grown in the absence of UV radiation (Lesser
1996). The ubiquitous haptophyte species, Phaeocvs-
tis pouchetii, contains UV-absorbing compounds o~ly
in the colonial stage of its life cycle, and the concen-
tration has been found to be higher in isolates from
the Antarctic region than in isolates from temperate
regions (Marchant et al. 1991 ). In contrast to temper-
ate Phaeocystis populations, in the Antarcti c isolate
the synthesis of the UV-absorbing compounds was en-
hanced under sublethal UV-B stress (Marchant et al.
199 1). In some freshwater phytoplankton species a
dramatic increase in MAAs content was found after
artificial UV-B irradiation which further increased af-
229
e
0
c
.g 15
..
e-o
D
<( Tlme(h)
400 345 335 320 305 295
Filter cut-off wavolongth (nm]
Figure 4. The ratio of UV peak absorption (ranging from 325 to
337 nm) to Chi a absorption (665 nm) in methanolic extracts from
Cwodinium dorsum exposed to simulated solar radiation under dif-
ferent cut-off tiltcrs (GG and WG series, Schott & Gen., Germany)
for up to 72 h.
ter exposure to natural sunlight (Xiong et al. 1997). In
long-term experiments with natural Antarctic phyto-
plankton assemblages MAA concentration s have been
shown to increase after exposure to PAR with UV but
not to PAR without UV (Villafaiie et al. 1995). The
spectral effect of radiation on MAA synthesis differs
largely between species. In Phaeocystis antarctica the
induction ofMAA synthesis is induced predominantly
by short wavelength UV-A but also by UV-B radiation,
while some diatom species respond predominantly
to long wavelength UV-A and short wavelength vis-
ible rad iation (Riegger & Robinson 1997). In the
dinoflagellate Gyrodinium dorsum the accumulation
ofMAAs is stimulated by PAR and UV radiation, but
the most pro minent induction is caused by radiation
below 345 nm (Figure 4 ). There are numerous stud-
ies revealing that synthesis of MAAs is influenced by
the irradiation conditions. However, in most of these
studies the spectral resolution is limited to broad wave-
length ranges with treatments such as PAR onl y or
PAR plus UY. So, it is evident that MAAs are ac-
cumulated in response to radiation, but a true action
spectrum fo r MAA synthesis is not yet available.
Effects of UV-8 radiation on macroalgae
Coastal areas all over the world are inhabited by
macroalgae (Liining 1985). As macroal gae grow from
supralittoral to sublittoral zones they are exposed to
vary ing levels of solar radiation. The absorption of so-
lar radiation in the water column begins in the lower
wavelength bands. Algae growing in the supralittoral
230
or in the eulittoral are exposed to solar radiation in-
cluding UV, while algae growing in the sublittoral are
exposed mainly to PAR. Many algae show a distinct
zonation at their growing site (Hanelt 1996; Franklin
& Forster 1997; Bischof et aL 1998). The influence
of increasing solar radiation affects the macroalgae di-
rectly and indirectly. A direct influence is damage of
DNA (Pakker & Breeman 1997), pigment composi-
tion and the photosynthetic apparatus (Aguilera et al.
1999). An indirect effect can be caused by changes in
the environment of the algae like desiccation, changes
in temperature and the release of toxic substances by
the influence of UV irradiation on chemicals in the
water.
In their natural environment photoinhibition de-
pends on the growth site of the algae. Macroalgae
growing at the surface or in the intertidal show much
higher photoinhibition compared to macroalgae grow-
ing in the subtidal or in crevices.
In the field, as well as under laboratory conditions,
the influence of UV-B radiation on photosynthetic
quantum yield, oxygen evolution and respiration as
well as the recovery has been investigated by several
workers (Hanelt et al. 1993; Larkum & Wood 1993;
Hader et al. 1996; Hader 1997; Perez-Rodriguez et al.
1998; Groniger et al. 1999). Algae growing in trans-
parent waters show a higher photoinhibition than algae
growing in turbid waters (Hanelt et al. 1993). A re-
duced photosynthetic activity of algae around midday
was shown under field as well as laboratory condi-
tions (Hader 1997). Depending on the stage in their
life cycle different photoinhibition and recovery lev-
els were found in Laminaria saccharina (Hanelt et al.
1997). Laboratory studies on Dasycladus vermicularis
exposed to PAR with or without UV radiation led to a
complete loss in oxygen evolution after 24 h of PAR
+ UV-A + UV-B radiation (Perez-Rodriguez et al.
1998). In Mastocarpus stellatus a drastic decline in the
ratio of the photosynthetic quantum yield was shown
when exposed for 3 days to a solar simulator (Fig-
ure 5). The decrease is mainly caused by PAR while
the effect of additional UV-A or UV-B does not affect
the algae as drastically as the PAR. The algae show
a recovery up to about 50% for PAR with or without
UV-A, while the thalli irradiated with PAR+ UV-A +
UV-B recover only to 40% (Groniger et al. 1999).
100
80
~
'C
60
Oi
>
0
40
~
a:
395
Filter treatment
exposure 42 h
E.xposurc timo
recovery
Figure 5. Ratio of the yield of dark-adapted (0 h) control sample
and a dark-adapted sample after 72 h of exposure and 42 h of re-
covery in Mastocarpus stellatus. The thalli were exposed to PAR
(395 nm cut-off filter) , PAR+ UV-A (320 nm cut-off filter) or PAR
+ UV-A + UV-B (295 nm cut-off filter) from a solar simulator in
a 12 h/12 h light/dark cycle. For recovery they were placed in low
light conditions in a 12 h/12 h light/dark cycle.
Photo protective substances in macroalgae
UV-absorbing substances in macroalgae were first re-
ported in 1961 (Tsujino & Saito 1961). In the last
few years qualitative and quantitative studies were car-
ried out to survey the distribution of MAAs among
macroalgae. MAAs are distributed in macroalgae from
polar to tropical habitats. Karentz et al. (1991c) re-
ported the presence of MAAs in macroalgae from
the Antarctic. A survey of Rhodophyta, Phaeophyta
and Chlorophyta from tropical to polar habitats gives
a broad overview of the distribution of MAAs in
macroalgae (Hader & Figueroa 1997; Karsten et al.
1998a, b, c). The percentage of investigated species
containing MAAs is much higher in red algae com-
pared to brown and green algae. Also the number of
different MAAs as well as the total amount of MAA
is higher in Rhodophyta compared to Phaeophyta or
Chlorophyta. A decreasing amount of MAAs is found
with increasing depth in the water column as well as in
species from higher latitudes. Deep water (more than
2m; depending on water turbidity) algal species do not
contain MAAs (Maegawa et al. 1993; Karsten et al.
1998c ). Therefore, the MAA content in algae varies
between classes and with growing depths. Differential
results in MAA analysis are also influenced exposure
to solar radiation.
Basically three patterns of MAAs distribution are
found among macroalgae; (a) high initial MAA con-
tent and no increase during light treatment, (b) low
 231
1.2

6
1.0 Oh
6h =' 5
ci .c
.!2'
Q. 0.8
31 h ~ 4
4> ~
u "C
c: 0.6
.!2' 3
-e"'
0 .s"' ~
rJ) 0.4
..0 ~
:;;:
72 h
<( Oh Ceramium rubrum
0.2
0
Mastocarpus stellatus
0.0
250 300 350 400 450

Wavelength [nm] Firter treatment

Figure 6. Absorption spectra of the MAAs of Gmcilaria mrnea Figure 7. Contents of M AAs in Masrocarpus srellm us and Ce-
atier increasing exposure time to UV-B. ramium ruhrum after three days of exposure ( 1211 2 h light/dark
cycle) to tndicated irrad iation treatments. The in itial value is set to
IOOo/c (n = 3; average standard deviation ±25% ).
MAA content with an increase during light treatment,
and (c) no initial MAA and no induction during light
tidal, does not produce MAAs as protection against
treatment. The red macroalga, Chondrus crispus, har-
harmful radiation. Bleaching of the thalli started on
vested from the subtidal zone shows an increase in
the second day of exposure. P rotundus might not have
the number and amount of MAAs (Karsten et al.
the possibility to produce MAAs as it is not necessary
1998a). Initial high levels but no significant increase
under normal growing conditions.
in the MAAs content was found in the upper intertidal
Rhodophyta, Porphyra umbilicalis (Groniger et a\.
1999). Similarly, no in vivo induction of MAAs was
recorded after exposure to either UV alone or in com-
Evolutionary significance of photoprotective
bination with PAR in the marine red alga, Gracilaria
compounds
cornea, which possesses a very high amount of natu-
Sagan ( 1973) first considered the possibility that or-
rally occurring MAAs having an absorption maximum
ganic molecules may have acted as a UV screen in the
at 334 nm. The MAAs were highly stable against UV-
aqueous environments of the early Earth. The nature
B irradiation (Figure 6) and heat treatment (Sinha et al.
and evolutionary origin of the first specific photopro-
2000). In the Chlorophyta Dasvcladus vermicularis
tective compounds on the Achaean Earth is unknown .
UV-absorbing substances were .found, but their ab-
But according to one assumption early aromatic con-
sorption spectra and retention times did not resemble
taining reaction centers were some of the earliest
those of known MAAs (Perez-Rodriguez et al. 1998).
UV-screens that over the period altered from a no n-
The intertidal rhodophytes, Mastocarpus stellatus
productive dissipative UV-screen lo a light harvesting
and Ceramium rubrum were exposed to a solar simu-
lator using different cut-offfilters to produce PAR on ly role in photosynthesis (Mulkicljanian & Junge 1997).
MAAs play a vital role as osmotic regulators in
or PAR + UV-A or PAR + UV-A + UV-B. Small
some cyanobacteria (Oren 1997) and such alternative
changes in the MAAs content were observed in both
rol es may have given rise to the first UV-screening
algae (Figure 7). As both algae were collected from the
MAAs (Cockcll 1998). MAAs evolution as specific
intertidal zone in Helgoland in July 1998 their initial
UV-protectants may represent an early innovation in
content of MAAs at the beginning of the experiment
dealing with Achaean UV-B flux. Certain MAAs such
might have been sufficient for protection to solar radi-
as mycosporine-glycine specificall y absorb in the UV-
ation or the maximum accumulation for these species
B range of the spectrum. It has been postulated that
had already been reached. In Polyides rotundus, ex-
later, as oxygen levels increased, UV-A screening
posed under the same conditions, no initial MAAs
MAAs became more important since many of the
were found and no MAAs were induced during the
effects of UV-A are mediated through oxygen free
experiment. P rotundus, growing strictly in the sub-
radicals (Garcia-Piche! 1998) and thus the role of UV-
232
A as a damaging agent in the biosphere increased. In
these compounds, the nitrogen atom replacing the ke-
tone function has a greater mesomeric effect on the
benzene ring and the absorbance is shifted towards
the UY-A. Most probably, a mutation in the earliest
UV-B screening compounds resulted in a UV-A screen
which became physiologically advantageous. Since
MAAs have been reported in several eukaryotic al-
gae, it is likely that they were passed to the eukaryotic
algae by cyanobacteria in the plastidic line (Cockell
& Knowland 1999 and references therein). Dillon &
Castenholz ( 1999) suggests that the protection against
UV radiation provided by scytonemin may have been
an important ecophysiological factor in cyanobacterial
evolutionary history. Scytonemin would have facili-
tated the ecological expansion of cyanobacterial mat
communities into exposed shallow-water and terres-
trial habitats during the early to mid-Precambrian de-
spite the high levels of UV radiation impinging on the
Earth's surface at that time.
Database
A database on photoprotective compounds in cyano-
bacteria, phytoplankton and macroalgae has been con-
structed (http://www.biologie.uni-erlangen.de/bota-
nikllindex.html). It contains information on the al-
gae, the reference, the MAAs found in the algae, the
collection site and depths. Further information on the
different MAAs like absorption maximum and extinc-
tion coefficient as well as experimental procedures are
available. The database allows to obtain information
on the different algae and to compare the results from
different growth sites or collection dates (Groniger
et al. 2000).
Conclusions
Increases in the level of UV-B radiation are likely to
induce changes in community structure since there
are great differences in susceptibility of species to
UV-induccd damage. Species with larger cells, hav-
ing the ability to accumulate UV-screening substances
or with more effective repair mechanisms will likely
be favored. There is ample evidence that the change
in the radiation climate originating from the deple-
tion in stratospheric ozone has significant adverse
etlects on aquatic organisms. However, the action of
defense mechanisms in organisms partially mitigates
the inhibitory effects of UV-B. Different sensitivity
of species and the subsequent changes in community
structures may lead to changes in steady state con-
ditions that still sustain a high primary production.
Changes in species composition might have conse-
quences propagating within aquatic food webs that are
at present difficult to predict.
The ecological significance of photoprotective
compounds in diverse organisms as screening agents
against UY-induced damage has yet to be elucidated.
Not much is known about the spatial distribution of
MAAs within the cells. The reports of specific dis-
tributions of absorption due to MAAs in the water
column indicate a role of these compounds in protec-
tion against UV radiation, but there are many reports
showing that the presence of MAAs in an organism is
no proof that this organism is UV-tolerant (Xiong et al.
1997). Higher MAAs concentrations in Prorocentrum
micans, adapted to UV radiation did not completely
mitigate the detrimental effects of UV on photosynthe-
sis (Lesser 1996). Literature survey reveals that MAAs
may serve at least three different functions: (a) they
may protect the cells from UV photodamage by play-
ing a sunscreen role, (b) they may to a certain extent
transfer radiant energy to the photosynthetic reaction
centers, which is supported by the fact that the emitted
fluorescence spectrum peaks at a wavelength near the
Soret band of chlorophyll absorption (Sivalingam et al.
1976; Sinha et al. 1999b), and (c) that they may aid
in osmotic regulation (Oren 1997). Thus, irrespective
of the fact that the UV protective potentials of MAAs
and scytonemin are a primary or secondary function
or they are synthesized/accumulated due to UV radia-
tion, the presence of these compounds in an organism
may provide protection to the internal organelles and
components from the full impact of deleterious UV
radiation.
Acknowledgements
The work outlined in this review was partially sup-
ported by the European Union (DG XII, Environ-
mental Programme, ENV4-CT97-0580). We thank M.
Schuster for excellent technical assistance.
References
Aguilera, J., Jimenez, C., Figueroa, r. L., Lebert, M. & Hader, D.-
P. 1999. Effect of ultraviolet radiation on thallus absorption and

photosynthetic pigments in the red alga Porphrra umbilicalis. J.
Photochem. Photobiol. B: Bioi. 48: 75-82.
Arrigo, K. R. 1994. Impact of ozone depletion on phytoplankton
growth in the Southern Ocean: large scale partial and temporal
variability. Mar. Ecol. Prog. Ser. 114: 1-12.
Bebout, B. M. & Garcia-Pichel. F. 1995. UV-B induced vertical
migrations of cyanobacteria in a microbial mat. Appl. Environ.
Microbial. 61: 4215-4222.
Bischof, K., Hanclt, D. & Wieneke. C. 1998. LTV-radiation can
affect depth zonation of Antarctic macroalgae. Mar. Bioi. 131:
597-605.
Blumthalcr, M. & Ambach, W. 1990. Indication of increasing solar
ultraviolet-B radiation flux in alpine regions. Science 248: 206-
208.
Bohm, G. A., Plleiderer, W., Bi)ger, P. & Scherer. S. 1995. Structure
of a novel oligosaccharide-mycosporine-amino acid ultravio-
let A/B sunscreen pigment from the ten·estrial cyanobacterium
Nostoc commune. J. Bioi. Chem. 270: 8536--8539.
Bornman, J. F. 1989. Target sites of UV-B radiation in photosyn-
thesis of higher plants. J. Photochcm. Photobiol. B: Bioi. 4:
145-158.
Bothwell. M. L., Sherbot, D. M. J. & Pollock. C. M. 1994.
Ecosystem response to solar ultraviolet-B radiation: influence of
trophic-level interactions. Science 265: 97-100.
Brenowitz, S. & Castenholz. R.W. 1997. Long-term efl'ects of UV
and visible irradiancc on natural populations of a scytonemin-
containing cyanobacterium (Ca!othrix sp.). FEMS Microbial.
Ecol. 24: 343-352.
Britt, A. B. 1995. Repair of DNA damage induced by ultraviolet
radiation. Plant Physiol. 108: 891-896.
Biidel, B., Karsten. LT. & Garcia-Piche], F. 1997. Ultraviolet-
absorbing scytonemin and mycosporine-likc amino acids deriva-
tives in exposed. rock-inhabiting cyanobacterial lichens. Oecolo-
gia 112: 165-172.
Burton. G. W. & Ingold. K. U. 1984. ;'l-Carotene: an unusual type
of lipid antioxidant. Science 224: 569-573.
Carrero, J. I., De Marco, S. G. & Lutz. Y. A. 1989. LTV-absorbing
pigments in the dinoflagellates Alexundriwn excuvatum and Pro-
rocenrrum micans. Effects of light intensity. Pp. 333-336. Tn:
Okaichi, T., Anderson. D. M. & Ncmoto, T. (eds). Red Tides.
Biology, Environmental Science and Toxicology. Elsevier. New
York.
Carreto. J. I., Lutz. Y. A .. De Marco, S. G. & Carignan. M. 0.
!990a. Fiuence and wavelength dependence of mycosporine-like
amino acid synthesis in the dinoflagellate Alexandrium excam-
tum. Pp. 275-279. In: Graneli, E., Edler. L., Sundstrom, B. &
Anderson, D. M. (eds). Toxic Marine Phytoplankton. Elsevier,
New York.
Can·eto, J. 1., Carignan. M. 0 .. Daleo, G. & De Marco. S.
G. 1990b. Occurrence of mycosporine-like amino acids in the
red tide dinoflagellate ;\/exandrium exutt'(/lum: UY-protcctivc
compounds'' J. Plankton Res. 12:909-921.
Castenholz, R. W. 1997. Multiple strategies for LTV tolerance in
cyanobacteria. Spectrum l 0: I 0-16.
Christopher. D. A. & Mullet, J. E. 1994. Separate photosensory
pathways co-regulate blue light/ultraviolet-A-activated psbD-
psbC transcription and light induced D2 and CP43 degradation
in barley (Hordeum m!gare) chloroplasts. Plant Physiol. I04:
1119-1129.
Cockell, C. S. I 998. The biological effects of UV radiation on early
earth-a theoretical evaluation. J. Theoret. Bioi. 193: 719-731.
Cockell, C. S. & Knowland, J. 1999. Ultraviolet radiation screening
compounds. Bioi. Rev. 74: 311-345.
233
Crutzen. P. J. 1992. Ultraviolet on the increase. Nature 356: 104-
105.
Cullen. J. J., Neale, P. J. & Lesser, M.P. 1992. Biological weighting
function for the inhibition of phytoplankton photosynthesis by
ultraviolet radiation. Science 258: 646--650.
de Chazal, N. M. & Smith, G. D. 1994. Characterization of a brown
Nos/oc sp. from Java that is resistant to high light intensity and
LTV. Microbiology 140: 3183-3189.
Dillon. J. G. & Castcnholz, R. W. 1999. Scytonemin. a cyanobacter-
ial sheath pigment, protects against UVC radiation: implications
for early photosynthetic life. J. Phycol. 35:673-681.
Donkor. Y. & Hader, D.-P. 1991. Effects of sol3r and ultraviolet
radiation on motility, photomovement and pigmentation in fil-
amentous, gliding cyanobacteria. FEMS Microbiol. Ecol. 86:
159-168.
Dunlap. W. C. & Chalker. B. E. 1986. Identification and quantifica-
tion of near-LTV absorbing compounds (S-320) in a hermatypic
sclcractinian. Coral Reefs 5: 155-159.
Dunlap, W. C., Chalker. B. E. & Oliver. J. K. 1986. Bathymethric
adaptations of reef-building corals at Davies Reef. Great Barrier
Reef, Australia. III. UV-B absorbing compounds . .1. Exp. Mar.
Bioi. Ecol. I 04: 239-248.
Dunlap, W. C. & Yamamoto. Y. 1995. Small-molecule antioxi-
dants in marine organisms: antioxidant activity of mycosporinc-
glycine. Comp. Biochcm. Physiol. 112: 105-114.
Dunlap, W. C., Rae. G. A., Helbling. E. W., Villafane. Y. E. &
Holm-Hansen. 0. 1995. UV-absorbing compounds in natural
assemblages of Antarctic phytoplankton. Antarct. J. U. S. 30:
323-326.
Dunlap. W. C. & Shick, J. M. 1998. Cltraviolet radiation-absorbing
mycosporine-like amino acids in coral reef organisms: a hio-
chemical and environmental perspective. J. Phycol. 34: 418-430.
Ehling-Schulz, M .. Bilger. W. & Scherer. S. 1997. UV-B-induced
synthesis of photoprotective pigments and extracellular polysac-
charides in the terrestrial cyanobacterium No.\·toc commune. J.
Bacteriol. 179: 1940-1945.
Favre-Bonvin. J., Bernillon. J., Salin. N. & Arpin. N. 1987.
Biosynthesis of mycosporine: mycosporine glutaminol in Tr;-
chorhecium roseum. Phytochem. 26: 2509-2514.
Franklin. L. A. & Forster. R. M. 1997. The changing irradiance
environment: consequences for marine macrophyte physiology,
productivity and ecology. Eur. J. Phycol. 32: 207-232.
Garcia-Piche!. F. & Castenholz. R. W. 1991. Characterization and
biological implications of scytonemin. a cyanobacterial sheath
pigment. J. Phycol. 27: 395-409.
Garcia-Piche!. F.. Sherry. N.D. & Castenholz, R. W. 1992. Evidence
for an ultraviolet sunscreen role of the extracellular pigment scy-
toncmin in the terrestrial cyanobacterium Chlorogloeopsis sp.
Photochem. Photobiol. 56: 17-23.
Garcia-Piche!. F. & Castenholz. R. W. 1993. Occurrence of LTV-
absorbing. mycosporine-like compounds among cyanobacterial
isolates and an estimation of their screening capacity. Appl.
Environ. Microbial. 59: 163-1()9.
Garcia-Piche!. F. Wingard, C. E. & Castcnholz. R. W. 1993. Ev-
idence regarding the UV sunscreen role of a mycosporine-like
compound in the cyanobacterium Cloeocapsa sp. Appl. Environ.
Microbiol. 59: 170-176.
Garcia-Piche], F 1996. The absorption of ultraviolet radiation by
microalgae: simple optics and photobiological implications. Sci.
Mar. 60: 73-79.
Garcia-Piche!, F. 1998. Solar ultraviolet and evolutionary history of
cyanobacteria. Origins of Life & Evolution of the Biosphere 28:
321-347.
234
Gerber, S. & Hader, D.-P. 1995. Etlccts of enhanced solar irradiance
on chlorophyll fluorescence and photosynthetic oxygen produc-
tion of five species of phytoplankton. FEMS Microbial. Ecol. 16:
33-42.
Ghetti, F., Herrmann, H., Hader, D.-P. & Seidlitz, H. K. 1999. Spec-
tral dependence of the inhibition of photosynthesis under sim-
ulated global radiation in the unicellular green alga Dunaliella
salina. J. Photochcm. Photobiol. B: Biol. 48: 166-173.
Gleason. D. F. 1993. Differential effects of ultraviolet radiation
on green and brown morphs of the Caribbean coral Porites
astreoides. Limnol. Oceanogr. 38: 1452-1463.
Griiniger, A .. Hallier, C. & Heider D.-P. 1999. Influence of UV radi-
ation and visible light on Porphyra umbilicalis: photoinhibition
and MAA concentration. J. Appl. Phycol. 11:437-445.
Griiniger, A., Sinha, R. P., Klisch, M. & Hader D.-P. 2000. Pho-
toprotective compounds in cyanobacteria, phytoplankton and
macroalgae- a database. J. Photochem. Photobiol. B: Biol. 58:
115-122.
Hader, D.-P. 1987a. Photomovement. Pp. 325-345. In: Fay, P. and
Van Baalen C. (eds), The Cyanobacteria. Elsevier, Amsterdam.
Hader, D.-P. 1987b. Photosensory behavior in procaryotes. Micro-
bioi. Rev. 51:1-21.
Hader, D.-P. 1988. Ecological consequences of photomovement in
microorganisms. J. Photochcm. Photobiol. B: Bioi. 1: 385-414.
Hader, D.-P., Herrmann, H. & Santas, R. 1996. Eflects of solar
radiation and solar radiation deprived of UV-B and total UV
on photosynthetic oxygen production and pulse amplitude mod-
ulated fluorescence in the brown alga Padina pavonica. FEMS
Microbiol. Ecol. 19: 53-61.
Hader, D.-P. 1997. Penetration and c!lects of solar UV-B on phyto-
plankton and macroalgae. Plant Ecol. 128: 4-13.
Hader, D.-P. & Figueroa, F. L. 1997. Photoecophysiology of marine
macroalgae. Photochem. Photobiol. 66: 1-14.
Hader, D.-P., Kumar, H. D., Smith, R. C. & Won·est, R. C. 1998.
Effects on aquatic ecosystems. J. Photochem. Photobiol. B: Bioi.
46: 53-64.
Hanelt, D., Huppertz, K. & Nultsch, W. 1993. Daily course of
photosynthesis and photoinhibition in marine macroalgae inves-
tigated in the laboratory and field. Mar. Ecol. Prog. Ser. 97:
31-37.
Hanelt. D. 1996. Photoinhibition of photosynthesis in marine
macroalgae. Sci. Mar. 60: 243-248.
Hanel!, D., Wieneke, C., Karsten, U. & Nultsch, W. 1997. Pho-
toinhibition and recovery after high light stress in different
developmental and life-history stages of Laminaria saccharina
(Phaeophyta). J. Phycol. 33: 387-395.
Helbling, E. W., Villafane, V. E., Ferrario, M. & Holm-Hansen, 0.
1992. Impact of natural ultraviolet radiation on rates of photosyn-
thesis and on specific marine phytoplankton species. Mar. Ecol.
Pro g. Ser. 80: 89-100.
Helbling, E. W, Villafane, V. E. & Holm-Hansen, 0. 1994. Etlects
of ultraviolet radiation on Antarctic marine phytoplankton pho-
tosynthesis with particular attention to the influence of mixing.
In: Weiler, C. S. and Penhale, P. A. (eds), Ultraviolet Radiation
in Antarctica: Measurements and Biological Effects. Antarctic
Res. Ser. 62: 207-227.
Helbling, E. W, Chalker, B. E., Dunlap, W C., Holm-Hansen, 0. &
Villafane, V. E. 1996. Photoacclimation of Antarctic diatoms to
solar ultraviolet radiation. J. Exp. Mar. Biol. Ecol. 204: 85-101.
Halfman, D. J. & Deshler, T. 1991. Evidence from balloon measure-
ments for chemical depletion of stratospheric ozone in the Arctic
winter of 1989-90. Nature 349: 300-305.
Ito, S. & Hirata, Y. 1977. Isolation and structure of a mycosporine
from the zoanthid Palythoa tuberculosa. Tctrah. Lett. 28: 2429-
2430.
Karentz, D., Cleaver, J. E. & Mitchell, D. L. 199la. Cell survival
characteristics and molecular responses of Antarctic phytoplank-
ton to ultraviolet-B radiation. J. Phycol. 27: 326--341.
Karentz, D .. Cleaver, J. E. & Mitchell, D. L. 1991b. DNA damage
in the Antarctic. Nature 350: 28.
Karcntz, D., McEuen, F. S., Land, M. C. & Dunlap, W. C. 199lc.
Survey of mycosporine-like amino acid compounds in Antarctic
marine organism: potential protection from ultraviolet exposure.
Mar. Bioi. 108: 157-166.
Karsten, U., Sawall, T. & Wieneke, C. 1998a. A survey of the
distribution of UV-absorbing substances in tropical macroalgac.
Phycol. Res. 46: 271-279.
Karsten, U., Franklin, L. A., Luning, K. & Wieneke, C. 1998b.
Natural ultraviolet radiation and photosynthetically active radi-
ation induce formation of mycosporine-like amino acids in the
marine macroalga Chondrus crispus (Rhodophyta). Planta 205:
257-262.
Karsten, U., Sawall, T., Hanelt, D .. Bischof, K., Figueroa, r.
L.. Flores-Moya, A. & Wieneke, C. 1998c. An inventory of
UV-absorbing mycosporine-like amino acids in macroalgae from
polar to warm-temperate regions. Bot. Mar. 41: 443-453.
Kerr, J. B. & McElroy, C. T. 1993. Evidence for large upward trends
of ultraviolet-B radiation linked to ozone depletion. Science 262:
1032-1034.
Kim, S.-T. & Sancar. A. 1995. Photorepair of nonadjacent pyrim-
idine dimers by DNA photolyase. Photochem. Photobiol. 61:
171-174.
Kirk, J. T. 0. 1994. Optics of UV-B radiation in natural waters.
Arch. Hydrobiol. 43: 1-16.
Kumar, A., Sinha, R. P. & Hader, D.-P. 1996a. Etlect of UV-B on
enzymes of nitrogen metabolism in the cyanobacterium NoshJC
calcicola. J. Plant Physiol. 148: 86--91.
Kumar, A .. Tyagi, M. B., Srinivas, G., Singh, N., Kumar, H. D.,
Sinha, R. P. & Hader, D.-P. !996b. UVB shielding role of FeCI3
and certain cyanobacterial pigments. Photochem. Photobiol. M:
321-325.
Larkum, A. W. D. & Wood, W. F. 1993. The etlect of UV-B radia-
tion on photosynthesis and respiration of phytoplankton, benthic
macroalgae and seagrasses. Photosyn. Res. 36: 17-23.
Lesser, M. P. 1996. Acclimation of phytoplankton to UV-B radia-
tion: oxidative stress and photoinhibition of photosynthesis are
not prevented by UV-absorbing compounds in the dinoflagellate
Prorocentrum micans. Mar. Ecol. Prog. Ser. 132: 287-297.
Llewellyn, C. A. & Mantoura, R. F. C. 1997. A UV absorbing com-
pound in HPLC pigment chromatograms obtained from Icelandic
Basin phytoplankton. Mar. Ecol. Prog. Ser. 158: 283-287.
Lubin, D. & Jensen, E. H. 1995. Effects of clouds and stratospheric
ozone depletion on ultraviolet radiation trends. Nature 377: 710-
713.
Liining, K. 1985. Meeresbotanik: Verbreitung, Okophysiologie und
Nutzung der marinen Makroalgen. Thieme Verlag, Stuttgart.
Maegawa, M., Kunicda, M. & Kida W. 1993. Ditlercncc of
the amount of UV absorbing substance between shallow and
deep-water red algae. Jpn. J. Ph yeo!. 41: 351-354.
Marchant, H. J., Davidson, A. T. & Kelly, G. J. 1991. UV-B protect-
ing compounds in the marine alga Phaeocystis pouchetti from
Antarctica. Mar. Bioi. 109: 391-395.
Middleton, E. M. & Teramura, A. H. 1993. The role of flavonol gly-
cosides and carotenoids in protecting soybean from ultraviolet-B
damage. Plant Physiol. 103: 741-752.

Mittler, R, & Tel-Or, E, 199], Oxidative stress responses in the uni-
cellular cyanobacterium Svnechococcus PCC7942. Free Radical
Res. Commun. 12: 845-850.
Mulkidjanian, A. Y. & .Junge. W. 1997. On the origin of photosyn-
thesis as infened from sequence analysis. Photosynth. Res. 51:
27-42.
Neale, P. J., Banaszak, T. & Jarriel, C. R. l998a. Ultravi-
olet sunscreens in G_vmnodinium sunguineum (Dinophyceae):
mycosporine-like amino acids protect against inhibition of pho-
tosynthesis. J. Phycol. 34: 928-938.
Neale, P. .I., Davis, R. A. & Cullen, .1 . .1. l998h. Interactive ef-
fects of ozone depletion and vertical mixing on photosynthesis
of Antarctic phytoplankton. Nature 392: 585-589.
Oren, A. 1997. Mycosporine-like amino acids as osmotic solutes in
a community of halophilic cyanobacteria. Gcomicrobiol. J. 14:
231-240.
Pakker, H. & Breeman, A. M. 1997. Etlects of ultraviolet B radi-
ation on macroalgae: DNA damage and repair. Phycologia 36:
82.
Perez-Rodriguez, E., Gomez, 1., Karsten, U. & Figueroa. F. L.
1998. Effects of U V radiation on photosynthesis and excre-
tion of UV-absorbing compounds of Dasvc!adus vermicularis
(Dasycladales, Chlorophyta) from southern Spain. Phycologia
37: 379-387.
Proteau, P. J., Gerwick, W. H .. Garcia-Piche!, F. & Castenholz, R.
W. 1993. The structure of scytonernin, an ultraviolet sunscreen
pigment from the sheaths of cyanobacteria. Experientia 49: 825-
829.
Quesada, A. & Vincent, W. F. 1997. Strategies of adaptation by
Antarctic cyanobacteria to ultraviolet radiation. Eur. 1. Phycol.
32: 335-342.
Quesada, A., Vincent, W. F. & Lean-David. R. S. 1999. Community
and pigment structure of Arctic cyanobacteria assemblages: the
occunence and distribution of UV-absorbing compounds. FEMS
Microbial. Ecol. 28: 315-323.
Rambler, M. B. & Margulis, L. 1980. Bacterial resistance to
ultraviolet radiation under anaerobiosis: implications for pre-
Phanerozoic evolution. Science 210: 638-640.
Reynolds, C. S., Oliver, R. L. & Walsby, A. E. 1987. Cyanobacter-
ial dominance: the role of buoyancy regulation in dynamic lake
environments. N. Z. J. Mar. Freshw. Res. 21: 379-390.
Richter, P., Krywult, M., Sinha, R. P. & Hader, D.-P. 1999. Calcium
signals from hctcrocysts of Anabaena sp. aticr UV irradiation.
154: 137-139.
Ricgger, L. & Robinson, D. 1997. Photoinduction of UV-absorbing
compounds in Antarctic diatoms and Phaeocysr;s antarcth:a.
Mar. Ecol. Prog. Ser. 160: 13-25.
Roger, P. A. & Kulasooriya, S. A. 1980. Blue-Green Algae and
Rice. International Rice Research Institute, Los Banos, Laguna,
Philippines.
Roos, .I. C. & Vincent, W. F. 1998. Temperature dependence of UV
radiation effects on Antarctic cyanobacteria. J. Phycol. 34: 118-
125.
Sagan, C. 1973. Ultraviolet radiation selection pressure on the
earliest organisms. J. Theoret. Bioi. 39: 195-200.
Scherer, S., Chen, T. W. & Boger, P. 1988. A new UV-A/B protect-
ing pigment in the tenestrial cyanobacterium Nostoc commune.
Plant Physiol. 88: 1055-1057.
Singh, R. N. 1961. Role of blue-green algae in nitrogen economy
of Indian agriculture. Indian Council of Agricultural Research,
New Delhi, India. Pp. 61-82.
Sinha, R. P., Kumar, H. D., Kumar, A. & Hader, D.-P. l995a. Ef-
fects of UV-B irradiation on growth, survival, pigmentation and
235
nitrogen metabolism enzymes in cyanohacteria. Acta Protozoal.
34: 187-192.
Sinha, R. P., Lebert, M., Kumar, A., Kumar, H. D. & Hader, D.-P.
l995b. Disintegration ofphycobilisomes in a rice field cyanobac-
terium Nostoc sp. following UV irradiation. Biochem. Mol. Bioi.
Int. 37: 697-706.
Sinha. R. P.. Lebert. M .. Kumar. A., Kumar. H. D. & Hader, D.-
P. l995c. Spectroscopic and biochemical analyses of UV etlects
on phycobiliproleins or Anobaeno sp. and Nostoc cormium. Bol.
Acta 180: 87-92.
Sinha, R. P. & Hader, D.-P. l996a. Photobiology and ecophysiology
of rice field cyanobacteria. Photochem. Photohiol. 64: 887-896.
Sinha. R. P. & Hader, D.-P. l996b. Response of a rice field
cyanobacterium Anabaena sp. to physiological stressors. Env.
Exp. Bot. 36: 147-155.
Sinha, R. P., Singh, N., Kumar, A .. Kumar. H. D., Hader. M.
& Hader, D.-P. 1996. Effects of UV irradiation on certain
physiological and biochemical processes in cyanobacteria. J.
Photochem. Photobiol. B: Bioi. 32: 107-113.
Sinha, R. P.. Singh, N., Kumar, A .. Kumar, H. D. & Hader, D.-
P. 1997. Impacts of ultraviolet-B inadiation on nitrogen-fixing
cyanobacteria of rice paddy fields . .I. Plant Physiol. ISO: 188-
193.
Sinha, R. P. & Hader. D.-P. 1998. Etlects of ultraviolet-B radiation
io three rice field cyanobacteria. J. Plant Physiol. 153: 763-769.
Sinha. R. P., Klisch, M., Grliniger, A. & Hader, D.-P. l998a.
Ultraviolet-absorbing/screening substances in cyanobacteria.
phytoplankton and macroalgae. J. Photochem. Photobiol. B:
Bioi. 47: 83-94.
Sinha, R. P., Vaishampayan, A. & Heider. D.-P. l998b. Plant-
cyanobacterial symbiotic somaclones as a potential bionitrogen-
fertilizer for paddy agriculture: biotechnological approaches.
.'vlicrobiol. Res. 153: 297-307.
Sinha, R. P., Singh. S.C. & Hader, D. P. l999a. Photoecophysiology
of cyanobacteria. Recent Res. Devel. Photochem. Photohiol. 3:
91-101.
Sinha. R. P., Klisch, M. & Hader, D.-P. l999b. Induction of a
mycosporine-like amino acid (MAA) in the rice-lield cyanobac-
terium Anabaena sp. by UV irradiation. J. Photochem. Photobiol.
B: Bioi. 52: 59-64.
Sinha. R. P., Klisch, M., Vaisharnpayan, A. & Hader, D.-P. l999c.
Biochemical and spectroscopic characterization of the cyanobac-
terium Lvngbro sp. inhabiting Mango (Mangifera indica) trees:
presence of an ultraviolet-absorbing pigment. scytonemin. Acta
Protozoal. 38: 291-298.
Sinha, R. P.. Klisch, M., Griiniger, A. & Hader, D.-P. 2000.
Mycosporine-like amino acids in the marine red alga Gracilarht
corneo-effects of UV and heal. Env. Exp. Bot. 43: 33-43.
Sivalingarn. P. M .. Ikawa, T. & Nisizawa, K. 1976. Physiological
roles of a substance 334 in algae. Bot. Mar. 19: 9-21.
Slaper, H., Velders. G. J. M., De Gruijl. F. R. & Van der Leun, J.
C. 1996. Estimates of ozone depletion and skin cancer incidence
to examine the Vienna Convention achievements. Nature 384:
256-258.
Smith. R. C. & Baker. K. S. 1979. Penetration of UV-B and
biologically etl'ective dose-rates in natural waters. Photochem.
Photohiol. 29: 311-323.
Smith, R. C., Prezelin, B. B., Baker. K. S .. Bidigare, R. R., Boucher,
N. P., Coley. T., Karentz, D., Macintyre, S., Matlick. H. A.,
Menzies. D., Ondrusek, M., Wan, Z. & Waters, K. J. 1992.
Ozone depletion: ultraviolet radiation and phytoplankton biology
in Antarctic waters. Science 255: 952-959.
236
Takano, S., Uemura. D. & Hirata, Y. 1978a. Isolation and struc-
ture of a new amino acid, palythene. from the zoanthid Palwhoa
tuberculosa. Tetrah. Lett. 26: 2299-2300.
Takano. S., Uemura, D. & Hirata, Y, 1978b. Isolation and struc-
ture of two new amino acids, palythinol and palythene, from the
zoanthid Palythoa tubercu/osa. Tetrah. Lett. 49: 4909-4912.
Takano, S., Nakanishi. A .. Uemura, D. & Hirata, Y. 1979. Isolation
and structure of a 334 nm UV-absorbing substance. porphyra-
334 trom the red alga Porphvra tenera Kjellman. Chern. Lett.
419-420.
Tandeau De Marsac, N. 1977. Occurrence and nature of chromatic
adaptation in cyanobacteria. J. Bacteriol. 130: 82-91.
Tirlapur, U., Scheucrlein, R. & Hader, D.-P. 1993. Motility and
orientation of a dinoflagellate, Gymnodinium, impaired by solar
and ultraviolet radiation. FEMS Microbial. Ecol. 102: 167-174.
Tsujino, I. & Saito, T. 1961. Studies in the compounds specific
for each group of marine algae. I. Presence of characteristic ul-
traviolet absorbing material in Rhodophyceae. Bull. Fac. Fish.,
Hokkaido Univ. 12: 49-58.
Tsujino, !., Yabe, K. & Sekikawa, I. 1980. Isolation and structure of
a new amino acid, shinorine, from the red alga Chondrus vendoi
Yamada et Mikami. Bot. Mar. 23: 65-68.
Vaishampayan. A., Sinha. R. P. & Hader, D.-P. 1998. Use of ge-
netically improved nitrogen-fixing cyanobacteria in rice paddy
fields: prospects as a source material for engineering herbicide
sensitivity and resistance in plants. Bot. Acta Ill: 176-190.
Valiela. I. 1995. Marine Ecological Processes, 2nd Edition,
Springer. New York.
Venkataraman. G. S. \981. Blue-green algae: a possible remedy to
nitrogen scarcity. Curr. Sci. 50: 253-256.
Verner, M., Neori, A. & Haxo, F. T. 19X9. Spectral properties and
photosynthetic action in red-tide populations of Prorocentrum
micans and Gonyaulax polyedra. Mar. Bioi. 103: 365-371.
Vernet. M. & Whitehead. K. 1996. Release of ultraviolet-absorbing
compounds by the red-tide dinoflagellate Lingulodinium polye-
dra. Mar. Bioi. 127: 35-44.
Villafane, V. E .. Helbling, E. W., Holm-Hansen, 0. & Chalker, B. E.
1995. Acclimatization of Antarctic natural phytoplankton assem-
blages when exposed to solar ultraviolet radiation. J. Plankton
Res. 17: 2295-2306.
Vincent. W. F. & Roy, S. 1993. Solar ultraviolet-B radiation and
aquatic primary production: damage, protection, and recovery.
Environ. Rev. I: 1-12.
Vincent, W. F. & Quesada, A. 1994. Ultraviolet radiation effects on
cyanobacteria: implication for Antarctic microbial ecosystems.
In: Weiler, C. S. & Penhale, P. A. (eds.). Ultraviolet Radiation
in Antarctica: Measurements and Biological Effects. Antarctic
Research Series, American Geophysical Union. Washington. 62:
111-124.
Watanabe, I. 1984. Use of symbiotic, free-living blue-green algae in
rice culture. Outlook Agri. 13: 166-172.
Watson, A. J. & Liss. P. S. 1998. Marine biological controls on cli-
mate via the carbon and sulphur geochemical cycles. Phil. Trans.
R. Soc. Land. B 353:41-51.
Williamson. C. E. 1995. What role does UVB radiation play in
freshwater ecosystems" Limnol. Oceanogra. 40: 386-392.
Xiong. F., Komenda, J., Kopecky, J. & Nedbal, L. 1997. Strategies
of ultraviolet-B protection in microscopic algae. Physiol. Plant.
100: 378-388.
Xiong, F., Kopecky, J. & Nedbal, L. 1999. The occurrence of UV-
B absorbing mycosporine-like amino acids in freshwater and
tenestrial microalgae. Aq. Bot. 63: 37-49.
 Section 5: Aquatic Plants and Aquatic Ecosystems

The experimental set-up in which charophytes of the species Chara aspera are being exposed to different levels of UV-B
radiation. (Photograph by N. de Bakker)
 Plant Ecology 154: 239-246, 200 L
239
© 2001 Kluwer Academic Puhlishers.

Effects of UV-B radiation on a charophycean alga, Chara aspera

N. V. J. de Bakker 1, A. P. van Beem, J. W. M. van de Staaij, J. Rozema & R. Aerts

Vrije Universiteit, Institute of Ecological Science, Dept. of' Systems 1'-'cology, De Boelelaan 1087, 1081 HV
Amsterdam, The Netherlands (e-mail: nancydb@bio. vu.nl)

Key words: Charophytes. Growth, Reproduction, UV absorbing pigments

Abstract
The charophycean algal species Chara aspera was exposed for 73 days to three levels of UV- B radiation (weighted
according to Caldwell's generalized plant action spectrum): 1.9 kJ m- 2 day- 1 ('no UV-B'), 6.4 KJ m- 2 day- 1
(ambient) and 10.5 kJ m- 2 day- 1 (enhanced UV-B), the latter level simulating 30% ozone reduction in The
Netherlands.
Charophycean algae are mainly freshwater organisms and are thought to be the algae most closely related to
higher land plants. Therefore we expected that responses of charophycean algae to UV-B radiation might be more
related to those observed in the higher land plants than those of other 'lower' algal groups.
U ndcr elevated UV-B radiation algal length was reduced. There was no induction of UV absorbing compounds
under enhanced UV-B. This might relate to a sensitive response to UV-B radiation. The charophyccan algae
show similar adaptations to UV-B radiation as terrestrial plants, while not having UV-screens as occur in many
angiosperms. Vegetative reproduction (bulbils) increased in the presence of UV-B radiation. while generative
reproduction (antheridia and oogonia) decreased.

Introduction protect themselves or reduce the UV-B damage by

The stratospheric ozone layer has declined since the
early 1970s, due to the anthropogenic emission of
halogen-containing compounds, such as chlorofluo-
rocarbons (CFC's). This decline in ozone thickness
has resulted in an increase of UV-B radiation (280-
315 nm) at the earth surface (Madronich 1992; Her-
man et al. 1996). The most pronounced effect of the
ozone depletion is the development of the Antarctic
ozone hole in the southern spring, but also at temperate
latitudes the ozone layer has declined (Herman et al.
1996; Madronich et al. 1998; SORG 1999).
Since the discovery of the ozone hole by Farman
ct al. ( 1985), much attention has been paid to the
effects of UV-B radiation on terrestrial and marine
organisms and ecosystems. UV-B radiation can cause
damage to DNA (Karentz et al. 1991 a; Burna et al.
1995; Taylor et a!. 1996), membranes (Kramer et al.
1991) and damage affecting photosynthesis (see Sul-
livan & Rozema 1999). Some organisms are able to
repairing the damage, e.g., by 'light' and 'dark' re-
pair of DNA damage (Sancar & Sancar 1988) or by
avoiding the damage. Avoidance can be realised via
changes in morphology (Tevini & Teramura 1989)
and by changes in chemical composition filtering out
deleterious UV-B, the so-called 'UV-B screens' (see
Meijkamp eta!. 1999).
Most research has focussed on effects of UV-B ra-
diation on marine and land organisms and ecosystems.
However, less attention has been paid to fresh water
systems (Williamson 1995). The charophytes, from
an evolutionary point of view, constitute an important
group of the green algae. These algae occur mainly
in fresh water systems. The evolution of the land
plants is believed to have taken place from algae, via
mosses to higher land plants (Bhattacharya & Medlin
1998). Rozema et al. ( 1997) assumed that it might
be of evolutionary importance that UV-B absorbing
phenolic compounds, acting among other functions
as UV-B screens, increase in complexity during evo-
240

lution. Among the green algae, charophytes appear N~ 0.06

~
to be most closely related to the higher land plants /'\
0.05 ./ "-.
(Devereux et al. 1990; Stafford 1991; Graham 1994 ). //./ "-
<!)
Therefore we expect that effects of or adaptations to (.) 0.04
I
~
UV-B radiation of charophycean algae might be in line I
with their evolutionary position: more close to higher ] O.D3
I
I
land plants than to other algal groups. As far as we ~ 0.02 I
know, no literature is available on the effects of UV-B
radiation on these algae.
b(.)
11.)
0..
rJJ
0.01
c../
/
I.
_/
Here we report results of a greenhouse experiment 0.00
on the etlects of UV-B radiation on charophycean 280 290 300 310 320
algae. This study focuses on morphological and chem- Wavelength (nm)
ical changes and reproductive characteristics in the
Figure 1. The average above water spectrum (280-320 nm) of the
charophyte, Chara aspera, in response to UV-B ra- treatments; solid line is the 'No UV-B" treatment; dotted line is
diation. the ·Ambient' treatment and dashed line is the "Enhanced UV-B'
treatment.

Methods
Experimental set-up
The effects of UV-B radiation on Chara aspera Deth.
Ex Wild. were investigated in a greenhouse exper-
iment. Sediment containing spores and bulbils was
collected from Lake Veluwe, NL, on April 29 1998
and stored dark at 4 oc until the start of the experi-
ment. Natural water of this Lake Veluwe was collected
on June 17 1998 and stored dark at 4 °C as well.
Lake Veluwe is a de-eutrophicated shallow lake with
a dense charophyte vegetation, where Chara aspera
is the dominant species. For a detailed description of
the Lake Veluwe see Van den Berg ( 1999). The ex-
periment was conducted from July 3 till September 14
1998.
Aquaria (length 39 em, width 21 em, and height
25 em) were filled with a 4 em thick layer of sed-
iment. Natural lake water was added to a height of
17 em above the sediment. Adding demineralised
water compensated for evaporation during the exper-
iment. Air temperature in the greenhouse was around
24 oc (day) and 16 oc (night). The aquaria were
wrapped in black plastic to prevent algal growth on
the sides of the aquaria. In addition to natural light,
170 ,'lmol m- 2 day- 1 PAR was supplied by Philips
HPI/T lamps (400 W) to the middle of each aquarium.
The light: dark cycle was 14: I0 hours.
In the middle of the light period the charophycean
algae were exposed to UV for 6 hours. Philips TL
40 UV tubes provided UV radiation. Three UV-
treatments were applied: no UV-B, ambient UV-B
and enhanced UV-B (Figure 1). The 'no UV-B' treat-
ment had a biologically effective dose (UVBE) of 1.9
(±0.7) kJ m- 2 day- 1, calculated using the generalized
plant action spectrum (Caldwell 1971) normalized at
300 nm. In this treatment, UV-B radiated by the UV
tubes was filtered out with 0.13 mm thick polyester
foil (mylar, Dupont Industries, USA), which absorbs
radiation < 313 nm. The 'ambient' treatment had a
UVBE of 6.4 (±0.5) kJ m- 2 day- 1 reflecting ambient
plus up to 4-7% ozone reduction levels around the
end of June in The Netherlands (52° N), calculated
using the model of Green ( 1980) and assuming cloud-
less sky conditions. The 'enhanced UV-B' treatment,
which simulated 30% ozone reduction, corresponded
to a UVsE of I 0.5 (± 1.2) kJ m- 2 day- 1. In both the
'ambient' and the 'enhanced UV-B' treatment the UV
tubes were covered with 0.1 mm thick cellulose ac-
etate foil (Tamboer and Co. Chemie, Haarlem, NL),
which absorbs radiation with wavelengths < 290 nm.
Differences between the treatments were obtained by
adjusting the height of the UV-B tubes above the
aquaria. Both the mylar and the cellulose acetate foils
were changed weekly to avoid changes in the radiation
regimes due to photodegradation of the foils. The UV-
A irradiance emitted by the Philips HPI/T lamps and
the Philips TL 40 UV tubes and the natural light was
on average 1.64 W m- 2 for the 'no UV-B' treatment
and 1.93 and 1.91 W m- 2 for the 'ambient UV-B'
and 'enhanced UV-B' treatment, respectively. Four
aquaria were used in every treatment. The spectral
irradiance of the UV-B lamps was measured with a
double-monochromator spectroradiometer (Optronics
Model OL 752, Orlando, FL, USA). With a portable
UV-X radiometer with a UV-X 31 sensor (San Gabriel,
CA, USA), prepared for underwater measurements,

UV-X
0 20 40 60 80 100 120 140
0
2
4
6 Chemical analysis of photosynthetic and UV
s absorbing pigments
~
~ 8
i3
0.,
IU 10
Ci
12
14
16
18
Figure 2. Below water UV conditions of the different treatments.
Attenuation of UV radiation within the water column, measured
with a broad band sensor; line with closed circles represents the 'No
UV-B' treatment; the line with open circles represents the 'Ambient'
treatment and line with closed triangles represents the 'Enhanced
UV-B' treatment; errors bars are shown (n = 4).
UV measurements in the aquaria at various depths
were performed after the vegetation was collected and
the sediment stabilised (Figure 2).
Harvest and morphological measurements
After four days charophycean algae started emerging
above the sediment in all aquaria. Electric conduc-
tivity (EC), a measure for total dissolved salts in the
water, and pH were measured weekly. After 73 days
the charophycean algae in the aquaria were harvested
from 187 cm 2 of the soil surface for measurements
of morphological characteristics. The other part of the
aquaria was harvested and used for chemical analysis.
Two species occurred in the aquaria: Chara a.1pera
and Chara contraria A. Braun ex Ki.itzing. About 94%
belonged to the species Chara aspera and 6% to the
species Chara contraria. The two species were sepa-
rated to measure characteristics of individuals of the
species Chara aspera. Length was only measured for
charophytcs longer than 25 mm. Charophytes shorter
241
than 25 mm were counted. Furthermore, the number of
branch whorls was measured. Dry weight of the har-
vested algal material was determined after drying for
4 days at 70 oc. The following reproductive character-
istics were measured: generative reproduction by the
presence of branch whorls containing oospores and/or
spores, and vegetative reproduction by the number
of bulbils, which are vegetative starch-rich organs
(Moore 1986).
Chlorophyll content was measured according to the
method of Amon ( 1949). For the extractions I 00 mg
of fresh Chara aspera material was put in test tubes.
After adding 5 ml of 80% acetone (Fiuka, Germany),
test tubes were sonicated for 15 min to break the cell
walls. After 3 hours of extraction at 4 oc in dark, test
tubes were centrifuged for I 0 min at 2500 rev min- 1•
The absorbance of the solution was then measured
with a spectrophotometer (Perkin-Elmer Lambda UV-
Vis) at 645, 652 and 663 nm.
For the determination of methanol soluble UV-
B absorbing compounds I 0 mg of lyophilised Chara
aspera was added to test tubes containing 6 ml extrac-
tant. The extractant was composed of 100% CH 0 0H
(.I. T. Baker. Deventer, NL), demineralised water and
37% HCI (Riedel-de Haen. Germany) in the ratio
79:20: I (v:v:v). After closing the test tube to prevent
evaporation, test tubes were sonicated for I 0 min af-
ter which they were placed in a water bath at 90 oc
for 1 h. Cooled test tubes were then centrifuged for
I 0 min at 2500 rev min -I. The absorbance of the re-
sulting solution was measured at 280, 300, 320, 340,
360, 380 and 400 nm (Perkin-Elmer Lambda UV-
Vis spectrophotometer) (Meijkamp et a!. 200 I : Visser,
1997).
Data analysis
For statistical analysis of treatment effects on the
length of the charophycean algae, only plants longer
than 25 mm were used. A Kolmogorov-Smirnov
test was performed to test for normality. The data
were logarithmically transformed. Although the vari-
ances of the data were not homogeneous (tested by
a Bartlett Box), the most suitable statistical test was
a nested ANOVA to analyse these data. The differ-
ent aquaria were nested within the UV-B treatments.
Length measurements were taken as replicates within
an aquarium.
242
Table I. ANOVA table of the statistics of length of Cham aspera.

Sum of df Mean F Significance

squares square

Between UV-B treatment 2. 140 2 1.070 7.8 10 = 0.01

Within UV-B treatment 1.237 9 0. 137 4.496 < 0.00 1
Error 59.330 1941 0.030

For data on dry weight, charophycean algal den- so

A B c
sity, both chlorophyll a and b concentrations and
number of bulbils, a one-way AN OVA was used, after
70
•
• a
a
60 c
testing for normality and homogeneity of variances of b b b
E' so ) ,.:t.,_...,_ r=-
the data with the tests mentioned above. The data on ~
5 ~
generative reproduction were first transformed using .c 40
the arcsinus square root transformation, after which a '"'
c0)
....l 30
one-way ANOYA was done. For the average number
20
of branch whorls a Kruskal-Wallis test was performed.
Significance level in all statistical tests was 0.05. All 10

statistical tests were performed using the program 0

SPSS (SPSS Inc., Chicago). oUV-B Ambient Enhanced UV-B
Figure 3. Average le ngth of Cham aspera per treatment (wide bars)
and the average le ngth (± S.E.) of Chara aspera per aq uarium
Results within a treatment (small bars; n varies from 9 1 to 232). D iffer-
ent capital letters indicate significant difference between treatments;
different lower-case letters indicate significant ditlerence within a
Experimental conditions treatment.

There was no significant difference in pH between the

applied treatments. At the start of the experiment the
average pH in the aquaria was 8.5 (± 0.1). During
the course of the experiment the average pH increased
to 9.4 (± 0.1 ) at the day of the harvest. The electric
conductivity (EC) decreased from 877 ± 10 to 770 ±
16 f.LS cm- 1 . There was also no significant difference
in EC between the aquaria in all treatments.
The radiation profile measured with the UV-X ra-
diometer (Figure 2) shows that UV is attenuated by the
water column.
Morphology
There was a significant effect of UV-B radiation on
length of Chara aspera (P = 0.0 I) at the end of the
experiment. The ' no UV-B' treatment resulted in the
largest charophycean algae, followed by the ambient
treatment. Under enhanced UV-B the charophycean
algae were the shortest. Besides this effect between
the UV treatments, there was also a significant differ-
ence between aquaria within some of the treatments
(P < 0.00 I; Figure 3, Table I). This might be related
to differences in irradiance, as the aquaria could not
be rotated in order not to disturb the growth of the
charophycean algae. Although there was some varia-
tion within the treatments, the effect ofUV-B radiation
is clear.
Biomass of the charophycean algae was not af-
fected by the different UV treatments, nor was there
a significant difference in charophycean algal density.
There was also no di fference in average number of
branches per plant (Table 2).
Reproduction
The ambient and enhanced UV-B treatment had more
bulbils (vegetative reproductive structures) than the
treatment without UV-B (P < O.Ol; Figure 4). How-
ever, in the no UV-B treatment the number of branches
containing generative reproductive organs was signifi-
cantly higher than in the ambient and enhanced UV-B
treatments ( P < 0 .0 1; Figure 5).
 243
300 Ta /Jle 2. The average (± S.E.) chlorophyll concentra-
tion ( mg I t ) in the charophyte samples per treatment
(n = 4).
250
b
Chlorophy ll a C hlorophyll b
200
~
No UV-B 6. 16±0. 10 2.38±0.08
:0 150
:; Ambient 6.0 I ±0.77 2.4 1±0.31
Ol
Enhanced UV-B 6.3 ~±0.~2 2.57±0.32
100

50

0.35
0
- NoUV-B
Ambient Enhanced UV-B
~
0.30 = Arnbieno UV-8
Enhanced UV-B
Figure 4. Average number of bulbils collected per aquarium per ~ 0.25
treatment (n = 3). Standard errors are shown; significant differences e)
(J
T
o f number of bulbils between treatments are indicated wi th difiCre nt c
lower-case letters. "'
.C> 0.20
~
.C> 0.15
"'
;:
e)
0.10
E
ell
a: 0.05

o.oo .J....__ .L,.LL_ .L,J-L_ .c,.U-_ .,..LL_ .,J-l-_ .,..LL_.,..LL---'

2 0 300 320 340 360 3 0 4()()

Wavelength (nm)
Figure 6. Absorbance of methanol soluble lJV-B absorb ing pig-
ments in Charo aspera. Bars represe nt average pigment absorbance
(± S.E.) per treatme nt at different wavelengths (n = 4).

No UV-8 Ambient Enhanced UV-8

Figure 5. Percentage (± S.E.; n = 4) of Charct ospera plants
that contain generative reproductive structures o n the branch whorls
as antheridia and/or oogonia (solid bars) or spores (dashed bars).
Significant differences of percentages of plants with anthcridia
and oogonia between treatments are indicated with ditTercnt cap-
itals; different lower-case letters indicate significant difference in
percentages of plants containing spores between treatments.
Photosynthetic and UV-B absorbing compounds
For the chemical analysis the charophyte samples
comprised both Chara aspera and Chara contraria .
There was no effect of UV-B on the chlorophyll a
and chlorophyll h concentrations in the samples (Ta-
ble 3), neither was there a significant difference in the
absorption of the methanol soluble UV-B absorbing
compounds between the treatments at the wavelengths
measured (Figure 6).
Discussion
Experimental conditions
T he pH and electric conductivity did not differ be-
tween aquaria during the experiment. Therefore, there
were no differences in growth conditions between
the UV treatments. Chara aspera grows under nat-
ural conditio ns at a pH of 6- 9 (Nat et al. 1994).
In the aquaria, the pH increased from 8.5 (±0.1 ) to
9.4 (±0.1 ). which slightly exceeds the range of nat-
ural occurrence. However, under c ultivated conditions
a higher pH than under natural conditions may be
observed (Van den Berg, 1999; J. Simons personal
comment).
Morphology
Charophytes, like Chara a.1pera, are composed of
multicellular nodes, from wh ich branch whorls are
formed, and si ngle celled internodes. These internodes
can be surrounded by modified latera ls (e.g., in Chara
aspera), forming a one cell layer thick cortex (Moore
1986). There was a negative effect of UV-B radiation
244
Table 3. Average(± S.E.) plant density, average number of branch whorls and average
biomass per treatment (n = 4 ).
Density (nr of plants cm- 2
Average number of branch whorls
Biomass (g DW cm-2)
on the length of Chara aspera plants. As there was
no effect of UV-B on plant density and on total bio-
mass or on the average number of branch whorls per
algae, it seems that UV-B negatively affects cell elon-
gation of the internodes. This is in contrast with the
way in which growth reduction under the influence
of UV-B occurs in unicellular marine algae. In these
organisms cells become larger, because cell division
is inhibited by UV- B radiation (Karentz 1994; Burna
et al. 1995). However, in higher land plants internode
length ( Meijkamp 2001) or shoot elongation can be
reduced as well, e.g., by changes in phytohormones
(Ros & Tevini 1995). Thus changes in morphology
can result in avoidance of UV-B radiation (Tevini &
Teramura 1989).
Reproduction
Chara aspera propagates both by the formation of
diploid oospores and by forming vegetative, starch-
rich organs, the bulbils (Moore 1986; Krause 1997).
There seemed to be a trade-off in reproduction strat-
egy. Under enhanced UV-B radiation Chara aspera
formed more vegetative reproduction structures than
in the absence of UV-B, while the opposite was found
for the generative reproduction. Bulbils can serve as
structures for propagation during the growing season,
but also play an important role in wintering (Krause
1997). More bulbils might perhaps result in a denser
vegetation in the next growing season, where compe-
tition with other algae and macrophytes might be influ-
enced. On the other hand less generative reproductive
structures, both antheridia, oogonia and spores, were
formed under enhanced UV-B radiation. Spores are
important for dispersion and recolonisation (Krause
1997). Therefore, enhanced UV-B might negatively
influence dispersion. This trade-off between gener-
ative and vegetative reproduction in Chara aspera
under enhanced UV-B has not been observed in higher
terrestrial plants. It is generally known that in higher
land plants the investment in generative reproduc-
.__
NoUV-B Ambient Enhanced UV-B
3.8 ±0.5 4.3 ±0.2 3.6 ±0.1
3.3 ±0.3 3.4 ±0.2 3.1 ±0.2
0.80±0.06 0.75±0.02 0.83±0.04
tivc structures might increase under stress conditions
(Grime 1989).
UV absorbing compounds
UV absorbing compounds are important in screen-
ing UV-B. By lowering UV-B levels within the plant
tissues damage to DNA, membranes, proteins and
photosynthetic tissue can be prevented or reduced
(Meijkamp et al. 1999). In Chara aspera no changes
were observed in methanol soluble UV absorbing
compounds under enhanced UV-B. This makes these
algae potentially sensitive to UV-B radiation. The ab-
sence of increased UV-B absorbing compounds under
elevated UV-B is remarkable, since both in cyanobac-
teria, algae and in higher land plants increased ab-
sorbance has been reported under influence of UV-B
(Bi.idel et al. 1994; Meijkamp 1999). In algae, both
in marine and fresh water, induction of mycosporine
like amino acids (MAA's) has been shown (Garcia-
Piche! & Castenholz 1991; Karentz et al. 1991; Xiong
et al. 1997), whereas in higher land plants, e.g.,
flavonoid concentrations may be enhanced under el-
evated UV- B (Meijkamp et al. 1999). Preliminary
measurements on the presence of flavonoids in Chara
aspera in our experiment did not show these types of
secondary metabolites (data not shown). Markham &
Porter ( 1969) reported the presence of flavonoids in
charophytes. However, this finding has not been repro-
duced since then (Harborne 1986; Wegner-Hambloch
1983). Thus, the presence of flavonoids in charophytes
is questionable (cf., Stafford 1990).
Ecological effects of changes in penetration l!{
UV-radiation
Light is attenuated in the water column (Kirk 1994 ).
The UV-B dose received by the charophycean algae
therefore was lower than applied at the water surface
(Figures I, 2). Under enhanced UV- B charophycean
algae were shorter. As UV-B radiation is absorbed
much more by water than in air this reduced algal

length will result in aquatic ecosystems in a lower
UV-B dose at plant level. Thus, the shorter charo-
phytes received a lower UV-B dose. This might be
advantageous to avoid UV-B radiation, but also pho-
tosynthetically active radiation (PAR) decreases with
depth and the growth rate of charophytes may be
reduced as well.
Penetration of UV into the water depends on local
water properties and may be influenced by e.g. the
amount of dissolved humic substances and the amount
of particles present in the water (Kirk 1994 ). De Lange
( 1999) measured UV-B radiation in Dutch lakes. Mea-
surements in the clearest lake showed that the depth
at which I% of the UV-B radiation was present was
0.5 m, while the euphotic depth reached to 5.5 m.
In Lake Veluwe Chara aspera occurs at depths of
30-80 em (Van den Berg 1999). Above charophyte
meadows in Lake Veluwe the water transparency for
PAR is high (a light attenuation coefficient, Kd (m- 1),
< I). However in unvegetated sites the Kd is > 4,
which is due to high detritus, inorganic suspended
solids and chlorophyll a (Van den Berg 1999). There-
fore Chara aspera might be exposed to solar UV-B
under natural conditions as well, depending on the
water transparency.
Besides receiving less PAR, another disadvantage
for short algae is lower nutrient uptake. Charophycean
algae take up nutrients both via the rhizoids and via the
shoots (Krause 1997). However, as charophytes were
smaller under enhanced UV-B, the total surface of the
charophycean algae was smaller as well and therefore
nutrient uptake can be limited. This might reduce the
competition capacity to algae and macrophytes that do
not suffer growth depression.
In general, it can be concluded that charophycean
algae show some adaptations to enhanced UV-B simi-
lar to terrestrial plants. However, charophycean algae
are potentially sensitive to UV-B radiation, as there
seems to be no induction of UV-B absorbing com-
pounds under influence of enhanced UV-B levels.
Avoiding UV-B by reducing length may be effective
(Tevini & Teramura 1989), but reduced length also af-
fects the competitive ability of the species, by reduced
light interception and perhaps lower nutrient uptake.
There was a change in reproduction strategy, which
might affect dispersion of the species.
245
Acknowledgements
Marcel van den Berg is acknowledged for collecting
lake Veluwe sediment. We would also like to thank the
fine mechanical engineering group for their technical
assistance during the experiment and Dr J. Bedaux for
his advice on the statistics. The authors also wish to
thank Dr J. Simons and an anonymous referent for
comments and suggestions on this manuscript. This
research was funded by EU (DG XII) within the pro-
gramme Environment and Climate (contract ENV4-
CT97-0580), which is gratefully acknowledged.
References
Arnon, D. I. 1949. Copper enzymes in isolated chloroplasts.
Polyphenyloxidase in Beta vulgaris. Plant. Physiol. 24: 1-15.
Bhattacharya, D. & Medlin, L. 1998. Algal phylogeny and the origin
of land plants. Plant Physiol. 116:9-15.
Biidel, B., Karsten. U., & Garcia-Piche!, F. 1994. Ultraviolet-
absorbing scytonemin and mycosporine-like amino acid
derivates in exposed, rock-inhabiting cyanobacterial lichens.
Oecologia. 112: 165-172.
Burna. A. G. J., Van Hannen, E. J., Roza. L.. Veldhuis, M. J. W.
& Gieskes, W. W. C. 1995. Monitoring ultraviolet-B-induced
DNA damage in individual diatom cells by immunofluorescent
thymine dimer detection. J. Phycol. 31: 314--321.
Caldwell, M. M. 1971 . Solar UV radiation and the growth and de-
velopment of higher plants. Pp. 131-268. In: Giese. A. C. (ed.).
Photophysiology, Vol. 6. Academic Press, New York.
De Lange, H. J. 1999. Effects of ultraviolet-B radiation on
phytoplankton-zooplankton interactions. Doctorate thesis. Wa-
geningen Agricultural University, Wageningen.
Devereux, R., Loeblich III, A. R. & Fox, G. E. 1990. Higher plant
origins and the phylogeny of green algae. J. Mol. Evol. 31: 18-
24.
Farman, J. C., Gardiner, B. G. & Shanklin, J. D. 1985. Large
losses of total ozone in Antarctica reveal seasonal CLOx/NOx
interaction. Nature 315:207-210.
Garcia-Piche!, F. & Castenholz, R. 1991. Characterization and
biological implications of scytonemin a cyanobacterial sheath
pigment. J. Phycol. 27: 395--409.
Graham, L. E. 1993. Origin of Land Plants. John Wiley & Sons,
New York.
Green A. E. S., Cross, K. R. & Smith, L.A. 1980. Improved analytic
characterization of ultraviolet skylight. Photochem. Photobiol.
31: 59--65.
Grime, J. P. 1989. Whole-plant responses to stress in natural and
agricultural systems. Pp. 31--46. In: H. G. Jones, T. J. Flowers
& M. B. Jones (eds.), Plants under Stress. Cambridge University
Press, Cambridge.
Herman, J. R., Bhartia, P. K., Ziemke, J., Ahmad. Z. & Larko,
D. 1996. UV-B increases (1979-1992) from decreases in total
ozone. Geophys. Res. Lett. 23: 2117-2120.
Karentz, D., Cleaver, J. E. & Mitchell D. L. 1991 a. Cell sur-
vival characteristics and molecular responses of phytoplankton
to ultraviolet-B radiation. J. Phycol. 27: 326-341.
Karentz, D., Me Euen, F. S., Land, M. C. & Dunlap, W. C. 1991b.
Survey of mycosporine-like amino acid compounds in Antarctic
246
marine organisms: potential protection from ultraviolet exposure.
Mar. Bioi. 108: 157-166.
Karentz, D. 1994. Prevention of ultraviolet radiation damage in
Antarctica marine invertebrates. In: Biggs, R. H. & Joyner,
M. E. B. (eds), Stratosperic Ozone Depletion/ UV-B Radiation
in the Biosphere, Vol. I 118. NATO AS! series. Springer-Verlag,
Berlin.
Kirk, J. T. 0. 1994. Light & Photosynthesis in Aquatic Ecosystems,
2nd edition. Cambridge University Press, Cambridge, UK.
Kramer, G. F., Norman, H. A., Krizek, D. T. & Mirecki, R. M. 1991.
Influence of UV-B radiation on polyamines, lipid peroxidation
and membrane lipids in cumcumber. Phytochemistry 30: 2101-
2108.
Krause, W. 1997. Charales (Charophyceae). G. Fisher, Jena.
Madronich, S. 1992. Implications of recent total atmospheric ozone
measurements for biologically active ultraviolet radiation reach-
ing the earth's surface. Geophys. Res. Lett. 19: 37-40.
Madronich, S., McKenzie, R. L., Bjorn, L. 0. & Calwell M. M.
1998. Changes in biologically active ultraviolet radiation reach-
ing the earth's surface. J. Photochem. Photobiol. B. 46: 5-19.
Markham, K. R. & Porter, L. J. 1969. Flavonoids in the green algae
(chlorophyta). Phytochem. 8: 1777-1781.
Markham, K. R. 1988. Distribution of flavonoids in the lower plants
and its evolutionary significance. In: Harborne, J. B. (ed.), The
Flavonoids. Chapman and Hall, London.
Meijkamp, B .. Aerts, R., Van de Staaij, J., Tosserams, M., Ernst, W.
& Rozema, J. 1999. Etlccts ofUV-B on secondary metabolites in
plants. Pp. 39-59. In: J. Rozema (cd.). Stratopheric Ozone De-
pletion, the Effects of Enhanced UV-B Radiation on Terrestrial
Ecosystems. Backhuys Publishers, Leiden.
Meijkamp, B. Doodeman, G., & Rozema. J. 2001. The response
of Vicia jaba to enhanced UV-B radiation under low and near
ambient PAR levels. Plant Ecol.: 154: 135-146 (this volume).
Moore, J. A. 1986. Charophytes of Great Britain and Ireland.
Botanical Society of the British Isles, London.
Nat, E .. Simons. J., De Ia Haye, M.A. A. & Coops, H. 1994. Ver-
spreiding van kranswieren in Nederland. RIZA report 94.148x,
Lelystad.
Ros, J. & Tevini, M. 1995. Interaction ofUV-radiation and IAA dur-
ing growth of seedlings and hypocotyls segments of sunflower. J.
Plant Physiol. 146: 295-302.
Rozema, J .. Van de Staaij, J., Bjorn, L. 0. & Caldwell, M. 1997. UV-
B as an environmental factor in plant life: stress and regulation.
Trends Ecol. Evol. 12: 22-28.
Sancar, A. & Sancar, G. B. 1988. DNA repair enzymes. Ann. Rev.
Biochem. 57, 29--67.
Sakal R. R. & Rohlf, F. J. 1998. Biometry, 3rd edition. W. H.
Freeman and C., New York.
SORG 1999. Stratospheric ozone 1999. Department of environment,
transport and the regions. U.K.
Stafford, H. E. 1991. Flavonoid evolution: an enzymatic approach.
Plant Physiol. 96: 680-685.
Sullivan J. & Ronma J. 1999. UV-B effects on terrestrial plant
growth and photosynthesis. Pp. 39-59 In: J. Rozema (ed.),
Stratopheric Ozone Depletion, the Effects of Enhanced UV-
B Radiation on Terrestrial Ecosystem. Backhuys Publishers.
Leiden.
Taylor, R. M., Nikaido, 0., Jordan, B. R .. Rosamond, J.. Bray, C. M.
& Tobin, A. K. 1996. Ultraviolet-B-induced DNA lesions and
their removal in wheat (Triticum aestivum L.) leaves. Plant Cell
Env. 19: 171-181.
Tevini, M. & Teramura, A. H. 1989. UV-B effects on terrestrial
plants. Photochem. Photobiol. 50: 4 79-487.
Van den Berg, M. S. 1999. Charophyte colonization in shallow
lakes: processes, ecological effects and implications for lake
management. Doctorate thesis. Vrije Universiteit. Amsterdam.
Visser, A. J. 1997. Growth and physiology of !triticum aestivuml
and lvicia fabal in response to increasing atmospheric C02
concentrations. Doctorate thesis. Vrije Universiteit, Amsterdam.
Wegner-Hambloch, S. 1983. Polyphenole aus der Phaeophycee
Cvstoseira granulata Agardh., sowie untersuchungen uher das
Flavonoidvorkommen in Chara contra ria Kutz und Nitella jlex-
ilis Agardh. Doctorate Thesis. Rheinischen Friedrich-Wilhelms-
Universitat, Bonn.
Williamson, C. E. 1995. What role docs UV-B radiation play in
freshwater ecosystems? Limnol. Ocean. 40: 386-392.
Xiong F., Komenda, J., Kopecky, J. & Nedbal, L. 1997. Strategies
of ultraviolet-B protection in microscopic algae. Physiol. Plant.
I 00: 378-388.
 Section 5: Aquatic Plants and Aquatic Ecosystems

UV research at Ny-Alesund (Spitsbergen. No rway). Kongsbreen at the kongsljord. (Photograph by B. A. Faafeng)

 Plant Ecology 154: 249-259, 2001.
249
© 2001 Kluwer Academic Publishers.

Differential sensitivity to natural ultraviolet radiation among

phytoplankton species in Arctic lakes (Spitsbergen, Norway)

E. van Donk 1, B. A. Faafeng2 , H. J. de Lange 3 & D. 0. Hessen 4

1N/00-Centre j(Jr Limnology, Rijkstraatweg 6, 363 I AC Nieuwersluis, The Netherlands; 2NIVA. P. 0. Box I 73,
Kjelsas, 041 I Oslo, Norway; 3 Department Earth & Environment Sciences, Lehigh University, Bethlehem,
PA 18015, USA; 4 Biological Institute, UniversitY of' Oslo, P.O. Box 1027, Blindem, 0316 Oslo, Norway

Key words: Cell size, Cyanobacteria, Daphnids, Foodweb, Grazers, Inedible algae. Svalbard, UV-B

Abstract
Incubation experiments demonstrated a differential sensitivity to natural UV-radiation among the dominant phy-
toplankton species from three Arctic lakes, situated near Ny-Alesund, Spitsbergen (79° N). The growth of small
chlorophytes, diatoms and picocyanobacteria from two oligotrophic lakes was inhibited primarily by the shorter
wavelength UV components, while the growth of the larger colony-forming species (cyanobacteria, Planktothrix
sp., Woronichinia sp. and the chrysophyte, Uroglena americana Calkins) apparently was stimulated. These colonies
(not easily eaten by daphnids) dominated at the end of the experiment in those treatments where the short wave-
length UV components were not excluded. For the two oligotrophic localities. 70 and 61 %, respectively, of total
phytoplankton biovolume were edible in the treatments excluding short wavelength UV, compared to only 13 and
19%, respectively, in the treatments including such radiation. For the third, more productive and less transparent
lake, the percentage of edible species in the treatments with and without short wavelength UV radiation did not
differ (ca. 75% for both treatments).

Introduction Typical characteristics of oligotrophic arctic lakes

The interest in UV photobiology during recent years
is a result of concern about increasing levels of ultra-
violet radiation (UV) that may influence terrestrial as
well as aquatic ecosystems (Rozema et al. 1997). A
rise in short wavelength UV-B (280-315 nm) radiation
level has been associated with ozone depletion at high
southern and northern latitudes (Crutzen 1992; Kerr &
McElroy 1993).
In aquatic environments with low primary produc-
tion and low input of allochthonous matter, shortwave
radiation may penetrate to significant depths (Kirk
1994 ). Effect studies of enhanced levels of UV- B ra-
diation have mainly focused on marine ecosystems
in the Southern Ocean (e.g., Smith et a!. 1992). Re-
ports of increases in UV-B radiation for both north-
ern and southern temperate latitudes have caused
increased interest in freshwater ecosystem response
(e.g., Williamson 1995).
are their high transparency, allowing UV radiation
to penetrate deeper, and their common shallowness,
offering no depth refugium for harmful radiation (Hes-
sen 1996). The plankton may be continuously exposed
to UV-radiation during the arctic summer. In these
arctic lakes, even present-day fluxes of ultraviolet
radiation may be a major environmental constraint af-
fecting nutrient flow, biogeochemical cycles and food
web structure ( Karentz et al. 1994; Siebeck et al.
1994).
Effects of UV-B radiation on phytoplankton have
been reviewed by Hader ( 1993), Karentz et al. ( 1994)
and Hessen et al. ( 1997). The most important effects
on phytoplankton are DNA damage, photosynthesis
inhibition, and decrease in growth rate (Karentz ct al.
1991: Buma et al. 1995a). Field measurements made
in the springtime in the antarctic marginal ice zone
clearly showed that as the ozone layer thinned. inhi-
bition of photosynthesis by UV-B increased (Holm-
250
Hansen eta!. 1993; Vincent & Roy 1993). Significant
effects of UV radiation have also been demonstrated
for phytoplankton production in lakes (Moeller 1994 ).
UV-B may affect cell morphology and biochemical
composition (Hessen eta!. 1997). The most important
effects are increase in cell volume (Karentz eta!. 1991;
Behrenfeld eta!. 1992; Burna eta!. 1995b; Van Donk
& Hessen 1995), thickened cell wall that may decrease
digestion by zooplankton (Van Donk & Hcsscn 1995),
and changes in biochemical composition. Generally
protein and lipid content decrease and carbohydrate
increase (Strict et al. 1994), while fatty acid profiles
may undergo major changes (Goes eta!. 1994; Wang
& Chai 1994 ). Clearly this will affect the nutritional
quality for zooplankton. In life history experiments
Daphnia pulex had a reduced growth rate and number
of offspring when feeding on UV-B-irradiated algae
(De Lange & Van Donk 1997).
Responses of phytoplankton to UV-B radiation
may be taxon specific (Worrest 1982; Gala & Giesy
1991; Cabrera eta!. 1997). Smaller species of diatoms
were found to be more susceptible to UV-B than larger
ones (Karentz et a!. 1991 ). Flagellated algae, which
are important primary producers of biomass in fresh-
water habitats, seem more susceptible than non-motile
algae (Hader & Hader 1988; Hader 1993). A decrease
in production of edible phytoplankton species may
alter trophic interactions and reduce transfer of en-
ergy from primary producers to consumers in aquatic
ecosystems.
In order to analyse the susceptibility of various
freshwater phytoplankton taxa to natural UV radia-
tion in the Arctic, we conducted a series of exper-
iments with the natural phytoplankton communities
from three high arctic lakes on Spitsbergen (79° N)
in the North Atlantic Ocean. The filters we used
did not have sharp euough spectral cutotTs to dis-
criminate between UV-A (315--400 nm) and UV-B
(280-315 nm), but we distinguished between short
wavelength and long wavelength ultraviolet radiation
because their ecological impacts may differ. The dam-
aging potential of UV photons increases almost ex-
ponentially with decreasing wavelength (e.g., Cullen
et a!. 1992), however, because longer wavelength
components dominate, they may have a greater over-
all impact on physiological processes (Karentz et a!.
1994 ). Moreover, longer wavelength radiation can
counteract the adverse effects of shorter wavelenth ra-
diation by activating repair processes (Quesada et a!.
1995).
Methods
Study sites
Lake Brandallaguna, Lake Storevatn and Lake Solvatn
are small, shallow lakes situated near Ny-Alesund,
Spitsbergen (79° N). Major characteristics of the lakes
are given in Table I. While the two former localities
were low in nutrients, lake Solvatn has by far the high-
est concentrations of nutrients (P and N) and organic
carbon due to fecal droppings from surrounding bird
colonies.
PAR and UV radiation
To assess daily incoming radiation during the experi-
mental period at Ny-Alesund, the band 370-695 nm,
henceforth called was measured with a GUV541 mul-
tichannel filter instrument (Biospherical Instrument)
and UV-B (280-315 nm) with an UV-Biometer So-
lar Light Company Model SL50 I, weighted with the
standard erythema action spectrum. The attenuation
coefficients of Lake Brandallaguna were calculated
from measuring extinction of UV-A and UV-B at 5
different depths (PUV5000 system, Biospherical in-
struments). Due to their shallowness, in-lake light
attenuation could not be obtained from Lake Storevatn
and Lake Solvatn. Hence UV-absorbing properties
from all localities were assessed by spectrophotome-
ter (Perkin-Elmer Lambda 5) readings in quartz cu-
vettes at 273 nm (Table I). The reason for choosing
273 nm is that this is the standard wavelength for wa-
ter quality assessment of levels of dissolved organic
carbon. This wavelength cannot directly be used as
a measure of in-situ UV penetration, but rather as a
way of characterising the levels of DOC between the
localities.
Chemical analyses
Water samples for chemical analyses were taken from
the upper 0.5 m of the three lakes on 21 July 1998 at 10
AM, two weeks after ice break up. The surface water
temperature of the lakes was about 7-8 oc. The chemi-
cal analyses were conducted at the Norwegian Institute
for Water Research (NIVA) according to their standard
methods. Total-P (TP) and total-N (TN) were analyzed
on a Skalar San Plus auto-analyzer with a Skalar Ma-
trix photometric detector SA 6250-02 after persulfate
digestion of non-filtered samples. TP was determined
with the molybdate-blue method at 880 nm, while TN
was determined as nitrate at 450 nm after reduction
 251
Table 1. Major characteristics of the three lakes.

Surface area Max. depth Tow\ P Total N TOC UV abs e m - 1

(hal lm ) (pg 1- 1 P l (pg J- 1 N) (mg J- 1C) 1273 nm )

Lake Brandallaguna 7.5 3 14 3 15 4.0 0.037

Lake S torevatn 2.5 19 420 2.6 0.05X
Lake Solvatn 1.0 62 X45 5.9 0.12.'i

Table 2. Attenuation coefficients PAR (370 - 695 nm)

(m - 1) and percentage of UV surface
200
irradialion still present at 0.2 min Lake - 1 80
Brandallaguna. ~E 160
~ 140
Wavele ngth I nrn) b 120
Lake walcr
m c;,. c100 .... ..,.
~ 80 ,.-

nn
~ 60
308 (LJVB) 2.9 56
8 40
320 (UVA) 2.3 63 20
0
340 (UYA) 1.7 71
~ u ~ ~ u u n ~ ~
380 (UYA) 0.9 84 J uly 1998

UVB (280- 315 nm)

120 80
- PAR+ UV2
70
~ 100 - PAR +UV1
- - PAR .;; 60
c:: 80
0
·u; ~ 50

840
·e 60
<I)

n
~ 30
<I) 40
c: ~ 20
t=."' 20 10
0
0
21 u ~ ~ ~ u n ~ ~
280 300 320 340 360 380 400
J uly 1998
Wavelength (nm)
Figure 2. Daily dose o f PAR and UV-B at Ny-Aiesund during the
Fig11re 1. Transmission spectra of the inc ubation bottles. PAR =
experimental period (2 1- 29 J uly 1998).
glass bottles, PAR + UV I = polyethyle ne bottles. PAR + UV2 =
polycarbonate bottles .

rather low extinctio n of UV, at the incubation depth

with cadmium. Total organic carbon was determined
on a Phoe nix XOOO TOC-TC analyzer.
Phytoplankton biomass and grvwth
Water samples for phytoplankton study were taken at
the same time and depth as the samples for chemi-
cal analyses. These samples were, however, filtered
th rough 90 ILm gauze to remove most of the crus-
tacean zoop lankton. The phytoplankton communities
were incubated simultaneously in bottles with differ-
ent transparency for short wave length and long wave-
length UV, placed horizontally within a frame fixed at
0.2 m depth in Lake Brandallag una. This lake had a
stil l 56%of UV-B and 65% of UV-A surface radiation
were prese nt (Table 2). Three treatments were used,
each with three replicates (9 bottles per lake): PAR ( I I
glass bottles), PAR + long wavelength UV ( 1.5 I poly-
ethylene bottles, 1.5 mm thick, hereafter referred to as
PAR + UV I treatment), and PAR + lo ng wave length
UV + short wavelength UV ( I I pol ycarbonate bot-
tles, I mm thic k, hereafter referred to as PAR + UV2
treatment). The growth rates in the di fferent bottles
should be re lated to spectral qualities alone. Prelim-
inary laboratory studi es showed similar growth rates
of algae cultured in these polycarbonatc , polyethy le ne
and glass hollies, when growing under artificial PAR
light.
252

Table 3. Total biovolume (mm 3ml- 1) of the dominant phytoplankton species from the three lakes
at the start of the incubation experiment (21 July 199~). Also are given the maximal axial length
(/lm) for oblong formed cells/colonies and max. diameter (/lm) for spherical cells/colonies.

Max. Max. Lake Lake Lake

axial diameter Brandallaguna Storcvatn Solvatn
length (J1m) Biovolume (mm3 ml- 1)
(/1)

Cyanobacteria (pico) 2 4 2 30
Woronichinia sp. (col) 70 86
Planktothrix sp. (col) 120 31 8 63
Uroglena americana (col) 75 65 97 69
Ochromonas sp. 10 9
Chlamydomonas sp. 15 66 II
Ankyra judavi 40 17 453
Kirclmeriella sp. 15 17
Diatmna tenuis 50 160 27 214
Fraf?i/aria sp. 20 41
Total biovolume (mm3 ml-1) 343 281 846

Transmission spectra of the ditlerent incubation
bottles were measured in full sunlight using a scan-
ning spectroradiometer (Macam SR 991 0) (Figure I).
These transmission experiments were performed with
pieces of clear polycarbonate and polyethylene from
the bottles immediately over the sensor, and with solar
radiation almost perpendicular to the filters.
Samples of 20 ml were taken every second day
from each bottle and fixed with Lugol's solution
for phytoplankton analysis. The cells were counted
using an inverted microscope with phase contrast,
and total biovolumes of the ditlerent phytoplankton
species were calculated according to Rott ( 1981 ). The
growth of the dominant phytoplankton species (bio-
volumes) was followed for 9 days, starting on 21
July 1998. Most phytoplankton species reached a sta-
tionary phase after 7 or 8 days of incubation in the
different treatments. We evaluated the responses to
UV irradiation of the different species from the ex-
ponential phase of the growth cu'rves, calculating the
growth rates by a least squares linear regression analy-
sis of In-transformed data. The regression was only
applied over the first seven or eight days, including
at least four points and R-squared values > 0.8. The
mean growth rates with standard deviation of the dif-
ferent species in the three treatments were calculated
from the three replicates. The growth rates of each
species for the different treatments were statistically
compared by one-way ANOVA and significant differ-
ences (p < 0.05) were distinguished by applying the
Tukey's test (Sokal & Rohlf 1995).
At the beginning and end of the experiment also the
dimensions of the cells were measured. Large phyto-
plankton cells, e.g., cells with a maximum axial length
> I 00 11m or equivalent spherical cellular or colonial
diameter > 30 11m, or with long spines or protective
coverings, were considered to be 'grazing-resistant'
to the most efficient large Daphnia species (Sterner
1989). The percentages of edible and non-edible phy-
toplankton biomass were estimated for the PAR , PAR
+ UVI and PAR+ UV2 treatments.
Results
PAR and UV radiation
The surface daily doses of PAR and UV-B at Ny-
Alesund during the experimental period (21-29 July)
are given in Figure 2. The highest values were mea-
sured on 24 July.
Phytoplankton composition and biomass
The biovolumes and dimensions of the different phy-
toplankton species from the three lakes at the start
of the incubation experiment are given in Table 3.
The highest phytoplankton biomass were found in
Lake Solvatn (846 x mm 3 ml- 1, i.e., approximately
 253

Table 4. Growt h rates (fl, day- 1) and mean growth rates plus standard deviation (SD) of the dominant phytoplankton species from
the th ree lakes in the different treatments (PAR, PAR + UV I. PAR + UV2).

Species PAR PAR+UV I PAR+UV2

2 3 Mean (SDI 2 Mean (SlJI 2 Mean (SDl

Brandallaguna

A. judavi 0. 19 0.1 3 0.15 0.16(.03) 0.14 0. 13 0.11 0.13 (.01 J 0,07 0.06 0.02 0.05 (.03)
Chlamydomonas. sp. 0.18 0. 18 0.23 0.20 (.03) 0.09 0.10 0. 12 0.10 (.02) 0.02 0.07 0.08 0.06 (.03)
D. tenuis 0.19 0.21 0.24 0.21 (.031 0.15 0.15 0.17 0.16 (.01) 0.00 -0.02 - 0.04 - 0.02 (.02)
Cyanobacteria (pico) 0.17 0 .22 0.21 0.20 (.021 0.06 0.03 0.02 0.04 (.02) - 0.11 -0.11 -0.08 - 0.10 (.01)
Planktothrix sp. 0.24 0.31 0.32 0.29 (.04) ()30 0 .31 0.33 0.31 (.02) 0.36 0.31 0.37 0.35 (.03)
U. americana 0.12 0.22 0.22 0.19 (.06) 0. 17 0. 12 0. II 0.13 ( .(13) 0.24 0.27 0.29 0.27 (.02)

Storevatn

Chlamydomonas. sp. 0.23 0.21 0.21 0.21 (.01) 0.16 0.1 7 0.16 0.16 (.005) 0.11 0.12 O.ll 0.12 (.001 I
Ochmmonas 0. 22 0.3 I 0.28 0.27 (.05) 0.24 0.23 0.20 0.22 (.02) 0.21 0.32 0.57 0.36 (.18)
D. tenuis 0.28 0. 30 0.25 0.28 (.02) 0.29 0.28 0.31 0.29 (.02) 0.06 0.09 0.09 0.08 (.02 )
Fragilaria sp. 0.27 0.2 1 0.25 0.24 (.03) 0.24 0.19 0. 18 0.21 (.03) 0.12 0.06 0 .09 0.09 (.(J3)
Cyanobacteria (pi co) 0.18 0.13 0.12 0.14(.03 ) 0.18 0 . 17 0.19 0.18 (.0 1) 0.0 1 0.01 0.05 0.03 (.02)
Planktothrix sp. 0.12 0.21 0.13 0.15 (.05) 0.09 0.22 0.14 0.15 (.07) 0.29 o.:n 0.52 0.39 (. II I
Woronichinia sp. 0.19 0. 15 0.22 0.19 (.03) 0.16 0.22 0.20 0.19 (.03) 0.39 0.48 0.51 0.44 (.06)
U. americana 0.21 0.21 0.24 0.22 (.02) 0.25 0.53 0.23 0.34 (.17) 0.40 0.35 0.41 0.38 (.03)

Solvatn

A.judayi 0.52 0.42 0.44 0.46 (.05) 0.54 0.45 0.46 0.48 (.05) 0.49 0.40 0.57 0.49 (.08)
Kirchneriel/a sp. 1. 84 1.39 1.58 1.60 (.22) 1.63 1.52 1.56 1.57 (.06) 1.57 1.41 1. 53 1.50 (.()9)
D. tenuis 0.43 0.55 0.52 0.50 (.06) 045 0. 37 0.44 0.42 (.04) 0.40 043 0.33 0.38 (.05)
Cyanobacteria (pico) 0.51 0.62 0.49 0.54 (.07) 0.39 0.45 0.31 0.39 (.07) 0.26 0.25 0.14 0.22 (.07)
Planktothrix sp. 0.27 0.54 0.40 0.40 (.14) 0.36 0.60 0.35 0.44 (.1 4) 0.33 0.36 0.41 0.36 (.04 )
U. americana 0.65 0.63 0.6 1 0.63 (.02) 0.58 0.65 0.6 1 0 .6 1 (.03) 0.64 0.62 0.62 0.62 (.01)

Brandallaguna

Figure 3. Mean growth rates (/L, day - 1) of the dominant phytoplankton species fro m Lake Brandallaguna as measured in the different treat-
ments (PAR, PAR+ UVl, PAR + UV2). Error bars represent I SD (n = 3). Similar symbols a.b,c per spec ies represent homogeneous groups
that are not significantly different at a 95% level (Tukey's test ).
254
0.7 ,--------;::========,--------
DPAR
0 .6
AAA
":'-: 0.5
"'
"0
-; 0.4
AAB
~
:5 0.3 A B C
:1
!:>
CJ 0.2
0.1
§>'I>
,_o
Q{>
'<''li
Figure 4. Mean growth rates (f-L , day- 1) of the dominant phytoplankton species from Lake Storevatn as measured in the different treatments
(PAR, PAR + UV I, PAR + UV2). Error bars represent I SD (n = 3). Similar symbols a,b.c per species represent homogeneous groups that
arc not significantly different at a 95% level (Tukey's test).
0.85 {Lg wet-weight per ml) dominated by the chloro-
phyte Ankyra judayi Fott and the diatom Diatoma
tenuis Agardh.. In Lake Brandallag una, with a to-
tal phytoplankton biomass of 0.34 {[g wet-weight per
ml, D. tenuis, the chlorophyte Chlamydomonas sp.
Ehrenberg and the chrysophyte Uroglena americana
Calkins were the dominant species. In Lake Storevatn
(total phytoplankto n biomass 0.25 {[g ml ·- l) U. amer-
ic:ana , the cyanobacterium Woronichinia sp. Elenkin,
Chlamydomonas sp. and the diatom Fragilaria sp.
Lyngbye were the most dominant species. Accord-
ing to the measured cell sizes (Table 3) three species,
U. americana, Woronichinia sp. and Planktothrix sp.
Anagnostidis & Komrek, are considered to be 'graz ing
resistant' (Sterner 1989).
Growth responses
Growth rates of the different phytoplankton species
in each bottle and mean growth rates with standard
deviations are given in Table 4. The growth response
to UV radiation of the different phytoplankton species
from Lake Brandallaguna varied (Figure 3). For five
of the six dominant species, a significantly different
response between the PAR and PAR + UV2 treat-
ments was obse rved. The growth rates of A. judayi
and Chlamydomonas sp., D. tenuis and picocyanobac-
terial phytoplankto n were lower in the PAR + UV2
treatment, while the colony-forming U. ame ricana ex-
hibits a significant stimulation compared to the PAR
Storevatn
- ---,
AAB
DPAR + UV1
.PAR+ UV2 AAB AABB
AAB AAB
,y.. -,..'1>
~'I>
,_oi_-$i !::-'1>
:.o--s
~'li ·~" ~!::- ~e
6~~ ~,CJ
v'o
f!-0 o., ~0
Ci~'l> ~0
and PAR + UV I treatments. Planktothrix sp. was
not affected by UV radiation (although it should be
noted that even in the PAR + UV2 treatment most of
the UV-B was filtered out). Chlamydomonas sp. and
picocyanobacterial phytoplankton were also inhibited
in the PAR + UV 1 treatment. For Chlamydomonas
sp. the PAR+ UVI and PAR+ UV2 treatments did
not differ significantl y, indicating that the inhibition in
growth rate of this species was caused mainly by long
wavelength UV radi ation. This species also showed a
pronounced loss of fl agella with increasing UV radi-
ation. At the end of incubation more than 70% of the
cell s were still with two flagella in the PAR treatments,
while in the PAR+ UV l and PAR + UV2 treatments
more than 50% had lost one or two tlagella.
The responses of the different species from Lake
Storcvatn to UV radiation were comparable with those
for Lake Brandallaguna (Figure 4). For seven of the
eight dominant species a significant difference be-
tween the PAR and PAR + UV2 treatment was ob-
served. In gene ral the most negative effects were found
in the small species. Chlamydomonas sp, D. tenuis
and Fragilaria sp. and pieocyanobacterial phytoplank-
ton showed a decrease in growth rate in the PAR +
UV2 treatment, while U. americana exhibits a signif-
icant increase compared to the PAR treatment. Also,
Planktothrix sp. and Woronichinia sp. showed a signif-
icant growth stimulation in the PAR+ UV2 treatment.
Again, both long and short wavelength UV had a neg-
 255

::,o1vam
2.00
A A
1.80
1.60
~ 1.40
"' 1.20
E.
e
0

.t:
1.00

~ 0.80
0 A A A
(; 0.60
0.40
0.20
0.00

Figure 5. Mean growth rates (/L, day- 1) of the domi nant phytoplankton species tfom Lake Solvatn as measured in the different treatments
(PAR, PAR+ UV I, PAR+ UV2). Similar symbols a,b,c per species represent homogeneous groups that are not significantly different at a
95%1evel (Tukey's test).

ative effect on the growth of Chlamydomonas and also
loss of flagella was observed.
The responses to UV of the dominant phytoplank-
ton species from Lake Solvatn are quite different from
the two other lakes (Figure 5). The total phytoplank-
ton biovolume, nutrient concentrations (TP and TN)
and UV absorbance in this lake are higher (Tables I
and 3). The highest growth rates were measured fo r
the chlorophyte Kirchneriella sp. Schmidle, a species
that is rather rare in the two other lakes (Table 3). No
significant differences between the treatments were
observed for A. judayi and Kirchnierella sp., D. tenuis,
Planktothrix sp. and U. americana. Only for the pica-
cyanobacterial phytoplankton, a significant inhibition
in growth could be accredited to short wavelength UV
radiation.
Biovolume of edible and inedible phytoplankton
Total biovolumes of the edible and inedible phyto-
plankton were estimated at the start (2 1 July) and at
the end of the incubation experiment (29 July 1998)
in the three treatments (Figure 6). For Lake Bran-
dallagu na and Lake Storevatn large differences were
found between PAR and PAR + UV2 treatments, at
the end 70 and 6 1%, respectively, of total biovolume
was considered to be edible in the PAR and only 13
and 19%, respectively, in the PAR + U V2 treatments.
For Lake Solvatn the fraction of edible species was
comparable in PAR, PAR + UV I and PAR + UV2
treatments (ca. 75%). Chlorophytes (Chlamydomonas
sp., Kirchneriella sp.) and diatom (FraJ;ilaria sp. ) in-
creased slightly in cell size due to short wavelength
UV, but not to an extent that should severely affect
grazing by zooplankton .
Discussion
The experiments demonstrated a differential sensi-
ti vity to short wavelength UV radiation among the
dominant phytoplankton species, yet with a consistent
pattern with regard to the species being sensitive or
resistant. An inhibition in growth rate due to short
wavelength UV radiation was observed for chloro-
phytes, diatoms and picocyanobacteria, but not for
the larger colony-forming species (Planktothrix sp.,
Woronichinia sp. and U. americana) and the small
chrysophyte Ochromonas sp. The growth of some
of the larger species were apparently stimu lated in
the presence of UV radiation . This could either he
due to release of nutrients, chelated metals or to re-
duced competition with more sensitive phytoplankton
species. Effects ofUV radiation were mainly observed
for the phytoplankton from Lake Brandallaguna and
Lake Storevatn, where low algal biomass and low
256

Brandallaguna
2500
~

'-
E 2000
"'E
.§.
Q) 1500
E
:I
0
> 1000
0
iii
500

0
LAKE(stort) PAR PAR+ UV1 PAR • UV2

Storevatn
20 00

. ; 1600
E
"'E 1200
.§.
Q)
E 800
:I
0
>
0
iii 400

0
LAKE (Start} PAR PAR+ UV1 PAR+ UV2

Solvatn
7500

.,~ 6000
E
"'.[ 4500
Q)
E
:I 3000
0
>
0
iii 1500

0
LAKE (stort) PAR PAR • UV1 PAR• UV2

Figure 6. The mean total biovolumes of the edible and inedible (by large zooplankters) phytoplankton at the end of the incubation experiment
(29 July 1998), calculated for the communities of the three lakes in the PAR, PAR + UVI and PAR + UV2 treatments. Error bars represent I
SD(n = 3).

concentrations of organic carbon compounds caused
a high penetration of shortwave radiation (Table I).
In both lakes, not only short wavelength UV, but also
long wavelength UV was a growth inhibiting factor for
Chlamydomonas sp. The pronounced loss of flagella
by Chlamydomonas sp. with increasing UV radiation
corresponds well with earlier UV-exposure studies,
using cultures of Chlamydomonas in laboratory (Hes-
sen et al. 1995) and field experiments (Van Donk &
Hessen 1996; Van Donk et al. in press), where a
strong relation was found between loss or withdrawal
of flagella and UV exposure.
Most phytoplankton species from Lake Solvatn
gave no significant response to UV radiation, although
these species (also incubated in Lake Brandallaguna)
probably received higher UV doses during the ex-
periment compared with their in situ conditions. The
high algal biomass and the higher concentration of
UV-absorbing organic carbon compounds in the incu-
bation bottles were probably sufficient to protect these
algae against short wavelength UV radiation. Only the
picocyanobacteria from this locality were inhibited.
Also phytoplankton from the two other lakes, prob-
ably experienced higher UV doses during incubation
than in situ, due to the lack of mixing to deeper water
layers.
Studies performed in enclosures in high-altitude
mountain lakes reported no effect of short wavelength
UV radiation on phytoplankton growth and species
composition (Halac et al. 1997; Vinebrooke & Leav-
itt 1999). The latter study reported only differential
sensitivity to UV radiation among algal species of
the littoral community. Even the picobacterial phy-
toplankton was unaffected by UV radiation. The UV
irradiance in our experiments was not higher than in
those lakes. There are, however, very few days with
bright sun at Spitsbergen. When one of these days oc-
cur, with continuous 24 h light exposure, we would
expect this to be a major stress. Also temperature
could be an important factor, and the prevailing low
temperature in our lakes (7-8 °C) could add to the
stress by preventing repair.
Cell size and morphology may determine the sus-
ceptibility of phytoplankton to damage by UV radia-
tion. Karentz et al. ( 1991) reported that the amount
of DNA damage in the antartic phytoplankton corre-
lated with the morphometric characteristics (ratio of
cell surface to cell volume) of individual species. In
smaller cells, the distance between the cell surface and
the nucleus (DNA) is shortened. Furthermore, Garcia-
Piche! ( 1994) stated that the absorbance of short-
257
wavelength radiation by intracellular 'sunscreens' will
be a function of the cell size. Thus, with equal in-
vestments in such photoprotective properties, a small
alga will be exposed more than a large one. Marchant
et al. ( 1991) found that colonial cells of the Antarc-
tic prymnesiophyte Phaeocystis pouchetti contained
significant amounts of mycosporine, a UV-absorbing
compound, whereas free-swimming single cells of the
same organism did not. U. americana in our experi-
ments may be protected against UV by a mucilaginous
substance around the colonies.
The observed decrease in growth of small-sized
species and increase in growth oflarge colony-forming
species due to short wavelength UV radiation may
affect the entire foodweb by cascading etlects. In
aquatic, pelagic ecosystems, numerous studies have
documented the grazing pressure by herbivorous zoo-
plankton on phytoplankton, and it is well known that
zooplankton feed with highly different efficiency on
various phytoplankton species. This is primarily re-
lated to parameters like size and shape of the cells,
as well as structure of the cell wall (e.g .. Burns 1968;
Lampert 1987; Sterner 1989; De Bernardi & Guissani
1990).
The increase in inedible species when short wave-
length UV radiation was not excluded, as found for
the phytoplankton community from Lake Brandalla-
guna and Lake Storevatn (Figure 6), may have impact
on the entire food web and adversely affect secondary
production at all levels.
Especially in the marine environment the response
of phytoplankton communities to short wavelength
UV radiation have been examined in several studies
(e.g., Santas et al. 1998a,b ). However. only a few stud-
ies have examined the effect of this radiation on the
whole aquatic community (e.g., Bothwell et al. 1994,
Cabrera et al. 1997; De Lange et al. 1999; Mostajir
et al. 1999 ). These studies show that indirect effects
may play an important role in ecosystem response to
UV. In enclosure experiments in a high altitude An-
dean lake (Cabrera et al. 1997) the diatoms Fragilaria
construens and F. crotonensis were more abundant in
the UV-B-excluded treatments, but the chlorophyte A.
judayi became more abundant in the UV-B treatment,
in contrast to our observations in Lake Branda! laguna.
The higher biomass ofA.judayi was explained by a di-
rect inhibition of grazing by zooplankton in the UV-B
treatment. Previous laboratory experiments indicated
that the grazing activity of melanic Daphnia midden-
dorffiana (the dominant zooplankter in our lakes) was
not directly influenced by UV radiation (Van Donk
258
et al. in press), but the digestion rates ofUV-irradiated
Chlamydomonas were significantly lower than those
of non-irradiated algae. The UV-exposed algae formed
a thicker cell wall and passed largely intact through
the Daphnia gut and were thus protected from heavy
grazing pressures (Van Donk & Hessen 1995).
In conclusion, the transfer of energy in olig-
otrophic arctic lakes from phytoplankton to zooplank-
ton may be negatively influenced by UV radiation.
This is not only caused by direct changes in phyto-
plankton cells (e.g., growth inhibition, cell wall thick-
ening and lipid composition), but also by a shift in
phytoplankton composition from small, edible forms
to larger inedible ones.
Acknowledgements
This study was supported by a grant of the Norwegian
Foundation for Science and Letters (project grant no.
112867/720). We would like to thank Pill Brettum and
Mark van Dijk for identification and counting of the
phytoplankton, Dr R. D Gulati for his usefull com-
ments on the manunuscript, the AWl (Alfred Wegener
Institute) for radiation measurements, and further our
colleagues in the project for continuous support and
cooperation.
References
Behrenfeld, M. J., Hardy, J. T. & Lee, H. 1992. Chronic ef-
fects of ultraviolet-B radiation on growth and cell volume of
Phaeodactylum tricomutum (Bacillariophyceae). J. Phycol. 28:
757-760.
Bothwell, M. L. Roberge, A. C. & Pollock, C. M. 1994. Ecosys-
tem response to solar ultraviolet B-radiation: influence of trophic
level interactions. Science 265: 97-100.
Burna, A. G. J., Van Hannen, E. J., Roza, L., Veldhuis, M. J. W.
& Gieskes, W. W. C. 1995a. Monitoring ultraviolet-B-induced
DNA damage in individual diatom cells by immunofluorescent
thymine dimer detection. J. Phycol. 31: 314--321.
Burna, A. G. J., Zemmelink, H. J., Sjollema, K. A. & Gieskes,
W. W. C. 1995b. Effect of UV-B on cell characteristics of the
marine diatom Cyclotel/a sp. Pp. 305-311. In: Bauer, H. &
Nolan, C. (eds), The Effects of Environmental UV-B Radiation
on Health and Ecosystems. European Commision EUR 15607.
Burns, C. W. 1968. The relatioship between body size of filter-
feeding Cladocera and the maximum size of particles ingested.
Limnol. Oceanog. 13: 675-678.
Cabrera, S., Lopez, M. & Tartarotti, B. 1997. Phytoplankton and
zooplankton response to ultraviolet radiation in a high-altitude
Andean lake: short- versus long- term effects. J. Plankton Res.
19: 1565-1582.
Crutzen, P. 1992. Ultraviolet on the increase. Nature 356: 104--105.
Cullen, J. J., Neale P. J. & Lesser, M.P. 1992. Biological weighting
function for the inhibition of phytoplankton photosynthesis by
ultraviolet radiation. Science 258: 646-650.
De Bernardi, R. & Guissani, G. 1990. Are blue-green algae suitable
food for zooplankton? An overview. Hydrobiologia 200/201:
29-41.
De Lange, H. J. & Van Dank, E. 1997. Effects of UVB-irradiated
algae on life history traits of Daphnia pulex. Freshw. Bioi. 38:
7ll-720.
De Lange, H. J., Verschoor, A. M., Gylstra, R., Cuppen, J. G. M.
& Van Dank, E. 1999. The effects of artificial UVB radiation on
experimental aquatic microcosms. Freshw. Bioi. 42: 545-560.
Gala, W. R. & Giesy, J. P. 1991. Effects of ultraviolet radiation on
the primary production of natural phytoplankton assemblages in
lake Michigan. Ecotoxicol. Environ. Safety 22: 345-361.
Goes, J. I., Handa, N., Taguchi, S., Hama, T. 1994. Effect ofUV-B
radiation on the fatty acid composition of the marine phytoplank-
ter Tetraselmis sp.: relationship to cellular pigments. Marine
Ecol. Progress Ser. 114: 259-274.
Gracia-Piche!, F. 1994. A model for internal self-shading in plank-
tonic organisms and its implications for the usefulness of ultra-
violet sunscreens. Limnol. Oceanography 39: 1704--1717.
Hader, D. P. 1993. Risks of enhanced solar ultraviolet radiation for
aquatic ecosystems. Pp. 1-45. In: Round, F. E. & Chapman, D. J.
(eds), Progress in Phycological Research, Vol. 9. Biopress Ltd.,
New York.
Hader, D.P. & Hader, M. A. 1988. Inhibition of motility and photo-
taxis in the green flagellate, Euglena gracilis, by UV-B radiation.
Arch. Microbial. 150: 20--25.
Halac, S., Felip, M., Camarero, L., Sommaruga-Wtigrath, S.,
Psenner, R., Catalan, J. & Sammaruga, R. 1997. An in situ en-
closure experiments to test the solar UVB impact on plankton
in an high-altitude mountain lake. I. Lack of effect on phyto-
plankton species composition and growth. J. Plankton Res. 19:
1671-1686.
Hessen, D. 0. 1996. Competitive trade-off stategies in Arctic
Daphnia linked to melanism and UV-B stress. Polar Bioi. 16:
573-579.
Hessen, D. 0., Van Dank, E. & Andersen, T. 1995. Growth
responses, P-uptake and loss of flagella in Chlamydomonas
reinhardtii exposed to UV-B. J. Plankton Res. 17: 17-27.
Hessen, D. 0., De Lange, H. J. & Van Dank, E. 1997. UV-induced
changes in phytoplankton cells and its effects on grazers. Freshw.
Bioi. 38: 513-524.
Holm-Hansen, 0., Helbling, E. W. & Lublin, D. 1993. Ultravi-
olet radiation in Antartica: inhibition of primary production.
Photochem. Photobiol. 58: 567-570.
Karentz, D., Cleaver, J. E. & Mitchell, D. L. 1991. Cell survival
characteristics and molecular responses of antartic phytoplank-
ton to ultraviolet-B radiation. J. Phycol. 27: 326-341.
Karentz, D., Bothwell, M. L., Coffin, R. B., Hanson, A., Herndl,
G. J., Kilham, S. S., Lesser, M. P., Lindell, M., Moeller, R.,
Morris, D. P., Neale, P. J., Sanders, R. W., Weiler, C. S. &
Wetzel, R. C. 1994. Impact of UB-B radiation on pelagic fresh-
water ecosystems: Report of working group on bacteria and phy-
toplankton. Arch. Hydrobiol. Beiheft ergebnisse der Limnologie
43: 31-69.
Kerr, J. B. & McElroy, C. T. 1993. Evidence for large upwardtrends
of ultravio1et-B radiation linked to ozon depletion. Science 262:
1032-1034.
Kirk, J. T. 0. 1994. Light and Photosynthesis in Aquatic Ecosys-
tems, 2nd edition. Cambridge University Press, Cambridge,
509 pp.

Lampert, W. 1987. Feeding and nutrition in Daphnia. Pp. 143-192.
In: Peters, R. H. & De Bernardi, R. (cds). 'Dapnict' memorie dell
Instituto Italiano di Idrobiologia.
Marchant, H. J., Davidson, A. T. & Kelly. G. J. 1991. UV-B protect-
ing compounds in the marine alga Phaeocystis pouchetti from
Antartica. Marine Bioi. 109: 391-395.
Moeller, R. E. 1994. Contribution of ultraviolet radiation (!!VA.
UVB) to photoinhibition of epilimnetic phytoplankton in lakes of
different UV transparency. Arch. Hydrobiol. Beiheft ergebnisse
der Limnologie 43: I 57-170.
Mostajir. B .. Demers, S .. De Mora. S .. Belzile, C .. Chanut. J.-
P., Gosselin, M., Roy, S .. Zulema Villegas. P., Fauchot. J. &
Bouchard, J. 1999. Experimental test of the effect of ultraviolet-
B radiation in a planktonic community. Limnol. Oceanography
44: 586-596.
Quesada. A .• Mouget. J. L., & Vincent. W. F. 1995. Growth of
antarctic cyanobacteria under ultraviolet radiation: UVA coun-
teracts UVB inhibition. J. Phycol. 31:242-248.
Rott, E. 1981. Some results from phytoplankton counting intcrcali-
brations. Schw. Z. Hydro!. 43: 34-62.
Rozema, J.. Gieskes. W. W. C .. Van de Geijn, S. C.. Nolan, C.
& De Boois, H. 1997. UV-B and Biosphere. Kluwer Academic
Publishers, Dordrecht.
Santas, R., Korda. A .. Lianou, C. & Santas. P. 199Ha. Community
responses to UV radiation. l. Enhanced UVB effects on bio-
mass and community structure of filamentous algal assemblages
growing in a coral reef mesocosm. Marine Bioi. 1.'\ I: I 53-162.
Santas. R .. Santas. P., Lianou. C. & Korda. A. 1998b. Community
responses to UV radiation. II. Effects of solar UVB on field-
grown diatom assemblages of the Carribean. Marine Bioi. 131:
163-171.
Siebeck. 0., Vail, T. L.. Williamson, C. E., Yetter, R .. Hessen. D. 0.,
Zagarese, H. E .. Little. E .. Balseiro, E .. Modenutti, B .. Seva. J. &
Shumate, A. 1994. Impact of UV-B radiation on zooplankton and
li'h in pelagic freshwater ecosystems. Arch. Hydrobiol. Beiheft
ergebnisse der Limnologie 43: 101-114.
Smith, R. C., Prb.elin, B. B .. Baker. K. S .. Bidigare. R. R., Boucher,
N. P., Coley, T.. Karentz. D., Macintyre. S .. Matlick, H. A.,
259
Menzies. D .. Ondrusek, M .. Wan, Z. & Water, K. J. 1992. Ozon
depletion: Ultraviolet radiation and phytoplankton biology in
Antarctic waters. Science 255: 952-959.
Sokal. R. R. & Rohlf. F. J. 1995. Biometry: the Principles and
Practice of Statistics in Biological Research. 3rd edition. W.C.
Freeman and Co .. New York.
Sterner. R. W. 1989. The role of grazers in phytoplankton succes-
sion. Pp. 107-170. In: Sommer. U. (ed.). Plankton Ecology:
Succession in Plankton Communities. Springer-Verlag, Berlin.
Strid, A .. Chow W. S. & Anderson J. M. 1994. UV-B damage and
protection at the molecular level in plants. Photosynt. Res. 39:
475-489.
Van Donk, E. & Hessen. D. 0. 1995: Reduced digestibility of
UV-B stressed and nutrient-limited algae by Daphnia magna.
Hydrobiologia 307: 147-151.
Van Donk. E. & Hcsscn, D. 0. 1996. Loss of nagella in the green
alga Ch/amrdomonas reinhardrii due to in situ UV-cxposure.
Sci entia Marina 60: I 07-112, 87-92.
Van Donk, E .. De Lange, H. J. & Faafeng. B. A. in press. Impact of
UV-radiation on grazing activity of Daphnia middendorfjiana.
and use of Chlamydomonas reinlwrdtii as UV-biodosimctcr in
an arctic freshwater lake (Spitsbergen. Norway). Yerhandlungen
Internationale Yereinigung Limnologic 27.
Vincent. W. F. & Roy. S. 1993. Solar ultraviolet-B radiation and
aquatic primary production: damage. protection. and recovery.
Environ. Rev. I: 1-12.
Vincbrooke, R. D. & Leavitt, P. R. 1999. Differential responses
of littoral communities to ultraviolet radiation in an alpine lake.
Ecology 80: 223-237.
Wang. K. S. & Chai, T. 1994. Reduction of omega-3 fatty acids by
\JY-B irradiation in microalgae . .!. Appl. Phycol. 6: 415-421.
Williamson, C. E. 1995. What role does UV-B radiation play in
freshwater ecosystems'' Limnol. Oceanography 40: 386-392.
Worrest. R. C. 1982. Review of literature concerning the im-
pact of UV-B radiation upon marine organisms. Pp. 309-346.
In: Calkins. J. (cd.), The Role of Solar Ultraviolet in Marine
Ecosystems. Plenum. New York.
 Section 5: Aquatic Plants and Aquatic Ecosystems

This study demonstrates the photoprotcclivc properties of aquatic humus. Both the green planktonic algae Selensas-
mon mpricornutuum (left) and the planktonic grazer Daphnia magna do benetit from U V-protection by humic matter.
(Photographs by Dag 0 . Hessen)
 Plant Ecology 154: 263-273, 200 I.
263
© 200 I Kilm·er Academic Publishers.

The photoprotective role of humus-DOC for Selenastrum and Daphnia

Dag 0. Hessen & Per J. Fxrpvig

University of Oslo, Department (~f Biology, P 0. Box 1027, Blindem, N-0316 Oslo, Norway
(E-mail: dag.hessen@bio.uio.no)

Key words: Daphnia, DOC, Humic substances, Phytoplankton, Selenastruum, UV-radiation, Zooplankton

Abstract
Cell numbers and fluorescence of the green algae Selenastrum capricomutuum and survival of Daphnia magna
exposed to simulated sun-light was assessed along a gradient of DOC (0, I, 5 10 and 50 mg C 1- 1). When exposed
to UV-doses and spectral distribution (295-750 nm) closely resembling surface solar radiation during mid summer,
Selenastrum showed major losses of cell fluorescence. In the absence of DOC, fluorescence was severely depressed,
with successively decreasing effects with increased DOC. Surviving cells also required an extensive recovery
period (10-12 d) for regrowth after exposure, while an almost immediate recovery was observed at concentrations
above 1 mg DOC 1- 1. For Daphnia, survival was reduced to less than I 0% after 4 h exposure, and almost zero after
8 h exposure in the absence of humus DOC, while no effects were observed in treatments with I 0 and 50 mg C 1- 1•
Selenastrum and Daphnia that were not directly irradiated, but exposed to UV-irradiated water with the same
concentrations of DOC did not reveal negative effects. This indicates negligible indirect effects mediated by long-
lived free radicals or other toxic compounds. Irradiation of Daphnia under increased oxygen concentration (200%
saturation) did not indicate acute effects, suggesting that effects of ambient radicals and oxidants would be of minor
importance relative to intracellular photoproducts.

Introduction less than 0.2 m- 1 in the clearest lakes to more than

A number of studies have clearly verified the detri-
mental role of short-wave solar radiation for fresh-
water plankton (Karentz et al. 1994; Siebeck et al.
1994; Williamson et al. 1994; Williamson 1995; Hes-
sen et al. 1997). The potential roles of stratospheric
ozone depletion and thus increased UV radiation have
caused considerable concern. For freshwater systems,
changes in the concentrations of dissolved organic car-
bon (DOC) may, however, have greater impact than
ozone depletion on the UV climate (Morris et al. 1995;
Williamson et al. 1994 ). Increased temperature and
reduced precipitation have caused major reductions of
DOC in North-American lakes, with a strong increase
in UV penetration (Schindler et al. 1996; Curtis &
Schindler 1997).
The profound effect of DOC, primarily humic sub-
stances (HS), on UV-penetration in lakes is well doc-
umented. The attenuation coefficient (Kd) is mainly a
function of DOC. and at 310 nm, Kd may vary from
I 00 m- 1 for typical bog lakes (Scully & Lean 1994;
Morris eta!. 1995). Expressed as the depth where l 0%
of subsurface UV-B remains (z[310 nm]), this covers
the range from less than I 0 em to more than I 0 m
(Kirk 1994). Many lakes worldwide have extremely
low levels of DOC, and thus a z(l 0%) of more than
5 m is not uncommon. Several recent papers have ex-
pressed the attenuation of UV-B as related to DOC
in more general terms, and pointed to the predomi-
nant role of humus DOC over particulate organic C
for UV attenuation in most oligo- and mesotrophic
lakes. Based on measurements in a number of lakes
and enclosures with different levels of DOC, Scully
& Lean (1994) found an UV-B integrated attenuation
coefficient largely related to DOC. In support of this,
Morris et al. (1995) found that among-lake variation in
Kd was mainly explained (87-96%) by differences in
DOC concentrations. Although both models describe
the relationship between DOC and Kd by power mod-
els, the data set of Morris et al. ( 1995) (65 lakes) also
264
gave a close fit by a linear model. Given the wide vari-
ability of HS qualities and photochemical properties,
the close agreement between Kd estimated from both
these surveys is quite remarkable.
Humic matter plays a dual role with regard to the
light effects. The strong absorption of photosynthet-
ically active radiation (PAR) implies a potential for
reduced photosynthesis. On the other hand, aquatic
humus effectively blocks harmful UV-A and UV-B.
Also, the suite of low molecular weight compounds
that originate from degradation of aquatic humus by
sunlight has attracted considerable interest, owing to
the potential effects on aquatic secondary production
(Lindell et a! 1995; Moran & Zepp 1997; Wetzel et a!.
1995; Herndl eta!. 1997). While a number of studies
reports enhanced bacterial growth upon inoculation in
water treated with ultraviolet radiation, this is not a
unifying response (Amon & Benner 1996). The pro-
nounced absorption of photons in the very surface
generates a complex photochemistry involving a set of
highly reactive, strong oxidants that interact strongly
with the chemistry of both detritus and living cells
(Cooperet al. 1989, 1994; Miller & Moran 1997; Lean
1998; Miller 1998). For photochemical breakdown,
the key issue is the quantum yield, which is the ra-
tio between number of photoproduced events and the
number of photons absorbed (Miller 1998). For most
humic compounds, or derivatives of these, quantum
yield peaks in the UV-B (when natural sunlight is ap-
plied) with a decreasing tail into the UV-A or PAR
region. Most of these photoproducts arc short-lived
strong radicals and reactive oxygen species as well as
more long-lived oxidants like H202, or carbon monox-
ide (Miller 1998). These photoproducts may induce a
set of negative effects on biota, primarily oxidation of
membrane fatty acids (lipid peroxidation), oxidation
of proteins and DNA damage (Fuchs & Packer 1991;
Cooper eta!. 1994 ).
Gjessing & Ki.illqvist ( 1991) found strong negative
effects on phytoplankton incubated in UV-irradiated
water with high concentrations of humic matter. Res-
sen & van Donk (1994) found an enhanced growth
rate in phytoplankton incubated in UV-irradiated hu-
mic water at low doses, while a strong negative effect
was detected when the water received higher doses.
They suggested a trade-off between initial effects that
could be caused by liberation of essential minerals or
elements upon photo-oxidation, and negative effects
caused by accumulation of long-lived oxidants or car-
bon monoxide at higher doses. High doses could also
liberate toxic metals or substances that were adsorbed
to humus aggregates. No such indirect effects were
found in corresponding tests on Daphnia magna. A
problem with these studies, however, is the lack of
realistic experimental UV-doses and spectral proper-
ties of the light sources. Also high levels of oxygen
could affect UV-induced oxidative damage, since the
rate of radical production in animals is proportional
to the rate of oxygen consumption (Barthalcmy et al.
1981; Abele ct a!. 1998).
There is a limited set of empirical data on the in situ
effects of DOC on phyto- and zooplankton. Moeller
(1994) found a marked effect of DOC on photoinhi-
bition of phytoplankton in lakes with different trans-
parency, and also noted a surprising lack of photoad-
aptation. Correspondingly, Williamson et a!. (1994)
and Zagarese et a!. ( 1994) found stronger effects of
UV-radiation on lake zooplankton in a clearwater lake
relative to lakes with higher UV-attenuation. The net
effect of UV radiation on primary producers will also
depend on the relative effect on the grazer relative to
the algae. If dominant grazers are relatively more sus-
ceptible to UV than the algae, the net effect of UV
on heavily grazed algae may in fact be positive (cf.,
Bothwell eta!. 1994 ).
Tn this study, we tested experimentally the modi-
fying effects of humic matter on direct UV-irradiation
of a green alga (Selenastrum capricomutuum) and the
cladoceran Daphnia magna along a gradient of DOC.
We also tested the indirect and potentially harmful
effects of UV-radiated DOC per se, by exposing al-
gae and zooplankton to UV-irradiated water along the
same concentration gradient of DOC. For the Daph-
nia exposures, we also manipulated the ambient 02
concentration, to see if elevated 02 induced more bi-
ologically harmful photoproducts. Care was taken to
mimic both the solar spectrum, doses and dose-rates
to yield realistic estimates.
Material and methods
The green alga Selenastrum capricornutuum (clone
NIVA CHL 10), was grown in batch culture in
a WC medium (Guillard & Lorentzen 1972) at
70 t-tmol m- 2 s- 1 PAR. A laboratory clone of Daph-
nia magna was grown in Elendt M7 medium (OECD
1996) with S. capricornutuum as the sole food source.
Care was taken to keep all animals in good shape
by providing continuous food concentrations above
500 {Lg C 1- 1 • All the experiments were conducted at
19.0 oc in a thermostated room.

1.4
-E
":'
1.2
c E
1.0
"!
E ~
E 0.8
~ -~
c 0.:
:e" 0.6
-~
0.4 - solar radiation
E - lamp with filter
t:~
0.2
Q.
"'
0
300 400 500 600
Wavelength, nm
Figure /. Spectral irradiance under the xenon-lamp used for the
experiments. A 93 11m cellulose acetate filter (Tamboer Inc.) was
applied
Light was provided by a I 00 W xenon lamp
(AMKO Mod. 02-Al020). Compared with mid-
summer outdoor radiation, a higher UV irradiance
was emitted. By use of a 93 11-m cellulose acetate fil-
ter (Tamboer Inc.) and by adjusting the distance to
80 em, the irradiance and spectral quality of the ar-
tificial light corresponded well to solar mid-summer
radiation (Figure 1). Irradiance in the 300-315 nm
band as revealed by a LICOR 1800 spectroradiometer,
was 1.096 and 1.092 W m- 2 for filtered light and sun,
respectively. For the 300-400 nm range, correspond-
ing values were 35.95 and 39.82 W m- 2 while for PAR
(400-700 nm) irradiance was somewhat lower for the
lamp (282 vs. 350 W m- 2 ). The cellulose acetate filter
remained optically stable for several days of exposure,
but was nevertheless replaced every second day during
the exposure experiments.
A gradient of DOC (0, 1, 5, 10 and 50 mg C l- 1)
was made by adding freeze-dried humus, isolated from
a humic lake. Water from Lake Skjervatjern, western
Norway, was filtered by a reverse osmosis procedure
following the method of Gjessing et a!. ( 1998). The
lake water was pumped through a pre-filter and feed
water was passed through an inline cation exchanger
for exchange of polyvalent and mono-valent cations
with Na+ The feed water was then passed through
a high-pressure pump to the reverse osmosis mem-
branes that separated the permeate from the retentate.
After repeated filtration, a total of concentrated hu-
mus in 5 I was freeze-dried to yield about 75 g solid
sample. The elemental composition of this highly
water soluble solid matter was 37.8% ash, 50.2% car-
265
mo
c< 500
::{ UV-B
400
c
300
l
"
.Si
"5
200
~
~
100
()
700
0 lO 20 30 40 50
DOC,mgl' 1
Figure 2. Attenuation coeJ'ficients !"or integrated UV-B and UV-A
respectively. as related to the gradient of DOC. Calculated using
equations I and 6 from Scully & Lean ( 1994).
bon, 4.8% hydrogen, 1.0% nitrogen and 1.6 % sulfur.
Spectrophotometric absorbance in the UV-B region
(31 0 nm) was linearly related to DOC concentrations,
and integrated UV-B attenuation (Kc~nlo) calculated
as Kt~ 1310 = 0.415 DOC 186 (Scully & Lean 1994) is
given in Figure 2.
Selenas/rum was added to four 150 ml quartz-
bottles along the gradient of DOC concentrations in
Guillard WC-medium. For exposure, two exposed bot-
tles were placed behind a quartz window inside a
modified water bath to keep the temperature constant
also during exposure (the lamp emitted some heat),
while two bottles were shielded from UV exposure.
During the 6 h exposure period the cells were homo-
geneously distributed by means of a magnetic stirrer.
For assays on indirect effects, nonirradiated cells were
inoculated in corresponding irradiated water qualities
(0-50 mg DOC l- 1 ) that had been exposed for 6 h. A
pre-assay was first run to test for the effect of exposure
time at zero DOC, where cell numbers were counted
for four days after UV-exposure periods of I 0 s, I min,
I hand 6 h.
For both the directly exposed algae and those ex-
posed to UV irradiated water, samples were collected
in 1.5 ml polypropylene tubes immediately before and
after exposure, and then daily for the next 3-4 weeks.
The samples were fixed with I% glutaraldehyde (final
concentration) for fluorescence determinations, and
cell counts by flow cytometry. These analyses were
performed on an Argus I 00 flow cytometer (Ska-
tron A/S, Norway) equipped with a 103 W Osram
mercury arc lamp (Nelex elektronikk, Norway). The
266
4 UV-exposed Control
10 .-------------~-----r~----------~----~--------------------,
Day 0 Day-! Day 1
' .:··.
:::·.·.
···"'
.
-::l:::::· .

10 4 +-================~==================F=================~
Day3 DayS DayS
.... ;:!/:- .
"(~~;::
.. . .
.. .:::·
.:;; ..:::::;:;:;:··
·_:r~~~~~;!i~~~~~-:·:
............

li

Light scatter (LSl)

Figure 3. Example of development of combined scatter and fluorescence diagram for light-exposed cells of Selenastrum capricornutuum (6 h)
at I mg DOC I- 1 over an 8-d period. For comparison, unexposed controls are displayed in the left panel. Grouped populations of signals are
visualized by contour plots. Numbers in the right panel denotes monodisperse fluorescent spheres for calibration (I). intact cells with 'normal'
fluorescence (2), and intact cells with strongly reduced fluorescence (3 ). Under and after exposure. there is a transition of cells from state 2 to
3, which then gradually recovers from day 4 onwards.

instrument was modified with gas pressure controlled
sheath flow and syringe-based sample delivery sys-
tem, which minimized contamination. For instrumen-
tal alignment/calibration, a range of monodisperse,
low CV particles was applied (FLCV=l.27%, no.
23517, Polysciences). Samples with high cell densities
were diluted immediately before analysis. Fcs Assis-
tant 1.3.4 converted list mode files before data analysis
in Fcap-list v.l.30 (Soft flow Hungary, Ltd.). The pos-
sibility to trigger for both scatter and fluorescensce by
separate channels allowed for a separation of cell num-
bers (scatter) and activity assessed as fluorescensce. A
lower threshold for cell specific level of fluorescence
was set in order to separate intact algal cells from
inactive (non-fluorescent) or broken cells and culture
debris. An example of a typical scattergram with com-
bined fluorescence output is given in Fig. 3 as a
selected time series for the exposure at I mg DOC 1- 1•
The unexposed control is given for comparison. Two
populations of cells can be identified, one with in-
tact cells and high fluorescence signal, another with
severely reduced fluorescence. Over several days after
 267
10(;-r-----.~~-----------
exposure, the UV-exposed cells gradually recovered to - - 1 0 second exp.
pre-exposure fluorescence. ---o---1 minute exp.
__...,_ control
For the Daphnia exposure experiments, 10
- -+-- 1 hour exp.
medium-sized (1.0-1.5 mm) individuals were placed w' -- .. -- 6 hours exp.
in four 150 ml quartz bottles equipped with silicone
plugs perforated by two syringe needles (in- and out-
let). A constant flow rate of fresh Elendt M7 medium

-·----- ----------------- -·
of I 0 ml min- 1 was provided to the exposed bottles 10" ,.... ~
with a peristaltic pump. These bottles were connected _.,._.,...,.~------
to an unexposed (shielded) pair of bottles by a 4 em
glass funnel attached to the needles. This provided a
continuous flow of exposed water to the unexposed
to":;-----+----'*====='====.d
bottles. For each assay, a fifth bottle was left unex- 0 2 3 4
posed as control. Parallel experiments with 4 or 8 h Time, days
exposure were performed. After irradiation, the ani- Fi!!,ure 4. Test of response in Se/enastrum capricornutuum exposed
mal were left in the bottles, with dim, blue-white light to various periods of UV irradiation without DOC added. The graph
( <20 pmol m- 2 s- 1). The mortality of the Daph- shows cell numbers as assessed by fiow cytometer scatter signals.
nia was recorded immediately and 24 h after each
exposure period.
ually weaker response with increasing concentrations
It should be noted that the indirect assays of phy-
of DOC. The effect was most pronounced for cell flu-
toplankton differed from those of the zooplankton,
orescence as a direct measure on photobleaching. For
since for the Daphnia assays there was a constant
the first 24 h during and after exposure, the fraction
flow from the irradiated bottles to the shielded bottles
of cells with intact fluorescence decreased to less than
with Daphnia. This procedure could not be applied for
I% of the controls in the absence of DOC and re-
Selenastrum, since the algae would pass through the
mained at this low level for some 5 days, until a rapid
needles, and could thus not be retained in the flow-
recovery occurred (Figure 5). The scatter signal indi-
through chambers. Selenastrum was thus inoculated
cated neither cell breakdown nor growth for 20 days
as a batch culture in previously irradiated water (6 h
after exposure, but rather arrested cell development.
pre-inoculation radiation).
At I, 5 and 10 mg DOC 1- 1 • there was also a pro-
To test for a possible effect of different 0 2 -
nounced and long lasting photobleaching, while both
conccntrations on UV-induccd mortality on Daphnia
the scatter and fluorescence recovery was gradually
along the same gradient of DOC. the 4-h exposure ex-
faster with increased DOC. At 50 mg C 1- 1• only mi-
periments were repeated with high oxygen saturation
nor e!Tccts were observed. There was no observable
(200% ). For this purpose a gas equilibration tank was
effect, neither on fluorescence nor on scatter in the in-
connected to the closed system. The equilibrium tank
direct treatments, i.e., cells inoculated in pre-irradiated
was filled with M7 medium containing the respective
water. These samples closely resembled those of the
DOC concentration. The oxygen concentration was
control (Figure 6).
kept constant by continuously bubbling a purified gas
For Daphnia, no mortality was observed immedi-
mixture of 40:60% 02:N2. 02 concentrations were
ately after 4 h inadiation, while after 8 h irradiation
checked hy Winkler titration.
there was < I 0% survival with no DOC additions
80% survival at 1 mg DOC 1- 1, while no mortality wa~
observed at higher DOC concentrations (Figure 7, up-
Results
per panel). Twenty-four h after terminated exposure,
animals in both the 4 and 8 h exposure treatments
For Selenastrum, brief UV-exposure periods (I 0 s and
sutTered almost complete mortality in the absence of
l min) apparently stimulated growth, while there was
DOC. At I mg DOC death was almost complete for
a transition towards decreased growth rate after I h
animals exposed for 8 h, while for those exposed for
irradiation. After 6 h of exposure no increase in cell
4 h, a near 60% survival was recorded (Figure 7, mid
numbers was recorded for 4 days (Figure 4 ).
panel). At 5 mg DOC. there was no mortality for an-
For the 6-h exposure experiments, there was a
imals exposed for 4 h, while survival decreased to
marked response in the absence of DOC and a grad-
268

10 8
Control 5 mgcl- 1

10 6

10 5

10 4

10 3 --LSI
--1i-- LS 1/FL2
10 2

10 I

Omgct· 1 10 mg C 1 -I
10 7.

';< 105
6
"' 104
~
u 103

w-0
10 I

I mgCI- 1
107

106

105

104

10 3
0
10-

10 I
0 10 20 0 10 20 30
Time, days
Fif?ure 5. Time series of population response of Selenastrum capricornutuwn to 6 h light exposure assessed by flow cytometer readings along
a gradient of DOC. Filled dots represent the forward scatter signal only (total cells number) and open dots represents the active cells with intact
tluorescence.

nearly 50% for those exposed for 8 h. At I 0 and 50 mg
DOC 1- 1 all animals survived even 24 h after expo-
sure. In the indirect exposure experiments (Figure 7,
lower panel), as well as in the controls, survival was
complete after both 6 and 24 h post-irradiation. This
suggests a possible minor effect of photoproducts in
the medium, although the transfer of irradiated water
to the non-exposed bottles with animals occurred at a
rate that would eliminate most of the short lived free
radicals.
The survival test with 200% oxygen saturation
yielded somewhat higher mortality than exposure un-
der ambient oxygen pressure (~ I 00% saturation)
(Figure 8). After 4 h exposure, survival was reduced
 269

10 K

10 7

10 6

10 5

10 4
--LSI
';' 10 3 ---1!!r- LS 1/FL2

s,$
10 2
Q)
u
10 7

10 6

lOS

10 4

103

10 2
0 5 10 15 0 5 10 15 20
Time, days
Figure 6. Time series of population response of Selenastrwn capricornutuum inoculated in pre-irradiated water (6 h light exposure) assessed
by flow cytometer readings along a gradient of DOC. Filled dots represent the forward scatter signal only (total cells number) and open dots
represents the active cells with intact fluorescence.

to nearly 30% relative to the I 00% at standard Oz
(Figure 8, left panel vs. Figure 7, upper panel), while
there were no survivors 24 h after exposure in either
of the two oxygen concentrations in the absence of
DOC. At 1 mg DOC, survival was almost complete
immediately after irradiation, while a lower proportion
of survivors was found after 24 h under high oxy-
gen (60% vs. 18% survival for standard and high Oz,
respectively). No effects of direct radiation exposure
were found at higher concentrations of DOC. For the
indirect treatments, i.e., unexposed Daphnia contin-
uously fed water from exposed bottles, all animals
survived also in the indirect exposure chambers under
high Oz concentration.
Discussion
The artificial light applied in these experiments did
not entirely reproduce the solar spectrum, but both
the spectral properties and total irradiance resem-
bled outdoor radiation. While brief exposure periods
of I 0 s and I min apparently yielded positive re-
sponses, there was a trend towards lower cell yields
at I h exposure, while a 6-h exposure caused arrested
growth for a period of almost I 0 days in the ab-
sence of light -protecting DOC. The stimulatory effects
should be judged with some caution due to the lack of
replicates, but nevertheless correspond to previously
reported growth enhancement under other kinds of
moderate stress (Hessen & Van Donk 1994 ). The ar-
rested growth after 6 h irradiation was confirmed by
the irradiation experiments along the DOC gradient.
These experiments unambiguously demonstrated the
photoprotcctivc role of humus DOC, both in terms of
cell fluorescence and cell numbers. Nearly 99% reduc-
tion in cell fluorescence for Se/enastrum immediately
after irradiation was recorded in all DOC concentra-
tions except for 50 mg, where a 70% reduction was
270

Direct ex posure, 0 hour o control Direct expOsur~ D ontrol

100 14h exp. 100

Dlmmcdintcly
anerexp.

80
18h exp. so
~ 24 hou"
aflercxp.

60

40
20
20 ;;
L
Indirec t exposure
u "
; 100

100 ~
~ ~0

80
~
.E
a 60

..
c
l:: 40 20
~
20
0 t 5 10 50
DO mg 1' 1
0
Figure 8 . Survival of Daphnia magna directly exposed to 4 h irradi-
100 ation (lett panel) or fe d irradiated water from exposed bottles (right
panel). Both experiments conducted less than 200% oxygen satura-
80 tion. Shaded bars represent recordings after 4 h exposure, black bars
is 24 h after terminated exposure.
60

40 (1995) examined the effects of UV-B and UV-A on

20
0 effects on PSII, whereas there was a rapid recovery af-
0 5 10 50
DOC, mgC I ' 1
Figure 7. Survival of Daphnia magna uireclly exposed to 4 h
(shaded bars) and 8 h irradiation (dark bars) along a gradient of
DOC. Upper panel represents survival immediately after terminated
exposure, mid panel represents 24 h after terminated exposure.
Lower pane l represe nts survival of Daphnia magna in unexposed
bottles continuously fed with irradiated water along a gradient of
DOC for 24 h.
found. At low DOC, there was a further decrease
in fluoresce nce during the days following irradiation,
suggesting delayed post-treatment damage.
Although DOC most efficiently attenuated the
shorter wavelengths, our experiments could not sep-
arate the effect of various parts of the spectrum.
Although PAR may cause photoinhibition (Cullen
& Lewis 1988 ; Vassiliev et al. 1994), short wave
radiation is the major cause of inhibition of cell
fluorescence (Forster et al. 1997; Cullen & Neale
1997). Time for fluorescence recovery after exposure
is clearly also wavelength dependent. Schofield et al.
photosystem (PS) I and PS II in Antarctic ice algae,
and found a very slow recovery of UV-B mediated
ter exposure to UV-A or PAR. Herrmann et al. ( 1997)
correspondingly reported pronounced effects of UV-B
on photosyntetic activity in the marine phytoplankton,
but comparatively minor effects of UV-A and PAR.
Also Forster et al. ( 1997) found slower fluorescence
recovery with decreasing wavelengths. Clearly, there
is also a strong species specific response in PAM fluo-
rescence, where generally small or filamentous species
tend to be more susceptible to UV-B than larger,
well protected species (Xiong et al. 1996 ). Most re-
searchers report fluorescence recovery within a couple
of days however, in contrast to the more long lasting
effect in our experiments. We assume that this may
be mostly accredited to different duration of expo-
sure. Forster et al. ( 1997), using similar xenon lamps
as we did, applied exposure periods of 20 min only,
compared with 6 h for our experiments.
The effect of DOC will not only be an overall re-
duction in UV and PAR, but a major decrease in the
UV:PAR ratio, since UV radiation is attenuated far
more efficiently than PAR by dissolved humic mat-

tcr (Kirk 1994; Scully & Lean 1994 ). There is little
direct evidence for the photoprotective role of humus
DOC for phyto- and zooplankton, however. Arrigo &
Brown ( 1996) made general model predictions on the
photoprotective role of DOC in marine areas, and con-
cluded that the presence of moderate concentrations of
dissolved matter could enhance primary production in
the upper 30m. Moeller ( 1994) found a strong impact
of natural sunlight on both photosynthesis (82-95%
inhibition) and chlorophyll a (57-79°;(, photobleach-
ing) in natural phytoplankton communities exposed to
sunlight. He also reported a major contribution from
UV-A to inhibition of photosynthesis, whereas UV-B
more clearly caused photobleaching.
Moeller (1994) also noted an apparent acclimati-
zation to high UV levels in phytoplankton from the
clearest lake. This could be important for the inter-
pretation of the response to irradiation in the cultured
clone of Selenastrum which had been cultured for
years in the absence of UY. Nevertheless. selection
over 10 cycles with repeated UV exposure of the
regrowth of surviving cells from this clone did not
invoke any increased tolerance to UV (unpubl. data).
While the relative effects as related to the DOC gra-
dient were clearcut, a 10 d period before recovery of
fluorescence and increase in cell numbers is never-
theless surprising. This delay suggests a pronounced
potential for a long term metabolic shut-down before
regrowth, but under natural conditions most of these
cells would probably be lost by sedimentation.
While previous studies have demonstrated negative
effects on phytoplankton inoculated in pre-irradiated
water (Gjessing & Kallqvist 1991; Hessen & Van
Donk 1994), no such indirect effect could be detected
in our experiments. This discrepancy could be caused
by the use of high pressure mercury lamps providing
unrealistic spectra, dose-rates and doses in the earlier
experiments. Also the time from irradiation to inocula-
tion could be important. Most of the free radicals and
oxidants produced by UV radiation of aquatic DOC
are present for only nano-seconds to seconds (Cooper
et al. 1989; Zepp 1988; Miller 1998; Lean 1998), and
the type of batch culture assay in pre-irradiated water
would not reflect the effects of these short- lived pho-
toproducts. Hydrogen peroxide and carbon monoxide
as well as liberated metals could be more long lasting.
The direct irradiation assays would represent a trade-
off between the photo-protective properties of DOC
and the potential production of harmful photoprod-
ucts. While the effect of these photoproducts cannot be
judged from these experiments, the data nevertheless
271
clearly demonstrate the overall net positive properties
of humus DOC under UV radiation.
The zooplankton assays gave support to the trend
found for phytoplankton. Increasing levels of DOC of-
fered an efficient protection from direct light exposure.
In the indirect assays where unexposed Daphnia were
exposed to irradiated water, there was no observed
photo-toxic effects, although the continuous flow of
irradiated water to the unexposed zooplankton cham-
bers should allow for a potentially stronger effect than
the corresponding batch cultures with algae. The an-
imals appeared healthy, and no distorted swimming
patterns were recorded during indirect exposure, in
contrast to direct exposure where animals displayed
anomalous behavior in the low DOC bottles during
exposure. As for the algal assays, this does not exclude
any sublethal or long lasting indirect effects, but the
overall positive net effect of DOC under UV-exposurc
is clear. Apparently, high 02 pressure induced slightly
increased negative effects, although only at 0 and
I mg DOC 1- 1 . Whether this is due to increased stress
caused by high 02 in itself, or increased production of
oxidants and radicals is not clear, but the lack of effects
at higher DOC concentrations suggest that interactions
between radiation, DOC and high oxygen per se did
not induce negative effects. This is further supported
by the findings of Borgeraas & Hessen (2000), who
reported no effect of various oxygen concentrations on
Daphnia magna under UV-exposure, and concluded
that effect of ambient photoproducts (in the water)
would be of minor importance relative to intracellular
products.
Acknowledgements
We are indebted to Egil Gjessing and Mike Perdue
for providing the freeze-dried humus isolate used for
the DOC gradient in these experiments. The reviewers
provided constructive comments on the manuscript.
References
Ahele, D .. Grozzpietsch. H. & Portner. H. 0. 1998. Temporal Auctu-
ations and spatial gradients of environmental P02, temperature.
H202 and H2S in its intertidal habitat trigger enzymatic anti-
oxidantprotection in the capitellid worm Heteromastusfiliformis.
Mar. Ecol. Pro gr. Ser. 163: I 79-191.
Amon, R. M. W. & Benner, R. 1996. Photochemical consumption
of dissolved organic carbon and dissolved oxygen in the Amazon
River system. Geochim. Cosmochim. Acta 60: 1783-1792.
272
Arrigo, K. R. & Brown, C. W. 1996. Impact of chromophoric dis-
solved organic matter on UV inhibition of primary productivity
in the sea. Mar. Ecol. Prog. Ser. 140: 207-216.
Barthelemy, L., Belaud, A. & Chaste!, C. 1981. A comparative study
of oxygen toxicity in vertebrates. Resp. Physiol. 44: 261-268.
Borgeraas, J. & Hessen, D. 0. 2000. UV-B induced mortality and
anti-oxidant enzyme activities in Daphnia magna at different
oxygen concentrations and temperatures. J. Plankton Res. 22:
1167-1183.
Bothwell, M. L., Roberge, A. C. & Pollock, C. M. 1994. Ecosystem
response to solar ultraviolet B-radiation: Influence of trophic-
level interactions. Science 265: 97-100.
Cooper, W. J., Zika, R. G., Petasne, R. G. & Fisher, A. M. 1989.
Sunlight-induced photochemistry of humic substances in natural
waters: Major reactive species. Adv. Chern. Ser. 219.
Cooper, W. J., Shao, C., Lean, D. R. S., Gordon, A. S. & Scully,
F. E. 1994. Factors affecting the distribution of HzOz in sur-
face waters. Pp. 391-422. In: Baker, L. A. (ed. ), Environmental
Chemistry of Lakes and Reservoirs.
Cullen, J. J. & Lewis, M. R. 1988. The kinetics of algal photoad-
aption in the context of vertical mixing. J. Plankton Res.IO:
1039-1063.
Cullen, J. J. & Neale, P. J. 1997. Effect of UV on short-term pho-
tosynthesis of natural phytoplankton Photochem. Photobiol. 65:
264-266.
Curtis, P. J. & Schindler, D. W. 1997. Hydrologic control of dis-
solved organic matter in low-order Precambrian Shield lakes.
Biogeochemistry 36: 125-138.
Forster, R. M., Haker, A. & Schubert, H. 1997. Wavelength de-
pendence of photoinhibition in the green alga Chiarella vulgaris.
Photosynthetica 33:541-552.
Fuchs, J. & Packer, L. 1991, Photooxidative stress in the skin.
Pp. 559-583. In: Sies, H. (ed.), Oxidative Stress: Oxidants and
Antioxidants. Academic Press, London.
Gjessing, E. T. & Kallquist, T. 1991. Algicidal and chemical effects
of UV-radiation of water containing humic substances. Wat. Res.
25: 491-494.
Gjessing, E. T., Alberts, J. J., Bruchet, A., Egeberg, P. K., Lydersen,
E., McGown, L. B., Mobed, J. J., Munster, U., Pempkowiak,
J., Perdue, M., Ratnawerra, H. Rybacki, D. Takacs, M. & Abbt-
Braun, G. 1998. Multi-method characterisation of natural organic
matter isolated from water: Characterisation of reverse osmosis
isolates from water oftwo semi-identical dystrophic lakes basins
in Norway. Water Res. 10: 3108-3124.
Guillard, R. R. L. & Lorentzen, C. J. 1972. Yellow-green algae with
chlorophyllide c. J. Physol. 8: 10-14.
Herrmann, H., Hader, D. P., & Ghetti, F. 1997. Inhibition of
photosynthesis by solar radiation in Dunaliella salina: relative
efficiencies of UV-B, UV-A and PAR. Plant Cell. Envir. 20:
359-365.
Hessen, D. 0. & Van Dank, E. 1994. Effects of UV-radiation of
humic water on primary and secondary production. Water, Air
Soil Poll. 75: 325-338.
Hessen, D. 0., De Lange, H. J. & Van Dank, El. 1997. UV-induced
changes in phytoplankton cells and its effect on grazers. Freshw.
Bioi. 38: 513-524.
Herndl, G. J., Brugger, A., Hager, S., Obernoster, I., Reiter, B.
& Slezak, D. 1997. Role of ultraviolet-B radiation on bacteria-
plankton and the availability of dissolved organic matter. Plant
Ecol. 128: 42-51.
Karentz, D., Bothwell, M. L., Coffin, R. B., Hanson, A., Herndl,
G. J., Kilham, S. S., Neale, P. J., Sanders, R. W., Weiler, C. S. &
Wetzel, R. G. 1994. Impact of UV-B radiation on pelagic fresh-
water ecosystems: report of the working group on bacteria and
phytoplankton. Arch. Hydrobiol. Beiheft. 43: 31-69.
Kirk, J. T. 0. 1994. Optics and UV-B radiation in natural wa-
ters.Arch. Hydrobiol. Beih. Ergeb. Limnol. 43: 1-16.
Lean, D. 1998. Attenuation of solar radiation in humic waters.
Pp. 109-124. In: Hessen, D. 0. & Tranvik, L. J. (eds), Aquaric
Humic Substances. Ecology and biogeochemistry. Springer-
Verlag, Berlin.
Lindell, M. J., Graneli, W. & Tranvik, L. J. 1995. Enhanced bacterial
growth in response to photochemical transformation of dissolved
organic matter. Limnol. Oceanogr. 40: 195-199.
Miller, W. L. 1998. Effects of UV radiation on aquatic humus: pho-
tochemical principles and experimental considerations. Pp. 125-
143. In: Hessen, D. 0. & Tranvik, L. J. (eds), Aquaric Hu-
mic Substances. Ecology and Biogeochemistry. Springer-Verlag,
Berlin.
Miller, W. L. & Moran, M. A. 1997. Interaction of photochem-
ical and microbial processes in the degradation of refractory
dissolved organic matter from a coastal marine environment.
Limnol. Oceanogr. 42: 1317-1324.
Moeller, R. E. 1994. Contribution of ultraviolet radiation (UV-A,
UV-B) to photoinhibition of epilimnetic phytoplankton in lakes
of differing UV transparency. Arch. Hydrobiol. Beiheft 43: 157-
170.
Moran, M.A. & Zepp, R. G. 1997. Role of photoreactions in the for-
mation of biologically labile compounds from dissolved organic
matter. Limnol. Oceanogr. 42: 1307-1316.
Morris, D. P., Zagarese, H., Williamson, C. E., Balseiro, E. G.,
Hargreaves, B. R., Modenutti, B., Moeller, R. & Queimalinos, C.
1995. The attenuation of solar UV radiation in lakes and the role
of dissolved organic carbon. Limnol. Oceanogr. 40: 1381-1391.
OECD 1996. OECD guidelines for testing of chemicals. Proposal
for updating guideline 202, part II. Daphnia magna reproduction
test.
Schindler, D. W., Curtis, P. J., Parker, B. R. & Stainton, M.P. 1996.
Consequences of climate warming and lake acidification for UV-
B penetration in North American boreal lakes. Nature 379: 705-
708.
Schofield, 0., Kroon B. M.A. & Pr'zelin, B. B. 1995. Impact of
ultraviolet-B radiation on photosystem II activity and its rela-
tionship to the inhibition of carbon fixation rates for antarctic ice
algae communities. J. Phycol. 31: 703-715.
Scully, N. M. & Lean, D. R. S. 1994. The attenuation of ultraviolet!
radiation in temperate lakes. Arch. Hydrobiol. Beih. 43: 134--
144.
Siebeck, 0. & Bohm, U. 1994. Challenges for an appraisal of
UV-B effect upon planktonic crustaceans under natural radiation
conditions with a non-magrating (Daphnia pu/ex obtusa) and a
migating cladoceran (Daphnia galeata). Arch. Hydrobiol. Beih.
Ergeb. Limnol. 43: 197-206.
Siebeck, 0., Vail, T., Williamson, C., Vetter, R., Hessen, D., Za-
garese, H., Little, E., Balseiro, E., Modenutti, B., Seva, J. &
Shumate, A. 1994. Impact of UV-B-radiation on zooplankton and
fish in pelagic, tfeshwater ecosystems. Arch. Hydrobiol. Beih.
Ergeb. Limnol. 43. 101-114.
Vassiliev, I. R., Prasil, 0., Wyman, K. D., Kolber, Z., Hanson
JR., A. K., Prentice, J. E. & Falkowski, P.G. 1994. Inhibition
of PS II photochemistry by PAR and UV radiation in natural
phytoplankton communities. Photosyn. Res. 42: 51-64.
Wetzel, R.G., Hatcher, P. G. & Bianchi, T. S. 1995. Natural
photolysis of recalcitrant dissolved organic matter to simple sub-
strates for rapid bacterial metabolism. Limnol. Oceanogr. 40:
1369-1380.

Williamson. C. E., Zagarese, H. E .. Schulze. P. C .. Hargreaves, B. R.
& Seva, J. 1994. The impact of short-term exposure to UV-B
radiationon zooplankton communities in north temperate lakes.
J. Plankton. Res. 16:205-218.
Williamson, C. E. 1995. What role docs UV-B radiation play in
fresh-water ecosystems. Lim no!. Oceanogr. 40: 386-392.
Xiong, F., Lederer, F.. Lukavsky. J. & Nedbal. L. 1996. Screening
of freshwater algae (Ch/orophvta. Chromophvlll) for ultraviolet-
B sensitivity of the photosynthetic apparatus. J. Plant Physiol.
148:42-48.
273
Zagarese. H. E .. Williamson, C. E., Mislevets, M. & Orr. P. 1994.
The vulnerability of Daphnia to UV-B radiation in Northeastern
United Slats. Arch. Hydrobiol. Beih. 43: 207-216.
Zepp. R. G., 1988. Enviromcntal photoprocesses involving natural
organic matter. Pp. 193-214. In: Frimmcl, F. H. & Christman,
R. F. (eds), Humic Substances and their Role in the Environment.
Wiley, New York.
* Plant Ecology 154: 275-276, 2001. 275

Subject Index

alga, 77 grass, 77, 125

AMF, 171 grasslands, 171
antarctic, 11 grazers, 257
antarctica, 77, 89, 103 growth, 67, 119, 239
apical dominance, 197
arbuscular mycorrhizal fungi, 171 historic UV-B radiation, II
arbuscules, 171 humic substances, 271
artificial wounding, 213
inedible algae, 257
biodiversity, 171
bio-indicator, 11 lichen, 77
biomass allocation, 197 light penetration, 125
- production, 197 - quality, 137

carbohydrates, 159, 197 macroalgae, 221

carotenoids, 89 maintenance respiration, 67
cell size, 257 mediterranean, 181, 213
charophytes, 239 mixed canopies, 125
climate change, I 03 morphology, 137
clover, 125 moss, 77
C02 enrichment, 197 mycorrhiza, 171
cyanobacteria, 221, 257 mycosporine-like amino acids (MAAs), 221

daphnids, 257 nutrient cycling, 29

decomposition, 29 nutrients, 159, 181
dehydrocholesterol, 3
DOC, 271 oxgygen evolution, 147
dune grassland ecosystem, 39, 159 ozone depletion, 29, 77,103,159

ecosystem, 103 PAR, 125

enhanced UV-B radiation, 39 phenolics, 213
ergosterol, 3 photochemical efficiency of PSII, 51
photodegradation, 29
Fv/Fm, 147 photoinhibition, 147
faba bean, 137, 197 photomorphogenic effects, 137
field experiment, 189 photoreceptor, 3
flavonoids, 171 photosynthesis, 89
floret surface, 59 photosynthetic acclimation, 197
flower demography, 59 - efficiency, 77
- properties, 59 phytoplankton, 221, 271
flowering, 119 pigments, 51
foodweb, 257 pollen, 11
polyphenolics, 11
glutathione, 147 protection, 67
276

protective compounds, 51 - radiation, 221

provitamin D, 3 understorey plant, 51
proxy, 11 UV absorbing pigments, 159, 239
- -radiation, 271
radiation, 51 UV-8, 3, 29, 51, 67, 103, 125, 147, 181, 189,213,257
reduced plant mass accumulation, 39 - absorbing compounds, 137
repair, 67 - filters, 77
reproduction, 239 - radiation, 59, 77, 89, 119, 159, 171, 197
root exudates, 171 - supplementation, 77, 89, 103
scytonemin, 221
selenastum, 271 YAM, 171
source-sink relations, 67 vitamin D, 3
sporopollenin, I I
stratospheric ozone depletion, 11, 39 water availability, 189
Svalbard, 257
tillering, 39 xanthophyll cycle, 147

ultraviolet 3 zooplankton, 271

ultraviolet-B. 29
* Plant Ecology 154: 277-278, 2001. 277

Species Index

Acer saccharum, 71, 172, 174 Daphnia middendorffiana, 257

Agrostis capillaris, 41 Daphnia pulex, 250
Alexandrium excavatum, 229 Dasycladus vermicularis, 230, 231
Alternaria so/ani, 216 Deschampsia antarctica, 9-22,45, 78, 83, 87-98,
Anabaena spp., 222, 226 101-113
Ankyrajudayi, 252 ff Anthemis arvensis, 57-63 Diatoma tenuis, 252 ff
Arabidopsis thaliana, 160 Dimorphotheca pluvial is, 164, 185
Asteroma microspermum, 35 Dinophyceae,228
Drepanocladus uncinatus, 78, 106
Bacillariophyceae, 228
Betula pubescens, 29-35 Emiliania huxleyi, 4
Brassica napus, 97
Bryum argenteum, 12, 22 Festuca rubra. 41
Fragilaria construens, 257
Calamagrostis epigeios, 32, 37--45, 169-175, 215 Fragilaria crotonensis, 257
Calamagrostis lapponica, 40 Fragilaria sp., 252 ff
Calamagrostis purpurea, 40, 67-71 Funts spiralis, 5
Calothrix sp., 226 Fucus vesiculosus, 4
Carex arenaria, 37-45, 169-175
Carexflacca, 174 Glycine max, 185
Ceramium rubrum, 231 Gracillaria cornea, 231
Ceratonia siliqua, 179-186, 187-193 Gyrodinium dorsum, 229
Cetraria islandica, 97, 98
Chara aspera, 237-245 Helleborus j(Jetidus, 14-18
Chara contra ria. 243 Heterocephalus glaber, 3
Chlamydomonas reinhardtii, 4
Chlamydomonas sp., 252 ff Kirchneriella sp., 252 ff
Chlorella spp., 4
Chlorogloeopsissp., 226 Lactuca sativa, 164
Chlorophyta, 230 Laminaria saccharina, 230
Chondrus crispus, 230 Laurus nobilis, 215
Cistus creticus, 60, 63 Lingulodiniwn polyedra, 229
Cistus lantifolius, 216 Lolium perenne, 6, 172
Cladonia mitis, 63, 90 Lycopersicon esculentum, 5
Cladosporium herbarum, 35 Lyngbya sp., 226
Cladosporium sp., 32, 34, 35
Colobanthus quitensis, 9-22, 78, 90, 91, 104 Mastocarpus stellatus, 230, 231
Cryptomys damarensis, 3 Mastodia tesselata, 78, 90, 106
Cucumis sativus, 185 Mentha spicata, 63
Cyanobacteria, 249-258 Mucor hiemalis, 35
Cystodendron sp, 32, 34, 35
Neophytium /olii, 6
Dactylis glomerata, 125-133 Neotyphodium lolii, 172
Daphnia magna, 261-273 Nerium oleander, 181, 185, 191
278
Nostoc commune, 222, 226
Nostoc spongiaeforme, 227
Nostoc sp., 222, 226
Ochromonas danica, 4
Ochromonas sp., 252 ff.
Oenothera biennis, 41
Penicillium simplicissimum, 35
Penicillium spinulosum, 35
Penicillium spp., 32
Phaeocystis antarctica, 29
Phaeocystis pouchetii, 29, 257
Phaeophyta, 230
Phaseolus vulgaris, 5, 49-155
Phlomis fruticosa, 179-186, 211-219
Phoma herbarum, 32, 35
Phoma nebulosa, 35
Pinuspinea, 181,185,191
Pisum sativum, 149-155
Planktothri.x sp., 249-258
Plantago lanceolata, 41, 157-167, 185
Poa spp., 112
Polyides rotundus, 231
Porphyra umbilicalis, 231
Prasiola crispa, 78, 83, 98, 106
Prorocentrum micans, 229
Pulmonaria officina/is, 49--63
Quercus robur, 32, 34, 172
Rhodophyta, 230
Rosmarinus officina/is, 117-122
Rubus caesius, 41
Rumex a/pinus, 164
Saniona uncinata, 78, 83, I 06
Scytonema hofrnanii, 227
Scytonema spp., 222
Selenastrum capricornutuum, 261-273.
Skeletonema menzelii, 4
Solanum glaucophyllum, 5
Stereocaulon alpinum, 75, 78
Trifolium repens, 125- 133
Turgidosculum complicatulum, 78, 87-98, 106
Umbilicaria americana, 90, 216
Uroglena americana, 249-258
Usnea antarctica, 79-82
Vaccinium myrtillus, 63
Vaccinium uliginosum, 34, 70, 215
Vaccinium spp., 172
Viciafaba, 9-22, 111, 137-144, 195-210
Vigna unguiculata, 167
Woronichinia sp., 249-258