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139
Key words: Development, Dune grassland ecosystem, Germination, Ozone depletion, Plant growth, Seedlings,
UV-absorbing pigments, UV-B
Abstract
The germination of seeds of seven plant species occurring in a dune grassland vegetation of the Netherlands, was
studied at four levels of UV-B radiation simulating unto 45% stratospheric ozone reduction during April. With the
exception of seeds of Senecio jacobaea, germination of the dune grassland species was not affected by enhanced
UV-B irradiance. Although a clear UV-B fluence-response relationship was not observed, the germination rate of
S. jacobaea seeds and maximal germination percentage were reduced at enhanced UV-B. Germination rate in the
dark was higher than germination in the light for Oenothera biennis, Plantago lanceolata, Rumex obtusifolius and
S. jacobaea. Total dry biomass accumulation of seedlings was not affected by increased UV-B radiation in any of
the species tested. Clear-cut differences in UV-absorbance of methanolic extracts were observed between species.
Enhanced UV-B irradiance stimulated UV-absorbance of seedling extracts of Holcus lanatus and Verbascum thapsus.
A clear UV-B fluence-response relationship was observed for both species. The results indicate that germination of
the studied plant species probably will not be adversely affected by the expected stratospheric ozone reduction in
The Netherlands.
Introduction
Since a decrease of the earths stratospheric ozone
column thickness was observed and reports on the
causes of this phenomenon were published, a considerable amount of research has focused on the implications of increased solar UV-B radiation for life on earth
(Caldwell & Flint 1994; Teramura & Sullivan 1994).
Growth chamber studies as well as greenhouse and
field experimentation have led to a better understanding
of how enhanced solar UV-B radiation may affect plant
life. There are considerable differences in the UV-B
response of different plant species (Krupa & Kickert
1989, Tosserams et al. 1997) and between cultivars
of the same species (Biggs et al. 1981; Teramura &
Murali 1986; Barnes et al. 1993; Dai et al. 1994).
UV-B radiation may reduce plant growth and yield
(Tevini & Teramura 1989; Teramura & Sullivan 1994).
To avoid damage by UV-B radiation, plants evolved
*122992*
140
and individual plants grown in pots. Other developmental stages like germination, flowering and seedset are less well documented. These stages in plant
development however, might prove to be susceptible to
UV-B. In a recent study by Musil (1995), dicotyledonous Asteraceae exhibited delayed flowering, decreased
flower production and reduced numbers of seeds set in
response to elevated UV-B. In addition, the latter study
as well as several earlier studies (Flint & Caldwell
1984; Feder & Shrier 1990), demonstrated the UVB sensitivity of pollen germination and pollen tube
growth. It is reasonable to assume that developmental
stages may have differential UV-B sensitivity, regarding that the maximal UV-B exposure of different developmental stages may vary considerably (Teramura &
Sullivan 1994).
In order to obtain a better understanding of the
consequences of increased solar UV-B radiation on
natural ecosystems it is important to evaluate effects of
UV-B radiation on all developmental stages during the
life history of a specific plant species. The aim of the
present study was to determine to what extent enhanced
UV-B radiation influences seed germination and initial
seedling development of different plant species of a
dune grassland ecosystem in The Netherlands.
141
model designed by Green et al. (1980), the lowest UVBBE level is comparable to the ambient UV-BBE (2.98
kJ m,2 day,1 ) on a clear day during April in The
Netherlands. The other UV-BBE levels simulate a situation of 17, 28 and 45% stratospheric ozone reduction
respectively in the same month. The UV-BBE received
by the seeds in the UV-A control groups was 0.033,
0.052, 0.070 and 0.105 kJ m,2 day,1 respectively.
In addition, four petri-dishes each with fifty seeds of
the same species, were wrapped in aluminium foil.
One of these dishes was placed underneath each lamp
system to test whether microclimate at the different
sites in the greenhouse influenced germination. The
400W HPI/T lamps provided 250 mol m2 s,1 additional photosyntheticallyactive radiation (PAR) during
16 hours a day. The maximal value of PAR measured
on a clear day was 800 mol m2 s,1 . Temperature
during the experiment varied between 13.9 C (night)
and 34.5 C (day).
Measurements. To determine the weight and the
water content of the seeds, fresh and dry weight (48 hr,
70 C) of 50 seeds per plant species were determined.
The number of germinated seeds was counted twice
a day. A seed was defined germinated at the moment
the radicula appeared. When all seeds had germinated or no further germination was observed, counting was stopped. After the germination experiment,
the UV absorbance of the seedlings was determined.
UV absorbing compounds were extracted with 5 ml of
an acidified methanolic solution (CH2 OH:HCl:H2 O,
79:1:20 v/v) from approximately 15 mg of fresh plant
material per petri-dish. Because seedlings of V. thapsus
were very small, all plant material was used for the
determination of UV absorbance. The extracts were
stored for one night (8 C) after which they were boiled
for 10 minutes at 90 C in a waterbath. The absorbance of the solution was measured with a Perkin-Elmer
Lambda 15 UV/VIS Spectrophotometer at either 300
or 310 nm. Total dry weight (48 hr, 70 C) per petridish was determined for all species except V. thapsus.
Statistics. All data were subjected to analysis of variance (ANOVA; Sokal & Rohlf, 1981). Dry weight data
were transformed to their natural logarithms. Analysis
of variance for repeated measurements was used for
the analysis of germination data.
Plant species
Monocotyledons
Bromus hordeaceus 204
Holcus lanatus
11.2
4.0
0.3
182
10.1
3.4
0.6
10.8
10.1
0.1
3.0
Dicotyledons
Oenothera biennis
Plantago lanceolata
Rumex obtusifolius
Senecio jacobaea
Verbascum thapsus
0.7
3.0
0.9
0.2
0.0
24.4
76.2
55.5
11.3
7.5
0.7
2.7
0.8
0.2
0.2
6.9
10.5
11.2
6.7
9.5
0.1
0.1
0.1
0.2
2.3
26.2
85.1
62.5
12.1
8.3
142
Figure 2. The effect of UV-B irradiance on germination of dune grassland plant species . In each treatment seeds either received 9.18 kJ m,2
day,1 UV-BBE ( ) or did not receive UV-B ( ). The other UV-BBE treatments are not presented because they did not significantly differ from
the presented data. Germination data of dark controls ( ) was only included when it was significantly different from the other treatments. All
presented data are means of 4 replicates SEM.
the seed coat of most species provides sufficient protection against deleterious UV-B effects (Krizek 1975).
Dark controls of O. biennis, P. lanceolata, R.
obtusifolius and S. jacobaea had a higher germination
rate as compared to germination in the light (Figures 2
and 3). Dark and light germination of the other plant
species tested was comparable (data not shown).
Total seedling dry weight of all species tested was
not affected by enhanced UV-B irradiance (Figure 4).
In accordance, Krizek (1975) reported no adverse
effects on dry weight accumulation of a variety of crop
species after 72 h of continuous UV-B irradiance. A
marked decrease of dry weight was reported by Tevini
et al. (1983) for bean and cucumber seedlings.
The accumulation of UV absorbing compounds
(e.g. flavonoids) in epidermal tissue appears to be
an important protective mechanism, which effectively reduces the detrimental action of UV-B irradiance on plants (Middleton & Teramura 1993; Kootstra
1994; Lois & Buchanan 1994; Stapleton & Walbot
1994). The absorbance of UV-B radiation by flavonoids and related phenolic compounds varies between
cultivars and species (Teramura et al. 1991; Day et
al. 1994; Wand 1995). In agreement, clear-cut differences in UV-B absorbance between seedling extracts
of the control treatments were observed in the present
experiment (Figure 5). Controls of O. biennis had the
highest absorbance ( 0.65) while the lowest absorbance (between 0.1 and 0.15) was observed in R. obtusifolius and V. thapsus seedlings.
In many plant species UV-B irradiation enhances
the accumulation of absorbing compounds (Tevini &
Teramura 1989). This UV-B-enhanced accumulation
is due to a higher activity and/or higher rate of biosynthesis of L-phenylalanine ammonia-lyase (PAL). This
enzyme regulates the diversion of L-phenylalanine into
precursors for secondary phenolics. In barley primary
leaves UV-B irradiance prolonged the activity of PAL
resulting in an enhanced accumulation of UV absorbing compounds (Liu & McClure 1995). The influence
of UV-B on the stimulation of UV-B absorbance varied between species. Absorbance of B. hordeaceus,
O. biennis, P. lanceolata, R. obtusifolius and S. jacobaea remained unaffected by increasing UV-B levels,
whereas the UV-B absorbance of H. lanatus and
V. thapsus was increased by UV-B enhancement. The
143
Figure 3. The effect of UV-B irradiance on germination of S. jacobaea seeds. Four UV-BBE levels were applied: (A) 2.46 kJ m,2 day,1 ; (B)
4.35 kJ m,2 day,1 ; (C) 5.75 kJ m,2 day,1 ; (D) 9.18 kJ m,2 day,1 . In each treatment seeds were either irradiated with UV-B ( ) or did not
receive UV-B ( ). Germination of dark controls ( ) is only included in D. All presented data are means of 4 replicates SEM.
UV-B absorbance of H. lanatus seedlings was stimulated by 37, 61, 74, and 99% when the different UVB treatments were compared to their respective controls. This is in agreement with results of Wellmann
(1985) who demonstrated the linear dependency of
flavonol accumulation with UV-B fluence. Seedlings
of V. thapsus also exhibited a clear UV-B fluenceresponse relationship. For this species the maximal
stimulation of absorbance was 28% at the highest UVB treatment.
Although large differences between species were
observed, the presence and/or accumulation of absorbing compounds may already protect the seedlings
against detrimental effects of UV-B radiation directly
after emergence from the soil. Enhanced UV-B fluence rates might however stimulate the accumulation
of phenolic compounds in some species, which may
affect herbivory (Coley et al. 1985), litter quality and
decomposition processes (Gehrke et al. 1995, Rozema
144
Figure 4. The influence of enhanced UV-B irradiance on dry matter accumulation of seedlings. Seedlings received no UV-B ( ) or additional
UV-B ( ). Values presented are means of 4 replicates SEM. Bars marked with the same letter are not significantly different (p 0.05). Data
for V. thapsus was not available because all seedlings were used for the determination of UV-absorbance.
Therefore, we conclude that the expected ozone reductions for The Netherlands (WMO 1994) will probably
not negatively influence germination of plant species
of the dune grassland ecosystem.
145
Figure 5. The influence of enhanced UV-B irradiance on UV-B absorbance of methanolic seedling extracts. Seedlings received no UV-B ( ),
additional UV-B ( ) or were kept in the dark ( ). Absorbance was measured at 300 (B. hordeaceus, O. biennis, S. jacobaea and V. thapsus) or
310 nm (H. lanatus, P. lanceolata and R. obtusifolius) and was recalculated to a concentration of 1 mg fresh material per ml extraction solution.
Values presented are means of 4 replicates SEM. Bars marked with the same letter are not significantly different (p 0.05).
146
Acknowledgements
We would like to thank dr Joe Sullivan and Professor
dr W.H.O. Ernst for their critical comments on the
manuscript. This work was supported by the Dutch
National Research Programme on Global Air Pollution
and Climate Change (NRP project number: 850022).
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