476 B. R, LOVEYS AND P. E, KRIEDEMANN Physiol, Plant, 28: 476-479.

1973

Rapid Changes in Abscisic Acid-like Inhibitors Following

Alterations in Vine Leaf Water Potential
By
B. R. LOVEYS and P. E. KRIEDEMANN ^
CSIRO Division of Horticultural Research, P.O. Box 350 G.P,O.,
Adelaide S,A, 5001 Australia
(Received November 22, 1972)

Abstract Experiments reported here were designed to test these

Endogenous levels of abscisic acid-like inhibitors doubled
within 15 minutes of leaf excision in Vitis vinifera under
laboratory conditions. At that stage water poceiitiai (^) Kad
fallen to -15 bars and stomatal closure was occurring. After
prolonged stress (6 days and final i(i -13 bars) inhibitor levels
in intact vine leaves increased by a factor of 44 but showed
a rapid decline following rewatering. Photosynthetic activity
was not immediately restored, and its gradual recovery was
not directly related to inhibitor level.
propositions. We excised vine leaves to follow any short
term increase in ABA-like inhibitors at the onset of
stomatal closure (10 min after excision) and then related
this information to changes in leaf water potential, and
gas exchange during prolonged moisture stress and sub-
sequent recovery. Our results emphasise the speed with
which levels of endogenous ABA-like inhibitors can alter
in response to changes in leaf water potential.

Introduction Material and Methods

Stomatal closure associated with plant moisture stress
probably depends upon the combined effects of reduced
turgor and abscisic acid (ABA) formation within the
leaves, Stomatal behaviour is known to be modified by
ABA (Little and Eidt 1968, Mittelheuser and van Steve-
ninck 1969, Jones and Mansfield 1970) while environ-
mental stress can lead to massive increases in endo-
genous levels. Wright and Hiron (1969) for example,
demonstrated a 40 fold increase after 4 hours following
a severe reduction in wheat leaf moisture content. Since
endogenous levels of ABA need only double for stomatal
closure to be initiated (Kriedemann et al. 1972), the
enormous increase during prolonged stress is likely to
impair subsequent stomatal function. This could be re-
lated to the widely documented "after effect" whereby
stomata fail to regain normal function for some days
despite immediate restoration of a favourable leaf water
potential. Moreover, stomatal closure which occurs dur-
ing the initial phases of moisture stress might also be
related to ABA formation provided the time course of
increase was sufficiently rapid (minutes rather than
hours or days).
Located at Merbein, Victoria, Australia.
Grape vines {Vitis vinifera L, cv. Sultana syn, Thomp-
son Seedless) were planted as rooted cuttings in 5 litre
containers of sterilised soil and grown in a shade house
(50 "h full sun). Shoots were trained vertically and
showed vigorous growth. The vines were well established
at the time of experimentation (12 weeks after planting).
Gas exchange rates (determined under laboratory con-
ditions) were measured repeatedly on certain "reference"
leaves, which remained attached to the vine and served
as an index of the vine's day to day performance, and
on additional leaves which were subsequently removed
for water potential measurement and inhibitor extrac-
tion,
ABA-like inhibitors were extracted in 80 Vo v/v
aqueous methanol containing sodium bicarbonate (Len-
ton et al. 1971) and cooled with solid carbon dioxide.
After a 24 hour period of extraction at 2°C the homo-
genate was filtered and the methanolic extract was
evaporated under reduced pressure at 30°C. The aque-
ous extract was then treated as described previously
(Kriedemann et al. 1972). Eluates from the thin layer
plates were dried onto filter paper discs, dissolved in
3 ml of water and assayed with the Avena coleoptile
PhiysioL Plant, 28,,--1973 MOISTURE STRESS AND ABSCISIC ACID 477
Table 1. Short term effects of water stress on inhibitor levels 40
in Sultana leaves. 15 minutes elapsed between excision and •—•ABA
homogenisation. X—X MOISTURE
»30 TENSION 14
Water potential Inhibitor I SE (dryl
Treatment ( ) g g 12 ^
(bariSE) initial fresh weight 10 -^

Control -2,63 ± 0,80 0,15 '

8 I
Stressed -15,72 + 2,80 10 6 I
1 Difference significant at the 0,05 level of probability. 4 I
Figures are the mean of 3 determinations.

straight growth test, Coleoptile sections 6 mm long and
cut 3 mm from the tips were incubated in the dark at
25 °C with the 3 ml of the aqueous inhibitor solutions
for 1 hour. One ml of an incubation medium contain-
ing glucose (2 X 10-2 M)^ KH2PO4 (10^2 M) and IAA
(2 X lO"* M) WHS then added to each dish and the in-
cubation continued for a further 6 hours. Inhibitor con-
centrations in the extracts were estimated by reference
to known concentrations of (±)-ABA. Gas-liquid chro-
matography of methylated extracts confirmed the pres-
ence of a compound with an identical retention time as
methyl-ABA,
Recovery of ABA
The efficiency of this extraction procedure was mea-
sured by adding approximately 0,2 [.ig (± )-2-'"'-C-ABA
(26 ±2,6 mCi/mmol) to the methanoi used to homo-
genise 20 g of vine leaves. This rate of addition repre-
sents approximately one tenth of the endogenous con-
centration in well watered vine leaves. In three separate
experiments the percentage recovery in the final fraction
was 76,1 ±2.5 (SE),
Photosynthesis and transpiration of single leaves was
measured under controlled conditions. Single attached
leaves were enclosed individually in a circular cuvette,
their COg and H3O vapour exchange was monitored by
infrared gas analysis at saturating irradiance (25 Wm'^)
and optimum leaf temperature (25 °C), The response of
vine leaves to environmental conditions together with
details of our measuring techniques have been described
previously (Kriedemann and Smart 1971, Kriedemann
1971),
Leaf water potential was measured with a pressure
bomb (Scholander et al 1965).
- '6 1600^
JC.
1200-i
o>
O
800 £
400 o
a
3 "a.
4 6 10 12
TIME days
Figure 1, Qhanges in leaf water potential, ABA-like inhibitor
levels, and gas exchange rates during prolonged moisture stress
and subsequent recovery.
stomatal resistance (routinely 2 to 4 s • cm ') were simply
excised. At the first suggestion of stomatal closure
(gauged from gas exchange measurements) we removed
the leaf from the cuvette, measured, its water potential
and immediately homogenised the lamina in cold meth-
anol. Stomatal closure was generally initiated within
10 minutes of petiole excision, and a further 5 minutes
elapsed before extraction. Over this period, inhibitor
levels virtually doubled. The increase is statistically
significant (Table 1), The figures take into account any
drop in fresh weight due to transpirational losses follow-
ing excision, (Both control and stressed leaves will have
lost some weight prior to homogenisation, the magnitude
of their fresh weight losses doe to transpiration will
differ. This loss generally amounted to only 5 or 6 %
of final weight, but data in Table 1 were nonetheless
corrected,)

Results Long term effects

Short term responses
The consequences of a rapid decline in leaf water
potential on levels of ABA-like inhibitors were examined
under laboratory conditions. Attached leaves which had
achieved a steady rate of gas exchange and minimal
In addition to the rapid increase following excision,
ABA-like inhibitors continue their increase during more
prolonged moisture stress. Figure 2 shows changes in
leaf water potential and inhibitor levels, together with
corresponding values for photosynthesis and transpira-
tion. These grapevines had remained unirrigated for
478 B. R. LOVEYS AND P. E. KRIEDEMANN
6 days and were then rewatered. Part of the population
was kept dry to act as a control for the rewatered group.
During droughting (day 0 —f day 6 in Figure 1) water
potential fell comparatively slowly due to combined
effects of mild weather and large moisture reserves in
the root medium. The penurbation between day 4 and
day 5 followed the low evaporative demand of over-
cast and humid conditions. Nonetheless, after 4 days,
water potential had fallen to -10 bars and inhibitor
levels had risen 26 fold. At the end of 6 days gas ex-
change had fallen to a very low level (leaf resistance
varied from 50 to 100 s - cm"'). Water potential had
fallen to -13 bars, and the concentration of ABA-like
inhibitors had increased by a factor of 44.
In the absence of supplementary water this situation
was maintained ("dry" curves in Figure 1) but inhibitor
levels fell precipitously after irrigation, and leaf mois-
ture status was rapidly regained. Inhibitor concentra-
tion nevertheless failed to reach its pre-stress value, and
endogenous levels remained about double the level at
day 0. Stomatal function did not recover immediately
and 5 days elapsed before gas exchange was back to
normal.
Discussion
The lag in photosynthetic recovery following mois-
ture stress is at least partly attributable to impaired sto-
matal function (transpiration curve Figure 1) which
could in turn result from the slightly elevated levels of
ABA-iike inhibitor. However, stomata tend to lose sen-
sitivity to applied ABA once a leaf has experienced
moisture stress (Kriedemann et al. 1972) and although a
final level of 2.08-2.25 mg (±)-ABA-kg"i dry weight
(compared to an initial value of 0.S9 mg (±)-ABA • kg"'
dry weight) might lead to stomatal closure in a plant
with no history of water shortage, "hardened" plants
(droughted vines in this instance) are less likely to re-
spond. In the present case, photosynthesis did recover
eventually, despite the continuing presence of ABA-like
inhibitors at a concentration twice that at day O.
As an alternative explanation the massive increase in
inhibitor by day 6 could have impaired synthesis of
carboxylative enzymes, the system requiring 5 days to
recover. This hypothesis derives support from the rela-
tively slow turnover which characterises RuDP car-
boxylase (Peterson et al. 1971). More detailed measure-
ments of component resistances to COj fixation during
this recovery period are required to separate stomata]
from such internal components. It is also possible that
photochemical reactions may offer some limitations dur-
ing the recovery period. Boyer (1971) found that in
sunflower photosynthesis was probably limited by photo-
chemical activity at leaf water potentials below —II to
-12 bars.
Physiol. Plant. 28. .1973
In the present instance we have demonstrated a sig-
nificant change in inhibitor levels in vine leaves within
15 minutes of petiole excision. Stomatal closure during
the initial phase of moisture stress may well be due to
the generation of inhibitor rather than to a fall in leaf
water potential per s.e. Equally rapid changes in growth
substance level have been demonstrated in the levels of
endogenous gibberellins in wheat leaves in response to
red light treatment (Beevers et al. 1970) and in the
cytokinin content of Rumex seeds following irradiation
with red light (Van Staden and Wareing 1972). The
rapidity of these responses highlights the plant's ability
to respond quickly to environmental change and empha-
sises the need for care and speed during the initial stages
of growth substance extraction.
The coupling system between cell turgor and ABA
synthesis is indeed fine but its exact nature obscure. The
increase in ABA following water stress appears due to
synthesis rather than release from a bound form (Mil-
borrow and Noddle 1970, Zeevaart 1971), and Mil-
borrow and Noddle propose that the site of regulation
may lie between the presumed ABA precursor (5-(l,2-
epoxy-2,6,6-trimethyI cyclohexyl}-3-niethyI penta-CM-2-
tMra-4-dienoic acid) and ABA.
Equally intriguing but as yet undocumented, is the
disappearance of ABA. Accumulation of massive levels
of inhibitor in leaves of droughted grapevines was
matched by a remarkable and virtually complete loss
24 hours after watering. A dissimilation mechanism
with this sort of capacity must occupy a key position in
ABA metabolism and the physiological effects of this
hormone.
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