Tree Physiology 21, 759–764
© 2001 Heron Publishing—Victoria, Canada
Abscisic acid in leaves and roots of willow: significance for stomatal
conductance
L. LIU,1 A. J. S. McDONALD,2,5 I. STADENBERG3 and W. J. DAVIES4
1
Department of Plant Breeding and Genetics, International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines
2
Department of Plant and Soil Science, University of Aberdeen, Cruickshank Bldg., St. Machar Drive, Aberdeen AB24 3UU, U.K.
3
Department of Production Ecology, Swedish University of Agricultural Sciences, P.O. Box 7042, Uppsala, Sweden
4
Biological Sciences Department, Institute of Environmental and Natural Sciences, Lancaster University, Bailrigg, Lancaster LA1 4YQ, U.K.
5
Author to whom correspondence should be addressed
Received July 21, 1999
Summary Excised leaves and roots of willow (Salix dasyclados Wimm.) accumulated abscisic acid (ABA) in response
to desiccation. The accumulation of ABA was greater in young
leaves and roots than in old leaves and roots. In mature leaves,
ABA accumulation was related to the severity and duration of
the desiccation treatment. Water loss equal to 12% of initial
fresh weight caused the ABA content of mature leaves to increase measurably within 30 min and to double in 2.5 h. The
drying treatment caused significant (P = 0.05) reductions in
leaf water potential and stomatal conductance. Recovery of
leaf water potential to the control value occurred within 10 min
of rewatering the dehydrated leaves, but recovery of stomatal
conductance took an hour or longer, depending on the interval
between dehydration and rewatering. The addition of ABA to
the transpiration stream of well-watered excised leaves was
sufficient to cause partial stomatal closure within 1 h and, depending on ABA concentration, more or less complete
stomatal closure within 3 h. When the ABA solution was replaced with water, stomatal conductance increased at a rate inversely related to the concentration of the ABA solution with
which the leaves had been supplied.
Keywords: ABA, dehydration, leaf, root, Salix dasyclados.
al. 1983, Cornish and Zeevaart 1984). High ABA concentrations have also been reported in specific leaf cells and tissues
(Brinckmann et al. 1990, Harris and Outlaw 1991). Endogenous ABA concentrations increase in root cultures (Hartung
and Abou-Mandour 1980) and excised roots under osmotic
stress (Walton et al. 1976, Behl and Hartung 1984, Zhang and
Tardieu 1996). Accumulation of ABA in response to tissue
desiccation has also been reported in intact leaves (e.g.,
Zeevaart 1971, 1977) and intact roots (e.g., Walton et al. 1976,
Zhang and Davies 1987, Zhang et al. 1987, Neales et al. 1989,
Masia et al. 1994, Liang et al. 1997).
Because ABA produced in roots may affect ABA concentrations in the transpiration stream, we studied the effect on
stomatal conductance of supplying excised willow leaves with
exogenous ABA in the transpiration stream. To examine other
aspects of the role of ABA in the control of stomatal conductance we carried out experiments with excised leaves and roots
to determine: (i) how tissue age and desiccation affect leaf and
root ABA content; (ii) the effect of duration of water stress on
leaf ABA accumulation; and (iii) the changes in water potential and stomatal conductance of fully expanded leaves in
response to tissue desiccation and rehydration.
Methods
Introduction
Abscisic acid (ABA) is a key component of signal-transduction pathways that result in physiological responses to environmental stress (Leung and Giraudat 1998). In particular,
water stress promotes the synthesis of ABA (Taylor et al.
2000) and regulates pH-dependent compartmentation of ABA
to the apoplast of stomatal guard cells (Hartung et al. 1998),
with resultant changes in stomatal aperture.
There are many reports on the accumulation of ABA in response to water stress. In bulk samples from excised dehydrating leaves, high endogenous ABA concentrations have been
reported (Wright and Hiron 1969, Wright 1977, Zeevaart
1977, 1980, 1985, Pierce and Raschke 1980, 1981, Hartung et
Plant material
Experiments were carried out on excised leaves and root segments of potted willow plants (Salix dasyclados Wimm.)
grown from clonal stem cuttings in a greenhouse. Roots
emerged from holes in the bottom of the plant pots and extended into a container of nutrient solution positioned immediately below (cf. Ericson 1981).
Harvesting plants
Leaves and roots were harvested at the end of the dark period.
Unless otherwise indicated, we took either the first or second
fully expanded leaf from the shoot apex. Roots were harvested
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LIU, MCDONALD, STADENBERG AND DAVIES
from among those growing from the base of the plant pot.
Incubation conditions
Unless otherwise indicated, excised leaves were incubated in
dim light (≤ 50 µmol m –2 s –1 of photosynthetic photon flux
density (PPFD)), whereas roots were incubated in darkness.
All incubations were at 23 °C.
Replication
For each measurement, there were five leaves or root segments
per treatment.
Measurements
Leaf water potential was measured with a custom-built pressure chamber. Stomatal conductance to water vapor was measured with a leaf porometer (AP4 Porometer, Delta-T Devices,
Cambridge, U.K.). Stomatal conductance was measured at a
PPFD ≥ 350 µmol m –2 s –1.
Leaf and root samples for ABA analysis (approximately
150 mg fresh weight) were wrapped in foil, frozen in liquid N
and stored at –20 °C. Before analysis, root and leaf samples
were freeze-dried for 48 h. Samples of dried material (60 mg)
were ground in 1.5 ml of sterile deionized water and shaken
for 8–10 h in darkness at 4 °C. After extraction, the samples
were stored at –20 °C. Before analysis, samples were thawed
at room temperature in darkness and a 50-µl aliquot removed
for ABA analysis by radioimmunoassay (Quarrie et al. 1988).
Sample concentrations were calculated by comparison with
calibration standards. The monoclonal antibody, MAC252,
was supplied by Steve Quarrie (John Innes Centre, Norwich,
U.K.).
Recovery in leaf water potential and stomatal conductance
Fully expanded excised leaves were allowed to lose 12% of
their initial fresh weight, transferred to clear plastic bags, and
incubated for various periods without further water loss. Subsequently, the petiole of each leaf was recut, immersed in a vial
of degassed, distilled water, and the leaf incubated in the light
(PPFD ≥ 350 µmol m –2 s –1). Successive measurements on different leaf samples were made to determine the time courses of
recovery in leaf water potential and stomatal conductance.
Stomatal response to an exogenous supply of ABA
Cis,trans (+/–) abscisic acid (Sigma) was dissolved in a few
drops of ethanol (99% solution) and diluted with degassed,
distilled water to 7 × 10 –5, 10 –4, 10 –3, 10 –2 and 10 –1 mol m –3.
A solution containing ethanol (zero ABA) at the highest molar
concentration was also prepared.
The petiole of each sample leaf was recut, immersed in a
vial of degassed distilled water for 12 h, and incubated in darkness to reduce evapotranspiration and embolisms. The petioles
were then recut under water and the leaves transferred to vials
containing one of the six ABA treatment solutions. After incubation for 1 h in the light (PPFD ≥ 350 µmol m –2 s –1) stomatal
conductance was measured.
Dynamics of stomatal response to exogenous ABA
Petioles of freshly cut leaves were immersed in ABA solutions
at one of three concentrations and incubated for 3 h in the light
(PPFD ≥ 350 µmol m –2 s –1). Thereafter, they were transferred
to vials containing degassed, distilled water and incubated for
a further 4 h. Stomatal conductance was measured throughout
the experiment.
Statistics
Severity of desiccation and ABA accumulation in leaves and
roots
Leaf samples were classified as (i) still folded; (ii) unfolded,
but still expanding and (iii) fully expanded. Roots were sampled at (i) 0–5 cm behind the tip; (ii) 5–10 cm from the tip and
(iii) > 10 cm from tip, (i.e., mature roots with laterals and root
hairs). Root samples were pooled to give five replicated samples per treatment. Excised leaves and roots were allowed to
dry slowly to different percentages of initial fresh weight.
Root fresh weights were recorded continuously until the sample weights had decreased by 0, 10, 20, 30, 35 and 40%. Leaf
samples were dried until the weight loss equaled 0, 2, 4, 6, 8
and 10% of initial fresh weight. The treated samples were
sealed in small glass vials, incubated for 5 h without further
water loss and then analyzed for ABA.
Treatment effects were assessed by analysis of variance (Microsoft Excel Version 5.0, single-factor ANOVA). The significance of treatment effects was assessed at P = 0.05.
Results
Severity of desiccation and ABA accumulation in leaves and
roots
The accumulation of ABA in leaves of all age classes increased with the amount of water loss. The range of leaf water
contents shown in Figure 1 was equivalent to a range in leaf
water potentials from –0.4 to –1.2 MPa. The greatest relative
increase in ABA content occurred in the youngest leaves. In
roots, the greatest increase in ABA concentration was also
found in the youngest age class of material (Figure 2). Abscisic acid concentrations were lower in roots than in leaves.
Duration of desiccation and ABA accumulation in leaves
Fully expanded excised leaves were allowed to lose 12% of
their initial fresh weight, transferred to clear plastic bags, and
incubated without further water loss for 0, 0.5, 1, 1.5, 2 and
2.5 h. Five leaves per incubation period were used for ABA
analysis.
Duration of desiccation and ABA accumulation in leaves
Accumulation of ABA depended on the duration of water
stress. Mature leaves allowed to lose 12% of their initial fresh
weight and incubated for 30 min without further water loss,
had a significantly higher (P = 0.05) ABA content than control
TREE PHYSIOLOGY VOLUME 21, 2001
ABA AND STOMATAL CONDUCTANCE IN WILLOW
Figure 1. Relationship between ABA concentration (nmol g –1 dw) in
excised leaves and leaf dessication (% of original fresh weight). The
dessicated leaves were incubated for 5 h. The leaf categories were:
young, unfolded leaves (䉱); young, extending leaves (䊐) and mature,
fully extended leaves (䉬); n = 5, and the bars indicate ± SE.
leaves (Figure 3). The amount of ABA in dehydrated leaves
continued to increase throughout the 2.5-h incubation period.
Recovery in leaf water potential and stomatal conductance
The water potential of mature leaves allowed to lose 12% of
their intial fresh weight and incubated without further water
loss for 10 to 60 min, matched that of control leaves (P = 0.05)
within 10 minutes of rewatering (Figure 4a). Stomatal conductance, however, recovered more slowly after rewatering (Figure 4b). Forty minutes after rewatering, leaves in all treatments
had a significantly lower (P = 0.05) stomatal conductance than
control leaves, and the degree of recovery was inversely related to the interval between desiccation and rehydration.
Twenty-four hours after rewatering, stomatal conductance of
leaves in all treatments matched (P = 0.05) that of the controls.
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Figure 3. Time course of ABA accumulation (nmol g –1 dw) in mature,
fully extended excised leaves following the loss of water equal to 12%
of their initial fresh weight (䉬) and 0% water deficit (䊐); n = 5, and
the bars indicate ± SE.
which was related in magnitude to the concentration of the
ABA solution (Figure 5). However, stomatal conductance in
leaves incubated at 7 × 10 –5 mol m –3 ABA (a concentration
typical of that found in the xylem sap of well-watered willow
plants) was not significantly different (P = 0.05) from that of
control leaves.
Dynamics of stomatal responses to exogenous ABA
Stomatal conductance decreased in a concentration-dependent
way in response to exogenous ABA (Figure 6). The response
approached a plateau after 2 to 3 h (Figure 6). When the ABA
Stomatal response to an exogenous supply of ABA
Leaves supplied through the cut end of the petiole with a solution of ABA showed a reduction in stomatal conductance,
Figure 2. Relationship between ABA concentration (nmol g –1 dw) in
excised roots and root water loss (% of original fresh weight). The
dessicated roots were incubated for 5 h. The root categories were:
0–5 cm behind tip (䉬); 5–10 cm from tip (䊐) and > 10 cm from tip
(䉱); n = 5, and the bars indicate ± SE.
Figure 4. Time course of recovery in (a) water potential (MPa) and (b)
stomatal conductance, (gs; mol m –2 s –1) after rewatering of mature,
fully extended leaves that had been excised, allowed to lose water
equal to 12% of their initial fresh weight and incubated for 0 min (䊐),
30 min (䉱), 60 min (×), 90 min (䊉) and 120 min (䊏). Control leaves
(䉬) experienced no water loss. Leaves were transferred to high PPFD
(≥ 350 µmol m –2 s –1) at time zero; n = 5, and the bars indicate ± SE.
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LIU, MCDONALD, STADENBERG AND DAVIES
Figure 5. Effect of a 1-h incubation in one of a range of ABA concentrations on stomatal conductance (gs; mol m –2 s –1) of mature,
well-watered excised leaves. Measurements were made at a PPFD of
≥ 350 µmol m –2 s –1; n = 5, and the bars indicate ± SE.
solution was replaced with water, stomatal conductance
showed no significant (P = 0.05) change for 2 h. Thereafter, it
increased at a rate that was inversely related to the concentration of the ABA solution with which the leaf had been
supplied. Recovery of stomatal conductance to control values
occurred within 4 h except in leaves treated with the highest
concentration of ABA.
Discussion
As shown for excised leaves (Wright and Hiron 1969, Wright
1977, Zeevaart 1977, 1980, Pierce and Raschke 1980, 1981,
Hartung et al. 1983, Cornish and Zeevaart 1984, 1985,
Brinckmann et al. 1990, Harris and Outlaw 1991) and root
segments (Walton et al. 1976, Hartung and Abou-Mandour
Figure 6. Time course of changes in stomatal conductance, gs (mol
m –2 s –1) of mature, well-watered excised leaves in response to addition and removal of ABA at different concentrations (mol m –3). The
solution concentrations were: water (䉬), 10 –4 (䊐), 10 –3 (䉱) and 10 –2
(䊏). The leaves were at zero water deficit and measurements were
made at a PPFD of ≥ 350 µmol m –2 s –1; n = 5, and the bars indicate
± SE.
1980, Behl and Hartung 1984, Zhang and Tardieu 1996) of
many other plant species, excised leaves and roots of willow
accumulated ABA when dehydrated (Figures 1 and 2). Our
finding that young willow leaves accumulated higher ABA
concentrations in response to desiccation than mature leaves is
consistent with the study of Cornish and Zeevart (1984).
Although ABA concentrations were lower in roots (Figure 2) than in leaves, root ABA concentration also increased in
response to tissue desiccation, indicating that dehydrating
roots may be a source of xylem sap ABA (cf. Davies and
Zhang 1991) with potentially important consequences for the
regulation of stomatal conductance and leaf extension in willow (Liu 1998, Liu et al. 2001). Consistent with our finding for
willow, Zhang and Tardieu (1996) reported relatively higher
synthesis of ABA in root tips than in older roots. However,
these authors pointed out that the contribution of ABA from
root tips to the total flux of root-sourced ABA may be small
relative to that from the greater mass of older root tissues.
The finding that ABA accumulation in willow increases
with the duration of water stress (Figure 3) is consistent with
previous reports (e.g., Pierce and Raschke 1980, 1981). The
delay in the onset of ABA accumulation in response to water
stress (≤ 30 min) in excised willow leaves was much shorter
than the 2 h reported in maize and sorghum (Beardsell and Cohen 1975) and wheat (Wright 1977) and the 1–2 h reported for
cocklebur (Zeevaart 1980). Harris and Outlaw (1991) detected
ABA accumulation in leaves of broad bean (Vicia faba L.)
40–50 min after excision, although accumulation in the guard
cells occurred within 15 min of the onset of dehydration.
Delays in stomatal response to rewatering after desiccation
occurred despite a full and rapid recovery in leaf water potential (Figure 4). In leaves allowed to lose 12% of their initial
fresh weight and then immediately rehydrated (0-min incubation, Figure 4), the delay in the onset of stomatal recovery
(10–30 min) and the time required (about 1 h) for full recovery of stomatal conductance to the value of control leaves,
suggest that the response was attributable, at least in part, to a
chemical component, not just a hydraulic stimulus. The delay
in stomatal response to rewatering may have been caused by
impaired stomatal functioning, although a flux of ABA into
guard cells (without necessarily any de novo synthesis of
ABA) may have occurred (cf. Harris and Outlaw 1991).
Abscisic acid redistribution between leaf compartments has
been reported in response to osmotic stress and drought stress
(e.g., Hartung et al. 1988, Daeter and Hartung 1990,
Wilkinson and Davies 1997). If the model of Slovik and
Hartung (1992) is relevant to dehydrated willow leaves, redistribution of ABA into the guard-cell wall may have been sufficient and fast enough to induce stomatal closure (in the 0-min,
incubation treatment) with little or no ABA synthesis. The delay in recovery of stomatal conductance following rewatering
of the leaf may have been caused by a delay in the subsequent
redistribution of ABA between the apoplast and symplast.
The accumulation of ABA in dehydrated willow leaves was
relatively rapid (measurable within 30 min after the onset of
water deficit, Figure 3). Newly synthesized ABA presumably
contributed both to stomatal closure and the longer recovery
TREE PHYSIOLOGY VOLUME 21, 2001
ABA AND STOMATAL CONDUCTANCE IN WILLOW
times following rewatering found in leaves that had been incubated for longer after desiccation (Figure 4b). The amount of
ABA accumulated in dehydrated leaves after longer (≥
60-min) incubation periods (Figure 3) led to persistently low
stomatal conductances after rewatering (Figure 4b). It was
only after 24 h (data not shown) that stomatal conductance
fully recovered to that of leaves never dehydrated or rewatered
immediately following dehydration (0-min incubation). These
results are consistent with large amounts of ABA being synthesized in leaves during prolonged periods of water stress and
with rates of ABA catabolism that were low enough to prevent
a rapid recovery in stomatal conductance. The dependence of
the recovery time on the length of the interval between desiccation and rewatering is consistent with the observations reported by Beardsell and Cohen (1975).
The addition of ABA to the transpiration stream was sufficient to cause more or less complete stomatal closure depending on ABA concentration (Figure 5). Similar responses of
stomatal conductance to exogenous ABA have been reported
in studies where ABA has been fed via roots to leaves of intact
plants (Zhang and Davies 1989, 1990, Tardieu et al. 1996) or
to excised, whole leaves (Gowing et al. 1993, Correia and
Pereira 1995, Correia et al. 1995, Tardieu et al. 1996, Jia et al.
1997). A similar result was also found when isolated epidermis was incubated in ABA solution (Trejo et al. 1995). The
ABA-induced reductions in stomatal conductance were reversed by removing ABA from the transpiration stream (Figure 6), and the recovery time increased with the concentration
of the ABA solution with which the leaves had been supplied.
Presumably, in leaves supplied with ABA at the highest concentration, the rate of ABA catabolism was too low to allow recovery of stomatal conductance during the 4 h after removal of
the ABA source.
In conclusion, ABA was produced in dehydrated leaves and
roots of willow and the accumulation of ABA depended on the
age of the plant material and the extent and duration of tissue
desiccation. Abscisic acid in the transpiration stream caused
decreases in stomatal conductance that were reversible on removal of the ABA source, although there was a considerable
lag phase in the full recovery of stomatal conductance. The
ABA concentration that caused 50% stomatal closure (Figure 6) is similar to that reported for 50% stomatal closure in
willow plants subjected to partial root drying (Liu 1998, Liu et
al. 2001). Thus, perturbations in the concentration of an exogenous supply of ABA alone, in a physiologically relevant
range, were sufficient to cause substantial changes in stomatal
conductance in willow. Our results are consistent with a model
of stomatal regulation in which the occurrence and dynamics
of ABA are of key importance in accounting for stomatal responses to dehydration in leaves or roots of willow (cf. Liu
1998, Liu et al. 2001).
Acknowledgments
This work was partly funded by a grant from The Swedish National
Board for Industrial and Technical Development.
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