Journal of Experimental Botany, Vol. 52, No. 355, pp. 301-308,
February 2001
© 2001 Oxford University Press
Original Papers |
Relationship between changes in the guard cell abscisic-acid content and other stress-related physiological parameters in intact plants
Department of Biological Science, Florida State University, Tallahassee, FL, 32306-4370, USA
Received 21 August 2000; Accepted 20 September 2000
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Abstract |
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The relationships of guard cell ABA content to eight stress-related physiological parameters were determined on intact Vicia faba L. plants that were grown hydroponically with split-root systems. Continuous stress was imposed by the addition of PEG to part of the root system. The water potentials of roots sampled after the addition of PEG were 0.25 MPa lower than the water potentials of other roots of the same plant, which were similar to the roots of untreated plants. The leaflet water potentials of plants sampled within 2 h of stress imposition were similar to those of control plants. However, leaf conductance was lower in plants sampled after only 20 min of stress imposition, and the root- and leaflet apoplastic ABA concentrations of these plants were higher than those of untreated plants. As the essence of this report, there was a linear relationship between guard cell ABA content and leaf conductance. Leaflet apoplastic ABA concentrations <150 nM were also linearly related to leaf conductance, but higher leaflet apoplastic ABA concentration did not cause equally large further declines in leaf conductance. It is suggested that evaporation from guard cell walls caused ABA to accumulate in the guard cell apoplast and this pool was saturated at high leaflet apoplastic ABA concentrations.
Key words: Abscisic acid, apoplast, polyethylene glycol, root signalling, split root, symplast.
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Introduction |
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Droughted roots synthesize ABA and export it to the shoot via the transpiration stream (Zhang and Davies, 1990
). Although specific results vary with species, leaf age, and duration and extent of drought, the general result is that elevation of leaf apoplastic ABA concentration correlates with a decline in leaf conductance (Davies and Zhang, 1991). Corroboratively, experimental alterations in the xylem sap ABA concentration yield the predicted change in leaf conductance (Tardieu et al., 1993). Thus, ABA is well established as a hormone that provides communication from the root to the shoot (Davies and Zhang, 1991; Jackson, 1993; Jackson, 1997).
The information encoded by ABA movement into the leaf is integrated with other information. First, the apoplast pH, [Ca2+] (Schurr et al., 1992; Mühling and Sattelmacher, 1995), and cytokinin concentration (Shashidhar et al., 1996) fluctuate. Second, stomatal sensitivity to ABA varies with leaf-water status (Tardieu and Davies, 1992; Peng and Weyers, 1994). Third, how to describe ABA movement into the leaf (e.g. rate of delivery of ABA into the leaf or absolute leaf apoplastic ABA concentration) is of current interest (Correia and Pereira, 1994; Gowing et al., 1993; Jokhan et al., 1996). In addition to these confounding factors, progress in describing intrafoliar movement of imported ABA has not been extended to the measurement of ABA in guard cells themselves. However, Trejo et al. inferred that metabolism and sequestration of ABA by mesophyll and epidermal cells lowers the ABA concentration delivered to guard cells (Trejo et al., 1993, 1995). Conversely, drought-induced elevation of apoplastic pH predicted a redistribution of ABA into the guard cell apoplast (Hartung et al., 1998) and has been shown to close stomata in an ABA-dependent manner, consistent with ABA accumulation in the guard cell apoplast (Wilkinson and Davies, 1997). Direct measurements of guard cell ABA content have been limited to detached leaves. These studies showed that water-stressing a detached leaflet results in a disproportionately rapid increase in guard cell ABA content (Harris and Outlaw, 1991), an effect that is present, albeit to a much lesser extent, in the absence of ABA biosynthesis (Popova et al., 2000).
This paper reports the ABA content of whole guard cells of intact plants. As a means of obtaining a range of conductance and ABA values, some plants were sampled after water stress, which was imposed by the addition of PEG to one-half of the root system. The 
Leaf of plants sampled during the first 2 h following PEG treatment were similar to those of control and pretreatment plants, but leaf conductance was less on the PEG-treated plants. As a basis for comparison, the ABA concentrations of whole roots, the root apoplast, whole leaf, and the leaf apoplast were also determined. The results are consistent with a conclusion that apoplastic ABA accumulates in the stomatal complex.
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Materials and methods |
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Plant materials
Seeds of Vicia faba L. cv. Longpod were surface-sterilized by immersing them in 0.05% (v/v) sodium hypochorite for 5 min. After they were rinsed to remove residual sterilizer, the seeds were imbibed in aerated water for 1 d at 22 °C. Then, they were transferred to a growth cabinet for germination inside a covered light-tight container. High relative humidity was maintained in this container by layers of moist paper towels between which the seeds were sandwiched. Ambient conditions in the growth cabinet were a 16 h day, a day/night temperature of 25/20 °C, a relative humidity of 60%, and a PFD (400–700 nm) of 600 µmol m-2 s-1 that was provided by a combination of incandescent and fluorescent lamps. The germinating seeds were removed daily, washed, and returned to the container; the moist towelling was renewed each day. After 1 week, each seedling was secured vertically by a foam-rubber plug through one of seven 2 cm ports in a 2.5 cm thick Styrofoam disc. The disc was floated so that the roots of the seven seedlings protruded into 4.0 l aerated quarter-strength Hoagland solution, pH 6.5, which was contained in an opaque 25 cm diameter cylindrical pot that was 21 cm deep. The nutrient solution was changed on alternate days. After 1 week, each 2-week-old seedling was transplanted to a final two-chambered container that was designed to permit imposition of water stress on one-half of the root system. This container consisted of two opaque parallel cylindrical nutrient chambers (internal diameter, 7 cm diameterx20 cm deep) with a removable lid. A flexible tube connected the bottoms of the two chambers during plant culture, but the tube was blocked during treatments. One-half of the root system was immersed in the nutrient solution in one chamber and the other half of the root system was immersed in the nutrient solution in the second chamber. The removable lid of the two-chambered container supported the plant and was opaque. The nutrient solution (1.0 l, described above) was continuously aerated, replenished daily, and replaced on a 2 d schedule. After 1 week of acclimation in the final container, the 3-week-old plants were subjected to water stress (see Experimental treatments). At several times during the ensuing period of continuous water stress, various samples were harvested and measurements were performed in the sequence described below.
Experimental treatments
Samples for ABA analysis and for measurements were taken from the third-youngest, fully expanded, bifoliate and from first-order-lateral roots. (ABA accumulation in response to a change in does not depend on age or branching order of roots (Simonneau et al., 1998).) First, a conductance measurement was made on one leaflet before the plant was otherwise disturbed. Second, the sister leaflet was harvested for ABA analysis. Third, the first leaflet was excised for measurement of water potential and collection of apoplastic sap. Fourth, whole-root tips (
0.3 cm) were harvested for ABA analysis. (The choice of using the apical 0.3 cm of the root was based on the large stress-induced increase in ABA concentration there (Zhang et al., 1996).) Fifth, first-order-lateral roots were excised for measurement and collection of apoplastic sap. Altogether, these procedures required about 5 min.
Water stress was imposed by adding 20% (g polyethylene-glycol-8000 ml-1 nutrient solution) PEG to one nutrient chamber of each two-chambered container. The PEG concentration was chosen on the basis of a series of preliminary experiments (three different plant-culture protocols, 5–30% PEG, 10 min–144 h imposition periods) aimed at effecting an elevation of apoplastic ABA concentration and a decline in conductance while Leaflet remained unchanged. Stress was imposed at 10.00 h (4 h after the onset of illumination), and all physiological evaluations and sampling were done 4–8 h after the onset of illumination. Alone, PEG was calculated to be -0.52 MPa (Michel et al., 1983) and Nutrient Solution was estimated to be -0.02 MPa (van't Hoff equation). During the continuous water-stress imposition on one-half of the root system, culture conditions were identical to those described (see Plant materials).
Use of PEG to impose water stress on plants is not without disadvantages (Krizek, 1985), but long-term growth studies (Pérez-Alfocea et al., 1993) have validated its use. Because of species-dependent effects of PEG (Fan and Blake, 1997) and of partially stressing the root system (Gallardo et al., 1994) it is shown that PEG addition caused predictable changes in leaf conductance and water potential in this study.
Water potential and stomatal-conductance measurements
Stomatal conductance was measured by use of a LI-1600 steady-state porometer (Li-Cor, Inc., Lincoln, NE, USA). Several reproducible conductance measurements on the same Vicia leaflet can be made by use of this instrument before damage is incurred (Lu et al., 1995).
Water potential was measured on leaflets and on first-order-lateral roots by use of a pressure chamber (PMS Instrument Co., Corvallis, OR, USA). The irregularly shaped succulent petiolule of Vicia required that modifications be made to the pressure chamber (Protocol C; Ewert et al., 2000).
Abscisic acid analysis
Organ samples:
A 1x1.5 cm rectangle of leaflet blade devoid of mid-rib was excised, frozen in liquid-N2 slurry, broken into 1–3 mm fragments, and stored at -80 °C until extraction. Whole-root tips were also frozen and stored at -80 °C until extraction. Fresh-frozen tissue, 4 mg, was homogenized successively three times in 80 µl 80% (v/v) aqueous methanol that contained 0.001% (w/v) 2,6-di-t-butyl-p-cresol. A complete homogenate, totaling 240 µl, was incubated in a covered, silanized borosilicate 6x50 mm tube overnight in darkness at 4 °C. Following a low-speed centrifugation, the supernatant was dried under a stream of N2. The residue was redissolved in nominally 150 µl of methanolic TRIS-buffered saline (10% (v/v) methanol in 50 mM TRIS, pH 8.1, 1 mM MgCl2, 150 mM NaCl). An aliquot, 0.6 µl, was used in the ABA assay.
Single-cell samples:
Fresh-frozen leaflet fragments obtained coincidentally with the whole-leaflet samples (see above) were freeze-dried and guard cell pairs were dissected from the abaxial epidermis (according to Hampp and Outlaw, 1987). Guard cell pairs were pooled, 50–100, in a single extract (90 nl 80% (v/v) aqueous methanol that contained 0.001% (w/v) 2,6-di-t-butyl-p-cresol, overnight, in darkness, at 4 °C) using the oil-well technique (Outlaw, 1980; Passonneau and Lowry, 1993). After addition of 2 µl of methanolic TRIS-buffered saline (see above), 0.6 µl was used in the ABA assay.
Apoplastic sap samples:
Apoplastic sap was collected by use of the pressure chamber and application of 0.2 MPa pressure excess of the water potential-balancing pressure. The initial 2–3 µl of extruded sap was discarded, and the second 2–5 µl of extruded sap was diluted nominally 3x with methanolic TRIS-buffered saline (see above). Of this, 0.6 µl was used in the ABA assay. (For a critique of this and other methods for collection of sap samples, see Jackson (1993).)
ABA assay:
The micro-scale ELISA of Harris et al. (Harris et al., 1988; Harris and Outlaw, 1990) with modifications (Zhang et al., 1991) was used to measure ABA in the 0.1–12 fmol range. All samples were analysed in triplicate; assay errors for whole leaflet, whole root or apoplastic samples were too small to show on the figures.
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Leaf conductance and water potential following PEG treatment
Leaf conductances and water potentials of roots and leaflets were measured in order to document the physiological effects of the experimental treatments on the particular plants used for guard cell ABA analysis.
The leaf conductance of the plant sampled under initial control conditions was 0.25 mol m-2 s-1 (Table 1). Subsequent measurements of leaf conductance on control plants were similar. The leaf conductances of all PEG-treated plants were lower than those of control plants. Thus, the leaf conductance of the plant sampled 20 min after the addition of PEG to one nutrient chamber was 0.18 mol m-2 s-1. The lowest leaf conductance was observed in plants sampled 2–24 h after PEG treatment; plants sampled later had increased leaf conductance, compared with the minimum.
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Leaflet of the plant sampled under initial conditions was -0.45 MPa (Table 1
). This value was similar to Leaflet in unstressed plants that were sampled up to 2 d later. Similarly, in plants sampled during the first 2 h or more than 48 h following stress imposition, the Leaflet was -0.45 MPa to -0.50 MPa, i.e. apparently the same as the values of unstressed plants. In the plants sampled 4–48 h after stress imposition, Leaflet appeared to be less than it was in the unstressed plants.
The initial Root was -0.20 MPa, similar to values of unstressed plants that were measured up to 2 d later (Table 1). Twenty minutes after PEG was added to one nutrient chamber, the water potential of the roots bathed in that chamber (Root–Stressed) was -0.45 MPa, but the water potential of the roots in the other nutrient chamber (Root–Nonstressed) was -0.25 MPa, near the pretreatment value. In the plants sampled after 4 d of stress, both Root–Nonstressed and Root–Stressed increased by nominally 0.1 MPa, compared with the initial pretreatment plant and mirroring the increase in Leaflet.
In addition to the preliminary experiments (Materials and methods), four replicates confirmed the leaf conductance results in Table 1. Altogether, there was a significant decline in conductance within 20 min of PEG treatment (P>0.95), a continued decline for 4 h (r2=0.71) followed by an increase. One complete replicate experiment confirmed the water potential results in Table 1. Overall, Leaflet were unaffected for 2 h following PEG treatment, suggesting the absence of foliar ABA synthesis during this period. Thereafter, the change in Leaflet was small and the plants did not wilt. Thus, plants treated in this manner are suitable for studying the relationship of the ABA content of guard cells and leaf conductance.
ABA concentrations in whole root, root apoplastic sap, whole leaflet, and leaflet apoplastic sap following PEG treatment
The ABA concentrations in whole root tips and in root apoplastic sap were determined for both nutrient chambers following imposition of water stress to part of the root system (Fig. 1). In addition, the ABA concentrations in whole leaflet and leaflet apoplastic sap were determined (Fig. 2). These were the identical plants used for leaf conductance and water potential measurements (Table 1), and, thus, provide a comprehensive characterization of the particular plants used for guard cell ABA analysis (Fig. 3).
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) of plants that were used for guard cell ABA analysis. Polyethylene glycol 8000 was added to the nutrient chamber of part of the root system (upper curves, ‘Stressed root’) whereas the other part of the root system remained in an unaltered nutrient solution (lower curves, ‘Non-stressed root’). The inset is an expansion of the data over the first 4 h.
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) and of tissue samples (
) of the youngest fully expanded bifoliates of plants that were used for guard cell ABA analysis. For other details, see Fig. 1
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The ABA concentration in the whole-root tip of the pretreatment plant was 46 nmol ABA kg-1 fresh mass (Fig. 1
). Although the whole-root tip ABA concentration in non-stressed roots varied from plant to plant (25–71 nmol ABA kg-1 fresh mass, n=20), there was no regular pattern of ABA concentration differences in plants sampled after stress imposition (Fig. 1). The ABA concentration was 92 nmol ABA kg-1 fresh mass in the stressed whole-root tip sample taken 20 min after stress imposition (Fig. 1). The whole root tip ABA concentration increased steeply in the plants sampled for the first 4 h after PEG treatment (36 nmol ABA kg-1 fresh mass h-1, r2=0.92). The maximum ABA concentration in the stressed whole-root tips, 292 nmol kg-1 fresh mass (Fig. 1), was observed at 24 h of PEG treatment.
The ABA concentration in root apoplastic sap of the plant sampled before the PEG treatment was 47 nM (Fig. 1). In unstressed roots, the ABA concentrations in the apoplastic sap (20–76 nM, n=18) showed no regular pattern with PEG treatment time and was attributed to plant-to-plant variation. The ABA concentration in the apoplastic sap of the stressed root taken after 20 min of PEG treatment was 5x higher (Fig. 1) than that of the plant sampled before the treatment began. The maximum root apoplastic sap concentration sampled was 430 nM ABA (Fig. 1), which coincided with 4 h of PEG treatment. The root apoplastic sap ABA concentration in the stressed root of the plant sampled 6 d after stress imposition was similar to the concentrations in unstressed plants (Fig. 1).
The ABA concentration in whole leaflet of the pretreatment sample was 65 nmol kg-1 fresh mass (Fig. 2). In plants that were not water stressed, the whole-leaflet ABA concentration range was 56–61 nmol g-1 fresh mass (Fig. 2). The ABA concentration in the leaflet sampled after 20 min of PEG treatment was 1.6x higher than that of the initial sample. The maximum whole-leaflet ABA concentration in stressed plants (273 nmol kg-1 fresh mass) was observed at 24 h after stress imposition. The whole-leaflet ABA concentrations in stressed plants sampled during the final 4 d of the experiment were 190–230 nmol kg-1 fresh mass.
In the plant sampled before PEG treatment, the ABA concentration in leaflet apoplastic sap was 37 nM ABA (Fig. 2). The values (31–44 nM ABA, n=6) were similar in untreated plants subsequently sampled (Fig. 2). The ABA concentration in the leaflet apoplastic sap of the stressed plant sampled 20 min after imposition of stress was 5x higher (193 nM, Fig. 2) than that in the pretreatment plant. The maximum ABA concentration in the leaflet apoplastic sap (306 nM, Fig. 2) was observed in the plant sampled 24 h after stress imposition. The leaf apoplastic ABA concentration in the plant sampled after 6 d of stress was only 76 nM, nominally 40 nM higher than that of unstressed plants.
A complete replicate experiment confirmed the results of Figs 1 and 2. The most relevant of the above organ-level ABA concentrations to the guard cell ABA content is the leaflet apoplast ABA concentration. Thus, it is noted that the values (Fig. 2) are in the mid-range for a number of species (4–100 nM for unstressed plants and from 30–1800 nM for stressed plants, Slovik and Hartung, 1992).
The ABA content of guard cells of intact PEG-treated plants
The essence of this report is Fig. 3, the guard cell ABA pool sizes of intact stressed plants that had been characterized with respect to leaf conductance, water potential and organ-level ABA pools sizes.
The ABA content of guard cells of the pretreatment plant was 1.6 fg ABA guard cell pair-1. This ABA content of whole guard cells from the pretreated plant was in the same range as those measured previously in intact leaflets of Vicia (Harris et al., 1988; Popova et al., 2000). The guard cell ABA pool was much higher in stressed plants (Fig. 3). In the plant sampled 2 h after stress imposition, the ABA content was 4.2 fg guard cell pair-1 or 2.6x control values for these hydroponically cultured plants.
As a broad perspective, the guard cell ABA content in the plant sampled 2 h after stress imposition was 8x that of the maximum leaflet apoplastic ABA concentration (for conversion factors, see Outlaw and Lowry, 1977; Ewert et al., 2000). Compared with the pretreatment plant, the percentage change in the guard cell ABA pool of plants stressed for 2 h was approximately the same as that of the whole leaflet, but it was considerably less than that of the leaflet apoplast (4.7x). There was no difference in the guard cell ABA pool in the plants sampled between 2 h and 24 h after stress imposition (Fig. 3). For reference, the maximum value of the guard cell ABA pool (24 h, 4.9 fg guard cell pair-1) was 40% less than the maximum found when whole Vicia leaflets were severely water-stressed by dehydration to 90% of fresh mass (Harris et al., 1988). The guard cell ABA contents of plants sampled in the final 4 d of stress were less than the maximum values.
The results on guard cell ABA pool sizes were confirmed in two ways. First, the assays were repeated on separate leaflet fragments of the same plants for two critical points (0 h and 24 h, Fig. 3). Second, in a replicate experiment using plants of a different growth lot, assays were conducted at 0, 4 and 24 h.
Relationship between guard cell ABA content, leaf apoplastic ABA content and conductance
Leaf conductance was inversely proportional to guard cell ABA content (Fig. 4) with the proportionality factor being (-0.021 mol m-2 s-1)/(fg ABA guard cell pair-1). This proportionality factor is equivalent to (-0.2 µm aperture)/(amol ABA). Thus, the minimum guard cell ABA content (t0, untreated plant) corresponded to the maximum leaf conductance and the maximum guard cell ABA content (t24) corresponded to the minimum leaf conductance.
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The relationship between leaf apoplastic sap ABA concentration and conductance was curvilinear (not shown), as reported in other studies (Tardieu et al., 1993
; Ismail and Davies, 1998). The data from the experiments reported in Figs 1–3 were combined with the replicate experiment and the 10 leaflet apoplastic ABA concentrations up to 150 nM fit a straight line, conductance (mol m-2 s-1)=-0.0006 (nM ABA)+0.26 (r2=0.76) whereas the 8 leaflet apoplastic ABA concentrations of 150 nM or greater showed a different relationship, conductance (mol m-2 s-1)=-0.0001 (nM ABA)+0.20 (r2=0.20). Analysis of variance of the data from the main experiment alone similarly indicated a biphasic relationship (P=0.02).
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Discussion |
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The primary objective of this study was to extend previous work on ABA localization during stress by determining the guard cell ABA content of intact plants that were subjected to continuous water stress. Although it is well known that ABA causes stomatal closure, the importance of interactions with natural ABA agonists or antagonists in the intact plant has not been assessed directly. Thus, a relationship (Fig. 4
) between guard cell ABA content (Fig. 3) and conductance (Table 1) was established over time since stress imposition, during which conductance decreased and then increased, and guard cell ABA content increased and then decreased. The correlation between these two parameters was convincing (r2=0.68) even though this study did not distinguish between the guard cell apoplastic pool and the guard cell symplastic pool. The values reported are a composite of these two pools and both an internal locus and an external locus for ABA perception have been reported. Thus, stomata close when guard cells are microinjected with ABA (Allan et al., 1994; Schwartz et al., 1994) and K-in channels are inhibited by ABA in the pipette during whole-cell patch clamp (Wang et al., 1998). Conversely, ABA microinjection has failed to cause stomatal closure (Anderson et al., 1994), and other data such as stimulation of inward Ca2+ conductance by external ABA and the presence of various elements of the IP3 signalling cascade (for references, see MacRobbie, 1997) indicate the presence of a plasma membrane-localized ABA receptor. Other work (Zhang and Outlaw, 2001) is directed toward identifying the importance of the two guard cell ABA pools under stress.
An increase in guard cell ABA content may result from synthesis by guard cells themselves (Cornish and Zeevaart, 1986) or by synthesis in other foliar compartments (Harris et al., 1988) and subsequent redistribution to guard cells (Popova et al., 2000).) Alternatively, ABA may be imported by the xylem as a signal from stressed roots (see Introduction). In the present experiments, stress was applied to the root, and the root apoplastic ABA concentrations (Fig. 1) and the leaflet apoplastic ABA concentrations (Fig. 2) were higher in all stressed plants than in any unstressed plants. These data, along with the absence of a detectable change in Leaflet of the plants sampled within 2 h of stress imposition (Table 1), are consistent with the leaflet apoplast, and ultimately, the root apoplast, as the source for guard cell ABA, at least during the initial phases of the experiment. However, the minimum values for conductance coincided with an apparent decline in Leaflet (Table 1), which opens the possibility that leaflet ABA after 2 h of stress may not be all imported. Thus, it is important to note (Fig. 4) that elevated guard cell ABA content in the first 2 h of the experiment diminished conductance.
Under ambient conditions like those used for this study, there is a positive relationship between the concentrations of sucrose in the leaflet apoplast and in the guard cell apoplast (Lu et al., 1997). Similarly, there is a positive relationship between the concentration of mannitol fed into the petiole and the concentration in the guard cell apoplast (Ewert et al., 2000). In both of these cases, the effect was to concentrate the solute by distillation at the guard cell wall. According to this mechanism, the accumulation of an apoplastic solute in the guard cell wall would depend on many factors such as the concentration of the solute in the leaf apoplast, the rate of transpiration, and the rate of removal of the solute from the guard cell apoplast. The simplest interpretation of the present data is that the apoplastic ABA behaves similarly to apoplastic sucrose and apoplastic mannitol. If this is the case, a concentration of 150 nM ABA in the leaflet apoplast would predict a guard cell apoplastic ABA concentration of perhaps 1.5–3 µM. For perspective, (a) 3 µM ABA in the guard cell apoplast is 3 fg ABA guard cell pair-1, which is the maximum observed increase in guard cell ABA content (Fig. 3) and (b) 1 µM (±) ABA in the presence of 0.1 mM Ca2+ caused a 50% diminution of stomatal aperture size (unpublished results) and 10 µM (±) ABA did not cause a further decrease. Thus, the guard cell apoplastic ABA content predicted on the basis of the leaf apoplastic ABA content is consistent with the effects of exogenous ABA on isolated guard cells and explains why leaflet apoplastic ABA concentrations >150 nM were saturating. Importantly, this conclusion does not argue against the physiological importance of an internal locus as it was found that most of the ABA that accumulated in guard cells of a water-stressed leaflet was symplastic (Harris et al., 1988). As mentioned above, circumstances under which internal or external loci for ABA perception function are not known.
With implicit reservations, it is possible to interpret changes in the organ-level ABA pools that may serve as donors to the guard cell ABA pool. The concentration of ABA was measured in the apoplastic sap of the root and the leaf (Figs 1, 2) and in the whole root and leaflet (Figs 1, 2). The difference between the ABA content of the apoplast and that of the whole organ is the symplastic pool. Thus, in the case of the leaflet, where the relative volumes of the apoplast and symplast are known (Ewert et al., 2000), the calculations are straightforward and only emphasize the displayed data (Figs 1, 2). In brief, the decline in leaflet apoplastic ABA content with time after 24 h (Fig. 2; r2=0.92) was not accompanied by a change in symplastic ABA content with time (r2=0.01). Indeed, the calculated symplastic ABA pool in the leaflet showed little, if any, change after 2 h. Thus, the leaflet symplastic ABA pool, which increased during ABA import (Fig. 2), is a potential source for the redistribution of ABA to guard cells when the leaflet is stressed (cf. Popova et al., 2000). As a result, root stress possibly poises the leaflet to respond to foliar stress, which deserves further study. Similar calculations indicated that root symplastic ABA content of stressed roots declined after reaching a maximum, mirroring the whole-root ABA content (Fig. 1). Finally, an estimate of the relative rate of delivery of ABA to the leaflet (Jackson, 1997) was calculated as conductancexleaflet apoplastic ABA concentration. The linear relationship between the estimate for the relative delivery of ABA into the leaflet and the ABA content of guard cells was weak (r2=0.31), indicating apoplastic heterogeneity, sequestration of ABA in other foliar pools or catabolism of ABA or other confounding factors. Quantitative studies of ABA delivery to guard cells per se via the transpiration stream will be required to understand root-source ABA signalling.
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Acknowledgments |
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EW Weiler provided the ABA antibody. M Temkit performed some of the statistical analyses. Anonymous reviewers provided thorough critiques and important suggestions. The research was supported by a grant from the US Department of Energy to WHO and by the National Key Basic Research Special Funds (G 1999011700) of the People's Republic of China.
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Notes |
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1 To whom correspondence should be addressed. Fax: +1 850 644 0481. E-mail: outlaw{at}bio.fsu.edu2
2 Present address: College of Biological Sciences, China Agricultural University, Beijing, China 100094.
3 Present address: Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA.
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