Journal of Experimental Botany, Vol. 53, No. 367, pp. 287-296,
February 1, 2002
© 2002 Oxford University Press
Original Papers |
Is the ABA concentration in the sap collected by pressurizing leaves relevant for analysing drought effects on stomata? Evidence from ABA-fed leaves of transgenic plants with modified capacities to synthesize ABA
Laboratoire d'Ecophysiologie des Plantes sous Stress Environnementaux (LEPSE), UMR INRA-ENSAM, 2 place Viala, 34060 Montpellier Cedex 1, France
Received 20 September 2001; Accepted 5 October 2001
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Abstract |
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Most studies on the role of ABA in the stomatal response of the whole plant to drought rely on a good estimate of ABA concentration in xylem sap. In this report, varying volumes of sap (Vsap) were collected by pressurizing leaves cut from several lines of N. plumbaginifolia with modified capacities to synthesize ABA. Leaves were fed with solutions of known ABA concentration ([ABA]solution from 0–500 µmol m-3) for 2–3 h before sap collection. ABA concentration in extruded sap ([ABA]sap) was compared with [ABA]solution. In low-volume extracts (less than 0.35 mm3 cm-2 leaf area) collected from leaves of well-watered plants, [ABA]sap was close to [ABA]solution. For all lines, [ABA]sap decreased with increasing Vsap. The same dilution effect was observed for leaves pressurized just after sampling on droughted plants, suggesting, as for detached leaves fed with ABA, that [ABA]sap in low-volume extracts approximated well with the concentration of ABA entering leaves still attached on droughted plants. However, ABA-fed leaves sampled from droughted plants yielded higher [ABA]sap than ABA-fed leaves sampled from well-watered plants. [ABA]sap was also increased, although very slightly, when leaves were preincubated in highly enriched ABA solution. This indicates that some leaf ABA contributed to the ABA concentration returned in the extruded sap. Consistently, [ABA]sap in medium-volume extracts (0.35–0.65 mm3 cm-2 leaf area) was lower for leaves sampled on under-producing lines than on the wild type. Despite these distortions between [ABA]solution and [ABA]sap in medium-volume extracts, stomatal conductance of ABA-fed leaves closely correlated with [ABA]sap with a similar relationship in all cases, whilst relationships with [ABA]solution were more scattered.
Key words: Abscisic acid, drought, Nicotiana plumbaginifolia, stomatal conductance, xylem sap.
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Introduction |
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Stomatal response to soil drying is partially mediated by abscisic acid (ABA) originating from roots and transported to the shoot via the transpiration stream (Davies and Zhang, 1991
). Stomata respond to the concentration of ABA in the guard cell apoplast (Harris and Outlaw, 1991; Hornberg and Weiler, 1984; Anderson et al., 1994), although mechanisms of perception remain poorly understood (Jacob et al., 1999). Because of the difficulties in measuring attomoles of ABA in guard cell apoplast (Zhang et al., 1991), ABA concentration in the xylem stream ([ABA]xyl) is usually substituted for the concentration in guard cell apoplast, both compartments being directly connected. Close relationships between stomatal conductance and [ABA]xyl of plants submitted to soil drying support this approximation (Liang et al., 1996; Tardieu and Simonneau, 1998). However, ABA can be removed from or released into the transpiration stream before reaching guard cells, depending on trans-membrane pH gradients around the ABA transport pathway (Daeter and Hartung, 1995; Wilkinson, 1999). Changes in transpiration rate are also suggested to influence the local accumulation of ABA in guard cell walls (Ewert et al., 2000), whilst metabolism of ABA can prevent build up (Trejo et al., 1995). Besides, the estimation of ABA concentration in the xylem stream can be distorted by the technique used to extract, store and analyse xylem sap (Munns et al., 1993; Sinclair et al., 1995; Dodd et al., 1996). To distinguish between these effects when studying the role played by ABA in plant response to drought, it is necessary to analyse the significance of [ABA]xyl.
Xylem sap is most usually collected by pressurizing plant parts enclosed in a Scholander pressure chamber to overcome the negative pressure that develops in the xylem vessels of transpiring plants (Schurr, 1998). The composition of xylem sap thus extruded from pressurized root systems varies with the exudation rate imposed by applying a given pneumatic pressure: the higher the exudation rate, the lower the concentration of most solutes (Schurr and Schulze, 1995; Else et al., 1995; Jokhan et al., 1996). A good estimate of solute concentration in xylem sap extruded from root systems requires that the exudation rate during sap collection should match the pre-existing transpiration rate of intact plant (Else et al., 1994). By contrast, submitting cut leaves to changes in pressure (and exudation rate) does not influence ABA concentration of leaf extracts (Liang et al., 1996; Dodd et al., 1996). This contrast between root and leaf extracts may be due to higher dilution of sap compounds by the water contained in root systems or surrounding them (Jackson, 1997). Pressurizing cut leaves thus appears more convenient for collecting undistorted xylem sap regardless of exudation rate. However, xylem sap of open vessels at the cut end of the leaf flows back into the leaf following excision and refills vessels during pressurization. Exchanges of solutes between apoplastic and symplastic compartments, including contamination by phloem sap (Correia and Pereira, 1994) or by the symplasm of wounded cells at the leaf section (Zhang and Davies, 1990), probably accompany this ebb and flow of apoplastic water (Canny, 1993; Sperry et al., 1996).
Insights into water and solute exchanges between leaf compartments during pressurization can be gained by fractionating sap collected from a single leaf into several successive extracts. ABA concentration in these extracts either increases with the volume of collected sap (Liang et al., 1996, on leaves of Acacia confusa and Litsea glutinosa), decreases (Triboulot et al., 1996, on oak shoots), or peaks for intermediate volumes (Hartung et al., 1988, on cotton leaves). Considerable work would be needed to understand how the dynamic contribution of the leaf compartments during pressurization could explain these contrasting influences.
The present report practically examines if sap collected by pressurizing cut leaves is relevant for characterizing the role played by ABA in stomatal response to drought. Two aspects are specifically addressed: (i) whether ABA concentration in sampled sap reflects the concentration fed to leaves of various genetic and environmental origins well and (ii) if stable relationships can be drawn between stomatal conductance and ABA concentration recovered in pressurized sap. The influence of the volume of extruded sap was analysed by fractionating sap collection. To examine whether ABA originating from different leaf compartments contaminated extruded sap, two specific sets of experiments were carried out. First, ABA concentration in sap extruded from ABA-fed leaves was compared between leaves sampled from well watered plants, and leaves sampled from water-stressed plants (with elevated ABA content). Second, transgenic lines of N. plumbaginifolia with modified capacities to synthesize ABA were compared to the wild type. The concentrations of ABA in the feeding solution and in extruded sap were compared for each line with regards to its capacity to synthesize ABA.
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Materials and methods |
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Plant material and growing conditions
Details on the wild type and transgenic lines of Nicotiana plumbaginifolia used in this study have been described earlier (Borel et al., 2001
). Briefly, N. plumbaginifolia var. viviani and the ABA-deficient zep mutant (aba2-s1 in Marin et al., 1996) were transformed with different sense or antisense ZEP cDNA constructs fused to a neomycin phosphotransferase sequence which conferred kanamycin resistance (Frey et al., 1999). Four transgenic lines together with the wild type (WT) and zep were retained: W-S1 and M-S2 were respectively obtained by transformation of WT and zep with a full-length ZEP cDNA sense construct, whereas W-AS15 was obtained by transformation of WT with a ZEP cDNA antisense fragment. All transgenic lines presented a single insertion of the ZEP construct (Borel, 1999).
Transgenic lines exhibited modified capacities to accumulate ABA. Compared to WT, M-S2 as well as W-AS15 under-accumulated ABA in seeds and presented, respectively, 2-fold and 5-fold lower ABA concentration in xylem sap in response to soil drying, whilst W-S1 overproduced ABA in seeds, but did not accumulate more ABA in xylem sap than WT (Frey et al., 1999; Borel et al., 2001). The mutant zep presented very low ABA content in all plant parts, and did not accumulate ABA in response to dehydration either in leaves or in roots (Audran et al., 1998; Borel et al., 2001).
Seeds were germinated for 4–6 weeks on agar medium (25/17 °C day/night, 16 h photoperiod) with kanamycin for the selection of transgenic plants. Zep plantlets were then hydroponically grown at 100% relative humidity in a growth chamber. All other lines were transferred to a greenhouse into 1.5 dm3 pots filled with a 1:1 (v/v) mixture of sieved peat and clay-sandy-loam soil from a field near Montpellier (France). Pots were wrapped with aluminium foil to minimize soil heating. Irrigation was automatically supplied four times a day (well-watered plants) using a one-tenth-strength modified Hoagland solution. Soil drying was imposed when stated (droughted plants) by withholding irrigation for 1–5 d starting at the flowering stage. The soil was covered with white perlite to avoid heating and direct evaporation during soil drying.
Sap extraction immediately after detaching leaves from droughted plants
Wild-type and transgenic (M-S2, W-S1, W-AS15) plants were submitted to various levels of soil dehydration as previously described, and placed in the dark for one night before leaf sampling. The first fully expanded leaf was cut on each plant, still in the dark, and placed into a Scholander-type pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, USA). The chamber gas pressure at which extruded sap first began to wet the cut surface (observed with a magnifying glass) was registered as the opposite of leaf water potential (
leaf). Osmotic potential of 20 sap extracts (about 50 mm3) extruded from 20 leaves of wild type and transgenic lines (M-S2, W-S1) was determined with a vapour pressure osmometer (Wescor 5520, Logan, Utah, USA), and ranged from -0.05 to -0.08 MPa without correlation with leaf (-0.4 to -1.7 MPa). Osmotic potential of extruded sap was therefore neglected, whatever the volume collected.
Sap collection from one same leaf was fractionated into several successive extracts (about 20 mm3 each) by applying step increases in pressure (up to 0.5 MPa over |leaf|). Sap was sequentially collected into 500 mm3 Eppendorf vials that fit onto the petiole, thus minimizing sap evaporation. The volume of each extract was determined by weighing (Precisa 100A-300M, 0.1 mg accuracy) assuming a density of 1 for the collected sap. Samples were then stored at -80 °C pending analyses.
ABA feeding to detached leaves with controlled evaporative demand
Plants at the flowering stage were placed in the dark for one night before each experiment. On the morning of experiments, the 12th leaf (fully expanded at this stage) was detached, recut in distilled water under reduced light, and immediately placed into plastic parallelepipedal flasks (50 cm3) containing 40 cm3 of a degassed buffer solution (KH2PO4 2 mol m-3, Ca(NO3)2 0.4 mol m-3, pH adjusted to 6.6 with NaOH) with varying concentrations (0–1000 µmol m-3) of synthetic (±)-ABA (Fluka). The concentration of (+)-ABA was calculated as half the applied concentration of (±)-ABA. Flasks were sealed with foam and covered with parafilm. Leaves were maintained inclined in the flasks by 45° to the horizontal, and placed 700 mm below metal halide lamps in the laboratory (800 µmol m-2 s-1 photosynthetic photon flux density, 28 °C air temperature and 40% relative humidity at the leaf level).
At the end of the feeding period (typically 3 h duration time unless otherwise stated), the turgid leaf was gently blotted on paper and rapidly placed into the pressure chamber. Sap collection from one same leaf was fractionated into several samples of 15–80 mm3 volume that were immediately stored at -80 °C for subsequent analyses. Samples of feeding solution were also collected at the end of the experiment, in order to check out ABA concentration. A few wilty leaves presented much lower leaf than the mean (-0.9 MPa compared to -0.4 MPa) and were discarded. Such behaviour, likely due to cavitation, was observed mainly for leaves fed with ABA-free solutions.
‘ABA-enriched’ leaves were first fed for 1.5 h with artificial sap containing 2000 µmol (+)-ABA m-3, then gently rinsed with water, and placed for the next 2.5 h in artificial sap containing from 0–500 µmol m-3 (+)-ABA.
‘Droughted’ leaves were harvested from wild-type plants subjected to soil water deficit as previously described. Pre-dawn leaf was measured before ABA feeding on a separate fully-expanded leaf of each droughted plant, and ranged between -0.6 and -1.3 MPa. Leaves were slightly wilty when placed in the flask for ABA feeding, but rapidly recovered a turgid aspect. leaf measured at the end of this experiment was similar to typical values measured on ABA-fed leaves of well-watered plants (-0.4 MPa), excepting rare cavitated leaves that were discarded.
Stomatal conductance of ABA-fed leaves
Every 20 min over the feeding period, flasks with leaves (or leaf replicas made from wet blotting paper) were weighed to determine transpiration rate (Jw), and leaf (or leaf replica) temperature (TL) was measured using an infrared thermometer (IR-74007 THI-300, Tasco, Osaka, Japan). Air temperature (T) and relative humidity were measured every 20 s (HMP35A, Vaisala, Finland), averaged and stored in a data logger every 10 min. Areas of leaves and leaf replicas (A) were measured by image analysis at the end of the experiment. Leaf area ranged from 80 to 180 cm2. The stomatal conductances (gs) of leaves and the boundary layer conductances (ga) of leaf replicas were calculated using the equation:
 | (001) |
is the gas constant.
ABA analysis and pH of xylem sap
After thawing and equilibration at room temperature, the volume of each sap sample was determined by weighing. Xylem pH was measured in sap samples with a microelectrode (Ingold, Mettler-Toledo, Switzerland). ABA concentration was then analysed in crude samples of xylem sap by radioimmunoassay (Quarrie et al., 1988) as previously described (Barrieu and Simonneau, 2000). Freezing, storage and thawing of sap samples did not alter pH nor ABA measurements (not shown). Specificity for ABA of the monoclonal antibody (MAC 252, provided by Dr SA Quarrie, Cambridge Laboratory, John Innes Centre, UK) was verified in xylem sap of N. plumbaginifolia by comparing RIA of crude sap samples with RIA of sap fractions selected by thin layer chromatography (Borel, 1999).
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Results |
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Preliminary experiments indicated that ABA concentration in the first 20 mm3 of sap expressed from ABA-fed leaves increased with the duration of feeding (Fig. 1
). Expressed sap of WT leaves fed for less than 1 h exhibited much lower ABA concentration ([ABA]sap) than imposed in the feeding solution ([ABA]solution). [ABA]sap was closer to [ABA]solution when ABA feeding lasted from 1.5–2 h, and was similar to [ABA]solution for feeding duration exceeding 2.5 h. Short feeding periods (40–60 min) allowed for the transpiration of about the same amount of water as contained in the leaf, whilst up to three times the total leaf water were transpired during the 3 h feeding periods. These amounts of transpired water were approximately reduced by half when [ABA]solution markedly increased which was accompanied by partial closure of stomata. In the following, only those duration times of ABA feeding of between 2.5 h and 3.5 h were retained.
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ABA concentration in sap extracted from pressurized leaves decreased as the volume of collected sap increased
By sequentially collecting sap from the same leaf, it was possible to analyse the influence of the volume of extruded sap on its ABA concentration. Typical results are presented for ABA-fed leaves of WT, W-S1 and M-S2 plants (Fig. 2a
). In all cases, [ABA]sap rapidly decreased below the concentration fed to the leaves, even for a moderate accumulated volume of sap, and stabilized around 100 µmol m-3 (half the concentration fed) in the last collected fractions, corresponding to the highest cumulated volume of collected sap (Fig. 2a). However, this relationship shifted among leaves. This was partly explained by differences in leaf size. Dividing the cumulated volume by leaf area (an indicator of leaf size independent of leaf water status) significantly reduced scattering among leaves (Fig. 2b).
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The influence of the volume of collected sap was similarly analysed in sap expressed from leaves just after sampling from intact plants at predawn. Irrigation of these plants was withheld from 1–4 d before leaf sampling, which resulted in predawn
leaf ranging from -0.36 MPa to -1.2 MPa. Overall, [ABA]sap exponentially decreased in sap fractions as the cumulated volume of extruded sap increased (Fig. 3). This result was clearer for leaves sampled from severely droughted plants (pre-dawn leaf ranging from -1.2 to -1.1 MPa, Fig. 3c), whilst the decrease in [ABA]sap was not significant for some leaves of mildly droughted plants (pre-dawn leaf higher than -0.9 MPa, Fig. 3a, b). This was partly explained by higher scattering of data points that accompanied low values of [ABA]sap in Fig. 3a. Changes in [ABA]sap with the volume of collected sap also appeared less pronounced for the under-producing-line W-AS15, whatever was the predawn water potential of the plant on which leaf was sampled.
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Parallel to changes in [ABA]sap with the volume, sap pH hardly varied (less than 0.2 pH unit) between all successive extracts from the same leaf pressurized just after sampling on the plant (data not shown). Drought stress slightly increased sap pH in the first extracts (about 0.1 pH unit increase as predawn leaf water potential decreased by 0.1 MPa) whilst the influence of drought was not clear for the following extracts.
ABA concentration in sap extruded from ABA-fed leaves was lower for lines with severely reduced capacities to synthesize ABA than for WT
Sap samples of 20–60 mm3 volume were collected from ABA-fed leaves of the different lines including zep. The volume of collected sap was adapted to the size of each leaf to keep the sap volume:leaf area ratio between 0.35 and 0.65 mm3 m-2. The overexpressing line W-S1 and the transformed mutant M-S2, exhibited the same [ABA]sap as the wild type (Fig. 4a). By contrast, the ABA-deficient mutant and, to a much lower extent, the under-producing line W-AS15, yielded lower [ABA]sap than imposed in the feeding solution (Fig. 4a). The same ranking in [ABA]sap among lines was conserved whatever the volume of collected sap (from about 0.1 mm3 m-2 (Fig. 4b) up to 3 mm3 m-2, not shown).
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In the first collected fraction of sap (25 mm3 maximal volume), differences in [ABA]sap among lines were less pronounced and very close to the concentration imposed in the feeding solution (Fig. 4b
). Only leaves of the under-producing line W-AS15 exhibited slightly lower [ABA]sap than [ABA]solution, specifically for the intermediate concentration of ABA in the feeding solution. The range of ABA concentrations imposed in the feeding solution was the same as those observed in the sap expressed from WT leaves sampled on droughted plants with predawn leaf water potential ranging from -0.2 MPa to -1.0 MPa.
ABA concentration in sap extruded from ABA-fed leaves was markedly higher when leaves were sampled from droughted rather than well-watered plants, but only slightly higher when leaves were preincubated in solution with elevated ABA concentration
The accumulation of ABA into the leaves being studied was imposed either endogenously, by submitting plants to soil water deficit before leaf sampling (‘droughted’ leaves), or exogenously by feeding leaves with 2000 µmol m-3 (+)-ABA solutions (‘ABA-enriched’ leaves). Further feeding of these leaves with less concentrated solutions (as before) for 2–3 h led to higher [ABA]sap than feeding control leaves sampled from well-watered plants without preincubation (Fig. 5). The most striking effect was noticed for ‘droughted’ leaves fed with low [ABA]solution (below 100 µmol m-3): in this case, [ABA]sap even exceeded the concentration imposed in the feeding solution. This was also observed when leaves were preincubated in ABA-enriched solutions then fed with ABA-free solutions, which yielded substantial non-zero [ABA]sap (Fig. 5). The influence of preincubation on [ABA]sap was not detectable (compared to control leaves) when [ABA]solution varied from 250 to 500 µmol m-3. Interestingly, changes in [ABA]solution similarly influenced [ABA]sap of control and droughted leaves, since increases in [ABA]sap with [ABA]solution were roughly parallel between treatments.
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Stomatal conductance of ABA-fed leaves better correlated with [ABA]sap than with [ABA] in the feeding solution
Stomatal conductance more closely correlated with [ABA]sap than with [ABA]solution particularly for ABA-fed leaves for which pretreatment (sampling on droughted plants or preincubation in elevated [ABA]solution) induced distortions between [ABA]sap and [ABA]solution (Fig. 6). For a given concentration of ABA in the feeding solution, [ABA]sap in single extracts (60–105 mm3, corresponding to 0.52–1.1 mm3 cm-2 leaf area) was systematically higher when leaves came from droughted plants or when they were preincubated in 2000 µmol ABA m-3 solution, compared to leaves sampled from well-watered plants (Fig. 6). Stomatal conductance exponentially decreased with [ABA]sap with a common relationship whatever the pretreatment of the leaf (Fig. 6a). By contrast, relationships between stomatal conductance and [ABA]solution differed among leaf treatments and were looser and more scattered for leaves sampled from droughted plants and leaves preincubated in elevated [ABA]solution (Fig. 6b).
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The concentration of ABA in sap extracted after incubating detached leaves in artificial sap of a known ABA concentration can be affected by the leaf itself
Leaves from the under-producing line W-AS15 and the ABA-deficient mutant zep returned sap with a lower [ABA] than WT, M-S2 and W-S1 (Fig. 4a
). Whatever caused the distortions between [ABA]sap returned from ABA-fed leaves and [ABA]solution, this was obviously related to the leaf capacity to synthesize ABA. However, lower [ABA]sap was only observed in lines with severely reduced capacities to synthesize ABA (22% of the capacity of wild type in W-AS15, and close to 0% in zep; Borel et al., 2001).
Intringuingly, the relationships between [ABA]sap and [ABA]solution were not parallel among transformants (Fig. 4a) whilst they were among the treatments of Fig. 5. If the difference in [ABA]sap between leaf extracts of WT and zep (Fig. 4a) were to come exclusively from the extraction of ABA synthesized, stored and returned by the leaf symplast, the same difference between WT and zep would be expected at any given [ABA]solution; that was not the case (Fig. 4a). More consistently, leaves of zep had a higher capacity to trap fed-ABA into symplastic compartments (presumably depleted in ABA at the beginning of feeding) than leaves of other genotypes. This can explain why the distortions among [ABA]sap of the different genotypes increased with the concentration fed to the leaves.
Experiments where leaves were preincubated into ABA-enriched solutions or sampled from droughted plants also supported that some ABA originating from leaf compartments can contaminate extruded sap. Notably, when detached leaves were supplied with ABA-free solutions, some ABA was recovered in sap extracts of moderate volume extruded from leaves sampled on water-stressed plants and from leaves preincubated in ABA-enriched solutions (Fig. 5). [ABA]sap also exceeded [ABA]solution when up to 100 µmol ABA m-3 was fed to leaves detached from water-stressed plants. Such leaves accumulated ABA in leaf compartments when they were still attached to the droughted plant, or when they were preincubated in ABA-enriched solution. The extrusion of leaf ABA during sap collection from these leaves can be the cause of the roughly constant distortion between [ABA]sap and [ABA]solution (Fig. 5). Leaf ABA was also shown to contribute to ABA present in the xylem sap of sunflower plants (Neales and McLeod, 1991).
The influence of the volume of collected sap on [ABA]sap was also clearly evidenced in this study. This induced markedly lower ABA concentrations in high-volume extracts than in the solution fed to detached leaves. The most simple interpretation of this distortion is based upon the contamination by water originating from a symplastic compartment in which ABA could be more concentrated but sequestered away from the apoplast, following the mechanism of alkaline trapping of the weak acid ABA (Hartung et al., 1998). Alternatively, the sustained sequestration of ABA by the leaf symplast may create an apoplastic compartment depleted of ABA and not immediately accessible by pressurization in the first extracts of sap, but extruded in the following extracts. Such gradients of solute concentration in the leaf apoplast have been evidenced for Ca2+ (De Silva et al., 1996) and have been suggested for ABA (Ewert et al., 2000). Dilution of fed ABA with the volume of collected sap is particularly well evidenced in ABA-fed leaves of the ABA-deficient mutant zep which yielded very low [ABA]sap (less than 150 µmol m-3) even when [ABA]solution reached up to 375 µmol m-3 (Fig. 4a). Whereas low leaf ABA content in zep leaves was consistent with the low ABA synthesis rate of this line (Audran et al., 1998), it should also be admitted that ABA was metabolized or trapped at high rates to counterbalance the high delivery rates of exogenous ABA that entered these ABA-fed leaves at the highest imposed [ABA]solution. High rates of ABA metabolism measured in other species (Gowing et al., 1993; Daeter and Hartung, 1995; Jia et al., 1996) support this interpretation.
Collecting low volumes of sap can limit the influence of leaf ABA and give a reliable estimate of ABA concentration in the xylem stream of most lines
ABA-fed leaves detached from well-watered plants of all lines except zep returned [ABA] in low-volume extracts of sap that were similar to [ABA]solution (Fig. 4). Higher ABA concentration in low-volume extracts compared to subsequent extracts from the same leaf was unlikely due to contamination by ABA released from wounded cells, since the osmotic potential of extruded sap remained very close to zero. The influence of the volume of collected sap may have been due to the dilution of the leaf apoplastic fraction by symplastic water (without symplastic ABA) that was extruded during leaf pressurization. Consistently, sap samples were of substantial volume compared to the apoplastic leaf water fraction. This fraction was estimated by analysis of pressure–volume curves and reached, respectively, 5% and 10% for wild type and W-AS15 (Borel, 1999). In the present study, up to 200 mm3 cumulated volume of sap were extruded from leaves, corresponding to about 2–3% of total leaf water (for typical leaf sizes used in this study), that was a significant part of the apoplastic fraction. By contrast, the first extract from N. plumbaginifolia leaves consisted of about 20 mm3 of extruded sap, that was less than 1% of the total leaf water, and was therefore hardly contaminated by symplastic water.
Contrary to ABA-fed leaves, ABA concentration in the xylem stream entering leaves of intact droughted plants was not known. In the absence of any reference method, it cannot be definitely concluded that ABA concentration in low-volume extracts from leaves just sampled on droughted plants also gave a good estimate of ABA concentration in the xylem stream. However, the same influence of the volume of collected sap on its ABA concentration was similarly observed whether leaves were pressurized just after sampling on droughted plants (Fig. 3), or detached and fed with solutions of known ABA concentration before sap collection (Fig. 2). It can be inferred that [ABA]sap in low-volume extracts approximated well with the concentration of ABA entering leaves still attached on droughted plants, as it was found with ABA-fed leaves. Other techniques have suggested that [ABA] in sap extruded from pressurized organs is a reliable estimate of xylem [ABA], notably those where [ABA] was similar in sap samples collected from pressurized shoots and from pressurized roots of the same plant (Correia and Pereira, 1994; Borel et al., 1997). However, the results of Fig. 1 showed that at least 2.5 h of feeding time at a given ABA concentration were needed to reach equilibrium in ABA concentration between the feeding solution and low-volume sap extracts. This suggests that rapid fluctuations of [ABA] in the xylem stream of intact plants may induce distortion between instantaneous [ABA] in the xylem stream and [ABA] recovered in sap extracts.
Special attention should be paid to the typical volume of sap required for ABA assays (about 150 mm3 in most studies). It is preferable to collect a standardized volume of sap since it influences the resulting [ABA]. The leaf size which varies with growing conditions and among species should also be taken into account. Therefore, the volume of sap should not be considered in absolute units, but should be related to the amount of water contained in each pressurized leaf.
ABA concentration measured in sap expressed from pressurized leaves is a relevant predictor of stomatal conductance
Another striking result of this study is that stomatal conductance of ABA-fed leaves better correlated with [ABA]sap than with [ABA]solution (Fig. 6). Notably, leaves that were harvested on droughted plants yielded significantly higher [ABA]sap, but had lower stomatal conductance, than control leaves sampled from well-watered plants and fed with the same [ABA]solution. Lower distortion between [ABA]solution and [ABA]sap of control leaves was observed when leaves were preincubated in ABA-enriched solutions (Fig. 5), but again, slightly higher [ABA]sap was associated with lower stomatal conductance than for control leaves. Similar results were obtained on vine (Correia et al., 1995): detached leaves fed with ABA-free solutions exhibited a 2-fold lower stomatal conductance when leaves were harvested on water-stressed plants than when they were sampled from well-watered plants.
The close relationship between [ABA]sap and stomatal conductance in Fig. 6 suggests that extravascular ABA which contaminated the collected sap also participated in the control of stomatal aperture. Specifically, the leaf ABA that was extracted from leaves of droughted plants (or from ‘ABA-enriched’ leaves, Fig. 5) must reflect the elevated ABA concentration in one (or more) leaf compartment(s) that was responsible for higher stomatal closure (compared to the single effect of exogenous ABA fed via the petiole in the xylem stream). Accordingly, stomatal conductance of plants acclimated to drought (that have accumulated ABA in leaf compartments) should be sensitized to further fluctuations in xylem ABA delivered into the leaves. Indeed, drought stress of 2–3 d increases stomatal sensitivity to ABA entering the leaves (fed via the petioles of detached leaves; Davies, 1978; Ackerson, 1980; Peng and Weyers, 1994). This strongly suggests that both leaf ABA, related to water-stress history, and xylem ABA currently feeding the leaf act together on stomatal conductance. Collecting a substantial volume of sap, as in Fig. 5, may be a way to take into account both sources. These sources co-evolve during a single soil-drying cycle, and therefore can similarly explain changes in stomatal conductance. By contrast, transient soil water deficit as well as rapid fluctuations in evaporative conditions are expected to uncouple changes in leaf ABA content from changes in xylem ABA concentration. In such situations, results of Fig. 6 suggest that [ABA]sap in medium-volume extracts (which is partly influenced by leaf ABA) can better explain changes in stomatal conductance than the concentration of ABA entering the leaves in the xylem stream.
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Conclusion |
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This study has defined the methodological precautions required for a good estimate of xylem sap [ABA] in pressurized leaf extracts. Specifically, the influence of the volume of collected sap was evidenced. The best estimate of [ABA] in the xylem stream entering the leaf was obtained in the lowest-volume samples. [ABA] in sap samples of higher volume was diluted compared to [ABA] fed to detached leaves, and also distorted by leaf ABA, but was a good predictor of stomatal conductance in all cases studied. The exact origin of the leaf ABA that contaminated the pressurized leaf extracts is not known, but the present results suggest that both leaf ABA and [ABA] in the xylem stream entering the leaf should be considered to account for changes in stomatal conductance. This can be achieved by collecting a substantial volume of sap (related to the leaf apoplastic water fraction) that contained extravascular leaf ABA together with xylem ABA. It can be of particular importance for predicting drought effects on stomatal conductance when uncoupling between leaf ABA content and xylem sap [ABA] is suspected (for example in plants rewatered after a soil drying episode). Overall, this study supports the use of the leaf pressurization technique for collecting xylem sap and assessing the role of ABA on stomata.
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Acknowledgments |
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The authors are grateful to Bertrand Muller and Francois Tardieu for helpful comments on the preliminary manuscript. Many thanks are also due to Philippe Barrieu and Isabelle Constant for precious experimental assistance, and to Anne Frey who provided the transgenic lines.
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Notes |
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1 To whom correspondence should be addressed. Fax: +33 4 67 52 2116. E-mail: simonneau{at}ensam.inra.fr
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Abbreviations |
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ZEP, zeaxanthin epoxidase; zep, N. plumbaginifolia ABA-deficient ZEP mutant; M-S2, zep complemented with a ZEP sequence; W-S1, wild-type N. plumbaginifolia transformed with an additional ZEP sequence; W-AS15, wild-type N. plumbaginifolia transformed with an antisense ZEP fragment; [ABA]xyl, ABA concentration in the xylem sap; [ABA]sap, ABA concentration measured in sap collected by pressurizing cut leaves; [ABA]solution, ABA concentration in the solution fed to detached leaves;
leaf, leaf water potential; RWClea, leaf relative water content; Vsap, cumulated volume of sap collected by pressurizing cut leaves. |
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