Abscisic acid in apoplastic sap can account for the restriction in leaf conductance of white lupins during moderate soil drying and after rewatering

We studied the effects of drought on leaf conductance (g) and on the concentration of abscisic acid (ABA) in the apoplastic sap of Lupinus albus L. leaves. Withholding watering for 5d resulted in complete stomatal closure and in severe leaf water deficit. Leaf water potential fully recovered immediately after rewatering, but the aftereffect of drought on stomata persisted for 2d. ABA and sucrose were quantified in pressurized leaf xylem extrudates. We assumed that the xylem sucrose concentration is negligible and hence that the presence of sucrose in leaf extrudates indicated that they were contaminated by phloem. To eliminate this interference, the concentration of ABA in leaf apoplast was estimated by extrapolation to zero sucrose concentration, using the regression between ABA and sucrose concentrations. The estimated apoplastic ABA concentration increased by 100-fold with soil drying and did not return to pre-stress values immediately following rewatering. g was closely related to the concentration of ABA in leaf apoplast. Furthermore, the feeding of exogenous ABA to leaves detached from well-watered plants brought about the same degree of depression in g as resulted from the drought-induced increase in ABA concentration. We therefore conclude that the observed changes in the concentration of ABA in leaf apoplast were quantitatively adequate to explain drought-induced stomatal closure and the delay in stomatal reopening following rewatering.     

ABA xylem concentrations determine maximum daily leaf conductance of field-grown Vitis vinifera L. plants

Differences in maximum leaf conductance in grapevine plants growing in soils with contrasting water availabilities during mid-summer in Portugal could be accounted for by differences in the concentration of ABA in xylem sap. This conclusion is reinforced by the observation that the relationship between leaf conductance and endogenous ABA concentration can be mimicked by the application of exogenous ABA to leaves detached from irrigated plants. During the day, leaf conductance decreased after a morning peak, even when the leaves remained in a constant environment at a moderate temperature and leaf-to-air vapour pressure difference. This decline in leaf conductance was not a consequence of an increase in the xylem ABA concentration or the rate of delivery of this compound by the transpiratory stream. The afternoon depression in leaf conductance was associated with an apparent limitation in stomatal opening potential, which persisted even when detached leaves were fed with water and rehydrated. The reason for this inhibition has still to be identified.     

Relationship between changes in the guard cell abscisic-acid content and other stress-related physiological parameters in intact plants

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.     

Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure

Petioles of water‐sufficient intact Vicia faba L. plants were infused with 1 µm abscisic acid (ABA) to simulate the import of root‐source ABA. This protocol permitted quantitative ABA delivery, up to 300 pmol ABA over 60 min, to the leaf without ambiguities associated with perturbations in plant–water status. The ABA concentrations in whole‐leaf samples and in apoplastic sap increased with the amount infused; ABA degradation was not detected. The ABA concentration in apoplastic sap was consistent with uptake of imported ABA into the leaf symplast, but this interpretation is qualified. Our focus was quantitative cellular compartmentation of imported ABA in guard cells. Unlike when leaves are stressed, the guard‐cell symplast ABA content did not increase because of ABA infusion (P = 0·48; 3·0 ± 0·5 versus 4·0 ± 1·2 fg guard‐cell‐pair^?1). However, the guard‐cell apoplast ABA content increased linearly (R² = 0·98) from ?0·2 ± 0·5 to 3·1 ± 1·3 fg guard‐cell‐pair^?1 (≈ 3·1 µm ) and was inversely related to leaf conductance (R² = 0·82). Apparently, xylem ABA accumulates in the guard‐cell wall as a result of evaporation of the apoplast solution. This mechanism provides for integrating transpiration rate and ABA concentration in the xylem solution.     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.     

How Do Stomata Read Abscisic Acid Signals?

When abscisic acid (ABA) was fed to isolated epidermis of Commelina communis L., stomata showed marked sensitivity to concentrations of ABA lower than those commonly found in the xylem sap of well-watered plants. Stomata were also sensitive to the flux of hormone molecules across the epidermal strip. Stomata in intact leaves of Phaseolus acutifolius were much less sensitive to ABA delivered through the petiole than were stomata in isolated epidermis, suggesting that mesophyll tissue and/or xylem must substantially reduce the dose or activity of ABA received by guard cells. Delivery of the hormone to the leaf was varied by changing transpiration flux and/or concentration. Varying delivery by up to 7-fold by changing transpiration rate had little effect on conductance. At a given delivery rate, variation in concentration by 1 order of magnitude significantly affected conductance at all but the highest concentration fed. The results are discussed in terms of the control of stomatal behavior in the field, where the delivery of ABA to the leaf will vary greatly as a function of both the concentration of hormone in the xylem and the transpiration rate of the plant.     

PH as a stress signal

The pH of the xylem sap of plants experiencing a range of environmental conditions can increase by over a whole pH unit. This results in an increased ABA concentration in the apoplast adjacent to the stomatal guard cells in the leaf epidermis, by reducing the ability of the mesophyll and epidermal symplast to sequester ABA away from this compartment. As a result the guard cell ABA receptors become activated and the stomata close, enabling the plant to retain water. Were it not for the low concentration of ABA ubiquitous to all land plants, the increase in the pH of the apoplast adjacent to the guard cell would induce stomatal widening, and cause excessive water loss. Not only does ABA prevent this potentially harmful phenomenon, but it also converts the pH increase to a signal which can bring about plant protection.     

ABA-based chemical signalling: the co-ordination of responses to stress in plants

There is now strong evidence that the plant hormone abscisic acid (ABA) plays an important role in the regulation of stomatal behaviour and gas exchange of droughted plants. This regulation involves both long-distance transport and modulation of ABA concentration at the guard cells, as well as differential responses of the guard cells to a given dose of the hormone. We will describe how a plant can use the ABA signalling mechanism and other chemical signals to adjust the amount of water that it loses through its stomata in response to changes in both the rhizospheric and the aerial environment. The following components of the signalling process can play an important part in regulation: (a) ABA sequestration in the root; (b) ABA synthesis versus catabolism in the root; (c) the efficiency of ABA transfer across the root and into the xylem; (d) the exchange of ABA between the xylem lumen and the xylem parenchyma in the shoot; (e) the amount of ABA in the leaf symplastic reservoir and the efficiency of ABA sequestration and release from this compartment as regulated by factors such as root and leaf-sourced changes in pH; (f) cleavage of ABA from ABA conjugates in the leaf apoplast; (g) transfer of ABA from the leaf into the phloem; (h) the sensitivity of the guard cells to the [ABA] that finally reaches them; and lastly (i) the possible interaction between nitrate stress and the ABA signal.     

Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L

The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the `apoplastic' ABA, increased before `bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.

Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period.

     

Plasmalemma protection by the apoplast as assessed from above-zero ozone concentrations in leaf intercellular air spaces

To evaluate reactive absorption of ozone (O3) in the leaf apoplast, amphistomatous leaves of Phaseolus vulgaris L. were allowed to take up O3 through the stomata on the lower leaf surface at high rates for 3-5 min. Up to 5% of the O3 taken up diffused through the leaf and emerged from the stomata on the upper surface, suggesting above-zero O3 concentrations in the leaf intercellular air space, [O3]i. Moreover, measurements revealed that [O3]i increased during exposure to the pollutant. Time patterns of O3 fluxes through the gas phase and into the aqueous apoplast indicated that the increase in [O3]i was the result of a decrease in the diffusion-reaction conductance of the aqueous apoplast, gaq. Under an intense O3 pulse, gaq approached the value of the pure diffusional conductance within 2.5 min of exposure, suggesting the exhaustion of protective resources in the leaf apoplast. Toward the end of the exposure gaq tended to increase, suggesting either a recovery in the protective resources in the leaf apoplast and/or the induction of new defences. The possibility of estimating the degree of protection afforded by apoplast constituents and the rate of recovery of these protective systems in intact leaves using brief O3 pulses is discussed.     

Xylem ABA controls the stomatal conductance of field-grown maize subjected to soil compaction or soil drying

Stomatal conductance of individual leaves was measured in a maize field, together with leaf water potential, leaf turgor, xylem ABA concentration and leaf ABA concentration in the same leaves. Stomatal conductance showed a tight relationship with xylem ABA, but not with the current leaf water status or with the concentration of ABA in the bulk leaf. The relationship between stomatal conductance and xylem [ABA] was common for variations in xylem [ABA] linked to the decline with time of the soil water reserve, to simultaneous differences between plants grown on compacted, non-compacted and irrigated soil, and to plant-to-plant variability. Therefore, this relationship is unlikely to be fortuitous or due to synchronous variations. These results suggest that increased concentration of ABA in the xylem sap in response to stress can control the gas exchange of plants under field conditions.     

REVIEW ARTICLE: Compartmental redistribution and long-distance transport of abscisic acid (ABA) in plants as influenced by environmental changes in the rhizosphere --a biomathematical model

Based on experimental data obtained in earlier studies on membranepermeabilities of abscisic acid (ABA) for cortex and stele cellsof roots and on measured com-partmental pH shifts after onsetor release of different types of soil-borne stresses, a biomathematicalmodel was developed which permits computer analysis of the dynamicsof compartmental ABA distribution within different root tissues(cortex, stele) and their compartments (apoplast, cytosol, vacuole),and in the xylem sap of the root stele. Metabolism and conjugationof ABA and its export from roots via the xylem and its importinto roots via phloem sap flow are also taken into consideration.We want to know which soil-borne stresses can biophysicallyprovoke a root-to-shoot signal of ABA. In this communicationwe describe the biomathematical structure of the root modeland present all necessary morphological (volumes, surfaces etc.)and physiological (pH, membrane conductances etc.) parametersof unstressed roots. This root model and an available leaf modelare integrated to a plant model (rosette plant). Simulationsreveal the fundamental role of the stele tissues, the rhizosphericABA concentration and the ABA synthesis in roots (root-to-shootcommunication). The shoot-to-root communication strongly dependson ABA synthesis in leaves, but hardly on ABA redistributioneffects after stress-induced compartmental pH-shifts in leaves. Key words: Abscisic acid, compartmental redistribution, computer model, pH shifts, root-to-shoot communication, shoot-to-root communication     

Maize stomatal conductance in the field: its relationship with soil and plant water potentials, mechanical constraints and ABA concentration in the xylem sap

Abstract. Stomatal conductance, leaf water potential, soil water potential and concentration of abscisic acid (ABA) in the xylem sap were measured on maize plants growing in the field, in two treatments with contrasting soil structures. Soil compaction affected the stomatal conductance, but this effect was no longer observed if the soil water potential was increased by irrigation. Differences in leaf water potential did not account for the differences in conductance between treatments. Conversely, the relationship between stomatal conductance and concentration of ABA in the xylem sap was consistent during the experiment. The proposed interpretation is that stomatal conductance was controlled by the root water potential via an ABA message. Control of the stomatal conductance by the leaf water potential or by an effect of mechanical stress on the roots is unlikely.     

Stomatal closure is induced rather by prevailing xylem abscisic acid than by accumulated amount of xylem-derived abscisic acid

We have studied the stomatal response in relation to the xylem-derived abscisic acid (ABA) accumulation in sunflower leaves. When ABA was introduced into detached leaves of the sunflower through xylem flux, stomatal conductance was regulated, water flux was changed as a result and at the same time the xylem-derived ABA was metabolised in the leaves. We computed the xylem-derived ABA accumulation in the leaves as a function of time by taking into account the variation of ABA flux into the leaves (the product of water flux and ABA concentration) and a continuing ABA metabolism. We found that ABA accumulation was rapid during an initial lag phase, much slowed down during the decreasing phase of stomatal conductance, but still substantial when stomatal conductance reached a new stable state. The results show a poor link between the kinetics of ABA-induced stomatal closure and the xylem-derived ABA accumulation. Xylem-derived ABA was metabolised rapidly in the leaves. Tetcyclacis, as an inhibitor, substantially inhibited this process. Two hours after ABA was fed into a leaf, about 70% of the fed ABA was metabolised, but when tetcyclacis was added into the feeding solution, less than 30% of ABA was metabolised, even after 24 h of incubation. The inhibition of ABA metabolism by tetcyclacis did not lead to more stomatal closure, which was still concentration-dependent. Since the accumulation of xylem-derived ABA was enhanced substantially by the presence of tetcyclacis, these results strongly indicate that stomata mainly respond to the prevailing ABA concentration in the xylem stream, rather than to the accumulated amount of xylem-derived ABA in the leaves.     

气孔导度对CO2浓度变化的模拟及其生理机制

基于气孔运动的生理生化机制重点进行了气孔导度(gs)对CO2浓度变化的响应机制分析,并推导得到气孔导度(gs)对CO2浓度变化响应模型,并以9种植物进行了模型验证。结果表明:随着CO2浓度的升高,气孔导度会逐渐降低,且下降的幅度会随着CO2浓度的升高而逐渐减弱。气孔导度对CO2浓度(Cs)变化的响应模型可以表达为gs=gmax/(1+Cs/Cs0),其中式中gmax是最大气孔导度和Cs0是实验常数。该模型较好地模拟了气孔导度随CO2浓度变化的规律,模型参数具有明确的生理意义,与Jarvis模型和Ball-Berry模型相比,该模型如何实现多种环境因子的耦合有待进一步突破。另外,模型是在短期改变叶片CO2浓度的条件下得出的,在CO2浓度长期胁迫下的适用性也有待进一步确认。     

Can stomatal closure caused by xylem ABA explain the inhibition of leaf photosynthesis under soil drying?

Effects of leaf water deficit and increase in endogenous ABA on photosynthesis of two tropical trees, t Acacia confusa and t Leucaena leucocephala, were investigated with two soil-drying methods, i.e. half or whole root system was subjected to soil drying. Half-root drying was achieved by allowing upper layer of soil column to dry and lower layer of soil column to remain watered. Half-root drying had little effect on leaf water potential, but when compared to the well-watered control, both methods of soil drying substantially increased the ABA concentration in xylem and reduced leaf conductance in both species. There was a significant relationship between leaf conductance and xylem ABA concentrations in both species, which was comparable to the same relationship that was generated by feeding ABA to excised twigs. The rate of photosynthesis was inhibited substantially in both soil-drying treatments and in both species, but photochchemical efficiency, measured as a ratio of variable fluorescence to a peak fluorescence emission of a dark-adapted leaf (F_v/F_m), was not reduced except in the whole root-dried t L. leucocephala plants where leaf water potential was reduced to –2.5 MPa. In all the cases where photosynthesis was inhibited, there was a concomitant reduction in both leaf conductance and calculated internal CO₂ concentration. After two days of rewatering, leaf water potential and xylem ABA concentration rapidly returned to pre-treatment levels, but leaf conductance and photosynthesis of both whole-root and half root dried t L. leucocephala remained inhibited substantially. Rewatering led to a full recovery of both stomatal conductance and photosynthesis in soil-dried t A. confusa, although its photosynthesis of whole-root dried plants did not recover fully but such difference was not significant statistically. These results suggest that drought-induced decline of photosynthesis was mainly a result of the stomatal factor caused by the increase of ABA concentration in the xylem sap. Non-stomatal factors, e.g. reduced photochemical activity and/or carbon metabolic activity, were species-specific and were brought about only at very low water potential.     

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Immunogold Localization and Quantification of Cellular and Subcellular Abscisic Acid, Prior to and During Drought Stress

An Immunogold labeling procedure and experimental data are presented, which demonstrate that antibodies produced against a bovine serum albumin-abscisic acid conjugate can be used both to characterize the cellular and subcellular localization of abscislc acid (ABA), and to permit quantitative comparisons of this hormone in the subcellular compartments prior to and at times of drought stress. At the control leaf water potential (approximately -0.45 MPa), a quantitatively similar positive labeling pattern was observed in the chloroplasts and apoplast. A twofold drought stress-induced increase in the apoplastic ABA concentration was observed in the drought stressed leaf tissue (i.e., at a leaf water potential of approximately -1.55 MPa), while the ABA concentration in the chloroplasts did not differ from that of the controls. Three histochemical controls and the physiological observations validated the specificity of the procedure. Based on the labeling patterns we observed and literature cited, the validity of the hypothesis that drought stress induces a release of chloroplastic ABA is questioned. We interpreted our results as providing indirect evidence for a drought stress-induced root source origin for the increased apoplastic ABA concentrations.     

Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO ₃ ^- pools and in NO ₃ ^- flux to the xylem, particularly in tomato which had smaller tissue NO ₃ ^- reserves. Even in barley, tissue NO ₃ ^- reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO ₃ ^- flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA abscisic acid - c_i leaf internal CO₂ concentration - L_p root hydraulic conductance     

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.

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 (V(sap)) 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 micromol 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 mm(3) 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 V(sap). 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 mm(3) 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.