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.     

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.     

Early ABA Signaling Events in Guard Cells

The plant hormone abscisic acid (ABA) regulates a wide variety of plant physiological and developmental processes, particularly responses to environmental stress, such as drought. In response to water deficiency, plants redistribute foliar ABA and/or upregulate ABA synthesis in roots, leading to roughly a 30-fold increase in ABA concentration in the apoplast of stomatal guard cells. The elevated ABA triggers a chain of events in guard cells, causing stomatal closure and thus preventing water loss. Although the molecular nature of ABA receptor(s) remains unknown, considerable progress in the identification and characterization of its downstream signaling elements has been made by using combined physiological, biochemical, biophysical, molecular, and genetic approaches. The measurable events associated with ABA-induced stomatal closure in guard cells include, sequentially, the production of reactive oxygen species (ROS), increases in cytosolic free Ca²⁺ levels ([Ca²⁺]_i), activation of anion channels, membrane potential depolarization, cytosolic alkalinization, inhibition of K⁺ influx channels, and promotion of K⁺ efflux channels. This review provides an overview of the cellular and molecular mechanisms underlying these ABA-evoked signaling events, with particular emphasis on how ABA triggers an “electronic circuitry” involving these ionic components.     

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.     

Are diurnal patterns of stomatal movement the result of alternating metabolism of endogenous guard cell ABA and accumulation of ABA delivered to the apoplast around guard cells by transpiration?

Abscisic acid (ABA) prevents opening of closed stomata and causes open stomata to close. A dual-source model is proposed linking ABA to diurnal stomatal movements. Darkness would favour guard cell biosynthesis of endogenous ABA and disfavour ABA catabolism. At first light, xanthophyll cycling, isomerization of ABA precursors, and activation of a cytochrome P450 mono-oxygenase (CytP450) would deplete endogenous guard cell ABA. The NADPH-requiring CytP450 would be activated by elevated O2 and reduced CO2 concentrations resulting from mesophyll photosynthesis. An increased O2-to-CO2 ratio would limit the Calvin cycle in guard cells, diverting NADPH produced by photosynthetic electron transport to the cytosol where, along with elevated O2, it would activate CytP450. Depletion of endogenous ABA would liberate guard cells to extrude protons and accumulate the ions and water needed to increase guard cell turgor and open stomata. By midday, stomata would be regulated by steady-state concentrations of ABA delivered to the apoplast around guard cells by transpiration. In temperate conditions, ABA would reach concentrations high enough to trigger ion efflux from guard cells, but too low to defeat the accumulation of sugars used to maintain opening. In dry conditions, ABA would reach effective concentrations by midday, high enough to trigger ion efflux and inhibit sugar uptake, reducing apertures for the rest of the day. At sunset, conditions would again favour biosynthesis and disfavour catabolism of endogenous guard cell ABA. The model can be used to reconcile proposed cellular mechanisms for guard cell signal transduction with patterns of stomatal movements in leaves.     

Transpiration rate. An important factor controlling the sucrose content of the guard cell apoplast of broad bean

Evaporation of water from the guard cell wall concentrates apoplastic solutes. We hypothesize that this phenomenon provides two mechanisms for responding to high transpiration rates. First, apoplastic abscisic acid is concentrated in the guard cell wall. Second, by accumulating in the guard cell wall, apoplastic sucrose (Suc) provides a direct osmotic feedback to guard cells. As a means of testing this second hypothesized mechanism, the guard cell Suc contents at a higher transpiration rate (60% relative humidity [RH]) were compared with those at a lower transpiration rate (90% RH) in broad bean (Vicia faba), an apoplastic phloem loader. In control plants (constant 60% RH), the guard cell apoplast Suc content increased from 97 +/- 81 femtomol (fmol) guard cell pair(-1) to 701 +/- 142 fmol guard cell pair(-1) between daybreak and midday. This increase is equivalent to approximately 150 mM external, which is sufficient to decrease stomatal aperture size. In plants that were shifted to 90% RH before daybreak, the guard cell apoplast Suc content did not increase during the day. In accordance, in plants that were shifted to 90% RH at midday, the guard cell apoplast Suc content declined to the daybreak value. Under all conditions, the guard cell symplast Suc content increased during the photoperiod, but the guard cell symplast Suc content was higher (836 +/- 33 fmol guard cell pair(-1)) in plants that were shifted to 90% RH. These results indicate that a high transpiration rate may result in a high guard cell apoplast Suc concentration, which diminishes stomatal aperture size.     

Fern and lycophyte guard cells do not respond to endogenous abscisic acid

Stomatal guard cells regulate plant photosynthesis and transpiration. Central to the control of seed plant stomatal movement is the phytohormone abscisic acid (ABA); however, differences in the sensitivity of guard cells to this ubiquitous chemical have been reported across land plant lineages. Using a phylogenetic approach to investigate guard cell control, we examined the diversity of stomatal responses to endogenous ABA and leaf water potential during water stress. We show that although all species respond similarly to leaf water deficit in terms of enhanced levels of ABA and closed stomata, the function of fern and lycophyte stomata diverged strongly from seed plant species upon rehydration. When instantaneously rehydrated from a water-stressed state, fern and lycophyte stomata rapidly reopened to predrought levels despite the high levels of endogenous ABA in the leaf. In seed plants under the same conditions, high levels of ABA in the leaf prevented rapid reopening of stomata. We conclude that endogenous ABA synthesized by ferns and lycophytes plays little role in the regulation of transpiration, with stomata passively responsive to leaf water potential. These results support a gradualistic model of stomatal control evolution, offering opportunities for molecular and guard cell biochemical studies to gain further insights into stomatal control.     

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.     

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.     

ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity

In most plant species, a decrease in atmospheric humidity at the leaf surface triggers a decrease in stomatal conductance. While guard cells appear to respond to humidity‐induced changes in transpiration rate, as opposed to relative humidity or vapour pressure difference, the underlying cellular mechanisms for this response remain unknown. In the present set of experiments, abscisic acid (ABA)‐deficient (aba1) and ABA‐insensitive (abi1‐1 and abi2‐1) mutants of Arabidopsis thaliana were used to test the hypothesis that the humidity signal is transduced by changes in the flux or concentration of ABA delivered to the stomatal complex in the transpiration stream. In gas exchange experiments, stomatal conductance was as sensitive to changes in vapour pressure difference in aba1, abi1‐1 and abi2‐1 mutant plants as in wild‐type plants. These experiments appear to rule out an obligate role for either the concentration or flux of ABA or ABA conjugates as mediators of the guard cell response to atmospheric water potential. The results stand in contrast to the well‐established role of ABA in mediating guard cell responses to decreases in soil water potential.     

Long-distance ABA Signaling and Its Relation to Other Signaling Pathways in the Detection of Soil Drying and the Mediation of the Plant’s Response to Drought

In this article we review evidence for a variety of long-distance signaling pathways involving hormones and nutrient ions moving in the xylem sap. We argue that ABA has a central role to play, at least in root-to-shoot drought stress signaling and the regulation of functioning, growth, and development of plants in drying soil. We also stress the importance of changes in the pH of the leaf cell apoplast as influenced both by edaphic and climatic variation, as a regulator of shoot growth and functioning, and we show how changes in xylem and apoplastic pH can affect the way in which ABA regulates stomatal behavior and growth. The sensitivity to drought of the pH/ABA sensing and signaling mechanism is emphasized. This allows regulation of plant growth, development and functioning, and particularly shoot water status, as distinct from stress lesions in growth and other processes as a reaction to perturbations such as soil drying.     

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.     

Pyrabactin, an ABA agonist, induced stomatal closure and changes in signalling components of guard cells in abaxial epidermis of Pisum sativum

Pyrabactin, a synthetic agonist of abscisic acid (ABA), inhibits seed germination and hypocotyl growth and stimulates gene expression in a very similar way to ABA, implying the possible modulation of stomatal function by pyrabactin as well. The effect of pyrabactin on stomatal closure and secondary messengers was therefore studied in guard cells of Pisum sativum abaxial epidermis. Pyrabactin caused marked stomatal closure in a pattern similar to ABA. In addition, pyrabactin elevated the levels of reactive oxygen species (ROS), nitric oxide (NO), and cytoplasmic pH levels in guard cells, as indicated by the respective fluorophores. However, apyrabactin, an inactive analogue of ABA, did not affect either stomatal closure or the signalling components of guard cells. The effects of pyrabactin-induced changes were reversed by pharmalogical compounds that modulate ROS, NO or cytoplasmic pH levels, quite similar to ABA effects. Fusicoccin, a fungal toxin, could reverse the stomatal closure caused by pyrabactin, as well as that caused by ABA. Experiments on stomatal closure by varying concentrations of ABA, in the presence of fixed concentration of pyrabactin, and vice versa, revealed that the actions of ABA and pyrabactin were additive. Further kinetic analysis of data revealed that the apparent K(D) of ABA was increased almost 4-fold in the presence of ABA, suggesting that pyrabactin and ABA were competing with each other either at the same site or close to the active site. It is proposed that pyrabactin could be used to examine the ABA-related signal-transduction components in stomatal guard cells as well as in other plant tissues. It is also suggested that pyrabactin can be used as an antitranspirant or as a priming agent for improving the drought tolerance of crop plants.     

A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000

Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.     

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations

A paradigm for the response of plants to stress is presented which suggests that plants move towards a state of minimal metabolic activity as a stress intensifies and remain in that state until that stress is relieved. The paradigm is based on the proposition that cells that interface with the transpiration stream employ variations on the following theme to move towards that state. Tension on the apoplastic water opens a mechanosensitive Ca2+ channel, a response that is augmented by apoplastic ABA. The resulting elevated cytoplasmic Ca2+ deactivates a plasmalemma H+/ATPase and also activates a K(+)-H+ symport. The inflow of K+ and H+ depolarizes the membrane and renders the apoplast less acidic, the protons being removed to the vacuole and the K+ ions being re-exported via the K+ outward rectifying channel. The onset of darkness in guard and mesophyll cells deactivates the plasmalemma H+/ATPase and then the events outlined above ensue except that these cells do not appear to utilize either Ca2+ or ABA during these changes. In stressed cells it is proposed that elevated cytoplasmic Ca2+ activates the release of an ABA precursor from a stored form. ABA is then released in the apoplast after export of the precursor if the activity of the K(+)-H+ symport has brought the apoplastic pH close to 7.0. It is proposed that aquaporins in the xylem parenchyma and mesophyll cells are opened by elevated cytoplasmic Ca2+ when the water potential of the transpiration stream is high so that water can be stored in the 'xylem parenchyma reservoir'. The water in this reservoir is then used to increase the water potential in the transpiration stream when the water column is under tension and to help repair embolisms by a mechanism that resembles stomatal closure.     

The stomata frontline of plant interaction with the environment-perspectives from hormone regulation

Plants have evolved elaborate mechanisms to perceive and integrate signals from various environmental conditions.On leaf surface,stomata formed by pairs of guard cells mediate gas exchange,water transp...     

Xylem Sap pH Increase: A Drought Signal Received at the Apoplastic Face of the Guard Cell That Involves the Suppression of Saturable Abscisic Acid Uptake by the Epidermal Symplast

Drought increased the pH of Commelina communis xylem sap from 6.1 to 6.7. Conductances of transpiring leaves were 50% lower in pH 7.0 than in pH 6.0 buffers, but bulk leaf abscisic acid (ABA) concentration and shoot water status were unaffected by pH. Stomatal apertures of isolated abaxial epidermis incubated on simple buffers increased with external pH, so in vivo this must be overridden by alternative pH effects. Reductions in leaf transpiration rate at pH 7.0 were dependent on the presence of 10-8 mol dm-3 ABA in the xylem stream. We inferred that at pH 7.0 leaf apoplastic ABA concentrations increased: pH did not affect distributions of ABA among leaf tissues, but isolated epidermis and mesophyll tissue took up more 3H-ABA from pH 6.0 than from pH 7.0 buffers. The apoplastic ABA increase at pH 7.0 may result from reduced symplastic sequestration. A portion of 3H-ABA uptake by the epidermis was saturable at pH 6.0 but not at pH 7.0. An ABA uptake carrier may contribute to ABA sequestration by the leaf symplast of well-watered plants, and its inactivity at pH 7.0 may favor apoplastic ABA accumulation in draughted plants. Effects of external pH on stomatal apertures in the isolated epidermis indicate that published data supporting a role for internal guard cell ABA receptors should be reassessed.