Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and abi1-1 mutant Arabidopsis

Cortical actin filaments in guard cells of Commelina communis L. show signal-specific organization during stomatal movements [S.-O. Eun and Y. Lee (1997) Plant Physiol 115: 1491–1498; S.-O. Eun and Y. Lee (2000) Planta 210: 1014–1017]. To study the roles of actin in signal transduction, it is advantageous to use Arabidopsis thaliana (L.) Heynh., an excellent model plant with numerous well-characterized mutants. Using an immunolocalization technique, we found that actin deployments in guard cells of A. thaliana were basically identical to those in C. communis: actin proteins were assembled into radial filaments under illumination, and were disassembled by ABA. In addition, we examined actin organization in an ABA-insensitive mutant (abi1-1) to test the involvement of protein phosphatase 2C (PP2C) in the control of actin structure. A clear difference was observed after ABA treatment, namely, neither stomatal closing nor depolymerization of actin filaments was observed in guard cells of the mutant. Our results indicate that PP2C participates in ABA-induced actin changes in guard cells. Received: 23 June 2000 / Accepted: 20 October 2000     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells.

Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt).     

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.     

Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid

The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abi1-1 protein phosphatase. Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCI and 15 mM tetraethylammonium chloride (TEA-CI) to eliminate K⁺ channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 ± 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (I_Cl) as indicated from tail current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics. Pronounced activation of I_Cl was recorded on stepping from a conditioning voltage of ?250 mV to voltages between ?30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (τ? 0.3 sec at ?150 mV). Challenge with 20 µM ABA increased the steady-state current conductance, g_Cl, near 0 mV by 1.2- to 2.8-fold and at ?150 mV by 4.5- to sixfold with a time constant of 40 ± 4 sec, and it slowed I_Cl deactivation as much as fourfold at voltages near ?50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations. Neither the steady-state and kinetic characteristics of I_Cl, nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on g_Cl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing I_Cl sensitivity to ABA three- to four-fold. Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. bentamiana, indicating that the abi1-1 protein phosphatase does not influence the ànion current or its response to ABA directly. These results demonstrate that ABA stimulates I_Cl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes I_Cl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of I_Cl together with guard cell K⁺ channels to effect solute loss and stomatal closure.     

Abscisic Acid Activation of Plasma Membrane Ca2+ Channels in Guard Cells Requires Cytosolic NAD(P)H and Is Differentially Disrupted Upstream and Downstream of Reactive Oxygen Species Production in abi1-1 and abi2-1 Protein Phosphatase 2C Mutants

The hormone abscisic acid (ABA) regulates stress responses and developmental processes in plants. Calcium-permeable channels activated by reactive oxygen species (ROS) have been shown recently to function in the ABA signaling network in Arabidopsis guard cells. Here, we report that ABA activation of these I(Ca) Ca(2)+ channels requires the presence of NAD(P)H in the cytosol. The protein phosphatase 2C (PP2C) mutant abi1-1 disrupted ABA activation of I(Ca) channels. Moreover, in abi1-1, ABA did not induce ROS production. Consistent with these findings, in abi1-1, H(2)O(2) activation of I(Ca) channels and H(2)O(2)-induced stomatal closing were not disrupted, suggesting that abi1-1 impairs ABA signaling between ABA reception and ROS production. The abi2-1 mutation, which lies in a distinct PP2C gene, also disrupted ABA activation of I(Ca). However, in contrast to abi1-1, abi2-1 impaired both H(2)O(2) activation of I(Ca) and H(2)O(2)-induced stomatal closing. Furthermore, ABA elicited ROS production in abi2-1. These data suggest a model with the following sequence of events in early ABA signal transduction: ABA, abi1-1, NAD(P)H-dependent ROS production, abi2-1, I(Ca) Ca(2)+ channel activation followed by stomatal closing.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

Gaseous fluxes of peroxyacetyl nitrate (PAN) into plant leaves

Peroxyactyl nitrate (PAN) is the most abundant of the gaseous organic nitrates produced from the photochemistry of hydrocarbons and NO_x (i.e. ozone and smog production). PAN is known to be toxic to plants and also as a reservoir for the transport nitrogen dioxide in the troposphere. Here, the effect of vegetation on PAN deposition was investigated in four plant species by measuring leaf fluxes of PAN in a dynamic leaf chamber using atmospheric PAN fumigations between 0.7 and 18 nmol mol^?1. A linear relationship was observed between PAN flux and ambient PAN mixing ratio for all species. Depending on the species, measured PAN flux varied between 11 and 24 pmol m^?2 s^?1. Measured fluxes of PAN accounted for 12–48% of the PAN flux predicted solely from modelled stomatal conductance to PAN, suggesting the presence of a mesophyllic resistance to PAN uptake. The brief (approximately 5–10 min) exposure to PAN during uptake measurements did not affect photosynthesis, transpiration or conductance to water vapour. Increasing stomatal resistance by varying the vapour pressure gradient between the leaf chamber and leaf internal air space led to a corresponding drop in PAN uptake. Varying leaf nitrogen and total leaf–ascorbate concentrations did not appear to influence PAN uptake as had been reported for other reactive odd‐nitrogen gases. Measured and model‐predicted PAN fluxes were offset, but correlated suggesting that PAN flux could be estimated using established stomatal conductance algorithms.     

PHO1 expression in guard cells mediates the stomatal response to abscisic acid in Arabidopsis

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up‐regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a pho1 shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.     

Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2-1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1-1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.     

Open stomata 1 (OST1) kinase controls R–type anion channel QUAC1 in Arabidopsis guard cells

Under drought stress, the stress hormone ABA addresses the SnR kinase OST1 via its cytosolic receptor and the protein phosphatase ABI1. Upon activation, OST1 phosphorylates the guard cell S–type anion channel SLAC1. Arabidopsis ABI1 and OST1 loss‐of‐function mutants are characterized by an extreme wilting 'open stomata′ phenotype. Given the fact that guard cells express both SLAC‐ and R–/QUAC‐type anion channels, we questioned whether OST1, besides SLAC1, also controls the QUAC1 channel. In other words, are ABI1/OST1 defects preventing both of the guard cell anion channel types from operating properly in terms of stomatal closure? The activation of the R–/QUAC‐type anion channel by ABA signaling kinase OST1 and phosphatase ABI1 was analyzed in two experimental systems: Arabidopsis guard cells and the plant cell‐free background of Xenopus oocytes. Patch‐clamp studies on guard cells show that ABA activates R–/QUAC‐type currents of wild‐type plants, but to a much lesser extent in those of abi1–1 and ost1–2 mutants. In the oocyte system the co‐expression of QUAC1 and OST1 resulted in a pronounced activation of the R–type anion channel. These studies indicate that OST1 is addressing both S–/SLAC‐ and R–/QUAC‐type guard cell anion channels, and explain why the ost1–2 mutant is much more sensitive to drought than single slac1 or quac1 mutants.     

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1

? The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. ? We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. ? We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. ? This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

ABA inhibits entry into stomatal‐lineage development in Arabidopsis leaves

The number and density of stomata are controlled by endogenous and environmental factors. Despite recent advances in our understanding of stomatal development, mechanisms which prevent stomatal‐lineage entry remain unclear. Here, we propose that abscisic acid (ABA), a phytohormone known to induce stomatal closure, limits initiation of stomatal development and induces enlargement of pavement cells in Arabidopsis cotyledons. An ABA‐deficient aba2‐2 mutant had an increased number/proportion of stomata within a smaller cotyledon, as well as reduced expansion of pavement cells. This tendency was reversed after ABA application or in an ABA over‐accumulating cyp707a1cyp707a3 doublemutant. Our time course analysis revealed that aba2‐2 shows prolonged formation of meristemoids and guard mother cells, both precursors of stoma. This finding is in accordance with prolonged gene expression of SPCH and MUTE, master regulators for stomatal formation, indicating that ABA acts upstream of these genes. Only aba2‐2 mute, but not aba2‐2 spch double mutant showed additive phenotypes and displayed inhibition of pavement cell enlargement with increased meristemoid number, indicating that ABA action on pavement cell expansion requires the presence of stomatal‐lineage cells.     

Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection

Oxalic acid is a virulence factor of several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, but the detailed mechanisms by which oxalic acid affects host cells and tissues are not understood. We tested the hypothesis that oxalate induces foliar wilting during fungal infection by manipulating guard cells. Unlike uninfected leaves, stomatal pores of Vicia faba leaves infected with S. sclerotiorum are open at night. This cellular response appears to be dependent on oxalic acid because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. In contrast to oxalate-deficient S. sclerotiorum, wild-type fungus causes an increase in stomatal conductance and transpiration as well as a decrease in plant biomass. Green fluorescent protein-tagged S. sclerotiorum emerges through open stomata from the uninfected abaxial leaf surface for secondary colonization. Exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening. Guard cells treated with oxalic acid accumulate potassium and break down starch, both of which are known to contribute to stomatal opening. Oxalate interferes with abscisic acid (ABA)-induced stomatal closure. The Arabidopsis (Arabidopsis thaliana) L. Heynh. mutants abi1, abi3, abi4, and aba2 are more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. We conclude that oxalate acts via (1) accumulation of osmotically active molecules to induce stomatal opening and (2) inhibition of ABA-induced stomatal closure.     

Ozone‐triggered rapid stomatal response involves the production of reactive oxygen species,and is controlled by SLAC1 and OST1

The air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS‐dependent stomatal signaling. We show that the ozone‐triggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S‐type anion channel function, and the protein kinase OST1 were required for the ROS‐induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1‐7 (S120F) and slac1‐8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass‐spectrometry analysis combined with site‐directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant‐negative mutants abi1‐1 and abi2‐1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS‐induced activation of the S‐type anion channel.     

Environmental regulation of stomatal response in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Cvi-0 ecotype

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO₂] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl⁻). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO₂]-induced and low humidity-induced stomatal closure.     

Stomatal and environmental control of transpiration in a lowland tropical forest tree

Stomatal control of crown transpiration was studied in Anacardium excelsum, a large-leaved, emergent canopy species common in the moist forests of Central and northern South America. A construction crane equipped with a gondola was used to gain access to the uppermost level in the crown of a 35-m-tall individual. Stomatal conductance at the single leaf scale, and transpiration and total vapour phase conductance (stomatal and boundary layer) at the branch scale were measured simultaneously using the independent techniques of porometry and stem heat balance, respectively. This permitted the sensitivity of transpiration to a marginal change in stomatal conductance to be evaluated using a dimensionless coupling coefficient (1-ω) ranging from zero to 1, with 1 representing maximal stomatal control of transpiration. Average stomatal conductance varied from 0.09 mol m^?2 s^?1 during the dry season to 0.3 mol m^?2 s^?1 during the wet season. Since boundary layer conductance was relatively low (0.4 mol m^?2 s^?1), 1-ω ranged from 0.46 during the dry season to only 0.25 during the wet season. A pronounced stomatal response to humidity was observed, which strongly limited transpiration as evaporative demand increased. The stomatal response to humidity was apparent only when the leaf surface was used as the reference point for measurement of external vapour pressure. Average transpiration was predicted to be nearly the same during the dry and wet seasons despite a 1 kPa difference in the prevailing leaf-to-air vapour pressure difference. The patterns of stomatal behaviour and transpiration observed were consistent with recent proposals that stomatal responses to humidity are based on sensing the transpiration rate itself.     

Stomatal control by both [ABA] in the xylem sap and leaf water status: a test of a model for draughted or ABA-fed field-grown maize

A model of maize stomatal behaviour has been developed, in which stomatal conductance is linked to the concentration of abscisic acid ([ABA]) in the xylem sap, with a sensitivity dependent upon the leaf water potential (Ψ₁). It was tested against two alternative hypotheses, namely that stomatal sensitivity to xylem [ABA] would be linked to the leaf-to-air vapour pressure difference (VPD), or to the flux of ABA into the leaf. Stomatal conductance (g_s) was studied: (1) in field-grown plants whose xylem [ABA] and Ψ₁ depended on soil water status and evaporative demand; (2) in field-grown plants fed with ABA solutions such that xylem [ABA] was artificially raised, thereby decreasing g_s and increasing Ψ₁ and leaf-to-air VPD; and (3) in ABA-fed detached leaves exposed to varying evaporative demands, but with a constant and high Ψ₁. The same relationships between g_s, xylem [ABA] and Ψ₁, showing lower stomatal sensitivity to [ABA] at high Ψ₁, applied whether variations in xylem [ABA] were due to natural increase or to feeding, and whether variations in Ψ₁, were due to changes in evaporative demand or to the increased Ψ₁ observed in ABA-fed plants. Conversely, neither the leaf-to-air VPD nor the ABA flux into the leaf accounted for the observed changes in stomatal sensitivity to xylem [ABA]. The model, using parameters calculated from previous field data and the detached-leaf data, was tested against the observations of both ABA-fed and droughted plants in the field. It accounted with reasonable accuracy for changes in g_s (r² ranging from 0.77 to 0.81). These results support the view that modelling of stomatal behaviour requires consideration of both chemical and hydraulic aspects of root-to-shoot communication.     

Stomatal response to humidity in sugarcane and soybean: effect of vapour pressure difference on the kinetics of the blue light response

Abstract. The effect of atmospheric humidity on the kinetics of stomatal responses was quantified in gas exchange experiments using sugarcane ( Saccharum spp. hybrid) and soybean ( Glycine max ). Pulses of blue light were used to elicit pulses of stomatal conductance that were mediated by the specific blue light response of guard cells. Kinetic parameters of the conductance response were more closely related to leaf-air vapour pressure difference (VPD) than to relative humidity or transpiration. Increasing VPD significantly accelerated stomatal opening in both sugarcane and soybean, despite an approximately five-fold faster response in sugarcane. In contrast, the kinetics of stomatal recovery (closure) following the pulse were similar in the two species. Acceleration of opening by high VPD was observed even under conditions where soybean exhibited a feedforward response of decreasing transpiration (E) with increasing evaporative demand (VPD). This result suggests that epidermal, rather than bulk leaf, water status mediates the VPD effect on stomatal kinetics. The data are consistent with the hypothesis that increased cpidermal water loss at high VPD decreases the backpressure exerted by neighbouring cells on guard cells. allowing more rapid stomatal opening per unit of guard cell metabolic response to blue light.