Copper amine oxidase and phospholipase D act independently in abscisic acid (ABA)-induced stomatal closure in Vicia faba and Arabidopsis

Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 1.4.3.6) and phospholipase D (PLD; EC 3.1.4.4) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H₂O₂ production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H₂O₂ generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H₂O₂ production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H₂O₂ production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.     

Connections between sphingosine kinase and phospholipase D in the abscisic acid signaling pathway in Arabidopsis

Phosphatidic acid (PA) and phytosphingosine 1-phosphate (phyto-S1P) both are lipid messengers involved in plant response to abscisic acid (ABA). Our previous data indicate that PA binds to sphingosine kinase (SPHK) and increases its phyto-S1P-producing activity. To understand the cellular and physiological functions of the PA-SPHK interaction, we isolated Arabidopsis thaliana SPHK mutants sphk1-1 and sphk2-1 and characterized them, together with phospholipase Dα1 knock-out, pldα1, in plant response to ABA. Compared with wild-type (WT) plants, the SPHK mutants and pldα1 all displayed decreased sensitivity to ABA-promoted stomatal closure. Phyto-S1P promoted stomatal closure in sphk1-1 and sphk2-1, but not in pldα1, whereas PA promoted stomatal closure in sphk1-1, sphk2-1, and pldα1. The ABA activation of PLDα1 in leaves and protoplasts was attenuated in the SPHK mutants, and the ABA activation of SPHK was reduced in pldα1. In response to ABA, the accumulation of long-chain base phosphates was decreased in pldα1, whereas PA production was decreased in SPHK mutants, compared with WT. Collectively, these results indicate that SPHK and PLDα1 act together in ABA response and that SPHK and phyto-S1P act upstream of PLDα1 and PA in mediating the ABA response. PA is involved in the activation of SPHK, and activation of PLDα1 requires SPHK activity. The data suggest that SPHK/phyto-S1P and PLDα1A are co-dependent in amplification of response to ABA, mediating stomatal closure in Arabidopsis.     

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H₂O₂ and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H₂O₂ levels as the wild type in response to ABA. However, ABA- or H₂O₂-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H₂O₂ in ABA-induced stomatal closure.     

Regulation of stomatal movement by cortical microtubule organization in response to darkness and ABA signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Microtubule dynamics are essential for plant cell development and in producing responses to external stimuli. However, little is known about the regulation of microtubule dynamics or crosstalk between microtubule and stomatal movement. Here we identified microtubule reorganization as a crucial factor determining guard cell responses to dark and abscisic acid (ABA) signaling. As stomata opened, guard cells exhibited radially arranged cortical microtubules, which depolymerized into the cytosol when exposed to darkness and ABA. Suppression of microtubule disassembly by paclitaxel, a microtubule-stabilizing drug, significantly enhanced stomatal aperture under light, and partially blocked ABA- or darkness-induced stomatal closure. However, treatment with only the anti-microtubule drug, oryzalin, did not affect stomatal movement with or without external stimuli. Phosphatidic acid (PA) bound to a clade A type 2C protein phosphatase (PP2C), PP2CA, and deletion of PP2CA partially inhibited PA-induced microtubule depolymerization and stomatal closure. Moreover, microtubule reorganization was altered in the ABA-insensitive mutant pldα1, but not in the ABA-hypersensitive mutant pp2ca. We propose that a faithfully balanced reorganization of microtubules fulfills fundamental functions to enable the fast change of stomata in plant adaptive responses to developmental and environmental cues.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

Involvement of phospholipase C-independent calcium-mediated abscisic acid signalling during <Emphasis Type="Italic">Arabidopsis</Emphasis> response to drought

The present study investigated whether Ca²⁺ mobilization independent of phosphoinositide-specific phospholipase C (PI-PLC) would delay wilting in Arabidopsis thaliana (L.) Heynh. cv. Columbia through mediating stomatal closure at abscisic acid (ABA) concentrations rising beyond a drought-specific threshold value. In wild type (WT) epidermis, the PI-PLC inhibitor (U73122) affected the stomatal response to 20 μM ABA but not to 30 μM ABA. Disruption in GTP-binding protein ά subunit 1 (GPA1) affected the stomatal response to 30 μM ABA, but not to 20 μM ABA. In the gpa1-4 mutant, the inhibitory effects of the Ca²⁺ buffer, 1,2-bis(0-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), the inactive mastoparan analogue, mas17 and the antagonist of cyclic ADP-ribose synthesis, nicotinamide, were differentially attenuated on 30 μM ABA-induced stomatal closure. By contrast, the NADPH oxidase atrbohD/F double mutation fully suppressed inhibition of 20 μM ABA-induced stomatal closure by BAPTA or U73122 as well as inhibition of 30 μM ABA-induced stomatal closure by BAPTA, mas17 or nicotinamide. On the contrary, The Al resistant alr-104 mutation modulated ABA-induced stomatal closure by a stimulatory effect of U73122 and an increased sensitivity to mas17, nicotinamide and BAPTA. Compared to WT, the atrbohD/F double mutant was more hypersensitive than the gpa1-4 mutant to wilting under the tested water stress conditions, whereas wilting was delayed in the alr-104 mutant. Since the atrbohD/F mutation breaks down ABA-induced Ca²⁺ signalling through fully preventing apoplastic Ca²⁺ to enter into the guard cells, these results showed that a putative guard cell GPA1-dependent ADP-ribosyl cyclase activity should contribute to drought tolerance within PI-PLC-independent-Ca²⁺-mediated ABA signalling.     

PLDα1介导ABA调控的拟南芥主根伸长并参与根毛生长

探讨了磷脂酶Dα1（PLDα1）在ABA抑制拟南芥主根伸长过程中的作用。PLOα1基因突变体pldα1主根伸长受ABA抑制小于野生型（WT）;根系PLDα1活性在ABA处理下升高;拟南芥根细胞原生质体中活性氧（ROS）含量在ABA处理下升高,但是pldα1升高小于WT;根系NADPH氧化酶活性在ABA处理下升高,pldα1升高小于WT,外源加入10μmol/L^-1 PA（磷脂酸,PLD水解产物）后,前者活性显著升高;外源加入H2O2可诱导WT和pldα1主根伸长都受到抑制,且二者差异不明显。结果表明,PLDα1产生的PA通过激活NADPH氧化酶产生ROS介导ABA调控的拟南芥主根伸长过程。此外,初步探讨了PLDα1在拟南芥根毛尖端生长中的作用：pldα1突变体根毛长度小于WT,根毛尖端ROS和Ca^2＋浓度低于WT。     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H₂O₂, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.     

The Roles of CATALASE2 in Abscisic Acid Signaling in Arabidopsis Guard Cells

We investigated the roles of catalase (CAT) in abscisic acid (ABA)-induced stomatal closure using a cat2 mutant and an inhibitor of CAT, 3-aminotriazole (AT). Constitutive reactive oxygen species (ROS) accumulation due to the CAT2 mutation and AT treatment did not affect stomatal aperture in the absence of ABA, whereas ABA-induced stomatal closure, ROS production, and [Ca²⁺]_cyt oscillation were enhanced.     

Stomatal morphology and physiology explain varied sensitivity to abscisic acid across vascular plant lineages

Abscisic acid (ABA) can induce rapid stomatal closure in seed plants, but the action of this hormone on the stomata of fern and lycophyte species remains equivocal. Here, ABA-induced stomatal closure, signaling components, guard cell K⁺ and Ca²⁺ fluxes, vacuolar and actin cytoskeleton dynamics, and the permeability coefficient of guard cell protoplasts (P_f) were analyzed in species spanning the diversity of vascular land plants including 11 seed plants, 6 ferns, and 1 lycophyte. We found that all 11 seed plants exhibited ABA-induced stomatal closure, but the fern and lycophyte species did not. ABA-induced hydrogen peroxide elevation was observed in all species, but the signaling pathway downstream of nitric oxide production, including ion channel activation, was only observed in seed plants. In the angiosperm faba bean (Vicia faba), ABA application caused large vacuolar compartments to disaggregate, actin filaments to disintegrate into short fragments and P_f to increase. None of these changes was observed in the guard cells of the fern Matteuccia struthiopteris and lycophyte Selaginella moellendorffii treated with ABA, but a hypertonic osmotic solution did induce stomatal closure in fern and the lycophyte. Our results suggest that there is a major difference in the regulation of stomata between the fern and lycophyte plants and the seed plants. Importantly, these findings have uncovered the physiological and biophysical mechanisms that may have been responsible for the evolution of a stomatal response to ABA in the earliest seed plants.

Physiological and biophysical evidence for insensitivity of stomata to abscisic acid in ferns and lycophytes supports stomatal responsiveness to abscisic acid evolved after the divergence of ferns.     

Two Arabidopsis guard cell-preferential MAPK genes,MPK9 and MPK12, function in biotic stress response

Abscisic acid (ABA) plays a major role in plant development and adaptation to severe environmental conditions. ABA evokes cellular events to regulate stomatal apertures and thus contributes to the plant’s ability to respond to abiotic stresses. Reactive oxygen species (ROS) are produced in response to ABA and mediate ABA-induced stomatal closure. We have shown that two MAP kinases, MPK9 and MPK12, are highly and preferentially expressed in guard cells and function as positive regulators of ROS-mediated ABA signaling in guard cells. Cell biological and electrophysiological analyses demonstrated that MPK9 and MPK12 act downstream of ROS and cytosolic Ca²⁺ and upstream of anion channels in the guard cell ABA signaling cascade. Plant pathogens use stomata as the primary gateway to enter into their hosts, and previous studies have indicated crosstalk between ABA and defense signaling. Here we show that mpk9-1/12-1 double mutants are highly susceptible to Pseudomonas syringae DC3000 compared to WT plants. These results suggest that the regulation of stomatal apertures by MPK9 and MPK12 contributes to the first line of defense against pathogens.     

Cucumber <Emphasis Type="Italic">Phospholipase D alpha</Emphasis> gene overexpression in tobacco enhanced drought stress tolerance by regulating stomatal closure and lipid peroxidation

Background

Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. Both PLD and PA play key roles in plant growth, development, and cellular processes. PLD was suggested to mediate the regulation of stomatal movements by abscisic acid (ABA) as a response to water deficit. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (CsPLDα, GenBank accession number EF363796) in the growth and tolerance of transgenic tobacco (Nicotiana tabacum) to drought stress.

Results

The CsPLDα overexpression in tobacco lines correlated with the ABA synthesis and metabolism, regulated the rapid stomatal closure in drought stress, and reduced the water loss. The NtNCED1 expression levels in the transgenic lines and wild type (WT) were sharply up-regulated after 16?days of drought stress compared with those before treatment, and the expression level in the transgenic lines was significantly higher than that in the WT. The NtAOG expression level evidently improved after 8 and 16?days compared with that at 0?day of treatment and was significantly lower in the transgenic lines than in the WT. The ABA content in the transgenic lines was significantly higher than that in the WT. The CsPLDα overexpression could increase the osmolyte content and reduce the ion leakage. The proline, soluble sugar, and soluble protein contents significantly increased. By contrast, the electrolytic leakage and malondialdehyde accumulation in leaves significantly decreased. The shoot and root fresh and dry weights of the overexpression lines significantly increased. These results indicated that a significant correlation between CsPLDα overexpression and improved resistance to water deficit.

Conclusions

The plants with overexpressed CsPLDα exhibited lower water loss, higher leaf relative water content, and heavier fresh and dry matter accumulation than the WT. We proposed that CsPLDα was involved in the ABA-dependent pathway in mediating the stomatal closure and preventing the elevation of intracellular solute potential.

     

Calcium ions as second messengers in guard cell signal transduction

Ca²⁺ is a ubiquitous second messenger in plant cell signalling. In this review we consider the role of Ca²⁺-based signal transduction in stomatal guard cells focusing on three important areas: (1) the regulation of guard cell turgor relations and the control of gene expression in guard cells, (2) the control of specificity in Ca²⁺ signalling, (3) emerging technologies and new approaches for studying intracellular signalling. Stomatal apertures alter in response to a wide array of environmental stimuli as a result of changes in guard cell turgor. For example, the plant hormone abscisic acid (ABA) stimulates a reduction in stomatal aperture through a decrease in guard cell turgor. Furthermore, guard cells have been shown to be competent to relay an ABA signal from its site of perception to the nucleus. An increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺]₁) is central to the mechanisms underlying ABA-induced changes in guard cell turgor. We describe a possible model of Ca²⁺-based ABA signal transduction during stomatal closure and discuss recent evidence which suggests that Ca²⁺ is also involved in ABA nuclear signal transduction. Many other environmental stimuli which affect stomatal apertures, in addition to ABA, induce an increase in guard cell [Ca²⁺]₁) This raises questions regarding how increases in [Ca²⁺]₁) can be a common component in the signal transduction pathways by which stimuli cause both stomatal opening and closure. We discuss several mechanisms of increasing the amount of information contained within the Ca²⁺ signal, including encoding information in a stimulus-specific Ca²⁺ signal or Ca²⁺ signature', the concept of the ‘physiological address’ of the cell, and the use of other second messengers. We conclude by addressing the emerging technologies and new approaches which can be used in conjunction with guard cells to dissect further the molecular mechanisms of Ca²⁺-mediated signalling in plants.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> Ca<Superscript>2+</Superscript>-dependent protein kinase CPK3 mediates relationship of putative inositol triphosphate receptor with slow-type anion channel

It has been suggested in Arabidopsis thaliana (L.) Heynh. cv. Columbia that, contrary to 30 μM abscisic acid (ABA), 20 μM ABA induces guard cell Ca²⁺ mobilization through activating phosphoinositide-specific phospholipase C (PI-PLC)-dependent inositol 1,4,5-triphosphate (IP₃) production. Here, it was investigated whether Ca²⁺-dependent protein kinase, CPK3 or CPK6 would mediate ABA-induced stomatal closure downstream of IP₃ production. In the knockout cpk3-1 mutant, the PLC inhibitor (U73122) adjusted 20 μM ABA-induced stomatal closure to the extent observed in the knockout cpk6-1 and cpk3-1cpk6-1 mutants and the wild type, whereas, in the wild type, the inhibitor of IP₃-induced Ca²⁺ mobilization, xestospongin C (XeC), adjusted this closure to the extent observed in the cpk3-1 mutant. The Ca²⁺ buffer, EGTA and XeC positively interacted with the slow anion channel blocker, anthracene-9-carboxylic acid (9-AC) to inhibit 20 μM ABA-induced stomatal closure, which was suppressed in the dexamethasone-inducible AtPLC1 antisense transgene or the knockout cpk3-1, cpk6-1, cpk3-1cpk6-1 and NADPH oxidase atrbohD/F mutants. Discrete concentrations of 9-AC or another slow anion channel blocker (probenecid) negatively interacted with the Ca²⁺ buffer, BAPTA or the inhibitor of cyclic ADP-ribose-induced Ca²⁺ mobilization, ruthenium red, to inhibit 30 μM ABAinduced stomatal closure in the wild type but not in the cpk6-1, cpk3-1cpk6-1 and atrbohD/F mutants. Based on so far revealed features of the tested compounds and plant materials, interpretation of the results confirmed that guard cell ABA concentration discriminates between two Ca²⁺ mediations and outlined that one of them sequentially implicates CPK6, PLC1, a putative IP₃ receptor homologue, CPK3, and the slow anion channel, whereas the other one excludes AtPLC1-dependent IP₃ production and CPK3.     

Phospholipases AtPLDζ1 and AtPLDζ2 function differently in hypoxia

Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane‐cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLDζ1 and PLDζ2 uniquely possess Ca²⁺‐independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pldζ1 and pldζ2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia‐induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca²⁺_cyt.], hydrogen peroxide (H₂O₂) and PA. The concentrations of cytosolic Ca²⁺ and H₂O₂ were determined by fluorescence microscopy and the fluorescent dyes Fura 2‐AM and CM‐H₂DCFDA, specific for calcium and H₂O₂, respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLDζ1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLDζ2 is involved in cytosolic Ca²⁺ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pldζ1, while the PA level is unchanged in pldζ2. Thus, it is likely that AtPLDζ2 is involved in PA production by a calcium signaling pathway, while AtPLDζ1 is more important in ROS signaling.     

Intracellular ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana

In Arabidopsis thaliana cell suspension,abscisic acid (aBa) induces changes in cytosolic calcium concentration ([Ca²⁺]_cyt) which are the trigger for aBa-induced plasma membrane anion current activation, H⁺-aTPase inhibition, and subsequent plasma membrane depolarization. In the present study, we took advantage of this model to analyze the implication of intracellular Ca²⁺ stores in aBa signal transduction through electrophysiological current measurements, cytosolic Ca²⁺ activity measurements with the apoaequorin Ca²⁺ reporter protein and external pH measurement. Intracellular Ca²⁺ stores involvement was determined by using specific inhibitors of CICR channels: the cADP-ribose/ryanodine receptor (Br-cADPR and dantrolene) and of the inositol trisphosphate receptor ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122). In addition experiments were performed on epidermal strips of A. thaliana leaves to monitor stomatal closure in response to ABA in presence of the same pharmacology. Our data provide evidence that ryanodine receptor and inositol trisphosphate receptor could be involved in ABA-induced (1) Ca²⁺ release in the cytosol, (2) anion channel activation and H⁺-ATPase inhibition leading to plasma membrane depolarization and (3) stomatal closure. Intracellular Ca²⁺ release could thus contribute to the control of early events in the ABA signal transduction pathway in A. thaliana.     

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.