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April 12, 2001 10:38 Annual Reviews AR129-22

Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:627–58Copyright c© 2001 by Annual Reviews. All rights reserved

GUARD CELL SIGNAL TRANSDUCTION

Julian I Schroeder, Gethyn J Allen, Veronique Hugouvieux,June M Kwak, David WanerUniversity of California, San Diego, Division of Biology, Cell and Developmental BiologySection and Center for Molecular Genetics, La Jolla, California 92093-0116;e-mail: [email protected]

Key Words stomatal movement, gas exchange, abscisic acid, ion channel,cytosolic calcium

■ Abstract Guard cells surround stomatal pores in the epidermis of plant leaves andstems. Stomatal pore opening is essential for CO2 influx into leaves for photosyntheticcarbon fixation. In exchange, plants lose over 95% of their water via transpirationto the atmosphere. Signal transduction mechanisms in guard cells integrate hormonalstimuli, light signals, water status, CO2, temperature, and other environmental condi-tions to modulate stomatal apertures for regulation of gas exchange and plant survivalunder diverse conditions. Stomatal guard cells have become a highly developed modelsystem for characterizing early signal transduction mechanisms in plants and for eluci-dating how individual signaling mechanisms can interact within a network in a singlecell. In this review we focus on recent advances in understanding signal transductionmechanisms in guard cells.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628LIGHT SIGNAL TRANSDUCTION AND STOMATAL OPENING. . . . . . . . . . . . 629

Early Events in Blue Light Signal Transduction. . . . . . . . . . . . . . . . . . . . . . . . . 630Cytosolic Factors That Regulate Inward-Rectifying K+in Channels . . . . . . . . . . . . 630Role of Actin Filaments in Stomatal Movements. . . . . . . . . . . . . . . . . . . . . . . . . 631Cloned Guard Cell K+in Channel Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632

MULTIPLE PHYSIOLOGICAL AND ABIOTIC STIMULI INDUCESTOMATAL CLOSING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633Model for Roles of Ion Channels in ABA-Induced Stomatal Closing. . . . . . . . . . 633CO2-Induced Stomatal Closing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634

STIMULI THAT INCREASE CYTOSOLIC CALCIUM IN GUARD CELLS . . . . . 634Roles of [Ca2+]cyt in Stomatal Closing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634Roles of [Ca2+]cyt in Stomatal Opening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

SECOND MESSENGER SYSTEMS REGULATING [Ca2+]cytIN GUARD CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

1040-2519/01/0601-0627$14.00 627

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Cyclic ADP-Ribose and Vacuolar [Ca2+] Release. . . . . . . . . . . . . . . . . . . . . . . . 635Phospholipase C and Inositol 1,4,5 Trisphosphate. . . . . . . . . . . . . . . . . . . . . . . . 636Phospholipase D and Phosphatidic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636Amplifying Calcium Signals by Calcium-Induced Calcium Release. . . . . . . . . . . 637Plasma Membrane Calcium Channels and Calcium Influx in Guard Cells. . . . . . . 637Cytosolic [Ca2+] Oscillations Are Necessary for Stomatal Closingin Guard Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638

ABA-INSENSITIVE PP2C MUTANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639ANION CHANNELS AND ABA-INDUCED STOMATAL CLOSURE. . . . . . . . . . 640

Regulation of Guard Cell ABA Signaling by ABC Proteins. . . . . . . . . . . . . . . . . 640Protein Kinases Function in ABA Signaling and Anion Channel Regulation. . . . . 640Okadaic Acid–Sensitive Phosphatases Regulate Anion Channels and

ABA Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641ACTIVITY OF PLASMA MEMBRANE K+out CHANNELS INSTOMATAL MOVEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642Regulation of K+out by Phosphorylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642Syntaxins and ABA Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643Osmolarity and Temperature Sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643Transient K+ Efflux Currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644

NEW GUARD CELL SIGNALING MUTANTS AND GENETICAPPROACHES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644New Genetic Screens and Reverse Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

FUTURE OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646

INTRODUCTION

Opening and closing of stomatal pores is mediated by turgor and volume changesin guard cells. During stomatal opening guard cells accumulate potassium, anions,and sucrose (130, 162, 198). Osmotic water uptake leads to guard cell swelling andstomatal opening. Stomatal closing is mediated by potassium and anion efflux fromguard cells, sucrose removal, and metabolism of malate to osmotically inactivestarch. Guard cells have become a popular system for dissecting the functions ofindividual genes and proteins within signaling cascades for the following reasons.

1. Guard cells control CO2 influx and water loss and thus critically affectwhole plant growth and physiology.

2. Guard cells respond cell-autonomously to well-known plant physiologicalsignals, including red and blue light (215), CO2, plant pathogens, thehormones abscisic acid, auxin, cytokinin and gibberellins, and otherenvironmental signals. Thus many specific receptors and early signalingmechanisms function at the single-cell level in guard cells.

3. Models for roles of guard cell ion channels (177, 178) and cytosolic [Ca2+]changes (134) during stomatal movements provide a basis for analyzingindividual mechanisms that contribute to signal transduction. These ion

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channels are targets of early signaling branches and provide molecularprobes to identify upstream regulators.

4. Hypotheses for mechanisms affecting signal transduction can be easilytested by analyzing stomatal opening and closing in response to variousstimuli. Furthermore, several powerful approaches have been adapted toguard cell signaling analyses allowing interdisciplinary time-resolved cellbiological, biophysical, molecular genetic, second-messenger imaging,physiological, and newly arising postgenomic analyses.

The central role of guard cells in regulating gas exchange is of importance forecological and biotechnological applications. Agricultural and horticultural use ofplants in climates to which these plants are not adapted, as well as short-term cli-mate changes, lead to dramatic crop losses or freshwater consumption under stressconditions such as drought. Recent studies inArabidopsishave demonstrated thatstomatal responses can be manipulated by modifying guard cell signal transduc-tion elements to reduce transpirational water loss and dessication during droughtperiods (53, 75, 154).

One might ask: Are guard cells different from many other plant cell types (asidefrom the obvious specializations) in terms of harboring many signal receptors in asingle cell? Most likely not, because most individual plant cells respond to manyclassical hormones, pathogens, and light signals. The combination of the above-listed attributes (1 to 4), however, renders guard cells a well-developed modelsystem for interdisciplinary and time-resolved characterizations of mechanisms orsegments of early plant signaling cascades.

In the present review we focus mainly on recent dissections of signaling trans-duction mechanisms in guard cells. Of further importance for stomatal movementsis signal-dependent modulation of starch-malate metabolism. The mechanisms bywhich signaling cascades described here tie into metabolic networks is an impor-tant frontier of future research and recent reviews on guard cell metabolic pathwayscan be found elsewhere (88, 149, 198). Furthermore, several reviews on aspects ofguard cell signal transduction have appeared in recent years (8, 12, 13, 21, 113, 132,136, 145, 180, 182, 207, 214).

LIGHT SIGNAL TRANSDUCTIONAND STOMATAL OPENING

Stomatal opening is driven by H+ extrusion through plasma membrane H+-ATPases that are activated by auxins (124), red light (189), and blue light (14, 193).Cell-autonomous light receptors in guard cells induce stomatal opening (215).Light-induced stomatal opening requires activation of plasma membrane proton(H+)-ATPases (14, 52, 94, 188, 193), causing plasma membrane hyperpolarizationproposed to drive K+ uptake into guard cells via inward-rectifying K+ (K+in) chan-nels (181, 202). Anion (Cl−) influx into stomatal guard cells is thought to occur

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via H+/anion symporters or anion/OH− antiporters in the plasma membrane. Inparallel, starch metabolism leads to accumulation of osmotically active malate inguard cells. In addition, sucrose levels in guard cells increase during light-inducedstomatal opening (125, 159, 165, 197, 198). Studies with intact leaves show that themain solute supporting stomatal opening at the beginning of a daily cycle was K+,whereas sucrose became predominant later in the daily cycle as guard cell K+

content decreased (197, 198).

Early Events in Blue Light Signal Transduction

Cytosolic Ca2+elevation reversibly inhibits blue light activation of the H+-ATPases(92) (K1/2 = 0.3µM Ca2+) (Figure 1). Furthermore, inhibitors of PP1- or PP2A-type (PP1/PP2A) protein phosphatases such as calyculin A and okadaic acid inhib-ited blue light–dependent H+pumping and light-induced stomatal opening inVicia,suggesting that PP1/PP2A phosphatases are positive regulators of light-inducedstomatal opening (93) (Figure 1). Abscisic acid (ABA) also inhibits blue light–dependent H+ pumping activity ofViciaguard cell protoplasts (52, 169, 193). ABAinhibition of apoplastic acidification was not observed in the ABA-insensitiveAra-bidopsismutantsabi1-1 andabi2-1 (Figure 1). Interestingly, the PP1/PP2A in-hibitor okadaic acid partially restored ABA inhibition of proton pumping inabi1-1guard cells, whereas the protein kinase inhibitorK-252apartially restored ABAinhibition of proton pumping inabi2-1guard cells (169).

14-3-3 proteins bind directly to the C-terminal domain and thus activate H+-ATPases, and fusiccocin stabilizes the 14-3-3–H+-ATPase complex (16, 49, 86).The direct mechanism of guard cell plasma membrane H+-ATPase regulation wascharacterized in an elegant biochemical study. Blue light activates H+-ATPasesvia phosphorylation of the C terminus (94). Coprecipitation of H+-ATPases withendogenous guard cell 14-3-3 proteins and binding of recombinant 14-3-3 pro-teins only to the phosphorylated H+-ATPase C terminus provide evidence for arole of 14-3-3 proteins as a positive regulator in physiological blue light signaltransduction (Figure 1).

Although two different types of blue light receptors, CRY proteins and NPH1,have been isolated from plants (1a, 28, 29), the blue light receptors in guard cellshave been proposed to include unique components. Stomata from the zeaxanthin-deficientArabidopsismutant,npq1(150), are impaired in blue light–induced stom-atal opening (47), and stomata from the blue light photoreceptor mutants,cry1,cry2, nph1, nph3, nph4, cry1cry2, andnph1cry1, showed a wild-type blue lightresponse (47, 101). These results have led to the proposal of a model in which zeax-anthins function as blue light receptors or receptor pigments in guard cells (47).

Cytosolic Factors That Regulate Inward-RectifyingK+in Channels

K+in channels in guard cells have been proposed to contribute to K+ uptake duringstomatal opening (178, 181). Reviews on the role of K+

in channels in guard cells

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and on the structure and function of these K+ channels have appeared elsewhere(35, 42, 65, 128, 183). Extracellular acidification increases the activity of guard cellK+in channels (20) and of cloned plant K+in channels expressed inXenopusoocytes (74, 78, 146, 204). Cytosolic Ca2+ ([Ca2+]cyt) elevation inhibits K+in chan-nels, thus limiting K+ uptake (108, 175) (Figure 1). Almost complete inhibition ofK+in channels has been measured inViciaguard cells when [Ca2+]cyt was buffered to≈1µM (54, 57, 90, 108, 175), or when InsP3 is uncaged in the guard cell cytoplasmto raise [Ca2+]cyt (23) (Figure 1). Interestingly, inVicia, K+in channels in abaxialguard cells were [Ca2+]cyt inhibited and stomatal movements were modulated byexternal Ca2+ and ABA, whereas K+in channels in adaxial guard cells were insensi-tive to [Ca2+]cyt and stomatal movements were less sensitive to external Ca2+ andABA (206).

In addition, protein phosphorylation has been suggested to play an importantrole in modulation of K+in channel activity. Inhibitors of calcineurin (PP2B), acalcium-dependent protein phosphatase, maintained K+

in channel activity in spiteof elevated [Ca2+]cyt in Vicia guard cells (126) (Figure 1). Cyclosporin A, aninhibitor of animal PP2B-type phosphatases, inhibits stomatal closure and reducesABA inhibition of stomatal opening inPisum, suggesting that PP2Bs might be neg-ative regulators of stomatal opening (70) (Figure 1). Note thatPP2Bhomologshave not yet been identified in plants. In contrast to PP2B inhibitors, inhibitors ofPP1/PP2A protein phosphatases downregulate K+

in channel activity in guard cells,suggesting that PP1 or PP2As are positive regulators of K+

in channels (121, 201)(Figure 1). Biochemical approaches identified aVicia guard cell Ca2+-dependentprotein kinase (CDPK) that phosphorylates theArabidopsisguard cell K+in channelsubunit KAT1 (118), and heterologous expression ofKAT1and a soybean CDPK inXenopusoocytes shows a reduction in KAT1-mediated K+ currents (17), suggest-ing a role of CDPK in Ca2+-mediated K+in channel inhibition (Figure 1). Combinedgenetic and cell biological analyses will be required to identify the kinases thatregulate K+in channels.

Stomatal aperture measurements show that linolenic acid and arachidonic acidpromote stomatal opening and inhibit stomatal closing (106). Furthermore, patch-clamp analyses show that these fatty acids activate K+

in currents and inhibit outward-rectifying K+out currents (106). These results are interesting as only a few agentshave been found that enhance K+in channel activity in guard cells. Furthermore,phospholipase A2 inhibitors decrease stomatal responses to light (196). Togetherthese data suggest that phospholipase A2 may be a positive regulator of the guardcell light response pathway (Figure 1).

Role of Actin Filaments in Stomatal Movements

Cytochalasin D, an actin filament-depolymerizing agent, activates K+in channels

and enhances light-induced stomatal opening (76). In contrast, inhibition of K+in

channel currents and light-induced stomatal opening was observed when an actinfilament stabilizer, phalloidin, was tested (76). These results imply a possible

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interaction of the actin cytoskeleton with guard cell plasma membrane K+in channels

in signal transduction (Figure 1).ABA treatments reorganize the actin structure from a radial pattern to a ran-

domly oriented and short-fragmented pattern (44). A small GTP-binding protein,AtRac1, can function as a negative regulator in ABA-induced actin reorganiza-tion (107). ABA causes AtRac1 inactivation (Figure 3). This ABA response wasimpaired inabi1-1 (107). In addition, transgenicArabidopsisplants expressinga dominant negative AtRac1 mutant mimic constitutive ABA-induced actin reor-ganization in guard cells and increase ABA sensitivity of stomatal closure (107),suggesting that inactivation of AtRac1 is required for actin reorganization andstomatal closure. Together these studies show important roles of actin and smallG-proteins in stomatal movements.

Cloned Guard Cell K+in Channel Genes

The ArabidopsisK+in channel geneKAT1 was cloned by complementation of ayeast mutant defective in K+ transport (10) and shown to mediate K+ currentsin Xenopusoocytes with typical properties of plant K+in currents (67, 73, 78, 173,204). Expression studies withArabidopsis KAT1and the potato orthologKST1show that these plant K+in channel genes are predominantly expressed in guardcells (146, 148). In animals, functional K+ channel proteins are composed of fourα–subunits (87) and additional regulatoryβ–subunits (45). AnArabidopsiscDNAencoding aβ–subunit homolog of K+ channels has been isolated and binding toKAT1 has been reported (199).

The model that K+in channels contribute to K+ uptake during stomatal opening(181) has been analyzed using molecular genetic approaches. In a study withtransgenicArabidopsisexpressingKAT1 mutants with a reduced sensitivity toCs+ block, transgenic plants exhibited partial light-induced stomatal opening inthe presence of Cs+ concentrations that inhibit stomatal opening in wildtype (77).In another study, transgenicArabidopsisexpressing a dominant negative mutantform of the guard cell K+in channel KAT1 showed 75% reduction in the activity ofguard cell K+in channel currents and a reduction in light-induced stomatal opening(100). As redundancy likely exists in K+ channel subunits in guard cells, thesedata show that dominant negativekat1can disrupt K+in channels. These data supportthe model that K+ channels constitute a central mechanism for K+ uptake duringstomatal opening (178, 181). These studies do not exclude the likely model thatother partially redundant (183) K+ uptake transporters function in parallel in guardcells. For example, there are>12 isoforms of theKT-KUP-HAKK+ transportergene family (48, 91, 161, 172) in theArabidopsisgenome and some of these arelikely to be expressed in guard cells. Furthermore, patch-clamp studies have shownthe activity of additional inward-conducting cation channels (69, 205, 212). Inconclusion, many positive and negative regulators of H+ pumps, K+in channels,and stomatal opening have been found (Figure 1). A combination of genetic, cellbiological, and biochemical studies will allow further testing and expansion ofthese models.

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MULTIPLE PHYSIOLOGICAL AND ABIOTIC STIMULIINDUCE STOMATAL CLOSING

Abscisic acid, produced in response to water deficit, causes stomatal closing. Fur-thermore, in C3 and C4 plants stomatal closing is induced by darkness and byelevated CO2 concentrations in the intercellular spaces in leaves, arising fromrespiration (12, 133). Stomatal closing and reduced transpiration also leads toelevated temperatures inside leaves. Pathogen elicitors also cause stomatal clos-ing, enabling plants to reduce access of pathogens to the inside of leaves (104).Aerial pollutants such as ozone and sulfur dioxide cause stomatal closing at highconcentrations (203), thus reducing further damage of leaf tissues by these pol-lutants. Thus multiple stimuli elicit stomatal closing. Although the receptionmechanisms for these stimuli remain unknown, the signaling pathways need toconverge on central guard cell ion channel and metabolic pathways.

Model for Roles of Ion Channels in ABA-InducedStomatal Closing

Stomatal closing requires ion efflux from guard cells. Models for roles of ionchannels during ABA-induced stomatal closing have been used as a basis for dis-secting upstream ABA signal transduction mechanisms (132, 175, 177, 207). Inbrief, ABA induces cytosolic Ca2+ increases (134) (Figure 2). Cytosolic cal-cium elevations, in turn, inhibit plasma membrane proton pumps (92) and K+

inchannels and activate two types or modes (39) of plasma membrane anion chan-nels that mediate anion release from guard cells (89, 175) (Figure 2). One ofthese anion channels shows slow and sustained activation (S-type anion channels)(122, 175, 179), whereas the other anion channel shows rapid transient activation(R-type or GCAC anion channels) (64). Channel-mediated anion efflux from guardcells causes either transient or sustained anion efflux and depolarization (Figure 2).Depolarization, in turn, deactivates inward-rectifying K+ (K+in) channels and ac-tivates outward-rectifying K+ (K+out) channels (181), resulting in K+ efflux fromguard cells (Figure 2). The ensuing long-term efflux of both anions and K+ fromguard cells contributes to loss of guard cell turgor and to stomatal closing (175)(Figure 2). Recent studies have shown a requirement for rapid ABA-induced Ca2+

influx and S-type anion channel activation forRAB18expression inArabidopsissuspension culture cells (49b, 49c) indicating that these mechanisms (Figure 2) areof general importance for early ABA signaling in other cell types.

Most ions released across the plasma membrane of guard cells need first to be re-leased into the cytosol from guard cell vacuoles (8, 130, 131, 207, 208). Models forthe roles of vacuolar K+ and anion channels during stomatal regulation have beenproposed previously and are described elsewhere (7, 8, 131, 157, 207, 208). How-ever, these models have not yet been tested as stringently as those for plasmamembrane ion channels. Combined molecular genetic (e.g. gene disruption) andcell biological approaches are needed to directly analyze these vacuolar ion channelmodels.

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CO2-Induced Stomatal Closing

Elevated CO2 concentrations arising from respiration in darkness stimulate stoma-tal closing (133). Increases in atmospheric CO2 concentrations are also predictedto reduce stomatal apertures and affect gas exchange (12, 41). CO2 signaling mech-anisms in guard cells have been reviewed recently (12, 41). Here, we briefly sum-marize some recent findings and signaling models. Elevated CO2 concentrationstrigger rises in [Ca2+]cyt (209), activate S-type anion and outward-rectifying K+

channel currents (24), and modulate R-type anion channels (K Raschke, personalcommunication). These data show that the ion channel targets of early CO2 sig-naling are to a degree shared with ABA signaling (Figure 2). However, upstreamCO2 sensing and transduction mechanisms have been reported to differ from ABAsignaling, because theabi1-1 andabi2-1 mutants show wild-type CO2-inducedstomatal closing (116). A CO2-induced increase in cell wall malate concentrationhas been proposed to cause stomatal closing (66). However,>20 mM externalmalate was required to produce stomatal closing in two independent studies un-der the same experimental conditions as the above study (31, 43). Furthermore,external malate counteracted CO2-induced stomatal closing (31), thus calling intoquestion the malate as CO2 sensor hypothesis. An alternative hypothesis suggeststhat the CO2 sensor is located in guard cell chloroplasts and functions via a CO2-induced decrease in zeaxanthin levels (216). Further understanding of CO2 sensingand signaling in guard cells will help in finding crucial links between the signal-ing pathways reviewed here and guard cell metabolic pathways. Foremost, futureresearch should have important implications for manipulating gas exchange andcarbon fixation in the face of rising atmospheric CO2 levels.

STIMULI THAT INCREASE CYTOSOLIC CALCIUMIN GUARD CELLS

Roles of [Ca2+]cyt in Stomatal Closing

Many stimuli that result in a change in stomatal aperture have been shown, at leastin part, to utilize signal transduction pathways involving changes in guard cell[Ca2+]cyt. ABA-induced stomatal closing is Ca2+-dependent (37, 51, 175, 186).Note that a Ca2+-independent pathway appears to also exist (2). ABA inducesrepetitive, transient increases or oscillations in guard cell [Ca2+]cyt (5, 50, 56, 135,176, 195). Genetic support for the importance of ABA-induced [Ca2+]cyt eleva-tions in guard cells has been obtained recently, as the ABA-insensitiveArabidopsismutantsabi1-1andabi2-1show greatly reduced ABA-induced [Ca2+]cytelevations,and theabi anion channel regulation and stomatal movement phenotypes are sup-pressed by experimentally elevating [Ca2+]cyt (4). Stomatal closure and guard cell[Ca2+]cyt oscillations can also be induced by increases in external (apoplastic)[Ca2+] (4, 5, 139). These Ca2+-induced [Ca2+]cyt oscillations include a repetitiveCa2+ influx across the plasma membrane coupled to Ca2+ release from intracellular

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stores for each separate Ca2+ transient (56, 139). Why [Ca2+]cyt regulation is sosensitive to changes in apoplastic Ca2+ is not fully understood, although manyspecies limit Ca2+ accumulation in the apoplast surrounding guard cells to preventaberrant stomatal regulation (38, 171). Increases in CO2 cause stomatal closureand [Ca2+]cyt elevations (209). Cold shock (3, 211a) and oxidative stress, inducedby application of H2O2 or methyl viologen, increase [Ca2+]cyt and result in stoma-tal closure (137, 156). The removal of extracellular Ca2+ using EGTA abolishesCO2- and H2O2-induced [Ca2+]cyt elevations, indicating that plasma membraneCa2+ influx occurs (156, 209).

Roles of [Ca2+]cyt in Stomatal Opening

Interestingly, stimuli that result in stomatal opening also induce [Ca2+]cyt ele-vations. Auxin promotes stomatal opening and direct [Ca2+]cyt measurements(80), and pharmacological studies (32) suggest a role for [Ca2+]cyt elevations. Fur-thermore, Ca2+-dependent protein kinases (CDPK) activate guard cell vacuoleCl− channels and malate uptake currents that have been implicated in vacuolar an-ion uptake during stomatal opening (157). Blue light promotes stomatal opening,and pharmacological experiments suggest the involvement of [Ca2+]cyt/calmodulinas a second messenger in this process (33, 194). However, increasing externalCa2+ can inhibit light-induced stomatal opening (4, 152, 192) (Figure 1). Cyclicnucleotides may also act in a Ca2+-dependent stomatal opening pathway as stom-atal opening can be stimulated by cAMP (33) or the membrane-permeable cGMPanalog 8-Br-cGMP (32). Cyclic GMP-induced stomatal opening is inhibited bychelation of external Ca2+ or by inhibitors of intracellular Ca2+ release (32).

How a single second messenger such as [Ca2+]cyt can control many diverse andopposing responses in a single cell type remains unknown but likely depends onthe Ca2+ channels and Ca2+ regulatory systems activated by each stimulus, thedownstream response elements expressed at a given time, and the characteristicsand dynamics of the elicited [Ca2+]cyt change (the Ca2+ signature) (138). Therecent demonstration that GFP-based “cameleon” calcium indicators (142, 143)function in Arabidopsiswill allow addressing of these questions by combinedgenetic and [Ca2+]cyt signaling studies (5).

SECOND MESSENGER SYSTEMS REGULATING [Ca2+]cyt

IN GUARD CELLS

Cyclic ADP-Ribose and Vacuolar Ca2+ Release

Recent experiments have implicated several second messenger systems in ABAand [Ca2+]cyt signaling in guard cells (Figure 3). In animal cells, cyclic ADP ri-bose (cADPR) is produced from NAD via the action of the enzyme ADP-Ribosylcyclase, and it mobilizes Ca2+ from intracellular stores by activation of an en-domembrane ion channel known as the ryanodine receptor (RYR) (103). In plant

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vacuoles, nanomolar cADPR concentrations can activate a Ca2+ permeable cur-rent (6). Microinjection of cADPR into tomato hypocotyl cells shows that cADPRcan function in ABA signaling (213). InCommelinaguard cells cADPR causes[Ca2+]cyt increases and elicits stomatal closing (102). However, microinjectionof the inactive cADPR analog 8-NH2-cADPR or noncyclic ADPR does not elicit[Ca2+]cyt increases or guard cell turgor loss. Ryanodine treatment of guard cellsalso reduces [Ca2+]cyt increases (57). Note that ABA-induced stomatal closureis only partly inhibited by either microinjection of 8-NH2-cADPR (102) or bynicotinamide, an inhibitor of cADPR production (85, 102), suggesting that addi-tional parallel [Ca2+]cyt elevation mechanisms are needed in the ABA signalingcascade.

Phospholipase C and Inositol 1,4,5 Trisphosphate

Various lines of evidence suggest that phospholipase C (PLC) is a component ofABA signal transduction in guard cells. Early experiments showed that releaseof caged InsP3 into the cytosol of guard cells could cause [Ca2+]cyt increases andstomatal closure (51) and inhibit K+in channels (23). Treating guard cell proto-plasts with ABA slightly elevates InsP3 levels (105, 151). Additionally, the PLCinhibitor U-73122 (but not the inactive analog U-73343) inhibits the activity ofrecombinant PLC from tobacco (195). ABA-induced stomatal closure was alsoinhibited by U-73122, but only by 20% (195). However, complete inhibition ofABA-induced stomatal closure can be achieved by treating stomata with a combi-nation of U-73122 and nicotinamide (85, 132a), suggesting that both cADPR andPLC signaling systems function in ABA signaling (Figure 3).

Note that other inositol-phosphates can also act as second messengers in ABAsignal transduction in guard cells. In a recent study, myo-inositol hexakisphosphate(InsP6) was identified as an intermediary of guard cell signal transduction. ABAstimulates production of InsP6 in guard cells, and InsP6 perfused into the cytosolvia a patch pipette inhibited K+in channels in potato guard cell protoplasts in a Ca2+-dependent manner. These data suggest that InsP6 production is also an importantcomponent of ABA signaling (109) (Figures 1, 3).

Phospholipase D and Phosphatidic Acid

Phospholipase D (PLD) has been implicated in ABA signaling in aleurone cells(164) and in guard cells (85). PLD generates phosphatidic acid (PtdOH), andABA treatment ofVicia guard cells caused PtdOH levels to transiently increase2.5-fold (85). PtdOH also promotes stomatal closure and inactivates K+

in currents.Guard cell [Ca2+]cyt did not increase following PtdOH treatment, suggestingthat PLD acts in a parallel pathway or downstream of Ca2+ mobilizing secondmessenger systems (Figure 3). An inhibitor of PLD activity, 1-butanol, causedonly a partial inhibition of ABA-induced stomatal closure whereas near-completeinhibition of stomatal closure resulted from adding 1-butanol together with

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nicotinamide (85), suggesting a parallel action of PLD to the cADPR-mediatedpathway.

Amplifying Calcium Signals by Calcium-InducedCalcium Release

Release of caged Ca2+ into the guard cell cytosol can induce [Ca2+]cyt increasesor oscillations that are larger than can be accounted for solely by the Ca2+ re-leased by photolysis (51, 139), suggesting that a [Ca2+]cyt-induced calcium release(CICR) mechanism exists in guard cells. Ca2+-permeable SV channels have beenproposed to amplify and propagate [Ca2+]cyt signals in guard cells by CICR fromvacuoles (208). Cytosolic Mg2+ sensitizes SV channels to physiological [Ca2+]cytelevations (158) and shifts the voltage-dependence of SV channels (27) such thatSV channel activity is enhanced. The ability of the SV channel to mediate CICRhas been questioned because in mesophyll vacuoles increasing the transvacuolarCa2+ gradient shifts the voltage-dependence to prevent channel opening underconditions that would otherwise allow Ca2+ to enter the cytosol (160). However,the voltage-dependence and pharmacology of radiolabeled Ca2+ release in vitrofrom vacuolar vesicles suggests that CICR can be mediated by SV channels (18).The role of SV channels in CICR in guard cells remains an important issue in signaltransduction, analysis of which best requires manipulation of SV channel genes.

Plasma Membrane Calcium Channels and Calcium Influxin Guard Cells

Experimental application of repeated hyperpolarizations negative of−120 mVinduced repetitive [Ca2+]cyt transients in guard cells (56). ABA application shiftedthe threshold of hyperpolarization-activated Ca2+ elevations to−80 mV, indi-cating that an early event in ABA signaling is the sensitization of Ca2+ influxto membrane potential. Furthermore, in 50 mM KCl buffers [in which the cellsare depolarized (168, 202)] ABA only induces [Ca2+]cyt increases in 30–60% ofguard cells (2, 4, 50, 135, 176). Transient ABA activation of Ca2+-permeable chan-nels was found during depolarizations in 37% of guard cells (176), which couldcontribute to ABA-induced [Ca2+]cyt elevations in depolarized guard cells. In-terestingly, in cells maintained in 5 mM KCl, guard cells are hyperpolarized andABA induces repetitive [Ca2+]cyt transients or oscillations in a higher proportion ofcells (80–90%) (5, 56, 195). These data indicate that changes in plasma membranepotential are a central component in ABA signaling.

The upstream second messenger mechanisms that activate guard cell plasmamembrane Ca2+ channels remained unknown. A recent study inArabidopsisguardcells shows that reactive oxygen species (ROS) can activate a hyperpolarization-activated Ca2+ influx current (ICa) and that ROS can act as a second messenger inABA signaling. ABA treatment enhances ROS production inArabidopsisguard

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cells (156). Interestingly, ABA activates guard cell ICa(156) only when NADPH ispresent in the cytosol, implicating NADPH oxidases in ABA signaling (Y Murata &J Schroeder, unpublished). Activation of ICa by hydrogen peroxide is impaired inthe recessive ABA-insensitive mutantgca2, whereas ABA-induced ROS produc-tion remains intact ingca2 (156), providing genetic evidence for roles of ROSand ICa in ABA signaling. These data lead to a model in which ROS production,GCA2, and Ca2+ channel activation represent a new signaling “cassette” in guardcells (Figure 3). A study in maize embryos has also shown that ABA enhancesROS production (60), indicating that ROS may be universal second messengers inplant ABA signaling.

Single channel recordings inVicia guard cell protoplasts have identified a Ca2+

channel that is hyperpolarization-activated and shows a 250-fold increased openprobability following addition of 20µM ABA (61). Currents exhibited by thischannel are very similar to ICa. The activation by ABA occurs in isolated patches,suggesting that ABA perception and channel activation are closely associated inVicia. Interestingly, the single channel open probability was reduced tenfold whenbuffering [Ca2+]cyt from 200 nM to 2µM, indicating that the channel is down-regulated during [Ca2+]cyt elevation and is therefore subject to negative feedbackcontrol during [Ca2+]cyt signaling (61).

Both at the single-channel level (61) and in whole cells (156), the activityof the Ca2+ influx channel can show spontaneous oscillatory behavior withoutexogenous ABA addition. Background ICa activity was inhibited by addition of0.1 mM DTT inArabidopsisguard cells (156). The spontaneous ICa activity maycontribute to spontaneous [Ca2+]cyt elevations found in hyperpolarized guard cells(5, 56, 195). Because ICachannels are regulated by ROS, various stress signals maycontrol Ca2+ influx by regulating the oxidative state of guard cells. For example,pathogen elicitors trigger ROS production in guard cells and stomatal closing (104),and ozone, which closes stomatal pores (203), might modulate ICa. In this regard,ROS production and ICa have been proposed to function as a shared “signalingcassette” of multiple stress signaling pathways (156).

Additional Ca2+ influx pathways may be provided by plasma membrane stretch-activated Ca2+ permeable channels (30) or via a Ca2+ permeability of transientand sustained K+out channels (153, 170). Clearly, Ca2+ influx, Ca2+ release, andsecond messengers are integrated in guard cells to produce a [Ca2+]cyt signal thatcontrols stomatal movements. How these separate processes may be integrated toproduce [Ca2+]cyt signals that encode information necessary for stomatal closureis an important focus of ongoing studies (see 132a).

Cytosolic [Ca2+] Oscillations Are Necessary for StomatalClosing in Guard Cells

In a few cases in animal cells, the frequency of [Ca2+]cyt oscillations has beenshown to control the efficiency and specificity of cellular responses (36, 40, 120).In plants, it remains to be investigated whether Ca2+ oscillations are an absolute

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requirement for eliciting physiological responses. Models for membrane poten-tial oscillations in guard cells have been generated (26, 59, 96) to analyze differ-ent mechanisms that may contribute to generation of [Ca2+]cyt oscillations. Usingguard cells of theArabidopsisV-ATPase mutantde-etiolated 3(det3) (185), whichhas reduced endomembrane proton pumping and energization, external Ca2+ andoxidative stress elicited prolonged Ca2+ increases (plateaus) that did not oscillate,whereas wild-type cells show [Ca2+]cyt oscillations (3). Unexpectedly, steady statestomatal closure was inhibited indet3in response to these stimuli. Conversely, coldand ABA elicited Ca2+oscillations indet3, and stomatal closures were not impaired(3). Moreover, indet3guard cells, experimentally imposing external Ca2+-inducedoscillations rescued steady state stomatal closure in response to external Ca2+, andimposing Ca2+ plateaus in wild-type guard cells prevented steady-state stoma-tal closing. These data provide genetic evidence that stimulus-specific Ca2+ os-cillations, rather than a mere plateau of [Ca2+]cyt, are necessary for long-termstomatal closure (3). These findings suggest that guard cells may provide an ex-cellent genetic system to study [Ca2+]cyt pattern-dependent responses.

ABA-INSENSITIVE PP2C MUTANTS

Genetic screens forArabidopsismutants insensitive to ABA inhibition of seedgermination yielded two dominant mutants that are impaired in ABA-inducedstomatal closure (97). The corresponding genes,ABI1 and ABI2, both encodetype 2C protein phosphatases and the dominant mutant allelesabi1-1andabi2-1have point mutations altering a conserved amino acid (112, 114, 140, 166). Sev-eral downstream responses to ABA are impaired in theseArabidopsismutantsincluding K+out and K+in channel regulation (11) and anion channel activation (155).These mutations also impair ABA-induced [Ca2+]cyt increases (4). Furthermore,experimental elevation in [Ca2+]cyt causes anion channel activation and stomatalclosure inabi1-1andabi2-1, thus bypassing the effects of theabi1-1andabi2-1mutations (4). These data demonstrate that the dominantabi PP2C mutants in-terfere with very early ABA signaling events that act upstream of [Ca2+]cyt (4)(Figure 3).

Because the only known alleles of these genes were dominant mutations, it hasbeen unclear whether the ABI1 and ABI2 phosphatases are positive or negativeregulators of ABA signaling or indeed whether they affect ABA signaling at allin wildtype. Recently, however, intragenic revertants of theabi1-1 and abi2-1mutants were isolated and shown to have reduced or no phosphatase activity invitro (53, 139a). Because a double mutant of both revertants shows hypersensitivityto ABA, ABI1 and ABI2 are likely negative regulators of ABA signaling (139a).In correlation, overexpression of wild-type ABI1 in maize mesophyll protoplastsblocks ABA regulation of gene expression (191). In spite of these advances,ABI1andABI2gene deletion or silencing mutants would be useful for a stringent test oftheir functions, because all intragenic revertant mutations lie downstream of the

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dominant mutant site (53, 139a), which might form an attachment to an essentialsignaling protein.

ANION CHANNELS AND ABA-INDUCEDSTOMATAL CLOSURE

Anion channel activation at the plasma membrane of guard cells has been proposedas an essential step during stomatal closure (Figure 2) (64, 175, 179). S-type anionchannel currents in guard cells are activated by increases in [Ca2+]cyt in Vicia (175)andArabidopsis(4). R-type anion currents are activated following an increase inexternal Ca2+ (64). Whether ABA regulates R-type anion channels remains to bedetermined. ABA activation of S-type anion channels in the plasma membraneof guard cells has now been demonstrated inArabidopsis(155), tobacco (58),andVicia (111, 119, 187) (Figure 3). This response is disrupted in theArabidopsisabi1-1 andabi2-1 mutants, providing genetic evidence that activation of S-typechannels contributes to stomatal closure (155). Putative anion channel genes havebeen cloned from tobacco (127) andArabidopsis(62) based on sequence homologywith the animal voltage-dependent CLC chloride channels [for review see (15)].Further work is needed to determine whether these genes encode components ofnative plant plasma membrane anion channels.

Regulation of Guard Cell ABA Signaling by ABC Proteins

Pharmacological studies have led to the model that guard cell S-type anion chan-nels may be encoded by or regulated by ATP binding cassette (ABC) proteins(110, 111) (Figure 3). ABC proteins comprise a large family of membrane prote-ins that actively translocate a wide spectrum of substrates. In addition, ABCproteins regulate the activity of other unrelated transporters. ABC proteins such asCFTR (cystic fibrosis transmembrane conductance regulator) or SUR (sulfonylureareceptor) show Cl− channel activity and/or regulate other channels [for review see(200)]. Inhibitors of SUR, such as glibenclamide, prevent ABA-induced stoma-tal closure inVicia andCommelina. Furthermore, the ABC protein inhibitors DPCand glibenclamide block slow anion currents inVicia guard cells (111). In con-trast, cromakalin, an antagonist of glibenclamide, triggers stomatal closing inCommelinaand reverses the inhibition of glibenclamide on S-type anion currentsin Vicia guard cells (111).

Protein Kinases Function in ABA Signalingand Anion Channel Regulation

Pharmacological approaches, using the serine/threonine protein kinase inhibitorK252a and cytosolic replacement of ATP, showed that phosphorylation events arecentral positive regulators in ABA-induced stomatal closure inVicia (174), as wellas inCommelina(43), Pisum(70), andArabidopsis(4). K252a abolishes both

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anion channel activity and ABA-induced stomatal closing (174). In correlationwith these results, ABA induction of gene expression (RD29aandKIN2) in tomatohypocotyls is inhibited by K252a (213), together with other studies, indicatingthat kinase-dependent transduction of ABA signaling (174) is of general signi-ficance (95, 190, 213). Note that the R-type anion currents are not regulated byphosphorylation events but that nucleotide binding activates these anion channels(64, 184).

Biochemical approaches led to the characterization of a Ca2+-independent,ABA inducible 48-kDa kinase (non-MAP kinase) inVicia guard cells (117, 144).The kinase activity was named AAPK or ABR. Recently, anAAPK gene wascloned (119). Transient expression of a dominant negative allele of AAPK, whichabolished kinase activity, prevents ABA activation of S-type anion currents andstomatal closing. In correlation with these findings, inVicia S-type anion currentsare activated at low [Ca2+]cyt and high ATP concentrations, suggesting that a finalphosphorylation event in anion channel activation can be Ca2+ independent (187)(Figure 3). Recessive loss-of-function mutations in AAPK will allow further anal-ysis of AAPK function in guard cells.

Two Ca2+-dependent protein kinases of 53 kDa and 58 kDa have been charac-terized inVicia guard cells (118, 144). Removal of Ca2+ with BAPTA in Viciaguard cell protoplast suspensions prevents ABR kinase activation and indicates thatCa2+ is required upstream for ABR activation (144) (Figure 3). These data andoverexpression studies on maize protoplasts suggest that CDPKs may be positiveregulators in ABA signal transduction (190).

In addition, MAP kinases have been reported to positively control ABA-inducedstomatal closure inPisum(25). ABA causes a transient activation of a 43-kDaMAP kinase named AMBPK. AMBPK exhibits all fundamental MAP kinaseproperties, including tyrosine phosphorylation (25). The MAPK kinase inhibitorPD98059 abolished ABA-induced stomatal closing (Figure 3) and ABA inductionof dehydrin mRNA (25). ABA activation of MAP kinases in barley aleurone waspreviously reported (68, 95).

In parallel to the above kinases that transduce ABA signals, other protein kinaseshave been suggested to act as negative regulators of ABA signaling in tobaccoandArabidopsisguard cells in theabi1-1 background (11, 155). Application ofkinase inhibitors partially restores ABA activation of anion channels and regulationof K+ channels and stomatal closing inabi1-1backgrounds (11, 155) (Figures 3and 4).

Okadaic Acid–Sensitive Phosphatases RegulateAnion Channels and ABA Signaling

Inhibitors of PP1 and PP2A protein phosphatases such as okadaic acid (OA) werefound to enhance S-type anion currents and ABA-induced stomatal closure inVicia(174, 187), as well as in tobacco (58),Commelina(43), andPisum(70), and en-hance ABA-induced gene expression in tomato hypocotyls (213), suggesting that

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PP1s or PP2As act as negative regulators in ABA signaling (Figure 3; PP1/PP2Aneg).Note that PP1/PP2Anegin Figure 3 and the PP1/PP2A shown in Figure 1 both pro-mote stomatal opening and therefore might be encoded by the same genes. InVicia, either OA or ABA maintain anion current activation without cytosolic ATP,indicating that ABA may indeed downregulate a PP1/PP2Aneg(187) (Figure 3).

In addition to negatively regulating PP1/PP2As, evidence suggests that otherPP1/PP2As can also act as positive regulators in ABA signaling (Figure 3; PP1/-PP2Apos). In Arabidopsis, OA partially inhibited ABA activation of S-type anionchannels and stomatal closing (155). A similar inhibitory effect of OA was also ob-served on ABA signaling during stomatal opening and ABA induction of dehydrinmRNA in Pisumepidermal peels (70). OA also inhibits ABA-induced expressionof thePHAV1gene in barley aleurone (99). Experiments inPisumshow that theactivity of either PP1/PP2Aposor PP1/PP2Anegcan be resolved depending on theaperture of stomates (70) (Figure 3).

These different studies bring to light that a complex phosphorylation and de-phosphoryation network exists in guard cells (Figure 3) and that significant diffe-rences can occur in ABA signaling depending on the physiological state of guardcells.

ACTIVITY OF PLASMA MEMBRANE K+out CHANNELS INSTOMATAL MOVEMENTS

Efflux of K+ from the cell during stomatal closing has been proposed to occurthrough outward rectifying K+ (K+out) channels that are activated by membranedepolarization (181). Guard cells respond to ABA by enhancing K+

out and reduc-ing K+in channel currents (19, 22). Unlike K+in channels, however, K+out channelsare largely insensitive to increases in [Ca2+]cyt that occur during ABA signaling(108, 175). A guard cell-expressedSKORK+ channel cDNA homologue, namedGORK, was isolated that when expressed inXenopusoocytes produces outward-rectifying K+ channels with properties similar to K+out channels (1).

ABA induces an increase in the cytosolic pH of guard cells (22, 54, 80). Ex-periments inVicia guard cells show that K+out currents are enhanced by increasedcytoplasmic pH (20). The pH stimulation of K+outchannels occurs in isolated mem-brane patches and is thus membrane delimited (141). ABA-induced increasesin K+out currents can be inhibited by acidification or buffering of the guard cellcytoplasmic pH, showing that cytosolic pH has a functional role in ABA signaltransduction (22) (Figure 4).

Regulation of K+out by Phosphorylation

Several lines of evidence suggest that protein phosphorylation plays a role inmodulation of K+out channel activity. Guard cells from tobacco plants transformedwith the dominant phosphatase mutant alleleabi1-1 from Arabidopsisshow K+outcurrents that are two- to fourfold lower than wild type, and both K+

in and K+out

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currents are insensitive to modulation by ABA, suggesting a role for PP2Cs inguard cell signaling (11). Despite the ABA insensitivity of K+out channels in theabi1-1-transformed guard cells, concurrent measurements of intracellular pH shownormal pH increases in response to ABA (55). This suggests that theabi1-1phosphatase acts downstream of, or parallel to, the cytosolic pH changes (Figure 4).If cytosolic pH directly modulates K+out channels (141), thenabi1-1must act in aparallel pathway to alter the responsiveness of the K+

out channel to pH.In addition to the PP2C phosphatases identified genetically, experiments using

the phosphatase inhibitor OA implicate PP1- or PP2A-type protein phosphatasesin the regulation guard cell K+out channels. OA downregulates both K+in and K+outchannel currents inVicia (201). However, another study showed that OA downreg-ulated only K+in, and not K+out, currents (121). Despite the experimental differences,one study indicates a putative role for PP1- and/or PP2A-phosphatases as positiveregulators of K+out channel activity in guard cells [(Figure 4); possibly related toPP1/PP2Apos in (Figure 3)].

Syntaxins and ABA Signaling

Syntaxins play important roles in membrane fusion. A tobacco cDNA encodinga homolog of human and yeast syntaxin (NtSyr1) was isolated using heterologousexpression of drought-stressed tobacco leaf mRNA inXenopusoocytes (115). Ex-pression of mRNA pools show ABA activation of endogeneous Ca2+-activatedCl− currents in oocytes (115) and ABA downregulation of KAT1-mediated K+

inchannels in oocytes (196a), suggesting that oocytes will provide an approachfor isolating and analyzing putative ABA receptor cDNAs. By subfractionationtheNtSyr1mRNA was isolated, which when expressed in oocytes constitutivelyactivated the Cl− currents without addition of ABA (115). WhetherNtSyr1con-tributes to the ABA response in oocytes remains to be determined (115). Voltageclamp recordings in tobacco guard cells provide pharmacological evidence for arole of syntaxins in ABA regulation of K+ channels and S-type anion channels.These data suggest that syntaxin acts as a positive regulator of ABA signaling inguard cells (115) (Figures 3 and 4). Guard cell volume is changed during stomatalmovements and is accompanied by changes in membrane surface area (72, 98).Syntaxins may link ABA signaling to membrane trafficking.

Osmolarity and Temperature Sensitivity

Guard cell measurements of86Rb+ efflux kinetics show that whereas a rapid tran-sient K+ release response to ABA was dependent on the concentration of ABA andduration of exposure, the end-state or final internal86Rb+ concentration reachedwas not (131). This led to the suggestion that guard cells have some means ofsensing their internal osmolarity that is linked to regulation of K+ efflux channels.Vicia guard cell plasma membrane patches exposed to osmotic gradients showK+out channels that are inactivated by hypotonic (guard cell swelling) conditionsand activated by hypertonic (guard cell shrinking) conditions (123).

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Recent work has suggested that K+out channels may be involved in the responseof guard cells to another environmental stimulus: temperature. At moderate tem-peratures (13◦ to 20◦C), both K+in and K+out channel conductances inVicia increasewith increasing temperature. At temperatures from 20◦ to 28◦C, K+out conductancedecreases with increasing temperature, whereas K+

in conductance continues to in-crease (79). This difference in temperature response between K+

in and K+outchannelsat higher temperatures would favor K+ influx and stomatal opening and thus couldallow increased transpirational cooling of leaves (79).

Transient K+ Efflux Currents

In addition to the slow activating K+outchannels discussed above, a rapidly activatedtransient outward K+ current, (IAP), has been observed inArabidopsisand tobaccoguard cells (11, 153, 167). In addition to being the first inactivating K+

out currentcharacterized in plants, IAP has several unusual characteristics. In contrast tothe slow K+out channels that are activated by alkaline cytosolic pH (20), IAP isslightly inhibited by alkaline pHcyt (153). IAP is also inhibited by increased [Ca2+]cyt(153), whereas guard cell K+out channels are not regulated by small cytoplasmicCa2+ changes. IAP channels show a significant Ca2+ permeability (153). Thephysiological role of this transient current is unknown, but IAP may contribute toshorter-term adjustments in stomatal aperture or to membrane potential oscillationsobserved in guard cells (59, 202).

NEW GUARD CELL SIGNALING MUTANTSAND GENETIC APPROACHES

Quantitative and mechanistic characterization (4, 155) of newArabidopsisguardcell signaling mutants is paramount to achieve a molecular understanding of theABA signaling cascade. Furthermore genes for many of the above proposed cell bi-ologically and pharmacologically derived mechanisms have not yet been identified.

Loss-of-function mutations in theArabidopsisERA1 farnesyltransferaseβ sub-unit cause an enhanced response to ABA in seeds (34). Moreover, theera1mutantshows ABA hypersensitive stomatal closing and ABA hypersensitive activationof S-type anion currents (154). Furthermore,era1plants show reduced water lossduring drought (154). Application of farnesyltransferase inhibitors to wild-typestomata mimics theera1 phenotype, suggesting that ERA1 functions in earlyguard cell signaling and illustrating the complementarity of “steady-state” geneknockouts and “short-term” inhibitor applications. In mammals and yeast, farnesy-lation of signaling proteins promotes their membrane location and protein-proteininteractions, suggesting that a negative regulator of guard cell ABA signaling istargeted via farnesylation (154) (Figure 3). ABA hypersensitive [Ca2+]cyt eleva-tions inera1show that ERA1 functions upstream of [Ca2+]cyt elevations (G Allen,Y Murata, & J Schroeder, unpublished) (Figure 3).

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A new ABA hypersensitive loss-of-function mutant,abcap, was isolated andcharacterized by screening for ABA hypersensitivity in seed germination in a pri-mary screen and identifying the subset of mutations that affect guard cell signalingin a secondary screen (75). Stomatal closing is hypersensitive to ABA inabcapand, consistent with this phenotype, ABA-induced [Ca2+]cyt elevations and guardcell plasma membrane anion currents are enhanced and K+

in currents are reducedin abcap. Suprisingly,ABCAPencodes a subunit of a nuclear RNA cap bindingcomplex (75) previously described in mammals and yeast, which regulates RNAprocessing and growth factor signal transduction (83, 84, 211). ABCAP may con-trol the strength of ABA signaling by modulating the expression of an early ABAsignal transduction element(s) (Figure 3).

New Genetic Screens and Reverse Genetics

To date, most of the ABA-insensitive mutations identified in guard cell signaling(abi1-1, abi2-1, andaapk) are dominant (46, 114, 119). This suggests that redun-dancy in phosphatase and kinase activities may limit the isolation of recessivemutations in such genes. Dominant mutations can also be generated inArabidop-sisby activation tagging or the random overexpression of wild-type genes (210).Such mutants can aid in identifying and isolating redundant genes involved in guardcell signaling pathways. However, dominant mutations can result from interactionswith unnatural partners, causing neomorphic responses. To identify unequivocallythe functions of such genes, it will be important to isolate loss-of-function mutants(71). Disruption or silencing of homologous redundant genes expressed in guardcells may allow more direct characterizations of in vivo functions of redundantgenes.

Isolating guard cell signaling mutants is not trivial, owing to the lack of easilyscorable phenotypes or markers, and is more difficult than isolating stomatal devel-opment mutants, which include cell-to-cell signaling mechanisms (16a, 49a). Thustheera1andabcapguard cell phenotypes were identified in secondary stomatalmovement screens (75, 154). A highly elegant stomatal movement screen has beendeveloped in which small differences in leaf temperature [due to stomatal transpi-ration (163)] were used to isolate newArabidopsisguard cell signaling mutants(J Giraudat & S Merlot, personal communication; 139a). In a different approach,mutations that affect circadian control of stomatal movements were identified byselectingArabidopsismutants with an enhanced sensitivity to sulfur dioxide atspecific times of day (R McClung, personal communication). This screen has ledto isolation of circadian timing-defective (ctd) and out-of-phase (oop) mutants.In another screen using luciferase as a reporter, many mutants were isolated thatare affected in osmotic, ABA, and cold stress–induced signal transduction (81). Asubset of these mutations will likely affect guard cell signaling. Use of novel cre-ative genetic screens will lead to identification of many new mechanisms affectingguard cell signaling. For example, new guard cell signaling mutants in stomatalresponses could be isolated based on variation in responses amongArabidopsis

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ecotypes and use of recombinant inbred lines to map quantitative trait loci (9) thataffect guard cell signaling.

Note that manyArabidopsislight and hormone signaling mutants have been iso-lated based on whole-plant or whole-tissue phenotypes, which can lead to isolationof genes that indirectly affect a signaling pathway via crosstalk or indirect feedbackamong signaling cascades. Analyses of ion channel regulation and [Ca2+]cyt sig-naling in mutants allows one to closely associate mutations with specific elementsin early signaling (4, 154, 155).

The completion of theArabidopsisgenome sequence will lead to reverse ge-netic functional characterizations of new guard cell signaling components usingbiophysical cell biological (4, 5, 155) and genomic methods developed and adaptedtoArabidopsisguard cells. The identification of the full complement of guard cell–expressed genes is now possible using DNA array and chip technologies, whichwill have profound influence on future research. Reverse genetic analyses of guardcell–expressed genes will be needed to identify genes and test the function andrelative contribution of the many proposed signal transducers reviewed here(Figures 1 to 4). Furthermore, many of the signal transducers reviewed here likelyform signal transduction complexes consisting of many proteins. Proteomic ap-proaches (119) will play an increasingly important role for identifying membersof guard cell signaling complexes.

FUTURE OUTLOOK

Guard cell research has revealed many new signal transduction components andled to models of early signal transduction elements and signaling cassettes inplants. Many of the proposed mechanisms summarized in this review can nowbe directly tested by reverse genetics. Furthermore, this research has led to aninitial understanding of how a large number of signaling mechanisms can interactin concert to produce a rapid physiological response. Future research on mu-tants will define molecular junction points of an integrated network comprisingFigures 1 to 4. As discussed in the introduction, most plant cells respond to theclassically known hormones and light signals in specific ways. In this sense, eachplant cell contains a microcosm of plant signaling cascades with intricate crosstalkand specificity mechanisms. The guard cell system lends itself to functional char-acterization of many new unknown early signaling mechanisms (see 1 to 4 inIntroduction). Interdisciplinary studies using cell biological, genomic, moleculargenetic, biophysical, reverse genetic, and proteomic approaches will define muchof the future research in this field.

Initial examples have shown that manipulation of guard cell signaling genesin Arabidopsiscan affect stomatal movements, leading to reduced water loss andslowing of dessication during transitory drought periods (53, 75, 154). Futureresearch in this area combined with inducible guard cell-specific gene expres-sion or cell-specific gene silencing will lead to identification of mechanisms for

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engineering improved gas exchange in response to drought, elevated atmosphericCO2, and other environmental stresses (180). In this respect, guard cell signalingresearch holds much promise at addressing major environmental and agriculturalproblems of the twenty-first century.

ACKNOWLEDGMENTS

We thank many colleagues for communicating new and unpublished findings.Majid Ghassemian, Nathalie Leonhardt, Pascal M¨aser, Jared Young, and othermembers of the Schroeder lab are gratefully acknowledged for comments on themanuscript. Research in the authors’ laboratory was supported by NSF, NIH, andDOE grants.

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LITERATURE CITED

1. Ache P, Becker D, Ivashikina N, Di-etrich P, Roelfsema MRG, Hedrich R.2000. GORK, a delayed outward rectifierexpressed in guard cells ofArabidopsisthaliana, is a K+-selective K+-sensing ionchannel.FEBS Lett.486:93–98

1a. Ahmad M, Cashmore AR. 1993. HY4 geneof A. thalianaencodes a protein with char-acteristics of a blue-light photoreceptor.Nature366:162–66

2. Allan AC, Fricker MD, Ward JL, BealeMH, Trewavas AJ. 1994. Two transductionpathways mediate rapid effects of abscisicacid inCommelinaguard cells.Plant Cell6:1319–28

3. Allen GJ, Chu SP, Schumacher K, Shi-mazaki C, Vafeados D, et al. 2000. Al-teration of stimulus-specific guard cellcalcium oscillations and stomatal clos-ing in Arabidopsis det3mutant.Science289:2338–42

4. Allen GJ, Kuchitsu K, Chu SP, MurataY, Schroeder JI. 1999.Arabidopsis abi1-1andabi2-1mutations impair abscisic acidinduced cytosolic calcium rises in guardcells.Plant Cell11:1785–98

5. Allen GJ, Kwak JM, Chu SP, Llopis J,Tsien RY, et al. 1999. Cameleon calciumindicator reports cytoplasmic calcium dy-

namics inArabidopsisguard cells.Plant J.19:735–47

6. Allen GJ, Muir SR, Sanders D. 1995. Re-lease of Ca2+ from individual plant vac-uoles by both InsP3 and cyclic ADP-ribose.Science268:735–37

7. Allen GJ, Sanders D. 1996. Control of ioniccurrents guard cell vacuoles by cytosolicand luminal calcium.Plant J.10:1055–69

8. Allen GJ, Sanders D. 1997. Vacuolar ionchannels of higher plants.Adv. Bot. Res.25:217–52

9. Alonso-Blanco C, El-Assal SE-D, Cou-pland G, Koornneef M. 1998. Analysisof natural allelic variation at floweringtime loci in the Landsberg erecta andCape Verde Islands ecotypes ofArabidop-sis thaliana. Genetics149:749–64

10. Anderson JA, Huprikar SS, Kochian LV,Lucas WJ, Gaber RF. 1992. Functionalexpression of a probableArabidopsisthaliana potassium channel inSaccha-romyces cerevisiae. Proc. Natl. Acad. Sci.USA89:3736–40

11. Armstrong F, Leung J, Grabov A, BrearleyJ, Giraudat J, Blatt MR. 1995. Sensi-tivity to abscisic acid of guard-cell K+channels is suppressed byabi1-1, a mu-tantArabidopsisgene encoding a putative

P1: FXY


648 SCHROEDER ET AL

protein phosphatase.Proc. Natl. Acad.Sci. USA92:9520–24

12. Assmann SM. 1999. The cellular basis ofguard cell sensing of rising CO2. PlantCell Environ.22:629–37

13. Assmann SM, Shimazaki K-I. 1999. Themultisensory guard cell. Stomatal re-sponses to blue light and abscisic acid.Plant Physiol.119:809–15

14. Assmann SM, Simoncini L, Schroeder JI.1985. Blue light activates electrogenic ionpumping in guard cell protoplasts ofViciafaba. Nature318:285–87

15. Barbier-Brygoo H, Vinauger M, Colcom-bet J, Ephritikhine G, Frachisse J-M,Maurel C. 2000. Anion channels in higherplants: functional characterization, mole-cular structure and physiological role.Biochim. Biophys. Acta1465:199–218

16. Baunsgaard L, Fuglsang AT, Jahn T, Ko-rthout HAAJ, De Boer AH, PalmgrenMG. 1998. The 14-3-3 proteins associatewith the plant plasma membrane H+-ATPase to generate fusicoccin bindingcomplex and a fusicoccin responsive sys-tem.Plant J.13:661–71

16a. Berger D, Altmann T. 2000. A subtilisin-like serine protease involved in the reg-ulation of stomatal density and distribu-tion in Arabidopsis thaliana. Genes Dev.14:1119–31

17. Berkowitz G, Zhang X, Mercier R, LengQ, Lawton M. 2000. Co-expression ofcalcium-dependent protein kinase withthe inward rectified guard cell K+ channelKAT1 alters current parameters inXeno-pus laevisoocytes.Plant Cell Physiol.41:785–90

18. Bewell MA, Maathuis FJM, Allen GJ,Sanders D. 1999. Calcium-induced cal-cium release mediated by a voltage-activated cation channel in vacuolar vesi-cles from red beet.FEBS Lett.458:41–44

19. Blatt MR. 1990. Potassium channel cur-rents in intact stomatal guard cells: rapidenhancement by abscisic acid.Planta180:445–55

20. Blatt MR. 1992. K+channels of stomatalguard cells: characteristics of the inward-rectifier and its control by pH.J. Gen. Phys-iol. 99:615–44

21. Blatt MR. 1999. Reassessing roles forCa2+ in guard cell signalling.J. Exp. Bot.50:989–99

22. Blatt MR, Armstrong F. 1993. K+channels of stomatal guard cells: abscisic-acid-evoked control of the outward-rectifier mediated by cytoplasmic pH.Planta191:330–41

23. Blatt MR, Thiel G, Trentham DR. 1990.Reversible inactivation of K+ channels ofVicia stomatal guard cells following thephotolysis of caged 1,4,5-trisphosphate.Nature346:766–69

24. Brearley J, Venis MA, Blatt MR. 1997.The effect of elevated CO2 concentra-tions on K+ and anion channels ofVi-cia faba L. guard cells.Planta 203:145–54

25. Burnett EC, Desikan R, Moser RC, NeillSJ. 2000. ABA activation of an MBP ki-nase inPisum sativunepidermal peels cor-relates with stomatal responses to ABA.J.Exp. Bot.51:197–205

26. Buschmann P, Gradmann D. 1997. Min-imal model for oscillations of membranevoltage in plant cells.J. Theor. Biol.188:323–32

27. Carpaneto A, Cantu AM, Gambale F. 2001.Effects of cytoplasmic Mg2+ on the slowlyactivating channels in isolated vacuoles ofBeta vulgaris. Planta.In press

28. Cashmore AR, Jarillo JA, Wu Y-J, Liu D.1999. Cryptochromes: blue light receptorsfor plants and animals.Science284:760–65

29. Christie JM, Reymond P, Powell GK,Bernasconi P, Raibekas AA, et al. 1998.Arabidopsis NPH1: a flavoprotein withthe properties of a photoreceptor for pho-totropism.Science282:1698–701

30. Cosgrove DJ, Hedrich R. 1991. Stretch-activated chloride, potassium, and cal-cium channels co-existing in the plasma

P1: FXY



membranes of guard cells ofVicia fabaL.Planta186:143–53

31. Cousson A. 2000. Analysis of the sens-ing and transducing processes impli-cated in the stomatal responses to carbondioxide inCommelina communisL. PlantCell Environ.23:487–95

32. Cousson A, Vavasseur A. 1998. Putativeinvolvement of cytosolic Ca2+ and GTP-binding proteins in cyclic-GMP-mediatedinduction of stomatal opening by auxin inCommelina communisL. Planta206:308–14

33. Curvetto N, Darjania L, Delmastro S. 1994.Effect of two cAMP analogs on stomatalopening inVicia faba: possible relation-ship with cytosolic calcium concentration.Plant Physiol. Biochem.32:365–72

34. Cutler S, Ghassemian M, Bonetta D,Cooney S, McCourt P. 1996. A protein far-nesyl transferase involved in abscisic acidsignal transduction inArabidopsis.Science273:1239–41

35. Czempinski K, Gaedeke N, ZimmermannS, Muller-Rober B. 1999. Molecular mech-anisms and regulation of plant ion chan-nels.J. Exp. Bot.50:955–66

36. DeKoninck P, Schulman H. 1998. Sensi-tivity of CaM kinase II to the frequency ofCa2+ oscillations.Science279:227–30

37. DeSilva DLR, Hetherington AM, Mans-field TA. 1985. Synergism between cal-cium ions and abscisic acid in preventingstomatal opening.New Phytol.100:473–82

38. DeSilva DLR, Hetherington AM, Mans-field TA. 1998. The regulation of apoplas-tic calcium in relation to intracellularsignalling in stomatal guard cells.Z.Pflanzenernaehr. Bodenkd.161:533–39

39. Dietrich P, Hedrich R. 1994. Interconver-sion of fast and slow gating modes ofGCAC1, a guard cell anion channel.Planta195:301–4

40. Dolmetsch RE, Xu K, Lewis RS. 1998.Calcium oscillations increase the effi-ciency and specificity of gene expression.Nature392:933–36

41. Drake BG, Gonzalez-Meler MA, LongSP. 1997. More efficient plants: a con-sequence of rising atmospheric CO2?Annu. Rev. Plant Physiol. Plant Mol. Biol.48:609–39

42. Dreyer I, Horeau C, Lemaillet G, Zim-mermann S, Bush DR, et al. 1999. Iden-tification and characterization of planttransporters using heterologous expres-sion systems.J. Exp. Bot.50:1073–87

43. Esser JE, Liao Y-J, Schroeder JI. 1997.Characterization of ion channel modula-tor effects on ABA- and malate-inducedstomatal movements: strong regulation bykinase and phosphatase inhibitors, andrelative insensitivity to mastoparans.J.Exp. Bot.48:539–50

44. Eun S-O, Lee Y. 1997. Actin filamentsof guard cells are reorganized in responseto light and abscisic acid.Plant Physiol.115:1491–98

45. Fink M, Deuprat F, Lesage F, HeurteauxC, Romey G, et al. 1996. A new K+ chan-nel beta subunit to specifically enhanceKv2.2 (CDRK) expression.J. Biol. Chem.271:26341–48

46. Finkelstein RR, Somerville CR. 1990.Three classes of abscisic acid (ABA)-insensitive mutations ofArabidopsisde-fine genes that control overlapping sub-sets of ABA responses.Plant Physiol.94:1172–79

47. Frechilla S, Zhu J, Talbott LD, Zeiger E.1999. Stomata fromnpq1, a zeaxanthin-lessArabidopsismutant, lack a specificresponse to blue light.Plant Cell Physiol.40:949–54

48. Fu H-H, Luan S. 1998. AtKUP1: a dual-affinity K+ transporter fromArabidopsis.Plant Cell10:63–73

49. Fullone MR, Visconti S, Marra M,Fogliano V, Aducci P. 1998. Fusicoc-cin effect on the in vitro interaction be-tween plant 14-3-3 proteins and plasmamembrane H+-ATPase.J. Biol. Chem.273:7698–702

49a. Geisler M, Nadeau J, Sack FD. 2000.

P1: FXY


650 SCHROEDER ET AL

Oriented asymmetric divisions that gen-erate the stomatal spacing pattern inAra-bidopsisare disrupted by the too manymouths mutation.Plant Cell12:2075–86

49b. Ghelis T, Dellis O, Jeannette E, BardatF, Cornel D, et al. 2000. Abscisic acidspecific expression ofRAB18involves ac-tivation of anion channels inArabidop-sis thalianasuspension cells.FEBS Lett.474:43–47

49c. Ghelis T, Dellis O, Jeannette E, BardatF, Miginiac E, Sotta B. 2000. Abscisicacid plasmalemma perception triggers acalcium influx essential forRAB18geneexpression inArabidopsis thalianasus-pension cells.FEBS Lett.483:67–70

50. Gilroy S, Fricker MD, Read ND, Tre-wavas AJ. 1991. Role of calcium in signaltransduction ofCommelinaguard cells.Plant Cell3:333–44

51. Gilroy S, Read ND, Trewavas AJ. 1990.Elevation of cytoplasmic Ca2+ by cagedcalcium or caged inositol triphosphate ini-tiates stomatal closure.Nature346:769–71

52. Goh C-H, Kinoshita T, Oku T, Shi-mazaki K-I. 1996. Inhibition of bluelight-dependent H+ pumping by abscisicacid inVicia guard-cell protoplasts.PlantPhysiol.111:433–40

53. Gosti F, Beaudoin N, Serizet C, WebbARR, et al. 1999. ABI1 protein phosph-atase 2C is a negative regulator of abscisicacid signaling.Plant Cell11:1897–910

54. Grabov A, Blatt MR. 1997. Parallel con-trol of the inward-rectifier K+ channel bycytosolic free Ca2+ and pH inViciaguardcells.Planta201:84–95

55. Grabov A, Blatt MR. 1998. Co-ordinationof signalling elements in guard cell ionchannel control.J. Exp. Bot.49:351–60

56. Grabov A, Blatt MR. 1998. Membranevoltage initiates Ca2+ waves and poten-tiates Ca2+ increases with abscisic acidin stomatal guard cells.Proc. Natl. Acad.Sci. USA95:4778–83

57. Grabov A, Blatt MR. 1999. A steep dep-

endence of inward-rectifying potassiumchannels on cytosolic free calcium concen-tration increase evoked by hyperpolariza-tion in guard cells.Plant Physiol.119:277–87

58. Grabov A, Leung J, Giraudat J, Blatt MR.1997. Alteration of anion channel kineticsin wild-type andabi1-1 transgenicNico-tiana benthamianaguard cells by abscisicacid.Plant J.12:203–13

59. Gradmann D, Blatt MR, Thiel G. 1993.Electrocoupling of ion transporters inplants.J. Memb. Biol.136:327–32

60. Guan LQM, Zhao J, Scandalios JG. 2000.Cis-elements and trans-factors that regu-late expression of the maize Cat1 antioxi-dant gene in response to ABA and osmoticstress: H2O2 is the likely intermediary sig-naling molecule for the response.Plant J.22:87–95

61. Hamilton DWA, Hills A, Kohler B, BlattMR. 2000. Ca2+ channels at the plasmamembrane of stomatal guard cells are ac-tivated by hyperpolarization and abscisicacid.Proc. Natl. Acad. Sci. USA97:4967–72

62. Hechenberger M, Schwappach B, FischerWN, Frommer WB, Jentsch TJ, Stein-meyer K. 1996. A family of putative chlo-ride channels fromArabidopsisand func-tional complementation of a yeast strainwith a CLC gene disruption.J. Biol. Chem.271:33632–38

63. Hedrich R, Becker D. 1994. Green circuits:the potential of plant specific ion channels.Plant Mol. Biol.26:1637–50

64. Hedrich R, Busch H, Raschke K. 1990.Ca2+ and nucleotide dependent regulationof voltage dependent anion channels in theplasma membrane of guard cells.EMBO J.9:3889–92

65. Hedrich R, Dietrich P. 1996. Plant K+channels: similarity and diversity.Bot.Acta109:94–101

66. Hedrich R, Marten I, Lohse G, Dietrich P,Winter H, et al. 1994. Malate-sensitive an-ion channels enable guard cells to sense

P1: FXY



changes in the ambient CO2 concentration.Plant J.6:741–48

67. Hedrich R, Moran O, Conti F, Busch H,Becker D, et al. 1995. Inward rectifierpotassium channels in plants differ fromtheir animal counterparts in response tovoltage and channel modulators.Eur. Bio-phys. J.24:107–15

68. Heimovaara-Dijkstra S, Testerink C, WangM. 2000. Mitogen-activated protein kinaseand abscisic acid signal transduction.Re-sults Probl. Cell Differ.27:131–44

69. Henriksen GH, Taylor AR, Brownlee C,Assmann SM. 1996. Laser microsurgery ofhigher plant cell walls permits patch-clampaccess.Plant Physiol.110:1063–68

70. Hey SJ, Bacon A, Burnett E, Neill SJ. 1997.Abscisic acid signal transduction in epider-mal cells ofPisum sativum L. Argenteum:Both dehydrin mRNA accumulation andstomatal responses require protein phos-phorylation and dephosphorylation.Planta202:85–92

71. Himmelbach A, Iten M, Grill E. 1998. Sig-nalling of abscisic acid to regulate plantgrowth.Philos. Trans. R. Soc. London Ser.B 353:1439–44

72. Homann U. 1998. Fusion and fissionof plasma-membrane material accommo-dates for osmotically induced changes inthe surface area of guard-cell protoplasts.Planta206:329–33

73. Hoshi T. 1995. Regulation of voltage-dependence of theKAT1channel by intra-cellular factors.J. Gen. Physiol.105:309–28

74. Hoth S, Dreyer I, Dietrich P, Becker D,Muller-Rober B, Hedrich R. 1997. Molec-ular basis of plant-specific acid activationof K+ uptake channels.Proc. Natl. Acad.Sci. USA94:4806–10

75. Hugouvieux V, Kwak JM, Murata Y,Schroeder JI. 2001. A nuclear mRNA capbinding protein, ABCAP, modulates earlyabscisic acid signal transduction inAra-bidopsis. Submitted

76. Hwang J-U, Suh S, Yi H, Kim J, Lee Y.

1997. Actin filaments modulate both stom-atal opening and inward K+-channel activ-ities in guard cells ofVicia fabaL. PlantPhysiol.115:335–42

77. Ichida AM, Pei Z-M, Baizabal-AguirreVM, Turner KJ, Schroeder JI. 1997. Ex-pression of a Cs+-resistant guard cellK+ channel confers Cs+-resistant, light-induced stomatal opening in transgenicArabidopsis. Plant Cell9:1843–57

78. Ichida AM, Schroeder JI. 1996. Increasedresistance to extracellular cation blockby mutation of the pore domain of theArabidopsisinward-rectifying K+ channelKAT1. J. Memb. Biol.151:53–62

79. Ilan N, Moran N, Schwartz A. 1995. Therole of potassium channels in the temper-ature control of stomatal aperture.PlantPhysiol.108:1161–70

80. Irving HR, Gehring CA, Parish RW. 1992.Changes in cytosolic pH and calcium ofguard cells precede stomatal movements.Proc. Natl. Acad. Sci. USA89:1790–94

81. Ishitani M, Xiong LM, Stevenson B, ZhuJ-K. 1997. Genetic analysis of osmotic andcold stress signal transduction inArabidop-sis: interactions and convergence of ab-scisic acid-dependent and abscisic acid-independent pathways.Plant Cell9:1935–49

82. Deleted in proof83. Izaurralde E, Lewis J, Gamberi C, Jar-

molowski A, McGuigan C, Mattaj IW.1995. A cap-binding protein complex me-diating U snRNA export.Nature376:709–12

84. Izaurralde E, Lewis J, McGuigan C,Jankowska M, Darynkiewicz E, Mattaj IW.1994. A nuclear cap binding protein com-plex involved in pre-mRNA splicing.Cell78:657–68

85. Jacob T, Ritchie S, Assmann SM, GilroyS. 1999. Abscisic acid signal transductionin guard cells is mediated by phospholi-pase D activity.Proc. Natl. Acad. Sci. USA96:12192–97

P1: FXY


652 SCHROEDER ET AL

86. Jahn T, Fuglsang AT, Olsson A, BruntrupIM, Collinge DB, et al. 1997. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma mem-brane H+-ATPase.Plant Cell9:1805–14

87. Jan LY, Jan YN. 1992. Structural elementsinvolved in specific K+ channel functions.Annu. Rev. Physiol.54:537–55

88. Jarvis AJ, Davies WJ. 1998. The coupledresponse of stomatal conductance to pho-tosynthesis and transpiration.J. Exp. Bot.49:399–406

89. Keller BU, Hedrich R, Raschke K. 1989.Voltage-dependent anion channels in theplasma membrane of guard cells.Nature341:450–53

90. Kelly WB, Esser JE, Schroeder JI. 1995.Effects of cytosolic calcium and limited,possible dual, effects of G protein modula-tors on guard cell inward potassium chan-nels.Plant J.8:479–89

91. Kim EJ, Kwak JM, Uozumi N, SchroederJI. 1998. AtKUP1: an arabidopsis geneencoding high-affinity potassium transportactivity. Plant Cell10:51–62

92. Kinoshita T, Nishimura M, ShimazakiK-I. 1995. Cytosolic concentration ofCa-2+ regulates the plasma membrane H+-ATPase in guard cells of fava bean.PlantCell 7:1333–42

93. Kinoshita T, Shimazaki K-I. 1997. Involve-ment of calyculin A- and okadaic acid-sensitive protein phosphatase in the bluelight response of stomatal guard cells.Plant Cell Physiol.38:1281–85

94. Kinoshita T, Shimazaki K-I. 1999. Bluelight activates the plasma membrane H+-ATPase by phosphorylation of the C ter-minus in stomatal guard cells.EMBO J.18:5548–58

95. Knetsch MLW, Wang M, Snaar-JagalskaBE, Heimovaara-Dijkstra S. 1996. Absc-isic acid induces mitogen-activated proteinkinase activation in barley aleurone proto-plasts.Plant Cell8:1061–67

96. Kolb HA, Marten I, Hedrich R. 1995.Hodgkin-Huxley analysis of a GCAC1

anion channel in the plasma membrane ofguard cells.J. Memb. Biol.146:273–82

97. Koornneef M, Reuling G, KarssenCM. 1984. The isolation of abscisicacid-insensitive mutants ofArabidopsisthaliana. Physiol. Plant61:377–83

98. Kubitscheck U, Homann U, Thiel G.2000. Osmotically evoked shrinking ofguard-cell protoplasts causes vesicular re-trieval of plasma membrane into the cy-toplasm.Planta210:423–31

99. Kuo A, Cappelluti S, Cervantes-Vervantes M, Rodriguez M, Bush DS.1996. Okadaic acid, protein phosphataseinhibitor, blocks calcium changes, geneexpression and cell death induced bygibberellin in wheat aleurone cells.PlantCell 8:259–69

100. Kwak JM, Murata Y, Baizabal-AguirreVM, Merrill J, Wang M, et al. 2001. Dom-inant negative guard cell K+ channel mu-tants reduce inward rectifying K+ cur-rents and light-induced stomatal openingin Arabidopsis. Submitted

101. Lasc`eve G, Leymarie J, Olney MA, Lis-cum E, Christie JM, et al. 1999.Ara-bidopsiscontains at least four indepen-dent blue-light-activated signal transduc-tion pathways.Plant Physiol.120:605–14

102. Leckie CP, McAinsh MR, Allen GJ,Sanders D, Hetherington AM. 1998. Ab-scisic acid-induced stomatal closure me-diated by cyclic ADP-ribose.Proc. Natl.Acad. Sci. USA95:15837–42

103. Lee HC. 1997. Mechanisms of cal-cium signaling by cyclic ADP-ribose andNAADP. Physiol. Rev.77:1133–64

104. Lee S, Choi H, Suh S, Doo I-S, OhK-Y, et al. 1999. Oligogalacturonic acidand chitosan reduce stomatal aperture byinducing the evolution of reactive oxy-gen species from guard cells of tomatoand Commelina communis. Plant Phys-iol. 121:147–52

105. Lee Y, Choi YB, Suh S, Lee J, AssmannSM, et al. 1996. Abscisic acid-inducedphosphinositide turn over in guard cell

P1: FXY



protoplasts ofVicia faba. Plant Physiol.110:987–96

106. Lee Y, Lee HJ, Crain RC, Lee A, Korn SJ.1994. Polyunsaturated fatty acids modu-late stomatal aperture and two distinct K+channel currents in guard cells.Cell. Sig-nal. 6:181–86

107. Lemichez E, Wu Y, Sanchez JP, ChuaN-H. 2000. Abscisic acid regulates stom-atal closure through the inactivation ofp21-Rac.6th Int. Congr. Plant Mol. Biol.

108. Lemtiri-Chlieh F, MacRobbie EAC.1994. Role of calcium in the modulationof Vicia guard cell potassium channelsby abscisic acid—a patch-clamp study.J.Memb. Biol.137:99–107

109. Lemtiri-Chlieh F, MacRobbie EAC,Brearley CA. 2000. Inositol hexakispho-phate is a physiological signal regulatingthe K+-inward rectifying conductance inguard cells.Proc. Nat. Acad. Sci. USA97:8687–92

110. Leonhardt N, Marin E, Vavasseur A,Forestier C. 1997. Evidence for the ex-istence of a sulfonylurea-receptor-likeprotein in plants: modulation of stom-atal movements and guard cell potassiumchannels by sulfonylureas and potassiumchannel openers.Proc. Natl. Acad. Sci.USA94:14156–61

111. Leonhardt N, Vavasseur A, Forestier C.1999. ATP binding cassette modulatorscontrol abscisic acid-regulated slow an-ion channels in guard cells.Plant Cell11:1141–52

112. Leung J, Bouvier-Durand M, Morris P-C,Guerrier D, Chefdor F, Giraudat J. 1994.ArabidopsisABA response geneABI1—features of a calcium-modulated proteinphosphatase.Science264:1448–52

113. Leung J, Giraudat J. 1998. Abscisic acidsignal transduction.Annu. Rev. PlantPhysiol. Plant Mol. Biol.49:199–222

114. Leung J, Merlot S, Giraudat J. 1997.TheArabidopsisabscisic acid-insensitive(ABI2) and ABI1 genes encode homol-ogous protein phosphatases 2C involved

in abscisic acid signal transduction.PlantCell 9:759–71

115. Leyman B, Geelen D, Quintero FJ, BlattMR. 1999. A tobacco syntaxin with arole in hormonal control of guard cell ionchannels.Science283:537–40

116. Leymarie J, Vavasseur A, Lasceve G.1998. CO2 sensing in stomata ofabi1-1andabi2-1mutants ofArabidopsis thali-ana. Plant Physiol. Biochem.36:539–43

117. Li J, Assmann SM. 1996. An abscisicacid-activated and calcium-independentprotein kinase from guard cells of favabean.Plant Cell8:2359–68

118. Li J, Lee Y-RJ, Assmann SM. 1998.Guard cells possess a calcium-dependentprotein kinase that phosphorylates theKAT1 potassium channel.Plant Physiol.116:785–95

119. Li J, Wang XQ, Watson MB, AssmannSM. 2000. Regulation of abscisic acid-induced stomatal closure and anion chan-nels by guard cell AAPK kinase.Science287:300–3

120. Li W-H, Llopis J, Whitney M, ZlokarnikG, Tsien RY. 1998. Cell-permeant cagedInsP3 ester shows that Ca2+ spike fre-quency can optimize gene expression.Na-ture392:936–41

121. Li WW, Luan S, Schreiber SL, AssmannSM. 1994. Evidence for protein phos-phatase and 2A regulation of K+ channelsin two types of leaf cells.Plant Physiol.106:963–70

122. Linder B, Raschke K. 1992. A slow anionchannel in guard cells, activation at largehyperpolarization, may be principal forstomatal closing.FEBS Lett.313:27–30

123. Liu K, Luan S. 1998. Voltage-dependentK+ channels as targets of osmosensing inguard cells.Plant Cell10:1957–70

124. Lohse G, Hedrich R. 1992. Characteri-zation of the plasma membrane H+ AT-Pase fromVicia fabaguard cells; modula-tion by extracellular factors and seasonalchanges.Planta188:206–14

125. Lu P, Zhang SQ, Outlaw WH, Riddle

P1: FXY


654 SCHROEDER ET AL

KA. 1995. Sucrose: a solute that accu-mulates in the guard cell apoplast andguard cell symplast of open stomata.FEBS Lett.362:180–84

126. Luan S, Li W, Rusnak F, Assmann SM,Schreiber SL. 1993. Immunosuppres-sants implicate protein phosphatase reg-ulation of K+ channels in guard cells.Proc. Natl. Acad. Sci. USA90:2202–6

127. Lurin C, Geelen D, Barbier-Brygoo H,Guern J, Maurel C. 1996. Cloning andfunctional expression of a plant voltage-dependent chloride channel.Plant Cell8:701–11

128. Maathuis FJM, Ichida AM, Sanders D,Schroeder JI. 1997. Roles of higher plantK+ channels.Plant Physiol.114:1141–49

129. MacRobbie EAC. 1981. Effects of ABAon “isolated” guard cells ofCommelinacommunisL. J. Exp. Bot.32:563–72

130. MacRobbie EAC. 1983. Effects oflight/dark on cation fluxes in guard cellsof Commelina communisL. J. Exp. Bot.34:1695–710

131. MacRobbie EAC. 1995. ABA-inducedion efflux in stomatal guard cells: mul-tiple actions of ABA inside and outsidethe cell.Plant J.7:565–76

132. MacRobbie EAC. 1998. Signal trans-duction and ion channels in guard cells.Philos. Trans. R. Soc. London Ser. B353:1475–88

132a. MacRobbie EAC. 2000. ABA activatesmultiple Ca2+ fluxes in stomatal guardcells, triggering vacuolar K+(Rb+)release, Proc. Natl. Acad. Sci. USA97:12361–68

133. Mansfield TA, Hetherington AM, Atkin-son CJ. 1990. Some current aspects ofstomatal physiology.Annu. Rev. PlantPhysiol. Plant Mol. Biol.41:55–75

134. McAinsh MR, Brownlee C, Hethering-ton AM. 1990. Abscisic acid-inducedelevation of guard cell cytosolic Ca2+precedes stomatal closure.Nature343:186–88

135. McAinsh MR, Brownlee C, Hethering-ton AM. 1992. Visualizing changes incytosolic-free Ca2+ during the responseof stomatal guard cells to abscisic acid.Plant Cell4:1113–22

136. McAinsh MR, Brownlee C, Hethering-ton AM. 1997. Calcium ions as secondmessengers in guard cell signal trans-duction.Physiol. Plant.100:16–29

137. McAinsh MR, Clayton H, MansfieldTA, Hetherington AM. 1996. Changes instomatal behavior and guard cell cytoso-lic free calcium in response to oxidativestress.Plant Physiol.111:1031–42

138. McAinsh MR, Hetherington AM. 1998.Encoding specificity in Ca2+ signallingsystems.Trends Plant Sci.3:32–36

139. McAinsh MR, Webb AR, Taylor JE,Hetherington AM. 1995. Stimulus-induced oscillations in guard cell cytoso-lic free calcium.Plant Cell7:1207–19

139a. Merlot S, Gosti F, Guerrier D, VavasseurA, Giraudat J. 2001. The ABI1 and ABI2protein phosphatases 2C act in a negativefeedback regulatory loop of the abscisicacid signalling pathway.Plant J.25:1–10

140. Meyer K, Leube MP, Grill E. 1994.A protein phosphatase 2C involved inABA signal transduction inArabidopsisthaliana. Science264:1452–55

141. Miediema H, Assmann SM. 1996. Amembrane-delimited effect of internalpH on the K+ outward rectifier ofVi-cia faba guard cells.J. Memb. Biol.154:227–37

142. Miyawaki A, Griesbeck O, Heim R,Tsien RY. 1999. Dynamic and quantita-tive Ca2+measurements using improvedcameleons.Proc. Natl. Acad. Sci. USA96:2135–40

143. Miyawaki A, Llopis J, Heim R, McCaf-fery JM, Adams JM, et al. 1997. Fluores-cent indicators for Ca2+ based on greenfluorescent proteins and calmodulin.Na-ture388:882–87

144. Mori I, Muto S. 1997. Abscisic acid

P1: FXY



activates a 48-kilodalton protein kinasein guard cell protoplasts.Plant Physiol.113:833–40

145. Muller-Rober B, Ehrhardt T, Plesh G.1998. Molecular features of stomatalguard cells.J. Exp. Bot.49:293–304

146. Muller-Rober B, Ellenberg N, ProvartN, Willmitzer L, Busch H, et al. 1995.Cloning and electrophysiological analy-sis of KST1, an inward rectifying K+channel expressed in potato guard cells.EMBO J.14:2409–16

147. Deleted in proof148. Nakamura RL, McKendree WL, Hirsch

RE, Sedbrook JC, Gaber RF, SussmanMR. 1995. Expression of anArabidop-sispotassium channel gene in guard cells.Plant Physiol.109:371–74

149. Netting AG. 2000. pH, abscisic acid andthe integration of metabolism in plantsunder stressed and non-stressed condi-tions: cellular responses to stress and theirimplication for plant water relations.J.Exp. Bot.51:147–58

150. Niyogi KK, Grossman AR, BjorkmanO. 1998.Arabidopsismutants define acentral role for the xanthophyll cyclein the regulation of photosynthetic en-ergy conversion.Plant Cell 10:1121–34

151. Parmar PN, Brearley CA. 1995. Meta-bolism of 3- and 4-phosphorylated phos-phatidylinositols in stomatal guard cellsof Commelina communisL. Plant J.8:425–33

152. Parvathi K, Raghavendra AS. 1997. Bluelight-promoted stomatal opening in abax-ial epidermis ofCommelina benghalensisis maximal at low calcium.Physiol. Plant101:861–64

153. Pei Z-M, Baizabal-Aguirre VM, AllenGJ, Schroeder JI. 1998. A transientoutward-rectifying K+ channel currentdown-regulated by cytosolic Ca2+ in Ara-bidopsis thalianaguard cells.Proc. Natl.Acad. Sci. USA95:6548–53

154. Pei Z-M, Ghassemian M, Kwak CM,

McCourt P, Schroeder JI. 1998. Role offarnesyltransferase in ABA regulation ofguard cell anion channels and plant waterloss.Science282:287–90

155. Pei Z-M, Kuchitsu K, Ward JM, SchwarzM, Schroeder JI. 1997. Differential ab-scisic acid regulation of guard cell slowanion channels inArabdiopsiswild-typeand abi1 and abi2 mutants.Plant Cell9:409–23

156. Pei Z-M, Murata Y, Benning G, ThomineS, Klusener B, et al. 2000. Calcium chan-nels activated by hydrogen peroxide me-diate abscisic acid signalling in guardcells.Nature406:731–34

157. Pei Z-M, Ward JM, Harper JF, SchroederJI. 1996. A novel chloride channel inViciafabaguard cell vacuoles activated by theserine/threonine kinase, CDPK.EMBO J.15:6564–74

158. Pei Z-M, Ward JM, Schroeder JI. 1999.Magnesium sensitizes slow vacuolar cha-nnels to physiological cytosolic calciumand inhibits fast vacuolar channels in fababean guard cell vacuoles.Plant Physiol.121:977–86

159. Poffenroth M, Green DB, Tallman G.1992. Sugar concentrations in guard cellsof Vicia fabailluminated with red or bluelight: analysis by high performance liquidchromatography.Plant Physiol.98:1460–71

160. Pottosin II, Tikhonova LI, Hedrich R,Schoenknecht G. 1997. Slowly activatingvacuolar channels can not mediate Ca2+-induced Ca2+ release.Plant J.12:1387–98

161. Quintero F, Blatt M. 1997. A new familyof K+ transporters fromArabidopsisthatare conserved across phyla.FEBS Lett.415:206–11

162. Raschke K. 1979. Movements of stomata.In Encyclopedia of Plant Physiology, ed.W Hale, E Feinleib, pp. 384–441. Berlin:Springer-Verlag

163. Raskin I, Ladyman JAR. 1988. Isolationand characterization of a barley mutant

P1: FXY


656 SCHROEDER ET AL

with abscisic-acid-insensitive stomata.Planta173:73–78

164. Ritchie S, Gilroy S. 1998. Abscisic acidsignal transduction in the barley aleuroneis mediated by phospholipase D activity.Proc. Nat. Acad. Sci. USA95:2697–702

165. Ritte G, Rosenfeld J, Rohrig K, RaschkeK. 1999. Rates of sugar uptake by guardcell protoplasts ofPisum sativunL. re-lated to the solute requirement for stom-atal opening.Plant Phsyiol.121:647–55

166. Rodriguez PL, Benning G, Grill E. 1998.ABI2, a second protein phosphatase 2Cinvolved in abscisic acid signal transduc-tion in Arabidopsis. FEBS Lett.421:185–90

167. Roelfsema MRG, Prins HBA. 1997. Ionchannels in guard cells ofArabidopsisthaliana(L) Heynh.Planta202:18–27

168. Roelfsema MRG, Prins HBA. 1998.The membrane potential ofArabidopsisthaliana guard cells: depolarizations in-duced by apoplastic acidification.Planta205:100–12

169. Roelfsema MRG, Staal M, Prins HBA.1998. Blue light-induced apoplastic acid-ification of Arabidopsis thalianaguardcells: inhibition by ABA is medi-ated through protein phosphates.Physiol.Plant.103:466–74

170. Romano LA, Miedema H, AssmannSM. 1998. Ca2+-permeable, outwardly-rectifying K+ channels in mesophyll cellsof Arabidopsis thaliana. Plant Cell Phys-iol. 39:1133–44

171. Ruiz LP, Mansfield TA. 1994. A pos-tulated role for calcium oxalate in theregulation of calcium ions in the vicin-ity of stomatal guard cells.New Phytol.127:473–81

172. Santa-Maria GE, Rubio F, Dubcovsky J,Rodriguez-Navarro A. 1997. The HAK1gene of barley is a member of a large genefamily and encodes a high-affinity potas-sium transporter.Plant Cell9:2281–89

173. Schachtman DP, Schroeder JI, Lucas WJ,Anderson JA, Gaber RF. 1992. Expres-

sion of an inward-rectifying potassiumchannel by theArabidopsis KAT1cDNA.Science258:1654–58

174. Schmidt C, Schelle I, Liao YJ, SchroederJI. 1995. Strong regulation of slow an-ion channels and abscisic acid signalingin guard cells by phosphorylation and de-phosphorylation events.Proc. Natl. Acad.Sci. USA92:9535–39

175. Schroeder JI, Hagiwara S. 1989. Cytoso-lic calcium regulates ion channels in theplasma membrane ofVicia faba guardcells.Nature338:427–30

176. Schroeder JI, Hagiwara S. 1990. Repet-itive increases in cystolic Ca2+ of guardcells by abscisic acid activation of non-selective Ca2+-permeable channels.Proc.Natl. Acad. Sci. USA87:9305–9

177. Schroeder JI, Hedrich R. 1989. Involve-ment of ion channels and active transportin osmoregulation and signaling of higherplant cells.Trends Biochem. Sci.14:187–92

178. Schroeder JI, Hedrich R, Fernandez JM.1984. Potassium-selective single chan-nels in guard cell protoplasts ofVicia faba.Nature312:361–62

179. Schroeder JI, Keller BU. 1992. Two typesof anion channel currents in guard cellswith distinct voltage regulation.Proc.Natl. Acad. Sci. USA89:5025–29

180. Schroeder JI, Kwak JM, Allen GJ. 2000.Guard cell abscisic acid signal transduc-tion network and engineering of plantdrought hardiness.Nature.In press

181. Schroeder JI, Raschke K, Neher E. 1987.Voltage dependence of K+ channels inguard cell protoplasts.Proc. Natl. Acad.Sci. USA84:4108–12

182. Schroeder JI, Schwarz M, Pei Z-M. 1998.Protein kinase and phosphatase regula-tion during abscisic acid signaling and ionchannel regulation in guard cells. InCel-lular Integration of Signaling Pathwaysin Plant Development, ed. RLF Schiavo,G Morelli, N Raikhel, pp. 59–69. Heidel-berg: Springer-Verlag

P1: FXY



183. Schroeder JI, Ward JM, Gassmann W.1994. Perspectives on the physiology andstructure of inward-rectifying K+ chan-nels in higher plants: biophysical implica-tions for K+ uptake.Annu. Rev. Biophys.Biomol. Struct.23:441–71

184. Schulz-Lessdorf B, Lohse G, Hedrich R.1996. GCAC1 recognizes the pH gradi-ent across the plasma membrane: a pH-sensitive and ATP-dependent anion chan-nel links guard cell membrane potentialto acid and energy metabolism.Plant J.10:993–1004

185. Schumacher K, Vafeados D, McCarthyM, Sze H, Wilkins T, Chory J. 1999. TheArabidopsis det3mutant reveals a cen-tral role for the vacuolar H(+)-ATPasein plant growth and development.GenesDev.13:3259–70

186. Schwartz A. 1985. Role of calciumand EGTA on stomatal movements inCommelina communis. Plant Physiol.79:1003–5

187. Schwarz M, Schroeder J. 1998. Abscisicacid maintains S-type anion channel ac-tivity in ATP-depletedVicia faba guardcells.FEBS Lett.428:177–82

188. Serrano EE, Zeiger E, Hagiwara S. 1988.Red light stimulates an electrogenic pro-ton pump inVicia guard cell protoplasts.Proc. Natl. Acad. Sci. USA85:436–40

189. Serrano R. 1988. Structure and functionof proton translocation ATPase in plasmamembranes of plants and fungi.Biochim.Biophys. Acta947:1–28

190. Sheen J. 1996. Ca2+-dependent proteinkinases and stress signal transduction inplants.Science274:1900–2

191. Sheen J. 1998. Mutational analysis of pro-tein phosphatase 2C involved in abscisicacid signal transduction in higher plants.Proc. Natl. Acad. Sci. USA95:975–80

192. Shimazaki K, Goh C-H, Kinoshita T.1999. Involvement of intracellular Ca2+in blue light-dependent proton pumpingin guard cell protoplasts fromVicia faba.Physiol. Plant105:554–61

193. Shimazaki K, Iino M, Zeiger E. 1986.Blue light-dependent proton extrusion byguard-cell protoplasts ofVicia faba. Na-ture319:324–26

194. Shimazaki K, Kinoshita T, NishimuraM. 1992. Involvement of calmodulin andcalmodulin-dependent myosin light chainkinase in blue light-dependent H+ pump-ing by guard cell protoplasts fromViciafabaL. Plant Physiol.99:1416–21

195. Staxen I, Pical C, Montgomery LT, GrayJE, Hetherington AM, McAinsh MR.1999. Abscisic acid induces oscillationsin guard-cell cytosolic free calcium thatinvolve phosphoinositide-specific phos-pholipase C.Proc. Natl. Acad. Sci. USA96:1779–84

196. Suh S, Park J-G, Lee Y. 1998. Possi-ble involvement of phospholipase A2 inlight signal transduction of guard cellsof Commelina communis. Physiol. Plant.104:306–10

196a. Sutton F, Paul SS, Wang XQ, AssmannSM. 2000. Distinct abscisic acid signal-ing pathways for modulation of guard cellversus mesophyll cell potassium channelsrevealed by expression studies inXenopuslaevis oocytes.Plant Physiol.124:223–30

197. Talbott LD, Zeiger E. 1996. Central rolesfor potassium and sucrose in guard cell os-moregulation.Plant Physiol.111:1051–57

198. Talbott LD, Zeiger E. 1998. The role ofsucrose in guard cell osmoregulation.J.Exp. Bot.49:329–37

199. Tang HX, Vasconcelos AC, BerkowitzGA. 1996. Physical association of KAB1with plant K+ channel alpha subunits.Plant Cell8:1545–53

200. Theodoulou FL. 2000. Plant ABC trans-porters.Biochim. Biophys. Acta1465:79–103

201. Thiel G, Blatt MR. 1994. Phosphatase an-tagonist okadaic acid inhibits steady-stateK+ currents in guard cells ofVicia faba.Plant J.5:727–33

P1: FXY


658 SCHROEDER ET AL

202. Thiel G, MacRobbie EAC, Blatt MR.1992. Membrane transport in stomatalguard cells: the importance of voltagecontrol.J. Memb. Biol.126:1–18

203. Torsethaugen G, Pell EJ, Assmann SM.1999. Ozone inhibits guard cell K+ chan-nels implicated in stomatal opening.Proc.Natl. Acad. Sci. USA96:13577–82

204. Very A-A, Gaymard F, Bosseux C, Sen-tenac H, Thibaud J-B. 1995. Expressionof a cloned plant K+ channel inXeno-pusoocytes: analysis of macroscopic cur-rents.Plant J.7:321–32

205. Very A-A, Robinson MF, Mansfield TA,Sanders D. 1998. Guard cell cation chan-nels are involved in Na+-induced stom-atal closure in a halophyte.Plant J.14:509–21

206. Wang XQ, Wu W-H, Assmann SM. 1998.Differential responses of abaxial andadaxial guard cells of broad bean to ab-scisic acid and calcium.Plant Physiol.118:1421–29

207. Ward JM, Pei Z-M, Schroeder JI. 1995.Roles of ion channels in initiation of sig-nal transduction in higher plants.PlantCell 7:833–44

208. Ward JM, Schroeder JI. 1994. Calcium-activated K+ channels and calcium-induced calcium release by slow vacuolarion channels in guard cell vacuoles impli-cated in the control of stomatal closure.Plant Cell6:669–83

209. Webb AAR, McAinsh MR, Mansfield TA,Hetherington AM. 1996. Carbon dioxide

induces increases in guard cell cytosolicfree calcium.Plant J.9:297–304

210. Weigel D, Ahn JH, Blazquez MA, Bore-vitz JO, Christensen SK, et al. 2000.Activation tagging inArabidopsis. PlantPhysiol.122:1003–13

211. Wilson KF, Fortes P, Singh US, OhnoM, Mattaj IW, Cerione RA. 1999. Thenuclear cap-binding complex is a noveltarget of growth factor receptor-coupledsignal transduction.J. Biol. Chem.274:4166–73

211a. Wood NT, Allan AC, Haley A, Viry-Moussa¨ıd M, Trewavas AJ. 2000. Thecharacterization of differential calciumsignalling in tobacco guard cells.PlantJ. 24:335–44

212. Wu W-H. 1995. A novel cation channelin Vicia faba guard cell plasma mem-brane.Acta Phytopathol. Sin.21:347–54

213. Wu Y, Kuzma J, Marechal E, GraeffR, Lee HC, et al. 1997. Abscisic acidsignaling through cyclic ADP-ribose inplants.Science278:2126–30

214. Zeiger E. 2000. Sensory transduction ofblue light in guard cells.Trends PlantSci.5:183–84

215. Zeiger E, Hepler PK. 1977. Light andstomatal function: blue light stimulatesswelling of guard cell protoplasts.Sci-ence196:887–89

216. Zhu J, Talbott LD, Jin X, Zeiger E.1998. The stomatal response to CO2 islinked to changes in guard cell zeaxan-thin. Plant Cell Environ.21:813–20

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Figure 1 A simplified working model for proposed functions of positive and negative reg-ulators in light-induced stomatal opening. Positive (in green,top) and negative regulators(in red,bottom) of light signaling in guard cells are shown. The sequence of events and epis-tasis among regulators remain largely unknown and requires further genetic, biochemical,and cellular signaling analyses. For simplicity, parallel signaling branches are not includedhere (see text for details).

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Figure 2 A guard cell model, illustrating the proposed functions of ion channels in ABAsignaling and stomatal closing (177). The right cell of the stomate shows ion channelsand regulators that mediate ABA-induced stomatal closing. The left cell shows the paralleleffects of ABA-induced [Ca2+]cyt increases that inhibit stomatal opening mechanisms.[Modified with permission from Schroeder et al, 2001 (180)].

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Figure 3 A working model for the proposed functions of positive and negative regulatorsof ABA-induced stomatal closing and of S-type anion channels in guard cells. Positivetransducers are shown at top and/or in green and negative regulators are shown below inred. The sequence of events and epistasis among regulators remains largely unknown andrequires further analysis. For simplicity, parallel signaling branches are not shown. Notethat some of the ABA signaling “casettes” (top, vertical cascades) may be activated by ABAin parallel (see text for details).

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Figure 4 A working model for the proposed functions of positive transducers and negativeregulators of K+out channels in guard cells. Positive transducers (green,top) and negativeregulators (red,bottom) are shown.

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