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Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:627–58
c 2001 by Annual Reviews. All rights reserved
Copyright °
GUARD CELL SIGNAL TRANSDUCTION
Julian I Schroeder, Gethyn J Allen, Veronique Hugouvieux,
June M Kwak, David Waner
University of California, San Diego, Division of Biology, Cell and Developmental Biology
Section and Center for Molecular Genetics, La Jolla, California 92093-0116;
e-mail: julian@biomail.ucsd.edu
Key Words stomatal movement, gas exchange, abscisic acid, ion channel,
cytosolic calcium
■ Abstract Guard cells surround stomatal pores in the epidermis of plant leaves and
stems. Stomatal pore opening is essential for CO2 influx into leaves for photosynthetic
carbon fixation. In exchange, plants lose over 95% of their water via transpiration
to the atmosphere. Signal transduction mechanisms in guard cells integrate hormonal
stimuli, light signals, water status, CO2, temperature, and other environmental conditions to modulate stomatal apertures for regulation of gas exchange and plant survival
under diverse conditions. Stomatal guard cells have become a highly developed model
system for characterizing early signal transduction mechanisms in plants and for elucidating how individual signaling mechanisms can interact within a network in a single
cell. In this review we focus on recent advances in understanding signal transduction
mechanisms in guard cells.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LIGHT SIGNAL TRANSDUCTION AND STOMATAL OPENING . . . . . . . . . . . .
Early Events in Blue Light Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . .
Cytosolic Factors That Regulate Inward-Rectifying K+
in Channels . . . . . . . . . . . .
Role of Actin Filaments in Stomatal Movements . . . . . . . . . . . . . . . . . . . . . . . . .
Cloned Guard Cell K+
in Channel Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MULTIPLE PHYSIOLOGICAL AND ABIOTIC STIMULI INDUCE
STOMATAL CLOSING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Model for Roles of Ion Channels in ABA-Induced Stomatal Closing . . . . . . . . . .
CO2 -Induced Stomatal Closing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
STIMULI THAT INCREASE CYTOSOLIC CALCIUM IN GUARD CELLS . . . . .
Roles of [Ca2+ ]cyt in Stomatal Closing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Roles of [Ca2+ ]cyt in Stomatal Opening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SECOND MESSENGER SYSTEMS REGULATING [Ca2+ ]cyt
IN GUARD CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Cyclic ADP-Ribose and Vacuolar [Ca2+ ] Release . . . . . . . . . . . . . . . . . . . . . . . .
Phospholipase C and Inositol 1,4,5 Trisphosphate . . . . . . . . . . . . . . . . . . . . . . . .
Phospholipase D and Phosphatidic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Amplifying Calcium Signals by Calcium-Induced Calcium Release . . . . . . . . . . .
Plasma Membrane Calcium Channels and Calcium Influx in Guard Cells . . . . . . .
Cytosolic [Ca2+ ] Oscillations Are Necessary for Stomatal Closing
in Guard Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ABA-INSENSITIVE PP2C MUTANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ANION CHANNELS AND ABA-INDUCED STOMATAL CLOSURE . . . . . . . . . .
Regulation of Guard Cell ABA Signaling by ABC Proteins . . . . . . . . . . . . . . . . .
Protein Kinases Function in ABA Signaling and Anion Channel Regulation . . . . .
Okadaic Acid–Sensitive Phosphatases Regulate Anion Channels and
ABA Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ACTIVITY OF PLASMA MEMBRANE K+
out CHANNELS IN
STOMATAL MOVEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulation of K+
out by Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Syntaxins and ABA Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Osmolarity and Temperature Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transient K+ Efflux Currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
NEW GUARD CELL SIGNALING MUTANTS AND GENETIC
APPROACHES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
New Genetic Screens and Reverse Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . .
FUTURE OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION
Opening and closing of stomatal pores is mediated by turgor and volume changes
in guard cells. During stomatal opening guard cells accumulate potassium, anions,
and sucrose (130, 162, 198). Osmotic water uptake leads to guard cell swelling and
stomatal opening. Stomatal closing is mediated by potassium and anion efflux from
guard cells, sucrose removal, and metabolism of malate to osmotically inactive
starch. Guard cells have become a popular system for dissecting the functions of
individual genes and proteins within signaling cascades for the following reasons.
1. Guard cells control CO2 influx and water loss and thus critically affect
whole plant growth and physiology.
2. Guard cells respond cell-autonomously to well-known plant physiological
signals, including red and blue light (215), CO2, plant pathogens, the
hormones abscisic acid, auxin, cytokinin and gibberellins, and other
environmental signals. Thus many specific receptors and early signaling
mechanisms 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 analyzing
individual mechanisms that contribute to signal transduction. These ion
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channels are targets of early signaling branches and provide molecular
probes to identify upstream regulators.
4. Hypotheses for mechanisms affecting signal transduction can be easily
tested by analyzing stomatal opening and closing in response to various
stimuli. Furthermore, several powerful approaches have been adapted to
guard cell signaling analyses allowing interdisciplinary time-resolved cell
biological, 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 for
ecological and biotechnological applications. Agricultural and horticultural use of
plants in climates to which these plants are not adapted, as well as short-term climate changes, lead to dramatic crop losses or freshwater consumption under stress
conditions such as drought. Recent studies in Arabidopsis have demonstrated that
stomatal responses can be manipulated by modifying guard cell signal transduction elements to reduce transpirational water loss and dessication during drought
periods (53, 75, 154).
One might ask: Are guard cells different from many other plant cell types (aside
from the obvious specializations) in terms of harboring many signal receptors in a
single cell? Most likely not, because most individual plant cells respond to many
classical hormones, pathogens, and light signals. The combination of the abovelisted attributes (1 to 4), however, renders guard cells a well-developed model
system for interdisciplinary and time-resolved characterizations of mechanisms or
segments of early plant signaling cascades.
In the present review we focus mainly on recent dissections of signaling transduction mechanisms in guard cells. Of further importance for stomatal movements
is signal-dependent modulation of starch-malate metabolism. The mechanisms by
which signaling cascades described here tie into metabolic networks is an important frontier of future research and recent reviews on guard cell metabolic pathways
can be found elsewhere (88, 149, 198). Furthermore, several reviews on aspects of
guard cell signal transduction have appeared in recent years (8, 12, 13, 21, 113, 132,
136, 145, 180, 182, 207, 214).
LIGHT SIGNAL TRANSDUCTION
AND 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 hyperpolarization
proposed to drive K+ uptake into guard cells via inward-rectifying K+ (K+in) channels (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. In
parallel, starch metabolism leads to accumulation of osmotically active malate in
guard cells. In addition, sucrose levels in guard cells increase during light-induced
stomatal opening (125, 159, 165, 197, 198). Studies with intact leaves show that the
main 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 = 0.3 µM Ca2+) (Figure 1). Furthermore, inhibitors of PP1- or PP2Atype (PP1/PP2A) protein phosphatases such as calyculin A and okadaic acid inhibited blue light–dependent H+ pumping and light-induced stomatal opening in Vicia,
suggesting that PP1/PP2A phosphatases are positive regulators of light-induced
stomatal opening (93) (Figure 1). Abscisic acid (ABA) also inhibits blue light–
dependent H+ pumping activity of Vicia guard cell protoplasts (52, 169, 193). ABA
inhibition of apoplastic acidification was not observed in the ABA-insensitive Arabidopsis mutants abi1-1 and abi2-1 (Figure 1). Interestingly, the PP1/PP2A inhibitor okadaic acid partially restored ABA inhibition of proton pumping in abi1-1
guard cells, whereas the protein kinase inhibitor K-252a partially restored ABA
inhibition of proton pumping in abi2-1 guard 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 was
characterized in an elegant biochemical study. Blue light activates H+-ATPases
via phosphorylation of the C terminus (94). Coprecipitation of H+-ATPases with
endogenous guard cell 14-3-3 proteins and binding of recombinant 14-3-3 proteins only to the phosphorylated H+-ATPase C terminus provide evidence for a
role of 14-3-3 proteins as a positive regulator in physiological blue light signal
transduction (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 cells
have been proposed to include unique components. Stomata from the zeaxanthindeficient Arabidopsis mutant, npq1 (150), are impaired in blue light–induced stomatal opening (47), and stomata from the blue light photoreceptor mutants, cry1,
cry2, nph1, nph3, nph4, cry1cry2, and nph1cry1, showed a wild-type blue light
response (47, 101). These results have led to the proposal of a model in which zeaxanthins function as blue light receptors or receptor pigments in guard cells (47).
Cytosolic Factors That Regulate Inward-Rectifying
K+
in Channels
K+in channels in guard cells have been proposed to contribute to K+ uptake during
stomatal 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 cell
K+in channels (20) and of cloned plant K+in channels expressed in Xenopus
oocytes (74, 78, 146, 204). Cytosolic Ca2+ ([Ca2+]cyt) elevation inhibits K+in channels, thus limiting K+ uptake (108, 175) (Figure 1). Almost complete inhibition of
K+in channels has been measured in Vicia guard cells when [Ca2+]cyt was buffered to
≈1 µM (54, 57, 90, 108, 175), or when InsP3 is uncaged in the guard cell cytoplasm
to raise [Ca2+]cyt (23) (Figure 1). Interestingly, in Vicia, K+in channels in abaxial
guard cells were [Ca2+]cyt inhibited and stomatal movements were modulated by
external Ca2+ and ABA, whereas K+in channels in adaxial guard cells were insensitive to [Ca2+]cyt and stomatal movements were less sensitive to external Ca2+ and
ABA (206).
In addition, protein phosphorylation has been suggested to play an important
role in modulation of K+in channel activity. Inhibitors of calcineurin (PP2B), a
calcium-dependent protein phosphatase, maintained K+in channel activity in spite
of elevated [Ca2+]cyt in Vicia guard cells (126) (Figure 1). Cyclosporin A, an
inhibitor of animal PP2B-type phosphatases, inhibits stomatal closure and reduces
ABA inhibition of stomatal opening in Pisum, suggesting that PP2Bs might be negative regulators of stomatal opening (70) (Figure 1). Note that PP2B homologs
have not yet been identified in plants. In contrast to PP2B inhibitors, inhibitors of
PP1/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 a Vicia guard cell Ca2+-dependent
protein kinase (CDPK) that phosphorylates the Arabidopsis guard cell K+in channel
subunit KAT1 (118), and heterologous expression of KAT1 and a soybean CDPK in
Xenopus oocytes shows a reduction in KAT1-mediated K+ currents (17), suggesting a role of CDPK in Ca2+-mediated K+in channel inhibition (Figure 1). Combined
genetic and cell biological analyses will be required to identify the kinases that
regulate K+in channels.
Stomatal aperture measurements show that linolenic acid and arachidonic acid
promote stomatal opening and inhibit stomatal closing (106). Furthermore, patchclamp analyses show that these fatty acids activate K+in currents and inhibit outwardrectifying K+out currents (106). These results are interesting as only a few agents
have been found that enhance K+in channel activity in guard cells. Furthermore,
phospholipase A2 inhibitors decrease stomatal responses to light (196). Together
these data suggest that phospholipase A2 may be a positive regulator of the guard
cell 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 actin
filament 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 randomly oriented and short-fragmented pattern (44). A small GTP-binding protein,
AtRac1, can function as a negative regulator in ABA-induced actin reorganization (107). ABA causes AtRac1 inactivation (Figure 3). This ABA response was
impaired in abi1-1 (107). In addition, transgenic Arabidopsis plants expressing
a dominant negative AtRac1 mutant mimic constitutive ABA-induced actin reorganization in guard cells and increase ABA sensitivity of stomatal closure (107),
suggesting that inactivation of AtRac1 is required for actin reorganization and
stomatal closure. Together these studies show important roles of actin and small
G-proteins in stomatal movements.
Cloned Guard Cell K+in Channel Genes
The Arabidopsis K+in channel gene KAT1 was cloned by complementation of a
yeast mutant defective in K+ transport (10) and shown to mediate K+ currents
in Xenopus oocytes with typical properties of plant K+in currents (67, 73, 78, 173,
204). Expression studies with Arabidopsis KAT1 and the potato ortholog KST1
show that these plant K+in channel genes are predominantly expressed in guard
cells (146, 148). In animals, functional K+ channel proteins are composed of four
α–subunits (87) and additional regulatory β–subunits (45). An Arabidopsis cDNA
encoding a β–subunit homolog of K+ channels has been isolated and binding to
KAT1 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 with
transgenic Arabidopsis expressing KAT1 mutants with a reduced sensitivity to
Cs+ block, transgenic plants exhibited partial light-induced stomatal opening in
the presence of Cs+ concentrations that inhibit stomatal opening in wildtype (77).
In another study, transgenic Arabidopsis expressing a dominant negative mutant
form of the guard cell K+in channel KAT1 showed 75% reduction in the activity of
guard 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, these
data show that dominant negative kat1 can disrupt K+in channels. These data support
the model that K+ channels constitute a central mechanism for K+ uptake during
stomatal opening (178, 181). These studies do not exclude the likely model that
other partially redundant (183) K+ uptake transporters function in parallel in guard
cells. For example, there are >12 isoforms of the KT-KUP-HAK K+ transporter
gene family (48, 91, 161, 172) in the Arabidopsis genome and some of these are
likely to be expressed in guard cells. Furthermore, patch-clamp studies have shown
the activity of additional inward-conducting cation channels (69, 205, 212). In
conclusion, many positive and negative regulators of H+ pumps, K+in channels,
and stomatal opening have been found (Figure 1). A combination of genetic, cell
biological, and biochemical studies will allow further testing and expansion of
these models.
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MULTIPLE PHYSIOLOGICAL AND ABIOTIC STIMULI
INDUCE STOMATAL CLOSING
Abscisic acid, produced in response to water deficit, causes stomatal closing. Furthermore, in C3 and C4 plants stomatal closing is induced by darkness and by
elevated CO2 concentrations in the intercellular spaces in leaves, arising from
respiration (12, 133). Stomatal closing and reduced transpiration also leads to
elevated temperatures inside leaves. Pathogen elicitors also cause stomatal closing, 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 high
concentrations (203), thus reducing further damage of leaf tissues by these pollutants. Thus multiple stimuli elicit stomatal closing. Although the reception
mechanisms for these stimuli remain unknown, the signaling pathways need to
converge on central guard cell ion channel and metabolic pathways.
Model for Roles of Ion Channels in ABA-Induced
Stomatal Closing
Stomatal closing requires ion efflux from guard cells. Models for roles of ion
channels during ABA-induced stomatal closing have been used as a basis for dissecting upstream ABA signal transduction mechanisms (132, 175, 177, 207). In
brief, ABA induces cytosolic Ca2+ increases (134) (Figure 2). Cytosolic calcium elevations, in turn, inhibit plasma membrane proton pumps (92) and K+in
channels and activate two types or modes (39) of plasma membrane anion channels that mediate anion release from guard cells (89, 175) (Figure 2). One of
these 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 guard
cells causes either transient or sustained anion efflux and depolarization (Figure 2).
Depolarization, in turn, deactivates inward-rectifying K+ (K+in) channels and activates outward-rectifying K+ (K+out) channels (181), resulting in K+ efflux from
guard cells (Figure 2). The ensuing long-term efflux of both anions and K+ from
guard 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 for RAB18 expression in Arabidopsis
suspension culture cells (49b, 49c) indicating that these mechanisms (Figure 2) are
of general importance for early ABA signaling in other cell types.
Most ions released across the plasma membrane of guard cells need first to be released into the cytosol from guard cell vacuoles (8, 130, 131, 207, 208). Models for
the roles of vacuolar K+ and anion channels during stomatal regulation have been
proposed previously and are described elsewhere (7, 8, 131, 157, 207, 208). However, these models have not yet been tested as stringently as those for plasma
membrane ion channels. Combined molecular genetic (e.g. gene disruption) and
cell biological approaches are needed to directly analyze these vacuolar ion channel
models.
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CO2-Induced Stomatal Closing
Elevated CO2 concentrations arising from respiration in darkness stimulate stomatal closing (133). Increases in atmospheric CO2 concentrations are also predicted
to reduce stomatal apertures and affect gas exchange (12, 41). CO2 signaling mechanisms in guard cells have been reviewed recently (12, 41). Here, we briefly summarize some recent findings and signaling models. Elevated CO2 concentrations
trigger rises in [Ca2+]cyt (209), activate S-type anion and outward-rectifying K+
channel currents (24), and modulate R-type anion channels (K Raschke, personal
communication). These data show that the ion channel targets of early CO2 signaling are to a degree shared with ABA signaling (Figure 2). However, upstream
CO2 sensing and transduction mechanisms have been reported to differ from ABA
signaling, because the abi1-1 and abi2-1 mutants show wild-type CO2-induced
stomatal closing (116). A CO2-induced increase in cell wall malate concentration
has been proposed to cause stomatal closing (66). However, >20 mM external
malate was required to produce stomatal closing in two independent studies under the same experimental conditions as the above study (31, 43). Furthermore,
external malate counteracted CO2-induced stomatal closing (31), thus calling into
question the malate as CO2 sensor hypothesis. An alternative hypothesis suggests
that the CO2 sensor is located in guard cell chloroplasts and functions via a CO2induced decrease in zeaxanthin levels (216). Further understanding of CO2 sensing
and signaling in guard cells will help in finding crucial links between the signaling pathways reviewed here and guard cell metabolic pathways. Foremost, future
research should have important implications for manipulating gas exchange and
carbon fixation in the face of rising atmospheric CO2 levels.
STIMULI THAT INCREASE CYTOSOLIC CALCIUM
IN GUARD CELLS
Roles of [Ca2+]cyt in Stomatal Closing
Many stimuli that result in a change in stomatal aperture have been shown, at least
in 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 induces
repetitive, 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 elevations in guard cells has been obtained recently, as the ABA-insensitive Arabidopsis
mutants abi1-1 and abi2-1 show greatly reduced ABA-induced [Ca2+]cyt elevations,
and the abi anion channel regulation and stomatal movement phenotypes are suppressed 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 repetitive
Ca2+ 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 so
sensitive to changes in apoplastic Ca2+ is not fully understood, although many
species limit Ca2+ accumulation in the apoplast surrounding guard cells to prevent
aberrant stomatal regulation (38, 171). Increases in CO2 cause stomatal closure
and [Ca2+]cyt elevations (209). Cold shock (3, 211a) and oxidative stress, induced
by application of H2O2 or methyl viologen, increase [Ca2+]cyt and result in stomatal closure (137, 156). The removal of extracellular Ca2+ using EGTA abolishes
CO2- and H2O2-induced [Ca2+]cyt elevations, indicating that plasma membrane
Ca2+ influx occurs (156, 209).
Roles of [Ca2+]cyt in Stomatal Opening
Interestingly, stimuli that result in stomatal opening also induce [Ca2+]cyt elevations. Auxin promotes stomatal opening and direct [Ca2+]cyt measurements
(80), and pharmacological studies (32) suggest a role for [Ca2+]cyt elevations. Furthermore, Ca2+-dependent protein kinases (CDPK) activate guard cell vacuole
Cl− channels and malate uptake currents that have been implicated in vacuolar anion uptake during stomatal opening (157). Blue light promotes stomatal opening,
and pharmacological experiments suggest the involvement of [Ca2+]cyt/calmodulin
as a second messenger in this process (33, 194). However, increasing external
Ca2+ can inhibit light-induced stomatal opening (4, 152, 192) (Figure 1). Cyclic
nucleotides may also act in a Ca2+-dependent stomatal opening pathway as stomatal opening can be stimulated by cAMP (33) or the membrane-permeable cGMP
analog 8-Br-cGMP (32). Cyclic GMP-induced stomatal opening is inhibited by
chelation of external Ca2+ or by inhibitors of intracellular Ca2+ release (32).
How a single second messenger such as [Ca2+]cyt can control many diverse and
opposing responses in a single cell type remains unknown but likely depends on
the Ca2+ channels and Ca2+ regulatory systems activated by each stimulus, the
downstream response elements expressed at a given time, and the characteristics
and dynamics of the elicited [Ca2+]cyt change (the Ca2+ signature) (138). The
recent demonstration that GFP-based “cameleon” calcium indicators (142, 143)
function in Arabidopsis will allow addressing of these questions by combined
genetic 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 ABA
and [Ca2+]cyt signaling in guard cells (Figure 3). In animal cells, cyclic ADP ribose (cADPR) is produced from NAD via the action of the enzyme ADP-Ribosyl
cyclase, and it mobilizes Ca2+ from intracellular stores by activation of an endomembrane ion channel known as the ryanodine receptor (RYR) (103). In plant
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vacuoles, nanomolar cADPR concentrations can activate a Ca2+ permeable current (6). Microinjection of cADPR into tomato hypocotyl cells shows that cADPR
can function in ABA signaling (213). In Commelina guard cells cADPR causes
[Ca2+]cyt increases and elicits stomatal closing (102). However, microinjection
of 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 cells
also reduces [Ca2+]cyt increases (57). Note that ABA-induced stomatal closure
is only partly inhibited by either microinjection of 8-NH2-cADPR (102) or by
nicotinamide, an inhibitor of cADPR production (85, 102), suggesting that additional parallel [Ca2+]cyt elevation mechanisms are needed in the ABA signaling
cascade.
Phospholipase C and Inositol 1,4,5 Trisphosphate
Various lines of evidence suggest that phospholipase C (PLC) is a component of
ABA signal transduction in guard cells. Early experiments showed that release
of caged InsP3 into the cytosol of guard cells could cause [Ca2+]cyt increases and
stomatal closure (51) and inhibit K+in channels (23). Treating guard cell protoplasts with ABA slightly elevates InsP3 levels (105, 151). Additionally, the PLC
inhibitor U-73122 (but not the inactive analog U-73343) inhibits the activity of
recombinant PLC from tobacco (195). ABA-induced stomatal closure was also
inhibited by U-73122, but only by 20% (195). However, complete inhibition of
ABA-induced stomatal closure can be achieved by treating stomata with a combination of U-73122 and nicotinamide (85, 132a), suggesting that both cADPR and
PLC signaling systems function in ABA signaling (Figure 3).
Note that other inositol-phosphates can also act as second messengers in ABA
signal transduction in guard cells. In a recent study, myo-inositol hexakisphosphate
(InsP6) was identified as an intermediary of guard cell signal transduction. ABA
stimulates production of InsP6 in guard cells, and InsP6 perfused into the cytosol
via 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 important
component 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), and
ABA treatment of Vicia guard cells caused PtdOH levels to transiently increase
2.5-fold (85). PtdOH also promotes stomatal closure and inactivates K+in currents.
Guard cell [Ca2+]cyt did not increase following PtdOH treatment, suggesting
that PLD acts in a parallel pathway or downstream of Ca2+ mobilizing second
messenger systems (Figure 3). An inhibitor of PLD activity, 1-butanol, caused
only a partial inhibition of ABA-induced stomatal closure whereas near-complete
inhibition 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-mediated
pathway.
Amplifying Calcium Signals by Calcium-Induced
Calcium Release
Release of caged Ca2+ into the guard cell cytosol can induce [Ca2+]cyt increases
or oscillations that are larger than can be accounted for solely by the Ca2+ released by photolysis (51, 139), suggesting that a [Ca2+]cyt-induced calcium release
(CICR) mechanism exists in guard cells. Ca2+-permeable SV channels have been
proposed to amplify and propagate [Ca2+]cyt signals in guard cells by CICR from
vacuoles (208). Cytosolic Mg2+ sensitizes SV channels to physiological [Ca2+]cyt
elevations (158) and shifts the voltage-dependence of SV channels (27) such that
SV channel activity is enhanced. The ability of the SV channel to mediate CICR
has been questioned because in mesophyll vacuoles increasing the transvacuolar
Ca2+ gradient shifts the voltage-dependence to prevent channel opening under
conditions that would otherwise allow Ca2+ to enter the cytosol (160). However,
the voltage-dependence and pharmacology of radiolabeled Ca2+ release in vitro
from 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 signal
transduction, analysis of which best requires manipulation of SV channel genes.
Plasma Membrane Calcium Channels and Calcium Influx
in Guard Cells
Experimental application of repeated hyperpolarizations negative of −120 mV
induced repetitive [Ca2+]cyt transients in guard cells (56). ABA application shifted
the threshold of hyperpolarization-activated Ca2+ elevations to −80 mV, indicating that an early event in ABA signaling is the sensitization of Ca2+ influx
to membrane potential. Furthermore, in 50 mM KCl buffers [in which the cells
are depolarized (168, 202)] ABA only induces [Ca2+]cyt increases in 30–60% of
guard cells (2, 4, 50, 135, 176). Transient ABA activation of Ca2+-permeable channels was found during depolarizations in 37% of guard cells (176), which could
contribute to ABA-induced [Ca2+]cyt elevations in depolarized guard cells. Interestingly, in cells maintained in 5 mM KCl, guard cells are hyperpolarized and
ABA induces repetitive [Ca2+]cyt transients or oscillations in a higher proportion of
cells (80–90%) (5, 56, 195). These data indicate that changes in plasma membrane
potential are a central component in ABA signaling.
The upstream second messenger mechanisms that activate guard cell plasma
membrane Ca2+ channels remained unknown. A recent study in Arabidopsis guard
cells shows that reactive oxygen species (ROS) can activate a hyperpolarizationactivated Ca2+ influx current (ICa) and that ROS can act as a second messenger in
ABA signaling. ABA treatment enhances ROS production in Arabidopsis guard
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cells (156). Interestingly, ABA activates guard cell ICa (156) only when NADPH is
present in the cytosol, implicating NADPH oxidases in ABA signaling (Y Murata &
J Schroeder, unpublished). Activation of ICa by hydrogen peroxide is impaired in
the recessive ABA-insensitive mutant gca2, whereas ABA-induced ROS production remains intact in gca2 (156), providing genetic evidence for roles of ROS
and 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 guard
cells (Figure 3). A study in maize embryos has also shown that ABA enhances
ROS production (60), indicating that ROS may be universal second messengers in
plant ABA signaling.
Single channel recordings in Vicia guard cell protoplasts have identified a Ca2+
channel that is hyperpolarization-activated and shows a 250-fold increased open
probability following addition of 20 µM ABA (61). Currents exhibited by this
channel are very similar to ICa. The activation by ABA occurs in isolated patches,
suggesting that ABA perception and channel activation are closely associated in
Vicia. Interestingly, the single channel open probability was reduced tenfold when
buffering [Ca2+]cyt from 200 nM to 2 µM, indicating that the channel is downregulated during [Ca2+]cyt elevation and is therefore subject to negative feedback
control during [Ca2+]cyt signaling (61).
Both at the single-channel level (61) and in whole cells (156), the activity
of the Ca2+ influx channel can show spontaneous oscillatory behavior without
exogenous ABA addition. Background ICa activity was inhibited by addition of
0.1 mM DTT in Arabidopsis guard cells (156). The spontaneous ICa activity may
contribute to spontaneous [Ca2+]cyt elevations found in hyperpolarized guard cells
(5, 56, 195). Because ICa channels are regulated by ROS, various stress signals may
control 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 “signaling
cassette” of multiple stress signaling pathways (156).
Additional Ca2+ influx pathways may be provided by plasma membrane stretchactivated Ca2+ permeable channels (30) or via a Ca2+ permeability of transient
and sustained K+out channels (153, 170). Clearly, Ca2+ influx, Ca2+ release, and
second messengers are integrated in guard cells to produce a [Ca2+]cyt signal that
controls stomatal movements. How these separate processes may be integrated to
produce [Ca2+]cyt signals that encode information necessary for stomatal closure
is an important focus of ongoing studies (see 132a).
Cytosolic [Ca2+] Oscillations Are Necessary for Stomatal
Closing in Guard Cells
In a few cases in animal cells, the frequency of [Ca2+]cyt oscillations has been
shown 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 potential oscillations in guard cells have been generated (26, 59, 96) to analyze different mechanisms that may contribute to generation of [Ca2+]cyt oscillations. Using
guard cells of the Arabidopsis V-ATPase mutant de-etiolated 3 (det3) (185), which
has reduced endomembrane proton pumping and energization, external Ca2+ and
oxidative stress elicited prolonged Ca2+ increases (plateaus) that did not oscillate,
whereas wild-type cells show [Ca2+]cyt oscillations (3). Unexpectedly, steady state
stomatal closure was inhibited in det3 in response to these stimuli. Conversely, cold
and ABA elicited Ca2+ oscillations in det3, and stomatal closures were not impaired
(3). Moreover, in det3 guard cells, experimentally imposing external Ca2+-induced
oscillations rescued steady state stomatal closure in response to external Ca2+, and
imposing Ca2+ plateaus in wild-type guard cells prevented steady-state stomatal closing. These data provide genetic evidence that stimulus-specific Ca2+ oscillations, rather than a mere plateau of [Ca2+]cyt, are necessary for long-term
stomatal closure (3). These findings suggest that guard cells may provide an excellent genetic system to study [Ca2+]cyt pattern-dependent responses.
ABA-INSENSITIVE PP2C MUTANTS
Genetic screens for Arabidopsis mutants insensitive to ABA inhibition of seed
germination yielded two dominant mutants that are impaired in ABA-induced
stomatal closure (97). The corresponding genes, ABI1 and ABI2, both encode
type 2C protein phosphatases and the dominant mutant alleles abi1-1 and abi2-1
have point mutations altering a conserved amino acid (112, 114, 140, 166). Several downstream responses to ABA are impaired in these Arabidopsis mutants
including 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 stomatal
closure in abi1-1 and abi2-1, thus bypassing the effects of the abi1-1 and abi2-1
mutations (4). These data demonstrate that the dominant abi PP2C mutants interfere 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 has
been unclear whether the ABI1 and ABI2 phosphatases are positive or negative
regulators of ABA signaling or indeed whether they affect ABA signaling at all
in wildtype. Recently, however, intragenic revertants of the abi1-1 and abi2-1
mutants were isolated and shown to have reduced or no phosphatase activity in
vitro (53, 139a). Because a double mutant of both revertants shows hypersensitivity
to ABA, ABI1 and ABI2 are likely negative regulators of ABA signaling (139a).
In correlation, overexpression of wild-type ABI1 in maize mesophyll protoplasts
blocks ABA regulation of gene expression (191). In spite of these advances, ABI1
and ABI2 gene deletion or silencing mutants would be useful for a stringent test of
their 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 essential
signaling protein.
ANION CHANNELS AND ABA-INDUCED
STOMATAL CLOSURE
Anion channel activation at the plasma membrane of guard cells has been proposed
as an essential step during stomatal closure (Figure 2) (64, 175, 179). S-type anion
channel currents in guard cells are activated by increases in [Ca2+]cyt in Vicia (175)
and Arabidopsis (4). R-type anion currents are activated following an increase in
external Ca2+ (64). Whether ABA regulates R-type anion channels remains to be
determined. ABA activation of S-type anion channels in the plasma membrane
of guard cells has now been demonstrated in Arabidopsis (155), tobacco (58),
and Vicia (111, 119, 187) (Figure 3). This response is disrupted in the Arabidopsis
abi1-1 and abi2-1 mutants, providing genetic evidence that activation of S-type
channels contributes to stomatal closure (155). Putative anion channel genes have
been cloned from tobacco (127) and Arabidopsis (62) based on sequence homology
with the animal voltage-dependent CLC chloride channels [for review see (15)].
Further work is needed to determine whether these genes encode components of
native 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 channels may be encoded by or regulated by ATP binding cassette (ABC) proteins
(110, 111) (Figure 3). ABC proteins comprise a large family of membrane proteins that actively translocate a wide spectrum of substrates. In addition, ABC
proteins regulate the activity of other unrelated transporters. ABC proteins such as
CFTR (cystic fibrosis transmembrane conductance regulator) or SUR (sulfonylurea
receptor) show Cl− channel activity and/or regulate other channels [for review see
(200)]. Inhibitors of SUR, such as glibenclamide, prevent ABA-induced stomatal closure in Vicia and Commelina. Furthermore, the ABC protein inhibitors DPC
and glibenclamide block slow anion currents in Vicia guard cells (111). In contrast, cromakalin, an antagonist of glibenclamide, triggers stomatal closing in
Commelina and reverses the inhibition of glibenclamide on S-type anion currents
in Vicia guard cells (111).
Protein Kinases Function in ABA Signaling
and Anion Channel Regulation
Pharmacological approaches, using the serine/threonine protein kinase inhibitor
K252a and cytosolic replacement of ATP, showed that phosphorylation events are
central positive regulators in ABA-induced stomatal closure in Vicia (174), as well
as in Commelina (43), Pisum (70), and Arabidopsis (4). K252a abolishes both
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anion channel activity and ABA-induced stomatal closing (174). In correlation
with these results, ABA induction of gene expression (RD29a and KIN2) in tomato
hypocotyls is inhibited by K252a (213), together with other studies, indicating
that kinase-dependent transduction of ABA signaling (174) is of general significance (95, 190, 213). Note that the R-type anion currents are not regulated by
phosphorylation 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) in Vicia guard cells (117, 144).
The kinase activity was named AAPK or ABR. Recently, an AAPK gene was
cloned (119). Transient expression of a dominant negative allele of AAPK, which
abolished kinase activity, prevents ABA activation of S-type anion currents and
stomatal closing. In correlation with these findings, in Vicia S-type anion currents
are activated at low [Ca2+]cyt and high ATP concentrations, suggesting that a final
phosphorylation event in anion channel activation can be Ca2+ independent (187)
(Figure 3). Recessive loss-of-function mutations in AAPK will allow further analysis of AAPK function in guard cells.
Two Ca2+-dependent protein kinases of 53 kDa and 58 kDa have been characterized in Vicia guard cells (118, 144). Removal of Ca2+ with BAPTA in Vicia
guard cell protoplast suspensions prevents ABR kinase activation and indicates that
Ca2+ is required upstream for ABR activation (144) (Figure 3). These data and
overexpression studies on maize protoplasts suggest that CDPKs may be positive
regulators in ABA signal transduction (190).
In addition, MAP kinases have been reported to positively control ABA-induced
stomatal closure in Pisum (25). ABA causes a transient activation of a 43-kDa
MAP kinase named AMBPK. AMBPK exhibits all fundamental MAP kinase
properties, including tyrosine phosphorylation (25). The MAPK kinase inhibitor
PD98059 abolished ABA-induced stomatal closing (Figure 3) and ABA induction
of dehydrin mRNA (25). ABA activation of MAP kinases in barley aleurone was
previously reported (68, 95).
In parallel to the above kinases that transduce ABA signals, other protein kinases
have been suggested to act as negative regulators of ABA signaling in tobacco
and Arabidopsis guard cells in the abi1-1 background (11, 155). Application of
kinase inhibitors partially restores ABA activation of anion channels and regulation
of K+ channels and stomatal closing in abi1-1 backgrounds (11, 155) (Figures 3
and 4).
Okadaic Acid–Sensitive Phosphatases Regulate
Anion Channels and ABA Signaling
Inhibitors of PP1 and PP2A protein phosphatases such as okadaic acid (OA) were
found to enhance S-type anion currents and ABA-induced stomatal closure in Vicia
(174, 187), as well as in tobacco (58), Commelina (43), and Pisum (70), and enhance 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/PP2Aneg in Figure 3 and the PP1/PP2A shown in Figure 1 both promote stomatal opening and therefore might be encoded by the same genes. In
Vicia, 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 other
PP1/PP2As can also act as positive regulators in ABA signaling (Figure 3; PP1/PP2Apos). In Arabidopsis, OA partially inhibited ABA activation of S-type anion
channels and stomatal closing (155). A similar inhibitory effect of OA was also observed on ABA signaling during stomatal opening and ABA induction of dehydrin
mRNA in Pisum epidermal peels (70). OA also inhibits ABA-induced expression
of the PHAV1 gene in barley aleurone (99). Experiments in Pisum show that the
activity of either PP1/PP2Apos or PP1/PP2Aneg can be resolved depending on the
aperture of stomates (70) (Figure 3).
These different studies bring to light that a complex phosphorylation and dephosphoryation network exists in guard cells (Figure 3) and that significant differences can occur in ABA signaling depending on the physiological state of guard
cells.
ACTIVITY OF PLASMA MEMBRANE K+
out CHANNELS IN
STOMATAL MOVEMENTS
Efflux of K+ from the cell during stomatal closing has been proposed to occur
through outward rectifying K+ (K+out) channels that are activated by membrane
depolarization (181). Guard cells respond to ABA by enhancing K+out and reducing K+in channel currents (19, 22). Unlike K+in channels, however, K+out channels
are largely insensitive to increases in [Ca2+]cyt that occur during ABA signaling
(108, 175). A guard cell-expressed SKOR K+ channel cDNA homologue, named
GORK, was isolated that when expressed in Xenopus oocytes produces outwardrectifying K+ channels with properties similar to K+out channels (1).
ABA induces an increase in the cytosolic pH of guard cells (22, 54, 80). Experiments in Vicia guard cells show that K+out currents are enhanced by increased
cytoplasmic pH (20). The pH stimulation of K+out channels occurs in isolated membrane patches and is thus membrane delimited (141). ABA-induced increases
in K+out currents can be inhibited by acidification or buffering of the guard cell
cytoplasmic pH, showing that cytosolic pH has a functional role in ABA signal
transduction (22) (Figure 4).
Regulation of K+out by Phosphorylation
Several lines of evidence suggest that protein phosphorylation plays a role in
modulation of K+out channel activity. Guard cells from tobacco plants transformed
with the dominant phosphatase mutant allele abi1-1 from Arabidopsis show K+out
currents 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 in
guard cell signaling (11). Despite the ABA insensitivity of K+out channels in the
abi1-1-transformed guard cells, concurrent measurements of intracellular pH show
normal pH increases in response to ABA (55). This suggests that the abi1-1
phosphatase acts downstream of, or parallel to, the cytosolic pH changes (Figure 4).
If cytosolic pH directly modulates K+out channels (141), then abi1-1 must act in a
parallel 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 phosphatases
in the regulation guard cell K+out channels. OA downregulates both K+in and K+out
channel currents in Vicia (201). However, another study showed that OA downregulated 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 positive
regulators of K+out channel activity in guard cells [(Figure 4); possibly related to
PP1/PP2Apos in (Figure 3)].
Syntaxins and ABA Signaling
Syntaxins play important roles in membrane fusion. A tobacco cDNA encoding
a homolog of human and yeast syntaxin (NtSyr1) was isolated using heterologous
expression of drought-stressed tobacco leaf mRNA in Xenopus oocytes (115). Expression of mRNA pools show ABA activation of endogeneous Ca2+-activated
Cl− currents in oocytes (115) and ABA downregulation of KAT1-mediated K+in
channels in oocytes (196a), suggesting that oocytes will provide an approach
for isolating and analyzing putative ABA receptor cDNAs. By subfractionation
the NtSyr1 mRNA was isolated, which when expressed in oocytes constitutively
activated the Cl− currents without addition of ABA (115). Whether NtSyr1 contributes to the ABA response in oocytes remains to be determined (115). Voltage
clamp recordings in tobacco guard cells provide pharmacological evidence for a
role 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 in
guard cells (115) (Figures 3 and 4). Guard cell volume is changed during stomatal
movements 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 of 86Rb+ efflux kinetics show that whereas a rapid transient K+ release response to ABA was dependent on the concentration of ABA and
duration of exposure, the end-state or final internal 86Rb+ concentration reached
was not (131). This led to the suggestion that guard cells have some means of
sensing their internal osmolarity that is linked to regulation of K+ efflux channels.
Vicia guard cell plasma membrane patches exposed to osmotic gradients show
K+out channels that are inactivated by hypotonic (guard cell swelling) conditions
and 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 response
of guard cells to another environmental stimulus: temperature. At moderate temperatures (13◦ to 20◦ C), both K+in and K+out channel conductances in Vicia increase
with increasing temperature. At temperatures from 20◦ to 28◦ C, K+out conductance
decreases with increasing temperature, whereas K+in conductance continues to increase (79). This difference in temperature response between K+in and K+out channels
at higher temperatures would favor K+ influx and stomatal opening and thus could
allow increased transpirational cooling of leaves (79).
Transient K+ Efflux Currents
In addition to the slow activating K+out channels discussed above, a rapidly activated
transient outward K+ current, (IAP), has been observed in Arabidopsis and tobacco
guard cells (11, 153, 167). In addition to being the first inactivating K+out current
characterized in plants, IAP has several unusual characteristics. In contrast to
the slow K+out channels that are activated by alkaline cytosolic pH (20), IAP is
slightly 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 cytoplasmic
Ca2+ changes. IAP channels show a significant Ca2+ permeability (153). The
physiological role of this transient current is unknown, but IAP may contribute to
shorter-term adjustments in stomatal aperture or to membrane potential oscillations
observed in guard cells (59, 202).
NEW GUARD CELL SIGNALING MUTANTS
AND GENETIC APPROACHES
Quantitative and mechanistic characterization (4, 155) of new Arabidopsis guard
cell signaling mutants is paramount to achieve a molecular understanding of the
ABA signaling cascade. Furthermore genes for many of the above proposed cell biologically and pharmacologically derived mechanisms have not yet been identified.
Loss-of-function mutations in the Arabidopsis ERA1 farnesyltransferase β subunit cause an enhanced response to ABA in seeds (34). Moreover, the era1 mutant
shows ABA hypersensitive stomatal closing and ABA hypersensitive activation
of S-type anion currents (154). Furthermore, era1 plants show reduced water loss
during drought (154). Application of farnesyltransferase inhibitors to wild-type
stomata mimics the era1 phenotype, suggesting that ERA1 functions in early
guard cell signaling and illustrating the complementarity of “steady-state” gene
knockouts and “short-term” inhibitor applications. In mammals and yeast, farnesylation of signaling proteins promotes their membrane location and protein-protein
interactions, suggesting that a negative regulator of guard cell ABA signaling is
targeted via farnesylation (154) (Figure 3). ABA hypersensitive [Ca2+]cyt elevations in era1 show 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 and
characterized by screening for ABA hypersensitivity in seed germination in a primary screen and identifying the subset of mutations that affect guard cell signaling
in a secondary screen (75). Stomatal closing is hypersensitive to ABA in abcap
and, consistent with this phenotype, ABA-induced [Ca2+]cyt elevations and guard
cell plasma membrane anion currents are enhanced and K+in currents are reduced
in abcap. Suprisingly, ABCAP encodes a subunit of a nuclear RNA cap binding
complex (75) previously described in mammals and yeast, which regulates RNA
processing and growth factor signal transduction (83, 84, 211). ABCAP may control the strength of ABA signaling by modulating the expression of an early ABA
signal 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, and aapk) are dominant (46, 114, 119). This suggests that redundancy in phosphatase and kinase activities may limit the isolation of recessive
mutations in such genes. Dominant mutations can also be generated in Arabidopsis by activation tagging or the random overexpression of wild-type genes (210).
Such mutants can aid in identifying and isolating redundant genes involved in guard
cell signaling pathways. However, dominant mutations can result from interactions
with unnatural partners, causing neomorphic responses. To identify unequivocally
the functions of such genes, it will be important to isolate loss-of-function mutants
(71). Disruption or silencing of homologous redundant genes expressed in guard
cells may allow more direct characterizations of in vivo functions of redundant
genes.
Isolating guard cell signaling mutants is not trivial, owing to the lack of easily
scorable phenotypes or markers, and is more difficult than isolating stomatal development mutants, which include cell-to-cell signaling mechanisms (16a, 49a). Thus
the era1 and abcap guard cell phenotypes were identified in secondary stomatal
movement screens (75, 154). A highly elegant stomatal movement screen has been
developed in which small differences in leaf temperature [due to stomatal transpiration (163)] were used to isolate new Arabidopsis guard cell signaling mutants
(J Giraudat & S Merlot, personal communication; 139a). In a different approach,
mutations that affect circadian control of stomatal movements were identified by
selecting Arabidopsis mutants with an enhanced sensitivity to sulfur dioxide at
specific times of day (R McClung, personal communication). This screen has led
to isolation of circadian timing-defective (ctd ) and out-of-phase (oop) mutants.
In another screen using luciferase as a reporter, many mutants were isolated that
are affected in osmotic, ABA, and cold stress–induced signal transduction (81). A
subset of these mutations will likely affect guard cell signaling. Use of novel creative genetic screens will lead to identification of many new mechanisms affecting
guard cell signaling. For example, new guard cell signaling mutants in stomatal
responses could be isolated based on variation in responses among Arabidopsis
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ecotypes and use of recombinant inbred lines to map quantitative trait loci (9) that
affect guard cell signaling.
Note that many Arabidopsis light and hormone signaling mutants have been isolated based on whole-plant or whole-tissue phenotypes, which can lead to isolation
of genes that indirectly affect a signaling pathway via crosstalk or indirect feedback
among signaling cascades. Analyses of ion channel regulation and [Ca2+]cyt signaling in mutants allows one to closely associate mutations with specific elements
in early signaling (4, 154, 155).
The completion of the Arabidopsis genome sequence will lead to reverse genetic functional characterizations of new guard cell signaling components using
biophysical cell biological (4, 5, 155) and genomic methods developed and adapted
to Arabidopsis guard cells. The identification of the full complement of guard cell–
expressed genes is now possible using DNA array and chip technologies, which
will have profound influence on future research. Reverse genetic analyses of guard
cell–expressed genes will be needed to identify genes and test the function and
relative contribution of the many proposed signal transducers reviewed here
(Figures 1 to 4). Furthermore, many of the signal transducers reviewed here likely
form signal transduction complexes consisting of many proteins. Proteomic approaches (119) will play an increasingly important role for identifying members
of guard cell signaling complexes.
FUTURE OUTLOOK
Guard cell research has revealed many new signal transduction components and
led to models of early signal transduction elements and signaling cassettes in
plants. Many of the proposed mechanisms summarized in this review can now
be directly tested by reverse genetics. Furthermore, this research has led to an
initial understanding of how a large number of signaling mechanisms can interact
in concert to produce a rapid physiological response. Future research on mutants will define molecular junction points of an integrated network comprising
Figures 1 to 4. As discussed in the introduction, most plant cells respond to the
classically known hormones and light signals in specific ways. In this sense, each
plant cell contains a microcosm of plant signaling cascades with intricate crosstalk
and specificity mechanisms. The guard cell system lends itself to functional characterization of many new unknown early signaling mechanisms (see 1 to 4 in
Introduction). Interdisciplinary studies using cell biological, genomic, molecular
genetic, biophysical, reverse genetic, and proteomic approaches will define much
of the future research in this field.
Initial examples have shown that manipulation of guard cell signaling genes
in Arabidopsis can affect stomatal movements, leading to reduced water loss and
slowing of dessication during transitory drought periods (53, 75, 154). Future
research in this area combined with inducible guard cell-specific gene expression 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 atmospheric
CO2, and other environmental stresses (180). In this respect, guard cell signaling
research holds much promise at addressing major environmental and agricultural
problems of the twenty-first century.
ACKNOWLEDGMENTS
We thank many colleagues for communicating new and unpublished findings.
Majid Ghassemian, Nathalie Leonhardt, Pascal Mäser, Jared Young, and other
members of the Schroeder lab are gratefully acknowledged for comments on the
manuscript. Research in the authors’ laboratory was supported by NSF, NIH, and
DOE grants.
Visit the Annual Reviews home page at www.AnnualReviews.org
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Figure 1 A simplified working model for proposed functions of positive and negative regulators 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 epistasis among regulators remain largely unknown and requires further genetic, biochemical,
and cellular signaling analyses. For simplicity, parallel signaling branches are not included
here (see text for details).
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Figure 2 A guard cell model, illustrating the proposed functions of ion channels in ABA
signaling and stomatal closing (177). The right cell of the stomate shows ion channels
and regulators that mediate ABA-induced stomatal closing. The left cell shows the parallel
effects 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 regulators
of ABA-induced stomatal closing and of S-type anion channels in guard cells. Positive
transducers are shown at top and/or in green and negative regulators are shown below in
red. The sequence of events and epistasis among regulators remains largely unknown and
requires further analysis. For simplicity, parallel signaling branches are not shown. Note
that some of the ABA signaling “casettes” (top, vertical cascades) may be activated by ABA
in parallel (see text for details).
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Figure 4 A working model for the proposed functions of positive transducers and negative
regulators of K+out channels in guard cells. Positive transducers (green, top) and negative
regulators (red, bottom) are shown.