The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid...

Journal of Experimental Botany, Vol. 60, No. 5, pp. 1439–1463, 2009Perspectives on Plant Development Special Issuedoi:10.1093/jxb/ern340 Advance Access publication 30 January, 2009

REVIEW PAPER

The guard cell as a single-cell model towards understandingdrought tolerance and abscisic acid action

Caroline Sirichandra, Aleksandra Wasilewska, Florina Vlad, Christiane Valon and Jeffrey Leung*

Institut des Sciences du Vegetal, Centre National de la Recherche Scientifique, UPR2355, 1 Avenue de la Terrasse, Bat. 23, 91190Gif-sur-Yvette, France

Received 26 September 2008; Revised 28 November 2008; Accepted 2 December 2008

Abstract

Stomatal guard cells are functionally specialized epidermal cells usually arranged in pairs surrounding a pore.

Changes in ion fluxes, and more specifically osmolytes, within the guard cells drive opening/closing of the pore,allowing gas exchange while limiting water loss through evapo-transpiration. Adjustments of the pore aperture to

optimize these conflicting needs are thus centrally important for land plants to survive, especially with the rise in

CO2 associated with global warming and increasing water scarcity this century. The basic biophysical events in

modulating membrane transport have been gradually delineated over two decades. Genetics and molecular

approaches in recent years have complemented and extended these earlier studies to identify major regulatory

nodes. In Arabidopsis, the reference for guard cell genetics, stomatal opening driven by K+ entry is mainly through

KAT1 and KAT2, two voltage-gated K+ inward-rectifying channels that are activated on hyperpolarization of the

plasma membrane principally by the OST2 H+-ATPase (proton pump coupled to ATP hydrolysis). By contrast,stomatal closing is caused by K+ efflux mainly through GORK, the outward-rectifying channel activated by

membrane depolarization. The depolarization is most likely initiated by SLAC1, an anion channel distantly related to

the dicarboxylate/malic acid transport protein found in fungi and bacteria. Beyond this established framework, there

is also burgeoning evidence for the involvement of additional transporters, such as homologues to the multi-drug

resistance proteins (or ABC transporters) as intimated by several pharmacological and reverse genetics studies.

General inhibitors of protein kinases and protein phosphatases have been shown to profoundly affect guard cell

membrane transport properties. Indeed, the first regulatory enzymes underpinning these transport processes

revealed genetically were several protein phosphatases of the 2C class and the OST1 kinase, a member of theSnRK2 family. Taken together, these results are providing the first glimpses of an emerging signalling complex

critical for modulating the stomatal aperture in response to environmental stimuli.

Key words: Abscisic acid, drought, guard cells, light, protein phosphorylation.

Introduction

A stone tablet, roughly 114 cm in height, 72 cm in width,

and 28 cm thick, weighing 760 kg, created a sensation in

1799 when it was unearthed at the Mediterranean harbour

Rashid, or Rosetta, during Napoleon Bonaparte’s cam-

paign in Egypt. The text on this stele dates back to 196 BC,

and part of the excitement was due to the fact that the

single passage was written in hieroglyphic, demontic, and in

classical Greek. This turned out to be very fortunate, as

comparative translation of the three languages by French

and British scholars was instrumental in deciphering many

previously impenetrable principles of hieroglyphic writing.

The term ‘Rosetta stone’ has since pervaded our every day

parlance as synonymous to a critical key to a process of

decryption or breakthrough understanding of a difficult

problem. Every intellectual barrier, regardless of its initial

enormity, has been breached eventually by the studies of

* To whom correspondence should be addressed: [email protected]ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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a judiciously chosen ‘Rosetta stone’. In biology, this

actually embodies diverse models, mainly in the guise of

bacteria, yeast, worms, flies, and for our intellectual quest in

plant biology, the model plant Arabidopsis (Simpson and

Dean, 2002). Being unrepentant reductionists in the era of

systems biology, our Rosetta stone—the stomatal guard

cell—that promises to enlighten our understanding of signal

integration between light, drought, and abscisic acid (ABA)measures merely 10–20 lm in its overall dimensions but it

possesses all of its mechanistic ‘languages’ decipherable by

genetics, molecular biology, and biophysics. The successful

cross-translation of these three ‘languages’ (i.e. as a multi-

disciplinary approach) will be needed to completely un-

derstand the syntax of each key component in the signalling

chain and how the guard cell turgor is dynamically adjusted

according to the environmental and hormonal cues. At itscrux, this is no more than a fundamental problem of the

signal transduction genre (on reversible cell expansion), but

this field has always held a special appeal to the collective

imagination of biologists, plant breeders, and policy mak-

ers alike because of the need to improve agricultural

productivity—for food, construction, medicine, etc.—in

view of diminishing fresh water supplies, population

growth, and global warming this century. The beckoningquestion for all of us who are intensely engaged in this

cross-disciplinary research area is how soon can these three

‘languages’ be articulated into a coherent model and how

many generalities can flow from the particularities of this

single cell. The limited results that allow a comparison of

similar work conducted in the guard cell and in cell culture

so far strongly suggest shared principles in stress signalling

(Jeannette et al., 1999; Brault et al., 2004).Genetics have revolutionized biology since the turn of the

20th century with the re-discovery of Mendel’s laws on

heredity. When molecular biology joined forces in the

1970s, it began more and more to be appreciated how the

life of a cell depends on the subtle details of the molecules

from which it is built. The small thale cress Arabidopsis

thaliana was ushered in the 1980s as the new plant model

precisely because of its accessibility to genetics and molec-ular probing. To this day, genetics screens—applicable in

both the forward and reverse modes—have been instrumen-

tal in identifying hundreds of mutations that altered

responses of the plant to drought and ABA. In addition to

such gene-by-gene approaches, high throughput tools that

allow monitoring gene expression across the whole genome

in the last decade have revealed networks of co-expressed

genes encoding elements of such diverse nature as todefy—at least at present—any reasonable effort to propose

a unifying signalling scheme. It is not clear why ABA

signalling and its link to drought-adaptive responses require

such complicated mechanistic schemes. This may be due to

the fact that drought adaptation has a large quantitative

component which can be influenced by the intensity of the

experimental drought, the method of drought application

(e.g. in vitro by mannitol, hair dryer on detached plants,etc.) and that the nature of the signalling networks may also

be modified by the cell’s particular developmental state. The

guard cell was developed as a simpler model system

(Armstrong et al., 1995) with the decided aim of decoding

the signalling network in which the membrane transport

system operates in a tight relationship with components in

ABA signalling (Wasilewska et al., 2008). Indeed, stomatal

closure in response to progressive drought has all of the

attributes for an excellent genetic model. Particularly

salutary, is the cell autonomy of the physiological responsedue to the absence of plasmodesmata (Wille and Lucas,

1984), the extreme robustness, and the all important virtue

of reducing the quantitative aspect of drought response into

an all-or-none phenotype (opening/closing).

The guard cells are specialized epidermal cells, located in

pairs on the aerial organs of the plant. Each pair of cells

forms the boundary of a small pore, or stoma, in the

epidermis. Due to the impermeable wax layer on theepidermal surface, the stomatal complex therefore plays

a major role in controlling gas exchange (photosynthetic

carbon dioxide uptake) between plant and the surrounding

atmosphere (Fig. 1a). The stomata are important regulators

of the global atmospheric environment (Hetherington and

Woodward, 2003), and, in fact, the developmental fate of

epidermal cells to differentiate or not into a pair of mature

guard cells—and therefore stomatal density—is intimatelylinked to environmental cues (Gray et al., 2000; Hether-

ington and Woodward, 2003; Masle et al., 2005). The

opening and closing of the stomatal pore are regulated by

osmotic pressure of the guard cells, brought about by the

dynamic changes in the intracellular concentrations of

inorganic and organic ions (K+ and malate2–) and sugars.

These events require interplay between ion channels,

vacuolar and membrane transporters, and metabolite con-versions (Roelfsema and Hedrich, 2005; Wasilewska et al.,

2008). The variations in the transpirational fluxes caused by

stomatal movements can alter leaf temperatures, the ampli-

tudes of which are sufficiently large to be semi-quantifiable

by remote infrared thermography (Raskin and Ladyman,

1988). This technology has therefore been applied to

identify mutants of Arabidopsis impaired in stomatal re-

sponse to experimental drought that is closer to fieldconditions than those based on rapid or chemical de-

hydration stress (Vartanian et al., 1994; Merlot et al., 2002)

(Fig. 1a). Below, first guard cell signalling as established by

biophysical and pharmacological approaches will be de-

scribed, followed by the more recent progress by genetics

and molecular biology in identifying the major components

that link guard cell functions to light and ABA signalling.

The biophysical framework of stomatalmovement

Throughout the 1980s and 1990s, a large body of physiolog-ical data showed that hyperpolarization of the plasma

membrane is stimulated by the activities of H+-ATPases

(proton pumps coupled to ATP hydroylsis), which then

initiates stomatal opening by allowing entry of K+ through

the voltage-dependent K+ inward-rectifying channels. In

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conditions of water stress, the increase in either cellular

ABA, or apoplastic ABA at the surface of guard cells,

triggers a reduction in turgor pressure caused by K+ efflux

and release of anions such as organic acids necessary forstomatal closure. The two distinct sites of ABA accumulation,

either in the cell or in the apoplast, correlate with the presence

of physiologically detectable perception sites or receptors

inside the cell, as well as on its surface. The enhanced

transpiration of ABA-biosynthetic mutants (Koornneef

et al., 1982; Leon-Kloorsterziel et al., 1996; Marin et al.,

1996; Merlot et al., 2002), transgenic plants expressing an

anti-ABA antibody (Artsaenko et al., 1995), and recentgenetic screens for mutants of Arabidopsis sensitive to

progressive drought, based solely on stomatal movement as

the phenotype (Merlot et al., 2002), have provided converg-

ing and incontrovertible evidence for this critical role of

endogenous ABA. As alluded to above, the reduction in

turgor pressure is achieved by the net efflux of both K+ and

release of anionic organic acids from the vacuole to the

cytoplasm and from the cytoplasm to the outside of the cells.One of the earliest responses to ABA frequently, but not

always, is a rise in cytosolic free Ca2+ ([Ca2+]i). The source of

Ca2+ required to elevate [Ca2+]i in response to ABA

originates from both voltage-activated influx across the

plasma membrane and release from internal stores as de-

duced by the use of pharmacological agents and Ba2+ (which

acts as a competitor of Ca2+ influx, internal release as well asthe transient) (MacRobbie, 2000). The uptake and release of

Ca2+ from internal stores, corresponding to various endo-

membranes and, particularly, the endoplasmic reticulum

(ER), are triggered by the intermediates cyclic ADP-ribose

(Allen et al., 1995; McAinsh et al., 1997; Wu et al., 1997),

inositol 1,4,5-trisphosphate (IP3) (Blatt et al., 1990; Gilroy

et al., 1990; Allen et al., 1995; Lee et al., 1996), hyperpolar-

ization of the plasma membrane in Vicia (Grabov and Blatt,1999) and, more recently, myo-inositol hexakisphosphate

(InsP6), which is about 1003 more effective than IP3 based

on inactivation of the K+ inward-rectifying channel (Lemtiri-

Chlieh et al., 2003) (Fig. 1b). The regulation of the Ca2+

signal relay seems highly redundant, as the changes in 32P

labelling of both inositol lipids and inositol phosphates argue

for ABA-induced phosphoinositide turnover in the guard cell

(Lee et al., 1996). However, the changes in label in putativeIP3 observed in these studies were surprisingly small if this

were the major activator of ABA-triggered Ca2+ release. The

aminosteroid, U73122, an inhibitor of phospholipase C, can

Fig. 1. Genetic screen of Arabidopsis mutants sensitive to progressive drought. (a) Closing (ABA, drought) and opening (light) of the

stomatal pore alter the transpirational flux (top) with amplitudes sufficiently large to be visualized by remote infrared thermography

(bottom). In comparison to wild-type plants, mutants that are impaired in stomatal closure in conditions of water deficit are therefore

cooler (plants noted by circles in the infrared images). Figures are modified from those in Merlot et al. (2002) and Roelfsema and Hedrich

(2005). (b) Summary of transport events at the plasma membrane of the guard cell. In responding to drought or ABA, the anion channel

(SLAC) is activated, which drives depolarization of the plasma membrane, while the activity of the proton pump ATPase (OST2), needed

for plasma membrane hyperpolarization, is inhibited. Both of these membrane transporters are regulated by Ca2+, and, in the case of

OST2, the inhibition is most likely mediated, at least in part, by the calcium-sensitive PSK5 kinase. Membrane depolarization also leads

to activation of the K+-outward-rectifying channel (GORK) to expel K+ and inhibition of the K+-inward-rectifying channels (KAT1 and

KAT2) for K+ influx, both of these events are coordinated in opposing fashion by a pH-sensitive pathway that is in part membrane limited.

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modify the form of the cytosolic Ca2+ oscillation induced by

ABA, but not externally applied Ca2+ (Staxen et al., 1999).

Accordingly, U73122 also only partially inhibits stomatal

closure by ABA but not by external Ca2+ (Jacob et al., 1999;

Staxen et al., 1999). Injection of 8-NH2-cADPR (a compet-

itive antagonist of cADPR) or blocking cADPR synthesis by

the addition of nicotinamide can also inhibit ABA-induced

stomatal closure, but again only partially (Leckie et al., 1998;MacRobbie, 2000).

In Arabidopsis, hyperpolarization of the guard cell

plasma membrane can lead to increased Ca2+ influx only in

the presence of applied H2O2, a by-product of superoxides

(Pei et al., 2000). Moreover, applied ABA can also induce

elevation of endogenous H2O2, and blocking the production

of such reactive oxygen species by diphenylene iodonium

chloride impairs the stomatal closing response (Pei et al.,2000). There are multiple sources of H2O2; perhaps the

most relevant to general ABA signalling is that originating

from the catalytic activities of two plasma membrane-

bound NADPH oxidase subunits, AtrbohD and AtrbohF

(Kwak et al., 2003). It is noteworthy though, that about

45% of the guard cells in the double mutant AtrbohD/F still

showed elevated Ca2+ signal in response to applied H2O2

(Kwak et al., 2003), indicating that there are additional andprobably equally important sources. Likewise, the range of

possible cellular targets of H2O2 is unknown, but, in vitro,

the protein phosphatase activities of both ABA-INSENSI-

TIVE1 (ABI1) and ABI2 can be inhibited by this radical

(Meinhard and Grill, 2001; Meinhard et al., 2002). On the

other hand, both the open stomata1 (ost1) (Mustilli et al.,

2002) and growth control by ABA2 (gca2) (Pei et al., 2000)

mutants, insensitive to stomatal closing in ABA, are eitherseverely deficient in H2O2 production or its perception,

respectively (Wang and Song, 2008).

Nitric oxide (NO), first discovered in 1978 as a gaseous

substance in endothelial cells that relaxes blood vessels,

turns out to function as an intermediate in ABA signal-

ling as well. NO—usually supplied in the form of NO

donors such as sodium nitroprusside or S-nitroso-N-

acetyl-penacillamine—was first shown to induce stomatalclosure and reduce transpiration in many plant species,

including Vicia faba, Salpichroa organifolia, Tradescantia

spp., pea, and Arabidopsis thaliana (Neill et al., 2008). NO

promotes intracellular Ca2+ release via cGMP-dependent

cyclic ADP-ribose synthesis (Garcia-Mata et al., 2003),

and this process can be blocked by large spectrum kinase

inhibitors such as K252A and staurosporine (Sokolovski

et al., 2005). The precise sources of NO production are stilldebatable but it is generally thought that nitrate reductase,

especially the isoform encoded by NIA1, may contribute to

this step (Neill et al., 2008).

The Ca2+ signal then triggers a network of effects,

including K+ efflux by vacuolar and plasmalemma K+

channels, release of anionic organic acids by the slow (S)-

type anion channels (Schroeder and Hagiwara, 1989;

Hedrich et al., 1990), while at the same time, inhibits theactivity of K+ inward-rectifying channels (Schroeder and

Hagiwara, 1989; Kelly et al., 1995).

Several observations suggest that Ca2+-independent path-

ways also exist. The physiological state of the guard cell

(e.g. in intact plants versus isolated protoplasts) or the

particular plant species require different rations of Ca2+ to

relay the ABA signal (Levchenko et al., 2005; Marten et al.,

2007). This had led to the alternative proposal of a pH-

sensitive pathway. The concept of pH as a second messen-

ger in ABA signalling has been parlayed from two relatedobservations made in the Blatt laboratory: (i) exposure to

ABA led to a 0.1–0.3 unit rise in pHi of guard cells (Irving

et al., 1992; Blatt and Armstrong, 1993); and (ii) artificially

decreasing the pHi by loading a weak organic acid (usually

butyric acid) suppressed potassium efflux (Blatt, 1992; Blatt

and Armstrong, 1993). pHi acts independently of Ca2+,

based on their effects on K+ influx as well as the differential

sensitivities of the K+ inward- and K+ outward-rectifiers(Grabov and Blatt, 1997). Miedema and Assmann (1996)

have shown that the effect of pHi was the result of proton

interaction with sites associated with the membrane, per-

haps K+ outward-rectifying channel or auxiliary proteins.

The action of pHi also extended to the control of K+ influx,

which was activated by acidity, opposite to that of the K+

efflux (Blatt, 2000). Although these electrophysiological

studies suggested membrane-delimited pathways, they didnot exclude the possibility that there might also be pH-

sensitive cytosolic regulators (see below in the discussion on

the sharp pH sensitivity of the protein phosphatase ABI1).

Merging biophysics with genetics andmolecular biology on stomatal opening: theroles of blue-light photoreceptor kinases,proton pumps, and K+ uptake channels

Light is captured by plants as a source of energy critical for

photosynthesis. But it also doubles as a trigger in signal

transduction pathways directing growth and development(photomorphogenesis). In particular, wavelengths in the blue

and UV-A regions of the electromagnetic spectrum control

phototropism, cotyledon and leaf expansion, stomatal open-

ing, and chloroplast movements in response to changes in

light intensity. Light in the blue spectral range is captured by

receptor kinases, known as phototropins, which then imple-

ment many of the above responses, including stomatal

opening for CO2 assimilation (Shimazaki et al., 2007). Bycontrast, photosynthetically active radiation seems unimpor-

tant for stomatal opening as guard cells that lack functional

green chloroplasts could still respond to blue light, CO2, and

ABA (Roelfsema et al., 2006). Phototropins are ubiquitous

among higher plants and Arabidopsis contains two: PHOT1

and PHOT2. Light sensing is mediated by two flavin-binding

motifs, known as LOV1 and LOV2 (for light, oxygen,

voltage), located at the N-terminal photosensory region ofthe proteins. Photoexcitation leads to activation of the C-

terminal kinase domain and, as a result, receptor autophos-

phorylation. The PHOT1 and PHOT2 are both required for

the activation of the H+-ATPases by blue light and mediate

stomatal opening in a redundant manner (Kinoshita et al.,



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2001). Although the signals from the blue-light receptors are

ultimatedly transduced to the H+-ATPases (see below), there

is no evidence that these proton pumps are direct phosphor-

ylation targets. In fact, their partially redundant functions in

stomatal opening via blue light may belie the actual complex

roles of PHOT1 and PHOT2. Both are critical for blue-light

stimulation of stomatal opening as the double mutant

phot1,phot2—but not their respective single mutants—iscompletely insensitive to blue light (Kinoshita et al., 2001).

These two photoreceptor kinases, however, also assume

distinct functions. PHOT1, but not PHOT2, can interact

directly with NON PHOTOTROPIC HYPOCOTYL3

(NPH3) and ROOT PHOTOTROPISM2 (RPT2), which are

plant-specific novel proteins required for PHOT1-

dependent phototropism (Motchoulski and Liscum, 1999;

Sakai et al., 2000; Inada et al., 2004). A protein interactingwith the N-terminal domain of the Vicia phot1a, but not

phot1b, was also isolated from guard cells (Emi et al., 2005).

It is therefore likely that PHOT1 and PHOT2 may in fact

process both common and separate information in the

transduction chain.

H+-ATPases, upon activation by the phototropin-mediated

signals, hyperpolarize the plasma membrane to ultimately

drive stomatal opening. Depending on the plant species, theplasma membrane potentials are hyperpolarized to different

potentials, but the important point is that they are always

slightly more negative than the activation potential of the K+

uptake channels (about –120 to –180 mV) (Dietrich et al.,

2001). This voltage gradient is therefore sufficient to account

for K+ uptake from the apoplastic space via the K+ inward-

rectifying channels (Blatt, 1991; Thiel et al., 1992; Roelfsema

and Prins, 1998). The functions of the H+-ATPases and theK+ inward-rectifying channels are therefore coupled electri-

cally. In addition to this electric coupling between the two

membrane transporters, other modes of regulations through

pump-driven apoplastic acidification are equally important in

assuring optimal K+ uptake capacity. Measurements on

guard cells in situ within epidermal strips and protoplasts

derived thereof have detected activation of K+ uptake

channels by low pH (Blatt, 1992; Ilan et al., 1996; Dietrichet al., 1998; Bruggemann et al., 1999). Functional coupling

between H+-ATPases and K+ inward-rectifying channels

underlying stomatal opening is therefore subjected to

selective control mechanisms relying on both voltage and

pH. Apoplastic acidication not only changes the voltage-

sensitivity, but also the number of K+ uptake channels as well

(Blatt, 1992; Ilan et al., 1996; Hoth et al., 1997; Dietrich et al.,

1998) (see below). Assuming one titratable pH-sensitive site,a maximum pH sensitivity (pKa) value of 5.3 was obtained

for K+ uptake channels in Vicia faba (Ilan et al., 1996). At

variance, a pKa value of 6.3 was instead obtained by Grabov

and Blatt (1997); with respect to pHi regulation of the K+

inward-rectifier, the central question may be the effective pH

within the fixed-acid matrix of the cell wall at the membrane

surface, rather than the apoplastic space. The pKa value of

K+ uptake channels in Arabidopsis thaliana was about pH4.8, whereas pH 6.2 was obtained for Solanum tuberosum and

Nicotiana tabacum (Dietrich et al., 1998; Bruggemann et al.,

1999). K+ uptake in the guard cell is therefore contingent on

the rate of the proton pump and the apoplastic pH buffering

capacity of the individual plant species.

Although K+ provides the major osmoticum, investiga-

tions have also reported that sucrose accumulates in guard

cells and replaces K+ in the afternoon to maintain stomatal

apertures (Tallman and Zeiger, 1988). Sugars are some of the

breakdown products from starch, but it is unlikely that theentire source of sucrose is derived from photosynthetic CO2

fixation within guard cell chloroplasts because the chloro-

phyll content in guard cells is <2% of that of mesophyll cells

on a cell basis and Rubisco activity is low on a chlorophyll

basis (Outlaw et al., 1979; Shimazaki et al., 1983; Gotow

et al., 1988; Shimazaki, 1989; Reckmann et al., 1990).

Because sucrose and hexose can accumulate to high levels in

the apoplast during the day, it is reasonable to assume thatthe source of sucrose originates mostly from import of this

reserve (Lu et al., 1997; Vavasseur and Raghavendra, 2004).

Consistent with this notion is that sucrose uptake by guard

cells was indeed detected when epidermal peels were over-

layed onto mesophyll tissues (Dittrich and Raschke, 1977);

moreover, the activation of H+-ATPase by the fungal toxin

fusicoccin was found to stimulate sucrose uptake in guard

cell protoplasts (Reddy and Das, 1986; Ritte et al., 1999).This suggests that H+-ATPase activity is also coupled to

sugar transporters, besides K+ channels. The sucrose trans-

porter AtSUC3 is expressed in Arabidopsis guard cells, and

also the AtSTP1 H+/monosaccharide symporter could con-

tribute to guard-cell specific accumulation of sucrose (Stadler

et al., 2003; Meyer et al., 2004).

Forward genetic screens for mutants in Arabidopsis with

enhanced transpiration under mild drought conditions haveidentified two allelic mutations, open stomata2 (ost2)-1 and

ost2-2, at the AHA1 locus encoding a proton pump (Merlot

et al., 2007) (Fig. 1a, b). Both of these mutations rendered

the AHA1 H+-ATPase constitutively activated. The family

of H+-ATPases is composed of 11 genes (and one likely

pseudogene) in the Arabidopsis genome, and their corre-

sponding transcripts are all detectable in guard cell proto-

plasts, whereas only four of them are detectable inmesophyll cells (Ueno et al., 2005). Arguably, identifying

two allelic mutations in the same gene out of 11 possible

targets could still be statistically fortuitous but it is nonethe-

less noteworthy that the genetic screens were accomplished

independently using Arabidopsis plants of different genetic

backgrounds and also under slightly varying experimental

drought conditions. Alternatively, the bias in only identify-

ing AHA1 could reflect the relatively dominant role of thisproton pump in regulating membrane hyperpolarization in

response to progressive water depletion and to ABA. In any

event, the fact that only constitutively activating mutations

have been recovered at this locus so far also indicates that

at least one other H+-ATPase in the gene family could

assume partially overlapping functions. Functional overlap

is consistent with the fact that an insertion mutation in the

AHA1 locus causes no obvious phenotype (data notshown). A candidate gene that functionally overlaps with

AHA1 might be the closest homologue AHA2.



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The current model on regulation of proton pump activity

suggests that an H+-ATPase is kept in an inactive state

by intramolecular contact between its autoinhibitory

C-terminal domain with certain critical amino acids in the

rest of the protein (Palmgren, 2001). This model is indeed

consistent with the data generated by the yeast two-hybrid

interaction system based on the split ubiquitin reporter

(Merlot et al., 2007) and by bimolecular fluorescencecomplementation (BiFC) in co-infiltrated living tobacco

epidermal cells (Fig. 2a) that these two domains can

interact, even in trans. When activated, the H+-ATPase is

phosphorylated on its penultimate threonine residue, and

the tight interaction of the C-terminal autoinhibitory

domain is released from the rest of the protein. Phosphor-

ylation by itself is not sufficient to activate the H+-ATPase,

but it requires the further binding of 14-3-3 proteins to thephosphorylated C-terminus (Kinoshita and Shimazaki,

1999; Emi et al., 2001). The kinase(s) that is critical for the

phosphorylation of the penultimate threonine residue has

not been identified, but, in Vicia, it is insensitive to general

protein kinase inhibitors (Kinoshita and Shimazaki, 2001),

and this upstream kinase in spinach has been demonstrated

to be associated with the plasma membrane (Svennelid

et al., 1999). The crystallization of the full AHA2 H+-ATPase monomer at a resolution of 5.5 A has been

achieved and the results suggested that the C-terminal

autoinhibitory domain assumed no particular structure

(Pedersen et al., 2007). In plant cells, however, the

physiologically quiescent form of an H+-ATPase may not

be a monomer but a dimer, with the C-terminal domains

arranged in a probable antiparallel configuration main-

tained by contacts between residues in the so-called RIIsubregion of the inhibitory domain (Ottmann et al., 2007).

Once activated, the dimer is said to be converted into

a wheel-like hexamer in a higher-order complex with three

14-3-3 dimers (Kanczewska et al., 2005; Ottmann et al.,

2007). The mechanisms by which interconversion between

these higher-order structures are mediated are not known.

ABA partially blocks the activation of H+-ATPases by blue

light. Reduction of this proton pumping activity in the plasmamembrane of the guard cell is necessary for subsequent

membrane depolarization leading ultimately to stomatal

closure. Our knowledge concerning how ABA inhibits the

proton pump remains fragmentary, but part of the effect

might be exerted indirectly through nitric oxide and H2O2

triggered by ABA (Zhang et al., 2004, 2007). The inhibitory

effect of ABA on proton pumping, H+-ATPase phosphoryla-

tion, and stomatal opening can be rescued by applying2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

(c-PTIO), a scavenger of NO (Zhang et al., 2007), but not by

H2O2. Instead, the stimulatory effect evoked by H2O2 to close

stomates may be directed through another yet-to-defined

pathway that implicates the histidine kinase AHK5 (Desikan

et al., 2008). This other pathway is thought to be divergent

from that of ABA despite the consistent observation of the

tight association of ABA and H2O2 production. An earlierobservation that the protein phosphatase inhibitor calyculin

was relatively more effective than okadaic acid in blocking

stomatal opening in response to blue light led to the

speculation, based on the known partial specificities of these

protein phosphatase inhibitors, that protein phosphatases 1

(PP1) might represent a key element in part of the blue-light

signalling chain (Kinoshita and Shimazaki, 1997). Transient

overexpression of a dominant negative form of the catalytic

subunit of a protein phosphatase 1 (VfPP1c) in the guard cells

of Vicia faba by bombardment was found to indeed blockstomatal opening in response to blue light, but only weakly so

to red light. The step inhibited appeared to be upstream of the

plasma membrane H+-ATPase, because the inhibition can be

circumvented by applied fusicoccin (which activates H+-

ATPases). That a PP1 is a key positive regulator in blue-light

signalling is reinforced by the treatment with the cell-perme-

able inhibitor tautomycin, which has a 10-fold higher potency

for PP1 than PP2A. Phototropins and H+-ATPases arephosphorylated when guard cells are exposed to blue light;

yet, only the phosphorylation of the proton pumping, but not

that of the phototropins, was observed to be reduced

dramatically in the presence of tautomycin (Takemiya et al.,

2006). There have been no corresponding mutants identified in

Arabidopsis because the PP1c is encoded by nine isogenes with

between 90% and 99% amino acid sequence homology. It

appears that a severe reduction in their gene expression byreverse genetic methods may also cause lethality as recovery of

this class of mutant has been problematic (Takemiya et al.,

2006). In addition, the inhibitory effect of ABA on H+-

ATPase is considerably lessened by mutations affecting

a homologous pair of protein phosphatases, 2C ABSCISIC

ACID-INSENSITIVE 1 and 2 (ABI1 and ABI2) (Roelfsema

et al., 1998), and that the C-terminus of AHA2 can complex

with PP2A scaffold subunits (Fuglsang et al., 2006). There is,however, no knowledge on whether these protein phospha-

tases directly inactivate the proton pumps. The most direct

evidence for a negative regulator of H+-ATPases comes from

the demonstration of a specific calcium-stimulated kinase,

PKS5 (a member of the SnRK3 kinase family), in phosphor-

ylating Ser931 of AHA2, which interfered with the binding of

a 14-3-3 protein to the penultimate threonine (Fuglsang et al.,

2007). In a functional assay based on yeast, AHA2 alone cancomplement the growth defect of a mutant disrupted in its

endogenous H+-ATPase. However, co-transformation of

AHA2 with PKS5 alone or PSK5 together with its calcium-

binding subunit SCaBP1 led to poor complementation of this

growth defect, consistent with the notion that the kinase and

its cognate calcium-binding subunit conjointly down-regulate

AHA2. Since the Ser931 is in a highly conserved block of

amino acid residues, phosphorylation by PSK5 (or homolo-gous SnRK3) has been proposed to represent a general mode

of negative regulation of H+-ATPases.

Dynamic regulation of the K+ inward-rectifying channels by changing itsactivation state

As mentioned above, the hyperpolarization of the plasma mem-

brane activates the voltage-dependent K+ inward-rectifying



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channels. K+ is in fact a macronutrient essential not only for

adjusting osmotic potential (as in the case of stomatal opening

through cell expansion), but it is also vital for the activity of

a large number of cytosolic enzymes, and for controlling

the membrane potential of cells. It is therefore not

surprising that the plant has elaborate uptake and

distribution systems for this cation (Very and Sentenac,

2003; Amtmann et al., 2004; Dreyer et al., 2004; Gierth and

Maser, 2007; Lebaudy et al., 2007). The functional form of

these channels is composed of four a-subunits (Daram

et al., 1997). In the guard cells of Arabidopsis, KAT1 and

KAT2 encode the majority of the a-subunits which proba-

bly assemble into homotetramers or heterotetramers at

different stoichiometric ratios (and with other minor iso-

forms) presumably to increase functional flexibility (Pilot

et al., 2001; Lebaudy et al., 2008a). Mutants disrupted for

Fig. 2. Confirmation of protein interaction by biomolecular fluorescence complementation (BiFC) based on reconstitution of the split

yellow fluorescent protein (YFP). (a) To test intramolecular interaction of the H+-ATPase OST2 in live plant cells, the N-terminal domain of

the proton pump (aa 1–842) was fused in-frame to the C-terminal half of the fluorescent protein YFP (YFc). Similarly, the C-terminal

autoinhibitory domain of OST2 (aa 838–949) was tagged with the N-terminal half of YFP (YFn) as a translational fusion. These fusion

constructs, along with the suppressor of transgene silencing HcPro, were propagated separately in the Agrobacterium tumefaciens

strain AGL0 and then the proteins were transiently and simultaneously expressed in the leaves of Nicotiana tabacum by co-infiltration.

Infiltrated plants were maintained in greenhouse conditions (22 �C, 60% humidity, 16 h light/8 h dark cycles) for 48–72 h. Infiltrated areas

from leaves were excised and examined by confocal microscopy (Leica TCS SP2). When the spectrum of the fluorescence was

measured, the reconstituted YFP emits a diagnostic frequency between 500 nm and 535 nm (a–c). (b) The well-characterized KAT1 was

served as the positive control in this protein interaction test based on BiFC. KAT1 is convenient because this K+ inward-rectifying a-

subunit can homotetramerize and form the characteristic aggregates of ;0.25–0.5 lm in the plane of the plasma membrane, which is

recapitulated here with the split YFP. (c) Like the yeast Snf1, the ABA-activated kinase OST1 can also homodimerize (as shown by the

particular strong fluorescence from the reconstituted YFP in the nuclei). The YFP fluorescence following the contour of the epidermal

cells most likely reflects cytoplasmic localization which is currently being pursued in detail (C Sirichandra, unpublished results) but it is

consistent with it being the target of negative regulation of ABI1, which is localized in both the nucleus and cytoplasm (Moes et al., 2008).

(d) Negative control based on co-infiltration of chimeric constructs expressing the C-terminal autoinhibitory domain of OST2 fused

translationally in-frame to YFPn and another unrelated transporter fragment similarly fused to YFPc (A Wasilewska, unpublished results).

Negative control interactions in most cases gave no fluorescence. For the purpose of illustration, however, the panel shown here

represents the highest level of background fluorescence that has been encountered in the entire series of experments. These occasional

irregularly shaped and randomly distributed spots are most likely due to autofluorescence. Although the background fluorescence is

visually also yellow, its peak emission frequency from these spots is ;600 nm, which distinguishes it from that of the reconstituted YFP.



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either one or a combination of both genes displayed hardly

perceptible K+ uptake defect. Increasingly severe pheno-

types were obtained, however, when dominant-negative

forms of these channels were introduced into the plants in

either the wild-type (as for the experiments reported for

KAT1) (Baizabal-Aguirre et al., 1999; Kwak et al., 2001)

or the corresponding null genetic background (as for

KAT2) (Lebaudy et al., 2008b). These combined resultsindicate that KAT1 and KAT2 are largely functionally

equivalent and are extremely efficient in K+ uptake to

assure an abundant supply of this macroelement esential

for plant development. The activity of the KAT1 seems to

be modulated by phosphorylation. A 48 kDa calcium-

independent kinase activity was detected in extracts of Vicia

guard cell protoplasts, but not in mesophyll cells, that can

phosphorylate in vitro a peptide containing the C-terminal356 amino acids of KAT1 (Mori et al., 2000). Interestingly,

this kinase was rapidly but transiently activated by exoge-

nous ABA in a dose- and time-dependent manner (Mori

et al., 2000). Since the KAT1 current is suppressed by

depolarization of the guard cell plasma membrane and

elevated Ca2+ triggered by ABA, these observations suggest

that phosphorylation of the C-terminus may bring about

a change in the voltage-dependent activation kinetics of thisK+ channel (Tang and Hoshi, 1999). The exact kinase from

Vicia that phosphorylated KAT1 is not known, but the

obvious candidate, AAPK, purified from ABA-treated

guard cell protoplasts (Li and Assmann, 1996; Li et al.,

2000) apparently did not support phosphorylation of KAT1

(Li et al., 1998). It has also been reported that a 57 kDa

calcium-dependent kinase activity from both guard cell

protoplast membrane and soluble fractions can phosphory-late KAT1 produced in a coupled transcription–translation

system (Li et al., 1998).

Evidence from earlier work shows that K+in channel

activities are regulated by GTP-regulatory proteins (G-

proteins) (Fairley-Grenot and Assmann, 1991). Similar to

the pH-sensitive pathway discussed above, at least part of

that regulated by G-proteins seems to be associated directly

with the plasma membrane as well because various GTPanalogues can alter K+

in channel opening/closing activities

in membrane patches isolated from Vicia faba guard cell

protoplasts (Wu and Assmann, 1994). Furthermore, toxins

such as pertusis and cholera, which covalently modify the

a-subunits of many G-proteins by catalysing the transfer of

ADP-ribose from NAD+ to specific amino acid residues can

also inhibit K+in channel activity (Wu and Assmann, 1994).

The precise mechanistic link between G-proteins and K+in

channels is still not known, however.

Controlling K+ inward current and H+-ATPase activity by endo- and exocytosis

As has been described, stomatal opening and closing are

reflected in the swelling and shrinking, respectively, of the

pair of stomatal guard cells. The volume changes can alter

the surface area by up to 40% (Raschke, 1979) but it was

not clear how the guard cell could achieve such dramatic

changes, especially in nature where fluctuation of micro-

environments is the norm that will demand continual but

small adjustments of the cell surface. Studies combining

biophysics and cell biology within the last few years have

shown that the guard cell can be surprisingly resourceful:

cell expansion can be driven by excursion and removal of

equivalent portions of plasma membrane material withremarkable rapidity (Homann, 1998; Homann and Thiel,

2002; Hurst et al., 2004). In Vicia guard cells, moreover,

a simultaneous measurement of membrane capacitance and

ionic currents suggested that the surface increase was

associated with insertion of both K+ inward- and K+

outward-rectifying channels (Homann and Thiel, 2002).

Parallels of this kind of mechanism of cell volume control

exist in animals, including the cAMP-induced insertion ofthe cystic fibrosis transmembrane conductance regulator

(CFTR, an ABC transporter with channel characteristics)

(Kleizen et al., 2000) and the delivery of epithelial Na+

channels to the apical membrane promoted by aldosterone

and insulin (Erlij et al., 1994; Blazer-Yost et al., 1998).

Further detailed studies on KAT1 by transient expression of

the protein tagged with the fluorescent protein GFP in Vicia

guard cell protoplasts (Hurst et al., 2004) or tobacco leafepidermal cells (Sutter et al., 2006) showed that the fusion

protein was organized as microdomains of ;0.25–0.5 lm in

diameter equivalent to clusters of roughly 50 tetrameric

channels (Hurst et al., 2004; Sutter et al., 2006) that were

positionally stable within the plane of the plasma membrane

(Sutter et al., 2006). A reservoir of KAT1 is therefore

available in the form of a vesicular pool in the cytoplasm,

and the channels are delivered into the plasma membraneon demand by vesicle exocytosis. Targeting KAT1 to the

plasma membrane was found to depend on SPY121,

a member of the Q-SNARE (soluble N-ethylmaleimide-

sensitive factor attachment protein receptor) subclass of

syntaxins. A fragment of SNARE derived from its cleavage

by Clostridum neurotoxin was shown to block, presumably

in a dominant-negative manner, responses of K+ and Cl–

channels to ABA (Leyman et al., 1999) and to have causednoticeable changes in the KAT1 distribution in the plane of

the plasma membrane (Sutter et al., 2006).

Insertion of KAT1 into the plasma membrane requires

exocytosis that is also encrypted by intrinsic sequence

signatures and structural contexts. Di-acidic motifs, D/E-X-

D/E, have been found to function as ER export signals in

plasma membrane proteins in yeast and animal cells

(Nishimura and Balch, 1997; Votsmeier and Gallwitz,2001), and this molecular cryptogram seems conserved in

plants. There are four similar di-acidic motifs (DXE/DXD)

in the C-terminal domain of KAT1, but only one of them

(DAE394–396 localized to the nucleotide-binding domain),

which also conserved in other plant K+in rectifiers, was

found to be required for export from the ER to the plasma

membrane, as mutation in this motif (to AAA) caused

nearly complete retention of KAT1 in ER-like endomem-branes in transfected guard cell protoplasts and human

embryonic kidney cells (Mikosch et al., 2006). These results



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would imply that ER export may constitute an important

mechanism in regulating channel density—and therefore

total K+ transport activity—in conjunction with cell volume

control. Conversely, inactivation of KAT1 by ABA was

accompanied by selective endocytosis from the plasma

membrane leading to their sequestration within an endo-

somal pool (Sutter et al., 2007). Although sequestration was

initiated in concert with detectable changes in K+ channelactivities evoked by ABA (Homann and Thiel, 2002;

Meckel et al., 2004; Homann et al., 2007), this process alone

is several orders of magnitude too slow to account for the

short-term control of K+ flux by this hormone. Remark-

ably, enodocytosis can still occur despite a high osmotic

pressure (Meckel et al., 2004).

By contrast to KAT1, less is known about trafficking of

the proton pump. In the same studies that examined KAT1transport by transient expression in tobacco epidermal cells,

the endogenous H+-ATPase PMA2 of tobacco was also

tracked but it seemed to be stationary during these same

studies (Sutter et al., 2006, 2007). However, the insertion of

H+-ATPases—exemplified by PMA2 (Lefebvre et al.,

2004)—into the plasma membrane was not by default, but

required cytosolic factors interacting with the nucleotide

binding site of the proton pump. In maize coleoptile,application of the auxin analogue 3-indole acetic acid can

stimulate ER exocytosis and, within minutes, an increase in

the number of H+-ATPases can be detected immunologi-

cally (Hager et al., 1991). Treatment of the coleoptile with

the protein synthesis inhibitor cyclohexamide suggested that

the half-life of an H+-ATPase in this fast-growing tissue was

;12 min. Thus, an increase in H+ extrusion necessary for

growth (the ‘acid-growth’ theory) can be accomplished incertain tissues by enhanced delivery of the proton pump

from the ER to the plasma membrane. Whether there might

be equally dynamic transport of certain H+-ATPases, apart

from PMA2, in the guard cell is not known.

ABA stimulation of stomatal closure:nuclear- and chloroplast-located receptors

Contrary to the effect of light, ABA is synthesized in

conditions of water deficit to trigger stomatal closure, thereby

limiting transpiration. It has already been mentioned above

that the activity of H+-ATPases can be inhibited, at leastpartially (to about 60%), by 10 lM exogenous ABA

(Roelfsema et al., 1998; Zhang et al., 2004). Multiple sites of

ABA perception, inside the cell as well as on the outside of

the cell surface, have been detected by electrophysiological

means for over 15 years (Leung and Giraudat, 1998;

MacRobbie, 1995). The possibility of multiple sites of ABA

perception seems to also correlate with the phenotypic

characterization of two tomato mutants, tos1 and tss2

(Borsani et al., 2002). The mutant tss2 was shown to be

hypersensitive to growth inhibition by exogenous ABA

(Borsani et al., 2001), while tos1, insensitive to ABA,

behaved in a rather similar way to the wild type when grown

on an alkaline medium buffered at pH 8 (Borsani et al.,

2002), which prevents almost completely the entry of ABA

into the cell (Jeannette et al., 1999). Alternatively, the

addition of ABA–BSA conjugate in the medium, which was

too large to penetrate the plasma membrane (Jeannette et al.,

1999), also reduced growth of the tos1 mutant as in the wild

type. The similar results obtained either with ABA–BSA

conjugate or by using pH 8 showed that the insensitivity

phenotype disappears when the ABA is kept extracellularly.The argument by the authors is that tos1 is altered in

intracellular ABA perception or signalling, but the nature of

their corresponding proteins is not known.

In the last few years, several serious candidate intracellu-

lar receptors, some with surprising characteristics, have

been proposed, based on their ability to bind ABA in vitro.

The first reported ABA-binding protein is the homologue of

the H subunit of the larger trimeric Mg2+-chelatase complex(CHLH) protein in Vicia faba (Zhang et al., 2002). The

Arabidopsis homologue is encoded by the ABAR gene which

turns out to be identical to GENOME UNCOUPLED5

(GUS5) (Shen et al., 2006), discovered earlier as being

essential for plastid–nucleus communication and, more

specifically, in the control of chlorophyll synthesis by either

the metabolism or sensing of the tetrapyrrole signal Mg2+-

protoporphyrin-IX. The GUS5/CHLH subunit has there-fore a dual function that generates independent downstream

physiological consequences: in binding to Mg2+-protopor-

phyrin in chlorophyll synthesis and binding to ABA during

adaptive response to dehydration. The knock-down abar

mutant (Shen et al., 2006) has normal chlorophyll content,

but the ABA-inducible genes were down-regulated except

for the the two homologous negative regulators of ABA

signalling, ABI1 and ABI2, which were up-regulatedinstead. Exogenously applied ABA stimulated ABAR ex-

pression, increased Mg2+-chelatase activity and Mg2+-

protoporphyrin content, but suppressed chlorophyll and

protoporphyrin synthesis.

Another candidate came from the biochemical character-

ization of a 52-kDa plasma membrane-localized protein

with a WW-domain from barley aleurone. This protein,

named ABAP1 has been shown to physically bind ABAin vitro (Razem et al., 2004) and this can be weakened by

the presence of H2O2 (Razem and Hill, 2007). Because there

is no detectable change in the overall protein structure

induced by H2O2, the authors have suggested that the

inhibitory effect may represent some form of feedback

regulation directly on the local ABA binding site of the

protein. The search for ABAP1 homologues in Arabidopsis

by the same research group turned up FCA as the closest(Razem et al., 2006), which is rather surprising considering

that this protein has long been recognized for its prominent

role in the control of flowering time by the so-called

autonomous pathway. In contrast to ABAP1 of barley, the

Arabidopsis FCA is not a plasma membrane-localized

protein, but rather a single-stranded RNA-binding protein

that is constitutively nuclear-localized (Macknight et al.,

1997). Binding of ABA to as yet undefined amino acidsinterfered with its interaction, via its WW domain, with

another RNA-binding protein FY, which is similar in



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sequence to the yeast polyadenylation and 3#-end processing

factor PsF2p (Simpson et al., 2003). Interaction of FCA with

FY is obligatory for the correct splicing of the FCA

transcript in a negative feedback loop. The disruption of this

interaction by ABA would thus correlate with a previous

observation that this hormone can cause delays in flowering

(Levy and Dean, 1998). Nonetheless, the purported inhibi-

tory effect of (+)ABA on the in vitro interaction of FCA-FY,and also their homologous rice counterparts has been

questioned recently (Jang et al., 2008). The mutant fca, in

addition, showed no substantial alteration in most of the

classical ABA responses (such as seed germination or

transpiration), except at the level of the lateral roots, which

were found to be reduced in sensitivity to the inhibitory

effects of applied ABA (Razem et al., 2006). There was also

no dramatically altered expression of genes related todrought nor ABA signalling in fca as ascertained by tran-

scriptome analysis (Marquardt et al., 2006). Lastly, there are

still the cell-surface receptors to account for the typical

physiological responses elicited by externally applied ABA on

guard cells (Anderson et al., 1994) and on cell cultures

(Jeannette et al., 1999). Although additional candidates have

surfaced, none possesses all of the characteristics expected of

a cognate ABA receptor, and, in recent months, a so-calledGCR2 has been particularly controversial (Gao et al., 2007;

Johnston et al., 2007; Liu et al., 2007a, b; Chen and Ellis,

2008; Illingworth et al., 2008).

The calcium signal coordinates membranetransporters in stomatal closure

ABA, at least in the guard cell, regulates repetitive cytosolic

free Ca2+ ([Ca2+]cyt) elevations (Mori and Muto, 1997). As

described above, this Ca2+ signal is likely to be responsible

for the inhibition of H+-ATPases possibly via the calcium-

sensitive kinase PKS5 (Fig. 1b). In addition, experimentallyimposed [Ca2+]cyt transients had previously revealed two

distinctive Ca2+-dependent stomatal closing responses:

a rapid so-called ‘Ca2+-reactive’ stomatal closing response,

and a long-lasting ‘Ca2+-programmed’ stomatal response,

which prevailed even after Ca2+ transients have been

terminated (Israelsson et al., 2006). The long-lasting Ca2+-

programmed response, but not the rapid Ca2+-reactive

stomatal closing response, was found to depend on theCa2+ transient pattern. In guard cells, the [Ca2+]cyt elevation

activated the slow- or S-type anion channels by reversible

protein phosphorylation (Schmidt et al., 1995), implying

that calcium-regulated kinases may also be involved in

decoding the Ca2+ signal. Two of such kinases, CPK3 and

CPK6, whose expressions are enriched in guard cells and

are induced by ABA, appear to be excellent candidates.

Indeed, disruption of either one or both of the kinasesimpaired the up-regulation of anion channel currents by

ABA, thereby confirming them as key transducers of the

Ca2+ signal in the guard cell (Mori et al., 2006). When

guard cell protoplasts were incubated in low extracellular

Ca2+, the S-type anion channels only responded moderately

to 2 mM Ca2+ in the pipette. This response can be enhanced

in the wild-type, but not in the mutant, cpk protoplasts by

the addition of ABA. The authors’ interpretation is that

ABA as a stomatal closing signal, mediates by an undefined

mechanism of ‘priming’ of guard cell Ca2+ sensors (among

them, CDPK) such that they can respond to an elevated

Ca2+ signal. These two CDPKs may also have other roles in

the ABA regulation of Ca2+-permeable channels (Israelssonet al., 2006; Mori et al., 2006).

Whether anion channels are direct targets of these Ca2+-

dependent kinases is not yet known. The hunt for the actual

slow anion channels that might be placed squarely in the

ABA signalling pathway itself had also been a long and

torturous quest until recently. The first ‘chloride’ channel

CLC reported turned out to be a nitrate/proton exchanger

(De Angeli et al., 2006). Earlier, there was some evidencebased on pharmacological tests that the anion channel

might correspond to an ATP-binding cassette (ABC) trans-

porter similar to the animal proteins, based on their

sensitivity to the known blocker glibenclamide (Leonhardt

et al., 1999). The mutant Atmrp5 affected in an ABC

transporter has been reported to show impaired Ca2+

signalling and partial ABA-induced anion current activity

as components of its pleiotropic phenotype (Klein et al.,2003; Suh et al., 2007). It is indeed very exciting to note that

independent direct genetic screens for mutants impaired in

stomatal response to either elevated CO2 (Negi et al., 2008)

and ozone (Vahisalu et al., 2008) have converged on slow

anion channel-associated 1 (slac1) as the closest candidate to

the long awaited S-type anion channel. These allelic

mutants turned out to be pleiotropic, being strongly di-

minished not only in anion current and in responding toCO2, ozone, but also to ABA, light/dark transition, Ca2+,

H2O2, and NO, but retained wild-type Ca2+ channel activity

(Vahisalu et al., 2008). Interestingly, slac1 did not affect

rapid (R)-type anion channel current. This observation is

important because it also clarifies the position as to whether

the slow and rapid anion channels are distinct entities or

alternate states of the same channel protein (Vahisalu et al.,

2008). The protein was specifically localized to the plasmamembrane of guard cells and loosely resembled the bacterial

and fungal C4-dicarboxylate/malic acid transporters. One

member of this family in Schizosaccharomyces pombe,

Mae1, had been functionally characterized as a malate

uptake transporter, although the amino acid identity is only

20% to that of SLAC1 (Grobler et al., 1995). Guard cell

protoplasts prepared from slac1 contained higher content of

not only malate, but also fumarate, K+, and Cl– ascompared with those of the wild type. This suggests a broad

default directly or indirectly caused by slac1 in the

homeostasis of organic and inorganic ions.

GORK is the major guard cell outward-rectifying K+ channel

By reverse genetics, a T-DNA insertion was identified in the

GORK gene encoding a potassium inward-rectifying



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channel (Hosy et al., 2003), which has been previously

shown to be highly expressed in the guard cell (Pilot et al.,

2001). Patch-clamp experiments on guard cell protoplasts

derived from gork showed only about 10% residual K+

outward current as compared with the native K+out channel.

A dominant-negative form of gork was produced by

replacing the hallmark motif GYGD (or gly-tyr-gly-asp),

expected to play a pivotal role in the formation of thechannel-conducting pathway (Nakamura et al., 1997), by

introducing the positively charged amino acid arginine (or

R) resulting in RRGD. The dominant-negative behaviour

of this channel was confirmed by heterologous expression in

Xenopus oocytes. These experiments, which involved in-

jection of cRNA produced in vitro, showed that the mutant

channel was electrically silent, and co-injection in parity

with the mutant and wild-type cRNA resulted in >76%inhibition of the GORK current. Similarly, expression of

this gene bearing the dominant negative mutation in stable

transgenic Arabidopsis blocked K+out current in the guard

cells by >90%. Stomatal movement assays in response to

ABA and light-to-darkness transition revealed that both the

disrupted GORK or its dominant negative counterpart

expressed in transgenic Arabidopsis led to reduced closing

response and higher transpirational fluxes in both excisedrosettes and intact plants submitted to progressive drought.

These observations provide clear genetic evidence for the

important function of this K+out channel in regulating

stomatal closure. Note that the knock-out and, particularly,

the dominant-negative form of the gene led to only partial

disruption of this response. This is likely due to the fact that

K+ channel current activities in guard cells are approxi-

mately an order of magnitude larger than physiological K+

fluxes during stomatal movements (Schroeder et al., 1987).

There might also be additional and perhaps more minor K+

efflux mechanisms. For example, the transiently activated

outward-rectifying K+ current IAP might contribute to K+

efflux, which is down-regulated by elevated Ca2+(Pei et al.,

1998).

ABA transport and distribution: theemergence of ABC transporters

Delivering ABA, like other hormones, to the right place

and at the right time is undoubtedly important inoptimizing plant growth in fluctuating environmental

constraints (e.g. light intensity and quality). As has been

epitomized by several excellent studies into auxin redistri-

bution, its gradients and maxima are orchestrated by the

PIN efflux carriers. PIN proteins direct this cell-to-cell

auxin transport, and orient plant development through

their asymmetric subcellular distribution (Robert and

Offringa, 2008). PIN polarity itself is regulated byPINOID and, surprisingly, by the phototropins as well, all

of which are members of the AGC protein serine/threonine

kinase family. In addition to the PIN efflux carriers, there

is also a wealth of evidence for the participation of several

ATP-binding cassette (ABC) proteins. Mutations in the

AtABCB19 locus have recently been found to release the

inhibitory effect of N-1-naphthylphthalamic acid (NPA)

on hypocotyl tropism (Nagashima et al., 2008). Also,

mutations in AtMDR1 and AtPGP1 have provided evi-

dence that relates auxin polar transport to photomorpho-

genesis and root development (Lin and Wang, 2005).

Unlike auxin, the mechanisms by which ABA is trans-

ported have not been extensively explored, probablyhindered by the lack of appropriate chemical inhibitors.

The enzymes that convert xanthoxin eventually into ABA

are expressed in the vascular parenchyma cells of roots and

shoots (Cheng et al., 2002; Koiwai et al., 2004), suggesting

that the vascular system constitutes the major conduit of

long-distance transport. Because not all cells express all

of the biosynthetic and modification enzymes of ABA

(Marion-Poll and Leung, 2006; Wasilewska et al., 2008),the intermediates in the synthetic pathway might be escorted

from one cell type to another for the successive maturation

of the hormone molecule. Using the luciferase gene driven

by promoters sensitive to ABA as a reporter, in vivo

imaging revealed that in transgenic Arabidopsis under ‘non-

stress’ conditions, the hormone is highly concentrated in the

apical meristem and in the guard cells (Christmann et al.,

2004). It is likely that these tissues are more than passivehormone repositories; rather, these observations compel the

question: does ABA have more subtle physiological func-

tions other than in abiotic stress? In responding to drought,

the ABA content increases ;10-fold, most of which will

likely come from de novo synthesis, as the delivery from

enzymatic release of the small pre-existing pool of inactive

ABA conjugate would not be significant to the overall

contribution (Neill et al., 1983; Priest et al., 2006). In fieldconditions, as the soil dries, ABA had been proposed to be

the long-distance messenger that spreads the alarm of water

deficit by moving from root to shoot, and to also play

a major role in orchestrating the subsequent physiological

responses, particularly reducing transpiration by stomatal

closure (Wilkinson and Davis, 2002). The argument in

favour of this root-to-shoot re-distribution of ABA was

inspired by split-root experiments with whole plants, inwhich only part of the root system was exposed to water

deficit. This is also consistent with results from experiments

in which the water status in leaves was maintained by

pressurization of drought-exposed roots that failed to

prevent stomatal closure in herbaceous species (Wilkinson

and Davis, 2002). Evidence to the contrary, however, also

exists. As noted above, the apical meristem and guard cells

in leaves, being the major detectable sites of ABA, alreadyhint at the incongruence with the strict view of root-to-

shoot transport in the moment of stress. Secondly, grafting

of tomato shoots onto root stocks deficient in ABA

biosynthesis indicated root-generated ABA was not re-

quired (Holbrook et al., 2002). Lastly, based on theoretical

considerations, the transport of a chemical signal from

roots to the canopy of tall trees may take >1 d if taking into

account the transpirational streaming speed of 2 m h�1 inconifer xylem (Zimmermann and Brown, 1971). Recently,

through a series of simple but elegant experiments,



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Christmann et al. (2008) tendered the provocative possibil-

ity that the drought alarm is more likely to be a hydraulic

signal instead. They showed that when water was directly

delivered to leaves, even a decrease in soil water potential

sufficient to elicit a strong loss of cell turgor pressure

(expected to be at or near 0 MPa) now proved to be

ineffective. Under these conditions, mesophyll cells

remained turgid (0.3 MPa), with cellular osmotic pressurescomparable to those from well-watered plants (0.38 MPa)

and, in particular, this response was dependent of neither

de novo ABA biosynthesis nor signalling (Christmann

et al., 2008).

What might be the ABA transporters? At this juncture,

we can only speculate. As described above, mutations

affecting certain ABC proteins lead to altered response to

auxin. They are also emerging as prominent integrators ofdiverse signalling pathways in guard cell physiology. These

transporters are encoded by large gene families with

memberships around 100, exist in eukaryotes and prokar-

yotes, and are structurally very diverse. They consist of

mechanochemically coupled integral membrane protein

complexes in which ATP binding and hydrolysis drive

solute transport across membranes. ABC proteins were first

discovered in animals owing to their capacity to exportxenobiotic substances such as toxic drugs, and hence, they

are also known as Multiple Drug Resistance (MDR) trans-

porters. A class of antidiabetic agents, collectively known as

sulphonylureas, are potent inhibitors of ABC transporters

and ATP-sensitive K+ channels in animal tissues (Higgins,

1995). Sulphonylureas can stimulate stomatal opening and

partially inhibit both outward K+ (Leonhardt et al., 1997)

and slow anion currents (Leonhardt et al., 1999) in Vicia

and Commelina. This inhibition is released by vasodilator

agents (e.g. cromakalin) collectively known as KCO which

open K+ channels in animals, or, alternatively, by ABA

treatment which can partially restore the slow anion current

(Leonhardt et al., 1999). In humans, the ABC transporter

CFTR functions as a chloride channel, thereby also blurring

the distinction between transporters and channels (Higgins,

1992, 1995).Three Arabidopsis mutants that are disrupted in genes

encoding specific ABC proteins leading to perturbed

stomatal physiology have been identified so far. Atmrp4 has

wider stomatal aperture as compared with the wild type, but

the mutant phenotype can be restored by ABA, indicating

that the phenotype is not due to a lesion in ABA signalling

(Klein et al., 2004). More recently, a screen of ;20,000 gene

trap lines designed to identify genes preferentially expressedin the guard cells led to the discovery of AtPDR3

(Pleiotropic Drug Resistance 3). Preliminary evidence

showed that disruption of this gene led to reduced

sensitivity of guard cells to ABA (Galbiati et al., 2008).

The relationship between the ATMRP5 and ABA signal-

ling in the guard cell is complex. The stomata of Atmrp5 are

insensitive to several stimuli, including auxin, Ca2+, and

ABA (Klein et al., 2003); more precisely, the mutationappears to impair activation of the anion channels by Ca2+

and ABA (Suh et al., 2007). The average stomatal aperture

of the mutant, despite de-regulated anion channels, is

unexpectedly smaller when compared with that of the wild

type (Klein et al., 2003). The Atmrp5 mutation is rather

pleiotropic (Gaedeke et al., 2001; Klein et al., 2003) and it

might be difficult to distinguish primary and indirect

phenotypic consequences.

Another ABC transporter has been recently reported to

play a role in CO2 sensing through malate transport. Ascompared with the wild type, guard cells on detached leaves

isolated from mutants AtABCB14 were shown to close

more rapidly in responding to high CO2 (800 ppm) and,

conversely, transgenic Arabidopsis plants overexpressing the

gene displayed the opposite phenotype (Lee et al., 2008). By

contrast, the mutant responded normally to light-to-

darkness transition, ABA, and Ca2+. The mutant guard

cells on isolated epidermal strips also close their stomatamore rapidly in malate (20 mM), while the overexpressors

not at all. The phenotype of the Atabcb14 mutant is

therefore roughly opposite to those in slac1. High CO2 has

been shown to increase apoplastic malate from 1 mM to

3 mM, and it was hypothesized that a default in malate

uptake (needed to maintain high osmotic pressure and

stomatal opening) in the mutant might be responsible for

the phenotype. AtABCB14 can partially restore growth ofthe dicarboxylate transporter mutant CBT315 of Escher-

ichia coli, which cannot use malate as a carbon source.

Similarly, expression of this ABC transporter in HeLa cells

can increase malate uptake by 80%. These results reinforce

the hypothesis that AtABCB14 controls stomatal movement

by importing malate from the apoplastic space into guard

cells, thereby increasing their osmotic pressure.

Reversible protein phosphorylation-basedpathways underpin rapid regulation ofstomatal movements: mechanistic insightsderived from genetic and molecular analysis

Correct interpretation of mutant phenotypes can sometimes

provide invaluable molecular insights into signalling or

developmental events. Recessive mutations are, as a general

rule, much easier to interpret because they must entail

a partial or full loss of function (usually catalytic activity).

Dominant mutations are, by comparison, more open todebate because their underlying causes can be multiple in

origin. One of the first regulators in ABA signal trans-

duction was identified by a single dominant mutation abi1-1

(Koornneef et al., 1984), which rendered the mutant highly

insensitive to exogenous ABA, reduced seed dormancy, and

hypersensitive to mild water deficit. The gene encodes a 2C-

type protein phosphatase (Leung et al., 1994; Meyer et al.,

1994; Bertauche et al., 1996; Leube et al., 1998) that canstably exist in monomeric forms (Leube et al., 1998).

Because the PP2C catalytic activity of the ABI1 is acutely

sensitive to pH and a shift from 7.2 (in prevailing cytosolic

condition) to pH 7.5 (during ABA signalling) would be

expected to double the ABI1 activity (Leube et al., 1998),



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this PP2C might be one of the important cytosolic factors

in the pH-dependent pathway in modulating K+ fluxes

(Blatt, 1992, 2000; Blatt and Armstrong, 1993) (see above).

ABI1 might also be the target of inactivation by the

second messenger H2O2 produced during ABA signalling,

thus implying the existence of a negative feedback loop

(Meinhard and Grill, 2001).

An important and intriguing publication has recentlyrevealed that the manifestation of the various ABA-

insensitivity phenotypes required nuclear localization of the

protein, and that the abi1-1 mutation resulted in the

preferential accumulation of the protein in the nucleus

(Moes et al., 2008). Application of the proteasome inhibitor

MG-132 led to both a preferential nuclear accumulation of

the wild-type ABI1 and an enhancement of PP2C-

dependent inhibitory action of the ABA responses. Theseobservations indeed contribute critically towards an under-

standing—admittedly still partial—of the ABI1 regulatory

function. At the whole plant level, numerous genetic

analyses had already defined a negative regulatory role for

the wild-type ABI1 in ABA signalling, and this function is

not altered but rather reinforced by the mutation abi1-1 in

that the mutant protein also blocks ABA sensitivity—even

constitutively. At the molecular level, the mutation renderedthe nuclear form of the PP2C more stable, which can be

recapitulated by MG-132 treatment of the wild-type ABI1

protein. A first conclusion is that the ABI1 is dynamically

regulated by proteolysis (and perhaps also inactivation by

H2O2, as mentioned above). That this dominant mutation

has a stabilizing effect against ubiquitin-mediated proteoly-

sis on the protein is indeed in accordance with an earlier

report of an extragenic revertant of abi1-1, named sleepy1

or sly1 (Steber et al., 1998). SLY1 encodes an F-box subunit

of an SCF E3 ubiquitin ligase complex (McGinnis et al.,

2003). Here, we further venture the prediction that the

mutant sly1 subunit in the E3 ligase complex might be more

efficient that its wild-type counterpart in promoting abi1-1

destruction, thereby relieving constitutive inhibition by this

mutated PP2C on the ABA signalling pathway. In survey-

ing the current literature on hormone signalling for clues, itwas found that this predicted mechanism bears striking

resemblance to the case of another sly allelic mutant (gar2-

1) E3 ligase subunit which, as compared with its wild-type

counterpart, has a higher affinity towards its substrates

which are certain members of the so-called DELLA

proteins, especially when they are phosphorylated (GAI

and RGA). These proteins function in growth restraints

during GA signalling. Presumably the mutant slygar2-1

promotes a much more efficient degradation of the phos-

phorylated DELLA proteins by the ubiquitin-proteasome

complex (Fu et al., 2004). A second and more tentative

conclusion drawing from GA signalling as an analogy is

that the Asp replacement in abi1-1, being more negatively

charged and mimicking a phosphorylated amino acid near

the catalyic site (Das et al., 1996), may increase its

degradation as it would be targeted more efficiently by themutant sly E3 ligase (this is why the mutant sly1 was

isolated as an extragenic suppressor of abi1-1). The simple

but elegant demonstration of Moes et al. (2008) portends

that the unbiquitin-mediated proteolysis, a rather common

mechanism in regulating other hormone signals up to now,

might also be an integral part of the ABI1 pathway.

Whether the mutation abi1-1 in itself causes an absolute

excess of catalytic activity in vivo as compared with its wild-

type counterpart—independent of the gain in protein

stability—is not clear, although this dominant mutationhas also been alternatively interpreted as ‘hypermorphic’

(Robert et al., 2006; Moes et al., 2008). This interesting

possibility is nonetheless entertained by the identification of

a particular 2A-type protein phosphatase, PP2Ac-2, that

when disrupted, can also partially suppress the various

elementary ABA-insensitivity phenotypes caused by the

abi1-1 mutation (Pernas et al., 2007). This possibility of

excess protein phosphatase activity is also corroborated bya second independent observation that numerous second-

site loss-of-function mutations dispersed within the mutant

abi1-1 gene (i.e. intragenic suppressor mutations) all re-

versed the ABA-insensitivity phenotype towards that of the

wild-type and, in some cases, even causing mild ABA

hypersensitivity (Gosti et al., 1999). Importantly, recombi-

nant abi1-1 proteins containing these intragenic second site

substitutions expressed in E. coli are enzyme-dead, or nearlyso, towards casein as the in vitro substrate (Gosti et al.,

1999). Thus any hypothetical excess PP2C activity caused

by abi1-1 is completely neutralized by either mutating

another phosphatase (PP2Ac-2) or second-site intragenic

mutations dispersed elsewhere in the same protein.

One major problem associated with the hypothesis

privileging excess PP2C activity caused by the abi1-1

mutation is that it has never been detected by the currentavailable in vitro biochemical tests. In fact, all these tests

used commercial casein as the classical surrogate substrate

and, paradoxically, a consistent partial loss of phosphatase

catalytic activity was rather observed (Bertauche et al.,

1996; Leube et al., 1998; Sheen, 1998). One could perhaps

reconcile that the presumed excess phosphatase activity

engendered by abi1-1 could not have been detected because

all phosphatase tests had relied on an animal protein as thesubstrate. The alternative possibility is that the presumed

excess of catalytic activity and the gain in protein stability is

in fact one and the same phenomenon. That is, the

dominant mutation might in fact cause: (i) a partial loss of

in vivo catalytic activity relative to that of the wild-type

counterpart, but at the same time (ii) this deficit is more

than offset by the gain in stability that it bestows on the

protein against ubiquitin-mediated proteolysis, as suggestedby the combined results of Steber et al. (1998), McGinnis

et al. (2003), and Moes et al. (2008).

In plants, the phenotype provoked by the loss-of-function

revertant alleles of abi1 (including those caused by T-DNA

insertions) is mild and hardly distinguishable from that of

the wild type. This can be explained readily by the largely

overlapping functions of at least three other homologous

PP2Cs in ABA signalling (Schweighofer et al., 2004),including the closest homologue ABI2, initially identified

through the same dominant mutation as that found in



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abi1-1 (Koornneef et al., 1984; Leung et al., 1997;

Rodriguez et al., 1998) that can compensate for one

another. Thus, a third conclusion based on analysis of these

loss-of-function alleles is that the ABI1-regulated part of

the signalling chain must be critical, as it is assured by at

least four functionally redundant PP2C, in addition to the

aforementioned one controlled by PP2Ac-2 (Pernas et al.,

2007). This is attested by the fact that a surprisingly largenumber of mutations affecting ABA perception, including

the putative receptor abar (see above), lead to perturbed

expression of the ABI1 gene, but presumably, any impact

would be largely buffered by genic redundancy. These

observations further suggest that these parallel protein

phosphatase pathways have common downstream targets.

The abi1-1 (as well as abi2-1) mutation impairs the rise in

Ca2+ (Allen et al., 1999) which blocks ABA responses of K+

and anion channels (Armstrong et al., 1995; Grabov et al.,

1997; Pei et al., 1997). The homologous AtPP2CA, one of

the redundant negative regulators of ABA signalling

(Sheen, 1998), has been shown to bind directly to the

potassium channel AKT2 in yeast, and the K+ channel

characteristics can be influenced by AtPP2CA observable by

co-expression in Xenopus oocytes (Cherel et al., 2002).

Similar interaction and functional tests with membranetransporters have not been carried out with ABI1. It is thus

not clear whether the impact of the abi1-1 mutation on ion

transport is direct or not.

One of the in planta cognate targets of the ABI1 is almost

certainly to be the ABA-activated kinase OPEN STO-

MATA1 (OST1), as these two proteins can interact in

a directed yeast two-hybrid assay (Yoshida et al., 2006).

Mutations in the OST1 locus led to guard cells that areunresponsive to the ABA signal and low humidity (Merlot

et al., 2002; Mustilli et al., 2002; Yoshida et al., 2002; Xie

et al., 2006), similar to some of the vegetative phenotypes

engendered by the abi1-1 mutation. As referred to earlier in

the section dealing with the Ca2+ signal, the ost1 mutation

blocks the production of the second messenger H2O2

(Mustilli et al., 2002). The OST1 belongs to the SnRK2

family with a membership of 10 in the Arabidopsis genome(Hrabak et al., 2003). The OST1 kinase (also known as

Srk2e and SnRK2.6) has a relatively conserved N-terminal

catalytic domain and a more divergent regulatory C-

terminal domain. A small stretch of amino acids, known as

domain II, localized to the extreme end of the C-terminus is

functionally required for ABA activation of the kinase

activity and, incidentally, is the docking site for the ABI1

(Yoshida et al., 2006). Although the biological consequen-ces of this interaction between ABI1 and OST1 have not

been pursued in these studies, examples abound in the

animal field that have shown PP2Cs to be negative

regulators of AMP-activated kinases (Moore et al., 1991;

Hardie, 2007) (which are homologues of Sucrose Non-

Fermenting kinase or Snf1 of yeast, and Snf1-Related

Kinases or SnRKs of plants). Moreover, these kinases in

yeast and animals assume important roles as energy sensorsto switch off ATP-consuming anabolic pathways in times of

stress while switching on ATP-producing catabolic path-

ways (Hardie, 2007). Since ABI1 has been defined as

a negative regulator of ABA signalling by genetics and by

in vitro reconstitution assays (Sheen, 1998; Gosti et al.,

1999), combining with the obvious parallel with the animal

systems, it is almost certain that ABI1 negatively regulates

OST1. There seems to be, however, an additional surprising

layer of negative regulation of the OST1, as suggested by

the crystal structure of the yeast homologue Snf1. Duringthe earlier directed yeast two-hybrid test between OST1 and

certain heteronuclear RNA-binding proteins as potential

substrates (Riera et al., 2006), it had been found that OST1

itself can homodimerize. It has been possible subsequently

to confirm this dimer formation in living plant cells by

BiFC analysis as well (Fig. 2c), although the meaning of the

homodimerization in the functional context was not obvi-

ous at the time. The crystal structure of the kinase domainof the Snf1 had been determined recently and, surprisingly,

it revealed a novel dimer (Nayak et al., 2006). These Sfn1

dimers exist in cells as determined by co-immunoprecipita-

tion using antibodies that could selectively recover one or

the other epitope-tagged Snf1. The particular conformation

of the conserved aC helix harbouring phosphate-binding

residues, and the masking of the activation segment, as well

as the substrate binding site within the groove formed bythe dimer, suggest that dimerization is a new mechanism

that maintains the inactive state of the kinase. Strikingly,

the interface was formed by the most-conserved residues

of the Snf1/AMPK kinases, being predominantly in the

T-loop activation segment and the loop-aG region of the

kinase domain. This implies that dimer formation is

a conserved feature of the Snf1/AMPK kinases and could

extend to OST1.In plants, the SnRK2 kinases have been characterized in

terms of their biochemical activities in most detail in

Arabidopsis and rice (Hrabak et al., 2003; Boudsocq et al.,

2004; Kobayashi et al., 2004). These kinases phosphor-

ylate context-dependent serine and threonine residues, and

representative members from Arabidopsis (OST1/Srk2e,

SnRK2.2, 2.3, 2.8) and from rice (SAPK 8, 9, 10) that have

been tested can all phosphorylate peptides derived from theconstant (C) domain in the b-ZIP class of transcription

factors containing the motif Arg–3–X–2–X–1–pSer/pThr

(where the pSer/pThr denotes phosphorylated serine or

threonine arbitrarily fixed at position ‘0’ in the degenerate

peptide, and the positions of the other amino acids in the

motif are denoted with a ‘–’ relative to this serine/threonine)

(Kobayashi et al., 2005; Furihata et al., 2006; Chae et al.,

2007). Thus, based on the in vitro phosphorylation of thesepeptides, SnRK2 kinases are thought to target b-ZIP

proteins to regulate gene transcription (Kobayashi et al.,

2005; Furihata et al., 2006; Chae et al., 2007). Physical

interaction between homologues of an SnRK kinase and

b-ZIP proteins from wheat has also been detected in yeast

two-hybrid screens (Johnson et al., 2002). Some b-ZIP

transcription factors have been shown to bind with avidity

in vitro to the core ABA-Responsive Element (ABRE,PyACGTGGC), enriched in certain promoters up-regulated

by ABA.



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On the basis of quantitative phosphorylation of an array

of semi-degenerate peptides using SnRK2-10 as test kinase,

the consensus sequence Leu–5–X–4–Arg–3–X–2–X–1–pSer/

pThr was derived (Vlad et al., 2008). The importance of the

Arg–3 was therefore independently confirmed (Furihata

et al., 2006; Vlad et al., 2008); moreover, this kinase also

has preference for hydrophobic amino acids at the –5

position, particularly Leu. The importance of Leu–5 can bein fact retrospectively confirmed from the study by others:

this amino acid was consistently present in the peptides

derived from Arabidopsis and rice b-ZIP transcription

factors that were phosphorylated efficiently by the SnRK2

(Kobayashi et al., 2005; Furihata et al., 2006), and was

absent from the single test peptide derived from a so-called

C3 domain that was also inefficiently phosphorylated

(Furihata et al., 2006). An undeniable advantage conferredby the technology of phosphorylation of soluble peptide

arrays (Hutti et al., 2004) is that it allows the prediction of

preferred or disfavoured amino acids at the variable

(denoted as X in the motifs) positions based on their

quantitative influence on the phosphorylation of the central

serine/threonine. This in turn allows the generation of

theoretical peptide sequences, ranked in hierarchical prefer-

ences, which can be used to match against proteins annotedin a given genome. The dehydrins, in addition to b-ZIP

transcription factors, stand out as another major category

of potential direct targets (Vlad et al., 2008), whose

functions are often attributed to be in osmotic adjustments

by binding ions or protection of membranes during re-

sponse to environmental stresses (Puhakainen et al., 2004;

Kariola et al., 2006).

A proteomics approach had also been used to identifytargets of SnRK2.8 in planta (Shin et al., 2007) by

comparing the profiles of the phosphoproteins in transgenic

plants ectopically expressing SnRK2.8 with those of a corre-

sponding insertion mutant. These experiments targeted

phosphoproteins migrating between pI values of 3.9–5.1

and pI 4.7–5.9 representing ;70% of the total phosphopro-

teins set by these defined limits of isoelectric points. Nine

candidate target proteins were retained, and further testsrevealed that seven of these were phosphorylatable in vitro

by SnRK2.8. Among the seven candidates, three were 14-3-

3 isoforms, and, by comparing their sites of phosphoryla-

tion, the deduced motifs were DYRPSKVE on 14-3-3j and

DYRPSKIE on 14-3-3v (Shin et al., 2007). Note that

SnRK2.8 was the identical test kinase in two of the above

independent studies carried out by Furihata et al. (2006)

and Shin et al. (2007), and yet no conservation in the motifswas observed between the 14-3-3 proteins and those in the

b-ZIP transcription factors or dehydrins. Particularly in-

congruent is Asp rather than Arg at position –3 in the 14-3-

3 proteins, because Asp had been predicted to be a very

disfavoured residue for this type of kinase (Vlad et al.,

2008). Perhaps the sample sizes are still too small to draw

any conclusion concerning a ‘consensus’ or ‘preferred’

phosphorylation site of SnRK2 and, moreover, the possibil-ity cannot be excluded that the two different experimental

approaches may have cryptic technical biases.

Transcription factors in regulating guard cellmovements

Transcriptional control in stomatal movement is a burgeon-

ing field of inquiry into ABA signalling, which will be

a valuable complement to the wealth of knowledge already

gleaned from that on volume control of guard cell through

active membrane transport mechanisms. Gene expression

has been traditionally considered as a relatively late event in

the unfolding sequence of guard cell signalling but, in

nature, where guard cells are exposed to continual fluctua-

tions of micro-meteological changes, the processes of cell

volume control by membrane transport and transcriptional

events are probably much more tightly integrated. There is

indirect but nonetheless tantalizing evidence for changes

in RNA composition in the guard cells that might be link-

ed to stomatal movements in response to environmental

stimuli. In brief, up-regulation of mRNA, particularly one

encoding the AtPP2CA (see above) that can be detected in

Arabidopsis guard cell protoplasts upon treatment with

ABA (Leonhardt et al., 2004), and a number of mutations

affecting guard cell response to drought as part of

their pleiotropic affects are impaired in RNA metabolism

(Wasilewska et al., 2008).

We have already alluded to the prominent role of some b-

ZIP domain proteins in ABA signalling by their affinity

towards promoters containing the ABRE motif to trigger

transcription. Very recently, the activity of several MYB

transcription factors has also been shown to influence

stomatal movements in response to environmental stimuli.

The name MYB is attributed to their structural similarity to

cellular proto-oncogenes in animals (Lipsick, 1996). The

MYB family in Arabidopsis comprises 163 genes, making it

one of the largest transcription factor families (Chen et al.,

2006). Three in the so-called R2R3 subclass, which has

a membership of 126, have been shown to influence stomatal

movements in response to different environmental stimuli.

The diagnostic feature in this subclass of MYB transcription

factors is the two to three 50- to 53-amino-acid imperfect

repeats that form the helix-turn-helix motifs R1, R2, and R3

(Rosinsky and Atchley, 1998). In plants, two-repeat (R2-

R3) MYB family members predominate. The transcript of

AtMYB44 was shown to be rapidly induced (within 30 min)

by dehydration, low temperature, high salinity, exogenous

JA, ethylene, and ABA (Jung et al., 2008). Overexpression

of the gene led to an overall hypersensitivity to ABA and

a more rapid stomatal response to ABA than the wild type.

However, there were pleiotropic effects as well, such as

smaller stomatal structures, but no difference in stomatal

density was noted, suggesting that AtMYB44 plays a role in

development. These transgenic plants showed reduced

activation of several key negative regulators of ABA signal-

ling, including ABI1 and ABI2 by stress conditions, most

notably high salt, and conversely, the transcript levels of

these key regulators were enhanced in the knock-out

Atmyb44 mutant.

Two other R2R3–MYB transcription factors were found

to participate in light/dark sensing of guard cells, and these



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results have been reviewed recently (Gray, 2005). Again in

brief, the two transcription factors, AtMYB60 and

AtMYB61, play opposing roles in response to light. The

expression of AtMYB60 is environmentally regulated, being

reduced by darkness and ABA, which are signals that

stimulate stomatal closure. There was no absolute correla-

tion with expression levels of this gene with stomatal closure

per se because CO2, while effective in inducing stomatalclosure, has no effect on gene expression. By contrast,

AtMYB60 was up-regulated by light (which stimulates

stomatal opening) (Cominelli et al., 2005). The other,

AtMYB61, seemed to be expressed only in darkness (closed

stomata), and not at all in the light. Very interestingly, this

expression seemed ‘specific’ to darkness, as neither ABA

nor drought exerted any impact and, in accordance,

darkness-induced stomatal closure, but not that from ABA,was also impaired in the Atmyb61 insertion mutant (Liang

et al., 2005). These findings thus provide additional evidence

that transcriptional activity in the guard cell is linked to cell

volume control via membrane transport.

Concluding remarks

Guard cells and their attendant mechanisms to withstand

bouts of water deficit have no doubt contributed to the

successful terrestrial adaptation of plants from their former

aquatic confines. The appropriate and continual ajustment

of stomatal aperture by these cells in land plants are vitalfor optimal CO2 uptake necessary for photosynthesis, yet

preventing excessive water loss by transpiration. Engineer-

ing guard cells to be more sensitive to low humidity has

proven successful in reducing water consumption of a crop

plant without noticeable sacrifice in seed yield (Wang et al.,

2005). Because guard cells have no plasmadesmata, the

adaptive responses are thus cell-autonomous, rendering

them essentially a biological ‘test tube’. These properties,and the indissociable link between stomatal closure and low

humidity, make the stomatal complex an ideal system for

inquisitive probing into drought-adaptative mechanisms by

biophysics, genetics, and molecular biology. The membrane

voltage in guard cells can undergo large (>100 mV) and

rapid (10 s period) oscillations between hyperpolarized

and depolarized values allowing K+ uptake and release,

respectively (Gradmann et al., 1993; Gradmann andHoffstadt, 1998). This led to the hypothesis that the control

of stomatal movement and steady-state aperture depends on

variations in the pattern of these oscillations, enabling the

net guard cell K+ content to increase or decrease as a result

of changes in the balance of, respectively, K+ uptake

through hyperpolarization-activated, and K+ release

through depolarization-activated channels (Gradmann

et al., 1993; Gradmann and Hoffstadt, 1998). As summa-rized in Fig. 1b, in Arabidopsis guard cells, K+ uptake is

mainly through KAT1 and KAT2, while K+ release is

largely mediated by GORK. In the last two years, the

identification of a specific H+-ATPase (OST2) and an anion

channel (SLAC1) has also been witnessed. These newer

additions complete the major categories of plasma mem-

brane transporters, whose existence in the guard cell plasma

membrane had been inferred from biophysics for more than

a decade. The next obvious challenge is to work out the

mechanistic details on how these transporters are individ-

ually regulated, as well as how their activities are globally

coordinated to bring about rapid membrane hyper- and

de-polarization. For instance, the activation mechanismsof H+-ATPases by structural conversion from a dimer to

a hexamer remain unclear. Crystallographic data based on

PMA2 of tobacco showed that the C-terminal inhibitory

domain can be further divided into ‘R’ subdomains

(Ottmann et al., 2007). There are still many open questions

concerning the function of the RI subdomain, to which

some mutations have been mapped (Merlot et al., 2007). It

remains unclear how RI can influence the phosphorylationat the penultimate Thr in the RII subdomain. Ottmann

et al. (2007) speculated that RI influences inter-RII in-

teraction between the two H+-ATPase units in the dimer

(the inactive form) to facilitate hexamer formation (the

active form). It seems, beyond the well-established model

of intramolecular interaction between the C-termini and

the rest of the protein (Palmgren, 2001), there may be

other parallel activation mechanisms. It is also unclearhow the mutation ost2-1D, predicted to locate in the first

transmembrane domain (Merlot et al., 2007), could in-

fluence accessibility to the penultimate Thr for phosphor-

ylation. On the other hand, the exact deactivation

mechanisms of H+-ATPases are also not completely

known, with the exception of the PSK5 kinase which

phosphorylates Ser931 of AHA2 to block 14-3-3 protein

binding. Whether this is the critical amino acid that alsodirectly governs the structural conversion of the complex

from hexamer to dimer is not clear.

It is also likely that more refined genetics screens will

reveal other novel types of transporters (and their regu-

lators) as they are encoded by large families in Arabidopsis.

The few ABC transporter mutants that are affected in

stomatal function display pleiotropic phenotypes and this

fact may reflect the possibility that each transporter iscapable of transporting a large spectrum of substances,

which is the hallmark property that contrasts them with

selective channels. Pinpointing the precise substance(s)

transported by each of individual ABC protein would thus

be an arduous task. Furthermore, these substances could

range from simple organic and inorganic ions to complex

macromolecules like lipids, or even nucleic acids. For

example, in Caenorhabditis elegans, 10 of the 60 ABCtransporters are required to propagate gene silencing by

RNA interference, suggesting that they may transport RNA

(Timmons, 2007). Even identification of the transported

substance(s) does not automatically give clues about their

specific physiological functions in a cellular context. Many

of the ABC proteins are said to be bifunctional; that is,

independent of the substance(s) transported, ABC proteins

can also impose specific regulatory functions, most likely bycomplexing with other structurally diverse proteins in the

membrane (Higgins, 1992).



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A partial reversion of the mutant phenotype of abi1-1 by

the presence of the pp2ac-2 mutation would strongly imply

that the overall and absolute amount of catalytic activities

contributed by members of these two protein phosphatase

families must be tightly balanced to down-regulate ABA

signalling properly. Also, although the immediate down-

stream cognate targets of the PP2Ac-2 and the PP2Cs might

be distinct, the genetic observations do suggest that theircorresponding sub-branches of the signalling pathways are

most likely to later converge. The fact that ABI1 exerts its

important physiological functions relevant to ABA signal-

ling in the nucleus suggests that the post-translational

modification in the nucleus is different from that in the

cytoplasm. One such modification would render the protein

a selective target of ubiquitin-mediated proteolysis. The

nuclear localization of ABI1 has been interpreted to meanthat transcription is required for the observable phenotypic

events, including stomatal closure to limit transpiration

evoked by ABA (Moes et al., 2008). Indeed, there had been

tantalizing evidence in the past that transcriptional activity

in the guard cell is important for volume control. For

example, the activities of the K+in channels are curtailed by

ABA, and this is also paralleled by the down-regulation of

the KAT1 transcript level (Leonhardt et al., 2004). Manytranscripts in guard cell protoplasts are also stimulated by

ABA, and are driven by promoters enriched in the ABRE

motif. Besides the b-ZIP domain transcription factors,

which have been known for almost 20 years to be involved

in ABA-mediated gene transcription (Guiltinan et al., 1990),

some members of the R2R3 MYB class have now surfaced.

How membrane transport and nuclear events are wedded to

execute stomatal responses to a variety of environmentalconstraints is not obvious, and this problem will have to be

solved if we are to procure a more integrated vision.

Membrane transport mechanisms and transcriptional events

in the guard cell have been treated traditionally as distinct

temporal events. For instance, the stimulatory effects on ion

transport in the plasma membrane of the guard cell that

lead ultimately to stomatal closure can be detected within

seconds upon ABA application (Levchenko et al., 2005).These rapid physiological responses at the plasma mem-

brane are thus unlikely to be a direct consequence of the

presumably time-consuming transcriptional events. A guard

cell in a laboratory setting, however, is invariably chal-

lenged by such sudden and high doses of applied hormones

(or other pharmaceutical agents). In a natural setting, the

guard cell is rather continually exposed to micro-meteolog-

ical fluctuations in CO2, light intensities, and ambianttemperatures. Gene transcription, mRNA translation, and

ion transport across endo- and plasma membranes are most

likely to be integrated events, at least in time if not in space.

Acknowledgements

The authors appreciate the thoughtful comments from the

two anonymous reviewers. We are also grateful to Alejan-

dro Ferrando Monleon for the unconditional use of his

unpublished vectors and sound advice in the BiFC experi-

ments, and Marie-Noelle Soler of the Imaging and Cell

Biology facility of the IFR87 for expert support with

confocal microscopy. Work carried out in the authors’

laboratory was generously funded by the Ministere de la

Recherche Doctoral Fellowship (CS, Universite Paris XI),

European Union Marie-Curie Early Stage Training Fellow-

ships MEST-CT-2005-020232-2 ADONIS (AW and FV),Genoplante GNP05037G ABRUPT (JL), and the Centre

National de la Recherche Scientifique (JL and CV).

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