Joshi-saha2011_A Brand New START_Abscisic Acid Perception and Transduction in the Guard Cell

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(201), re4. [DOI: 10.1126/scisignal.2002164]4Science Signaling Archana Joshi-Saha, Christiane Valon and Jeffrey Leung (29 November 2011)Guard Cell

A Brand New START: Abscisic Acid Perception and Transduction in the`

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www.SCIENCESIGNALING.org 29 November 2011 Vol 4 Issue 201 re4 1

IntroductionLand plants, being rooted in place, must

sense and adapt to their incessantly fluctuat-

ing surroundings. At one time or another, wehave all noticed a plant neglected in a shady

corner exhibiting heliotropism: leaning out

and orienting its leaves perpendicularly to-

ward the Sun’s rays to maximize photosyn-

thesis. In contrast to laboratory settings,

plants in heterogeneous field conditions

must continuously cope with the constraints

in their environments to optimize growth.

In a given day, the plant will have endured

transient and local differences in light qual-

ity and intensity, fluctuations in temperature,

humidity, CO2, and possibly uneven water

distribution in the soil. Carbon dioxide and

water are among the most important ingre-dients contributing to plant biomass [6CO2

+ 6H2O + light → sugar + 6O2]; in return,

plants recycle CO2 and H2O, with O2 release

as a photosynthetic by-product (1). Because

land plants are protected by a waxy layer,

transpirational water loss and gas exchange

with the surrounding atmosphere are pos-sible almost exclusively through stomatal

pores (Fig. 1A). In most plants, these pores

are flanked by a pair of kidney-shaped guard

cells, whose volumes can expand or contract

by changes in turgor pressure to control the

opening and closing of the pore, respective-

ly (Fig. 1B). Sunlight stimulates stomatal

opening to allow diffusion of atmospheric

CO2 to photosynthetic tissues. This, how-

ever, exacts a trade-off in water loss through

transpiration, which will compromise growth

(2). Stomates also close in response to el-

evated CO2, the cause of global climate

warming, and to high concentrations of pol-lutants, such as ozone (O3), presumably to

prevent oxidative damage (3, 4). Guard cells

are, therefore, multisensorial and integrate

diverse cues in the leaf environment with

endogenous growth signals to optimize the

plant’s conflicting needs.

The turgor pressure within the guard

cells is modulated by the dynamic changes

in intracellular concentrations of inorganic

and organic ions (K +, Cl – , NO3 – , malate) and

sugars. Depending on the nature of the in-

put stimuli, coordinated ionic fluxes across

membranous compartments of the guard

cell will be assured by teams of different

channels and transporters. The electrical

signals generated by ion fluxes across the

plasma membrane are then converted bythe cell into chemical messages to shape the

final physiological output (in this case, the

binary decision of stomatal opening or clos-

ing). Because the guard cell is accessible

to studies by pharmacological approaches,

genetics, and molecular biology, it serves

as an excellent higher plant cell model for

unraveling signal integration between the

environment and endogenous growth fac-

tors. Furthermore, understanding the major

mechanistic aspects of abscisic acid (ABA)

signaling, which promotes stomatal closure

in guard cells, will not be unique to this cell

type but can provide a base to extend toother plant tissues or organs that respond to

this hormone.

A Retrospective on Abscisic AcidSignaling in the Guard Cell—TheCircuitry of Ion FluxesWater deficit stimulates the synthesis and,

to a much lesser extent (~5%), release from

storage of the hormone ABA to promote sto-

matal closure. The early physiological intri-

cacies of ABA signaling in guard cells were

teased out largely by pharmacological and

biophysical approaches that provided the

first sketch of the circuitry (Fig. 1B). Thesestudies showed that an early detectable event

triggered by ABA is the production of reac-

tive oxygen species [(ROS), sometimes also

known as oxidative burst], which stimulates

Ca2+ release from internal stores and influx

across the plasma membrane (5). The Ca2+-

dependent release of anions (often simply

referred to as Cl – in the early days) into the

apoplast, which is formed by the continuum

of cell walls of adjacent cells as well as the

extracellular spaces, causes depolarization

of the plasma membrane (6 – 10). At the

same time, Ca2+ also prevents membrane

hyperpolarization by inhibiting H+ –ad-enosine triphosphatases [(ATPases), proton

pumps coupled to ATP hydrolysis] required

to drive stomatal opening (11). Two distinct

types of anion efflux currents are detectable

in the guard cell, designated as slow (S) or

rapid (R) (12, 13). The S-type current is car-

ried by a range of anions that include NO 3 – ,

Cl – , and malate (14, 15). It was proposed

that the S-type, and not the R-type, current

was responsible for ABA-mediated stomatal

P L A N T B I O L O G Y

A Brand New START: Abscisic AcidPerception and Transduction in the

Guard CellArchana Joshi-Saha,1 Christiane Valon,1 Jeffrey Leung1*

*Corresponding author. E-mail, [email protected]

1Institut des Sciences du Végétal, Centre Na-tional de la Recherche Scientifique, Unité Pro-pre de Recherche 2355, 1 Avenue de la Terrasse,Bâtiment 23, 91198 Gif-sur-Yvette, France.

The soluble receptors of abscisic acid (ABA) have been identified in Arabidopsisthaliana . The 14 proteins in this family, bearing the double name of PYRABACTINRESISTANCE/PYRABACTIN-LIKE (PYR/PYL) or REGULATORY COMPONENTSOF ABA RECEPTOR (RCAR) (collectively referred to as PYR/PYL/RCAR), con-tain between 150 and 200 amino acids with homology to the steroidogenic acuteregulatory-related lipid transfer (START) protein. Structural studies of these re-ceptors have provided rich insights into the early mechanisms of ABA signaling.The binding of ABA to PYR/PYL/RCAR triggers the pathway by inducing struc-tural changes in the receptors that allows them to sequester members of theclade A negative regulating protein phosphatase 2Cs (PP2Cs). This liberates theclass III ABA-activated Snf1-related kinases (SnRK2s) to phosphorylate varioustargets. In guard cells, a specific SnRK2, OPEN STOMATA 1 (OST), stimulatesH2O2 production by NADPH oxidase respiratory burst oxidase protein F and in-hibits potassium ion influx by the inward-rectifying channel KAT1. OST1, the ki-nase CPK23, the calcium-dependent kinase CPK21, and the counteracting PP2Csmodulate the slow anion channel SLAC1, a pathway that contributes to stomatalresponses to diverse stimuli, including ABA and carbon dioxide. A minimal ABAresponse pathway that leads to activation of the SLAC1 homolog, SLAH3, andpresumably stomatal closure has been reconstituted in vitro. The identificationof the soluble receptors and core components of the ABA signaling pathway pro-vides promising targets for crop design with higher resilience to water deficitwhile maintaining biomass.


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closure. However, it was not clear at the time

whether the two types of anion currents oc-

curred through distinct channels or through

modification of the same channel. ABA alsoinduces the net efflux of both K + and Cl –

from the vacuole, which occupies 90% of

the guard cell volume, to the cytoplasm and

from the cytoplasm to outside of the guard

cell. At least one other signaling branch of

the pathway is independent of Ca2+, instead

requiring cytoplasmic alkalinization as an

intermediate (6 ). This pH-sensitive signal-

ing branch modulates K + efflux through

outward-rectifying channels.

In the early 1990s, indirect evidence

was obtained for ABA perception sites on

the “outside” (cell surface), as well as on

the “inside” of the guard cell. Stomatal clo-sure can be evoked by exogenously applied

ABA, hinting at an “outside” perception

site; however, the protonated form of the

weak acid ABA can readily permeate the

lipid bilayer of the cell membrane, which

could still suggest the possibility of cyto-

solic receptors. Furthermore, the guard cells

of Commelina communis have substantial

carrier-mediated uptake of ABA (16 , 17 ),

which could deliver externally applied ABA

to intracellular reception sites. The require-

ment for an extracellular receptor was sug-

gested by the ability of ABA at high pH,

when it is charged and can no longer crossthe plasma membrane, to induce stomatal

closure in Valerianella locusta (18) and the

failure of ABA to inhibit stomatal opening

when microinjected directly in the cytosol

of Commelina guard cells (19). Externally

applied ABA to barley aleurone protoplasts

(single cells without the cell wall derived

from barley ovules) reversed the stimulation

of α-amylase synthesis by gibberellic acid,

whereas microinjecting up to 250 µM ABA

was ineffective (20). A cell surface–local-

ized receptor was also concordant with K +

fluxes (21) and reporter gene expression in

either Arabidopsis or rice cell cultures thatwere stimulated by ABA coupled to carri-

ers that could not penetrate membranes (21,

22). Immunolocalization of presumptive ex-

tracellular ABA binding sites has also been

reported (23). On the other hand, there was

accumulating evidence for internal ABA

reception sites. In Vicia faba, externally ap-

plied ABA was not effective in maintaining

slow anion channel current in ATP-depleted

V. faba guard cell protoplasts (24). In Com-

melina, extracellular ABA was compara-

tively less effective in regulating stomatal

aperture at higher versus lower pH, the latter

of which favors uptake by passive diffusionof the protonated form of ABA, providing

indirect evidence for an intracellular recep-

tor (17 , 19, 25, 26 ). The presence of an in-

tracellular receptor was also supported by

reports that stomatal closure was triggered

in Commelina by release of caged ABA in

the cytosol of guard cells (27 ) and by ABA

directly microinjected into guard cells (17 ).

The Essential Components of theCore Signaling Complex: SolubleReceptor, Protein Phosphatase 2C,and the Kinase SnRK2

To dissect the underlying mechanisms bywhich ABA rapidly causes stomatal clos-

ing, guard cell signaling came under joint

assault in the early 1990s by genetics and

molecular biology. Several putative and

somewhat controversial ABA receptors

have been proposed intermittently since the

first report more than 25 years ago of bind-

ing proteins in the plasmalemma of V. faba

guard cells (28, 29). After many candidates

that have been described as false starts (30),

Fig. 1. Biophysics of stomatal movement. (A) One-week-old Ara- bidopsis rosette leaf is shown with an image of a single stomate.The microscopic pores contoured by the two flanking guard cellsdefine a stomatal opening. The fluorescent round structures arechloroplasts. (B) Stomatal opening (left) and closing (right). Increas-ing turgor pressure inside the cells causes the two cells to swelland bow out from each other, resulting in the opening of the pore.Stomatal opening requires hyperpolarization of the plasma mem-brane and entry of K+. ABA accumulates in response to droughtand fosters stomatal closing. The earliest detectable signal is thepresence of reactive oxygen species (H2O2) and then a transient

increase in Ca2+. A second signaling intermediate is sensitive to pHand stimulates K+ efflux through K+-outward rectifying channels. Be-cause 90% of the volume of the guard cell is the vacuole, the effluxof ions must first cross the vacuolar membrane (pale green oblongstructure), then the plasma membrane, and finally move into theapoplastic space. For simplicity, the left cell shows the plasma mem-brane proteins that are active during stomatal opening, and the rightcell shows the plasma membrane proteins that are active during sto-matal closure. Red, H+-ATPase; yellow, K+ inward-rectifying channel;light blue, Ca2+ permeant channel; dark blue, anion channels; lightgreen, K+ outward-rectifying channel.

Ca2+

A B

Low humidity

ABA

High CO2

Darkness

ABA

ABA

Fusicoccin

Light

High humidity

Low CO2

Light / Low CO2

A-

Depolarization

K +

K +

H+

Hyperpolarization

H2O2

H2O

2

pH

C R E D I T S : ( A ) J E F F R E Y L E U N G ; ( B ) Y . H A M M O N D / S C I E N C E S I G N A L I N G


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those with properties matching

the required physiological and

molecular profiles were identi-

fied in 2009. This momentous

discovery, celebrated as one of

the top 10 discoveries of the

year (31), represents an awaken-ing in our understanding of the

initial ABA signaling events.

This soluble receptor family

has 14 members in Arabidopsis

thaliana, and this high degree

of functional redundancy may

have cloaked their identity from

being revealed by standard ge-

netic screens (at least for the

loss-of-function category of mu-

tations). However, the discovery

that the synthetic compound,

4-bromo-N-[pyridin-2-yl meth-

yl]naphthalene-1-sulfonamide, known as pyrabactin (Fig. 2), partially mimicked

the inhibitory effects of ABA on seed ger-

mination, early seedling growth, and gene

expression program (32), presumably by

binding and modifying the activities of sev-

eral of the soluble ABA receptors simulta-

neously, allowed Park et al . to side-step the

functional redundancy barrier. By selecting

for mutagenized seeds that germinated in

the presence of inhibitory concentrations

of pyrabactin, followed by map-based clon-

ing of one such locus named PYRABACTIN

RESISTANCE 1 ( PYR1), they succeeded in

isolating a gene encoding a homolog to thesteroidogenic acute regulatory lipid trans-

fer (START) proteins. These proteins are

characterized by a structural scaffold that

can accommodate a large spectrum of hy-

drophobic ligands, such as lipids, antibiot-

ics, and hormones (33). Importantly, PYR1

interacted in a yeast two-hybrid screen, in

an ABA-dependent manner, with several

phosphatases of the protein phosphatase 2C

(PP2C) family (namely, ABI1, ABI2, and

HAB1) that had previously been established

as key negative regulators of the ABA sig-

naling cascade (34 – 40). Moreover, the pro-

tein-protein interaction pattern established by the yeast two-hybrid system related to

the phenotypes of the respective mutant

plants. For example, the loss-of-function

mutations Pro88 to Ser 88 (P88S) and Ser 152

to Leu152 (S152L) in PYR1 that confer the

receptor’s property of pyrabactin resistance

and, conversely, the gain-of-function Gly168

to Asp168 (G168D) mutation in ABI2, which

defined this negative regulator and confers

ABA resistance in the mutant plants (abi2-

1), all reduced the interaction between thereceptor and PP2C. Independently, Ma et al .

isolated a protein named REGULATORY

COMPONENT OF ABA RECEPTOR 1

(RCAR1), as a partner of ABI1 and ABI2

(41). The mutation G168D in ABI2 (abi2-1)

or the equivalent mutation G180D in ABI1

(abi1-1) also abolished its interactions with

RCAR1 (also known as PYL9).

The crystal structures of several recep-

tors were rapidly accomplished, and fine

structural details were obtained in quick suc-

cession for (i) ABA-bound, (ii) pyrabactin-

bound, (iii) and ligand-free (PYR1, PYL1,

PYL2) apo-receptors, as well as (iv) thetertiary complex ABA-PYL/RCAR-PP2C.

These structural studies have uncovered

a wealth of mechanistic insights into the

early events of ABA signaling (42 – 50). The

receptor protein has a central cradle that is

formed by the alignment of seven-stranded

antiparallel β sheets wrapped around by along α helix from the C-terminal end of the

protein. The bottom of the cradle is created

by two other α helices situated between thefirst and second β sheets. The ABA moleculeis held inside the cavity by a combination

of nonpolar and polar interactions. Among

the charged interactions, the carboxylgroup of the ABA is plunged deep within

the pocket, and it is in direct contact with

Lys59 of PYR1 (or Lys86 of PYL1 and Lys64

of PYL2) (Fig. 3, A, C, and D). This lysine

is conserved in the gene family, with the ex-

ception of PYL13 in which this residue is

occupied by a glutamine. The access to the

ABA molecule from outside the receptor is

controlled by two important structures: The

first is called the proline “gate” (with the

signature amino acid motif SGLPA; A, Ala),

which is conserved in all of the receptors

except, again, PYL13, in which the leucine

is replaced by phenylalanine. The second

functionally important domain is called the

leucine “latch” GG(E/D)HRL (where the

slash means “or”; E, Glu; H, His; R, Arg),again with PYL13 as the outgroup having

the E/D residue substituted by glutamine.

The cyclohexane ring of the ABA molecule

(Fig. 2A) extends toward the opening of the

binding cavity and stabilizes the gate in the

closed conformation by interactions with a

number of hydrophobic amino acids, which

are also conserved in all 14 receptor mem-

bers (51). This closing of the gate is further

secured by the positioning of the latch and

the extension of an α-helical loop (“recoil”region) (50). This recoil region encompass-

es 13 amino acids (Met147 to Phe159 in PYR,

which align with Val177

to Phe189

in PYL1 inFig. 3) that, after ABA binding, coil into the

C-terminal α helix of the receptor.In the absence of ABA, the receptor

(PYR as the model) exists as an asymmet-

ric dimer (50) with ~10° deviation from atwofold (180°) symmetry. These mono-meric subunits are held together through

bonds between their gates. The binding of

ABA leads to conformational changes in the

gate to allow the dimer to assume a perfect

twofold symmetry, resulting in a more com-

pact structure with a biconcave disc shape

resembling a red blood cell. Coimmunopre-

cipitation assays confirmed the existenceof dimer in vivo both with and without ex-

ogenous ABA. It seems that ABA binding

causes the dimer to dissociate into mono-

mers and each monomer then binds a PP2C

(52). Although the chemical structure of the

agonist pyrabactin is very different from

that of ABA (Fig. 2), pyrabactin binds (as

a folded structure resembling π) PYR1 andat least several other member receptors to

form a “productive” complex in which the

gate is closed. However, pyrabactin does not

activate PYL2; instead, it binds and forms

a “nonproductive” complex. Thus, this syn-

thetic compound could theoretically antago-nize activation of PYL2 by ABA (45, 49).

In the productive ABA receptor–pyrabactin

configurations [derived from the structural

studies of PYL1-pyrabactin (45), PYR1-

pyrabactin, and PYL1-pyrabactin-ABI1 (45,

49)], the orientation of the bound pyrabactin

provides the necessary van der Waals inter-

actions to induce gate closure. In contrast,

in the nonproductive mode [deduced from

the PYL2-pyrabactin structure (45, 49)], the

N

O OH

OH

OO=S=O

Br

NH

O=S=O

Br

NH

Fig. 2. ABA and pyrabactin used in classical and chemi-cal genetic screens, respectively, to identify signalingcomponents. (A) Chemical structure of ABA, highlightingits active carboxyl group that directly binds to a conservedLys residue (Lys86 in PYL1) deep in the pocket of the re-ceptors (except PYL13). (B) Structures of the syntheticchemical pyrabactin (left) and its analog apyrabactin(right). The pyradyl nitrogen (arrow) is important for thepyrabactin agonist effect, because apyrabactin is inactive.

C R E D I T : Y . H A M M O N D / S C I E N C E S I G N A L I N G


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relative orientation of the pyrabactin along

the length of the molecule is flipped by 180°

in PYL2. The pyrabactin in this inverted ori-

entation can no longer supply the necessary

van der Waals forces to maintain gate clo-

sure. Thus, the binding of the ligand (ABA

or pyrabactin) within the cavity is not itselfsufficient to trigger the downstream path-

way. These structural comparisons revealed

that the formation of a productive signaling

complex also depends on the ability of the

bound agonist to maintain the gate in the

closed conformation. The maintenance of a

closed gate and latch configuration is need-

ed to create a binding surface to tether and

inhibit the clade A PP2Cs.

Comparisons of the hormone-bound

receptor (ABA-PYL1) (44) to that of the

tertiary structures ABA-PYL1-ABI1 and

ABA-PYL2-HAB1 (48, 51, 53) revealed no

structural difference in the receptor moietyof the complex, suggesting that the recep-

tor is a rigid structure. An important insight

informed by the tertiary complex is that

amino acid Ser 112 of PYL1, or the equivalent

Ser 89 of PYL2, make contact with Glu142 and

Gly180 of ABI1 (Fig. 3). Thus, the Ser 112 or

Ser 89 of the receptor functions like a plug

and physically obstructs the entrance to the

catalytic site of the phosphatase. In light

of the data from structural (53), in vitro,

and yeast two-hybrid analyses (32, 41), the

G180D mutant abi1-1 and the analogous

G168D abi2-1, with their glycine residues

replaced by the bulkier and charged asparticacid, would permit escape from repression

by receptor binding during ABA signaling,

which would explain the dominant or con-

stitutive nature of these mutations. Again,

the structural data with these soluble recep-

tors and the PP2Cs fit those from the genetic

analysis of the mutants and molecular prop-

erties of their proteins.

The dissociation constants ( K d ) of four

representative receptors (expressed as re-

combinant proteins in bacteria) are unex-

pectedly high, near or in the micromolar

range. However, their affinity for ABA in-

creases to the nanomolar range when com- patible PP2Cs are present (Table 1). This

increase in receptor affinity for ABA in the

presence of PP2C was also reflected by in

vitro assays of the inhibition of protein phos-

phatase activity by the receptors, which was

sensitive to the ratios of the two components

as well as the particular homolog of the clade

A PP2C (Table 2). The efficiency of ABA-

mediated inhibition of phosphatase activity

in vitro was generally higher for ABI1 than

for ABI2, and in terms of the receptors, ABA

was more effective with RCAR3 than with

RCAR1. For example, at the ratio of one

PP2C to four receptor molecules, the me-

dian inhibitory concentration (IC50) of either

ABI1 or ABI2 by RCAR3 was between 15

to 40 nM ABA; in comparison, RCAR1/ABI2 revealed a two- to threefold higher

IC50 value of roughly 60 to 95 nM ABA (Ta-

ble 2) (47 , 54). These observations suggest

that the combination of particular RCARs

and PP2Cs behaves as a coreceptor complex

for ABA (although PP2Cs are not widely

known to bind ABA, as would be expected

by a classical coreceptor) and that together,

different receptor-PP2C combinations might

activate the drought adaptive response path-

ways differently (54). The mechanistic basis

of this enhanced ABA sensitivity displayed

by the receptors in the presence of PP2Cs is

not obvious, because ABA is cloistered deepwithin the cavity of the receptor. However,

Trp300 of ABI1 (or Trp385 of HAB1), some-

times referred to as the Trp lock (42), plays

a unique structural role in the receptor-PP2C

complex. It is the only amino acid residue in

the phosphatase that bridges indirectly with

the ABA molecule and the receptor simul-

taneously through a combination of water-

mediated and hydrophobic interactions,

respectively (Fig. 3). Mutational analysis

showed that this tryptophan is not essential

for phosphatase activity, but only its affin-

ity with the receptor and, as a consequence,

ABA-dependent inhibition of ABI1 (53) orHAB1 (55) is affected when this residue

is mutated. Conformational changes in the

receptor induced by its interaction with this

key tryptophan residue facilitate the fasten-

ing of the receptor’s gate and latch into the

closed configuration. Whether this could

provide a structural rationale for the more

than 10-fold increase in ABA binding affini-

ty observed for the PYL-PP2C complexes as

compared with the apo-receptors still needs

to be confirmed (39, 41, 44, 51, 53, 54).

Several research groups have indepen-

dently, and by different experimental ap-

proaches, identified an ABA-activated andcalcium-independent kinase in wheat (56 ),

the broad bean V. faba (57 , 58), and its or-

tholog in Arabidopsis (59, 60) that acts as

a positive regulator of this stomatal closing

pathway. It is this kinase that is muted by the

PP2Cs when the ABA signaling pathway

is off. The ABA-ACTIVATED PROTEIN

KINASE was purified biochemically from

V. faba guard cells. When a catalytically

dead variant of the kinase was expressed

transiently in wild-type (WT) Vicia guard

cells, ABA-mediated activation of anion

channels required to close stomates was

abolished (58). Likewise, mutations in the

homologous kinase SnRK2 in Arabidop-

sis, known variously as OPEN STOMATA

1 (OST1), SRK2E, and SnRK2.6 (59, 60),also blocked the typical stomatal closing re-

sponse to ABA and to progressive drought.

Yoshida et al . demonstrated a direct interac-

tion between ABI1 and OST1 by the yeast

two-hybrid approach. Further, they delineat-

ed a small amino acid motif, called domain

II, at the noncatalytic C terminus of OST1

as the direct docking site for ABI1 (61).

This domain II is also found in the C termini

of SnRK2.2/2D and SnRK2.3/2I, two other

closely related ABA-activated SnRK2s in

the same clade as OST1 (60, 62). Of the 10

members in the entire family, these three

SnRK2s seem to regulate all of the knownelementary ABA responses (63 – 65). Sev-

eral serines within the activation loop of

OST1 become phosphorylated in vivo in

response to ABA (66 ). In vitro, ABI1 and its

mutant counterpart abi1-1 dephosphoryl-

ated the ABA-stimulated OST1 recovered

from cell extracts (65, 66 ). Also, relative to

WT plants, the ABA-activated kinase ac-

tivity from plant extracts was lower in the

dominant gain-of-function PP2C mutants

(for example, abi1-1); conversely, it was

higher in the PP2C loss-of-function mutants

(65, 66 ), consistent with the notion that in

vivo the three kinases are negatively regu-lated by these PP2Cs. Finally, OST1 (66 ),

SnRK2.2, and SnRK2.3, along with 9 of the

14 members of the soluble receptors (67 ),

coimmunoprecipitate with ABI1 in Arabi-

dopsis protein extracts. The composition of

the copurified proteins did not change re-

gardless of whether or not exogneous ABA

was added. This suggests that at least ABI1,

the three ABA-activated SnRK2s, and at

least nine members of the soluble receptors

might be stable constituents of a core ABA

signalosome (67 , 68). Because whole plants

were used in the coimmunoprecipitation

studies, all components may not be part ofthe same signalosome simultaneously, but

different combinations of the constituents

could exist in different tissues.

Regulation of Ion Transport Acrossthe Plasma Membrane by the ABACore Signaling ComplexRelative to its closest homologs SnRK2.2

and SnRK2.3, mutations in the OST1 locus

have the most negative impact on guard cell


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response to environmental stress (59, 60,

69, 70). OST1 may thus be the key guard

cell kinase regulating a large roster of tar-

gets. A substantial fraction of the transcrip-

tome responsive to ABA is regulated by

b-ZIP transcription factors (71), which are

potentially activated by OST1 (72, 73). The

minimal pathway activated by ABA, which

presumably leads to altered gene expression

in vivo, has been reconsituted in vitro. The

components consist of PYR1, ABI1, and

OST1, which produced the ABA-dependent

phosphorylation of the b-ZIP transcription

factor ABA RESPONSIVE ELEMENT

BINDING FACTOR 2 (ABF2) (more cor-

Fig. 3. The mechanistic basis of ABA-mediated inhibition of PP2Cactivity. (A) Ribbon models of superimposed apo-PYL2 (gray) andABA-bound PYL2 (green) [generated using Protein Data Bank(PDB) accession codes 3KAZ and 3KBO (44 )]. The orientation of

ABA (ball model) in the ligand binding pocket is shown. Magenta,apo-latch; blue, ABA-bound latch; pink, apo-gate; yellow, ABA-boundgate. (B) The PYL1-ABA-ABI1 tertiary complex [modified with per-mission from (53 )]. The Trp lock of ABI1 is shown as yellow spacefill.(Right) Close-up view of the intermolecular interactions that explainreceptor sequestration of PP2C upon ABA binding. (C) A general-ized scheme of the receptor-ABA-PP2C complex highlighting the es-sential serine residue (Ser112 in PYL1) in the receptor that tethers thePP2Cs by interacting with a Glu (Glu142 in ABI1) and a Gly residue(Gly180 in ABI1). A conserved Trp in the PP2Cs (Trp300 in ABI1) inter-acts with the ABA through a water molecule (blue dot). The carboxylgroup of ABA is in contact with a Lys residue (Lys86 in PYL1) deep in

the pocket. N, N terminus. (D) In PYL1, amino acids participating inABA binding are underlined in black (86, 171, 116 to 121, and 143 to149). The START homology spans from amino acids 50 to 206. Reddots denote amino acids that dock onto the catalytic regions of ABI1.

Residues underlined in blue denote α-helical structures (aminoacids 34 to 47, 69 to 77, 82 to 84, and 183 to 208); orange underlinesindicate β strands (57 to 67, 89 to 93, 105 to 110, 117 to 122, 135to 137, and 148 to 175); and the red underline denotes the helicalturn at amino acids 128 to 131 (127 ). Various mutant alleles of thePYR1 locus have been transposed onto the equivalent amino acidsof PYL1. (E) ABI1; blue residues represent the catalytic region. Yel-low dots mark the amino acids that, when mutated, decreased PYL1binding. Orange overlines indicate E142, G180 (abi1-1), and W300(Trp lock), which define the entrance to the ABI1 catalytic center inthe crystal structure. Mutations around W300 show an eight–aminoacid insert specific to plant PP2Cs (42 ).

ABA

PYL1

Gate pyr1-9 pyr1-3

Latch pyr1-6 pyr1-5 pyr1-8

1-4 pyr1-2

ABI1

ABI1

PYL1ABA receptor

Clade A PP2C

Apo-latch

Apo-gate

Latch

Latch

Recoil

Recoil

ABA-bound

gate

ABA

Glu142

Gly180

Ser112

gate

Trp300

GluGly

Trp

Catalytic

center

Lys

SerGateLatch

N

N

C R E D I T S : ( A ) Y . H A M M O N D / S C I E N C E S I G N A L I N G ; ( B ) M O D I F I E D W I T H P E R M I S S I O N F R O M N A T U R E 4 6 2 , 6 0 9 – 6 1 4 ( 2 0 0 9 )


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rectly, a ~80–amino acid protein fragment)

to represent the transcriptional output of

this minimal pathway (68).OST1 also modifies membrane trans-

port properties in the guard cells by phos-

phorylating and inactivating one of the ma-

jor potassium inward-rectifying channels,

KAT1, which was shown in vitro and in

the Xenopus oocyte heterologous expres-

sion system (Fig. 4) (74). The ensuing re-

duction in K + influx is consistent with the

known electrophysiological effect of ABA

on modifying guard cell membrane trans-

port (Fig. 1). ABA stimulates the produc-

tion of H2O2 (Fig. 1) through plasma mem-

brane–localized NADPH oxidases, which

are encoded by 10 genes in the Arabidopsis genome. Although H2O2 is similar in struc-

ture and chemical properties to H2O (75),

unlike water, it is a powerful oxidant, or

ROS. During signaling, the increases in

concentration of H2O2 must be maintained

above a certain threshold (estimated to be

between 10 to 100 µM)—long enough tooxidize its effector molecules, but not so

long as to cause cellular damage. Hence, to

curb rampant H2O2 cytotoxicity, the activi-

ties of the NADPH oxidases are thought to

be tightly regulated by numerous cytosolic

factors that include Ca2+, protein kinases,

and small guanosine triphosphatases (76 ).Kwak et al . (77 ) identified mutations in two

genes encoding NADPH oxidase catalytic

subunits, AtrbohD and AtrbohF (respira-

tory burst oxidase homolog D and F), that

abolished ABA-induced stomatal closure,

ABA-mediated promotion of ROS produc-

tion, and ABA-induced increase in cyto-

solic Ca2+. In vitro, OST1 phosphorylates

AtrbohF, but not AtrbohD (Fig. 4) (78),

which is also consistent with the lack of

ABA-mediated ROS production in the ost1

mutant guard cell (60).

The target of OST1 that has attracted mostof the attention is involved in the regulation

of anion efflux critical for ABA-mediated

stomatal closure. Anion efflux is controlled

by a balance between phosphorylation and

dephosphorylation events (79). Two groups

independently converged on the locus

SLOW ANION CHANNEL–ASSOCIATED

1 (SLAC1) as the gene encoding the most

likely S-type anion channel involved in sto-

matal closure (80, 81) (Fig. 4). Both groups

used impaired stomatal closure in response

to either high CO2 (80) or hypersensitiv-

ity to damage of photosynthetic tissues by

ozone (O3) (81) as the phenotypic criterionfor the mutant screen. The putative SLAC1

protein is a distant relative of bacterial and

fungal C4-dicarboxylate transporters and a

weak homolog (20% amino acid identity)

of Mae1 of Schizosaccharomyces pombe,

which has been functionally characterized

as a malate uptake transporter. Guard cell

protoplasts derived from slac1-2, compared

with those from the WT plants, contain

higher amounts of organic anions, notably

malate and fumarate, and the inorganic ions

Cl – and K +, possibly as an indirect conse-

quence of perturbed ionic homeostasis (80).

There are three related SLAC1 homologs(SLAH) in the Arabidopsis genome. Re-

verse transcriptase polymerase chain reac-

tion (RT-PCR) coupled to histochemical

analysis of the β-glucuronidase (GUS ) reporter under the control of the individual

SLAH promoters revealed that all of them

are expressed in various tissues besides

guard cells in plants grown under the specif-

ic conditions used for this analysis (80). De-

spite the differences in their tissue-specific

expression patterns, at least two of these are

functionally interchangeable in guard cells,

because ectopic expression of either SLAH1

and SLAH3 under the control of the SLAC1

guard cell–specific promoter complements

the phenotypes of CO2 insensitivity and ac-

cumulation of organic and inorganic ions of slac1-2 (80). The slac1 mutant is phenotypi-

cally pleiotropic: The guard cells are only

moderately sensitive to light and humidity,

and they exhibit a pronounced indifference

to ABA, NO, O3, and H2O2.

These phenotypes associated with slac1

mutant plants and the rescue by SLAH1 or

SLAH3 are important for several reasons.

The first is that they provide genetic evi-

dence that the CO2, O3, low humidity, and

ABA perception pathways are interconnect-

ed and that SLAC1 has an important role in

integrating these environmental and endog-

enous signals. Two of the transferred DNAinsertion mutant alleles of slac1 that were

studied by Saji et al . (82), which they called

ozs, are identical to slac1-3 and slac1-4. The

ozs mutants were initially isolated on the

basis of the appearance of necrotic lesions

on leaves when exposed to O3 (~200 parts

per billion or 0.2µl/l). At the stomatal level,however, the responses of the ozs mutant

plants to ABA, CO2, H2O2, and O3 measured

by Saji et al . were indistinguishable from

those of WT plants (82). The reasons for the

discrepant observations are unknown.

A second reason that the phenotypes of

the slac1 plants are biologically importantis that, in contrast to WT plants, the char-

acteristic slow and sustained anion current,

which is weakly voltage-dependent, is bare-

ly detectable in the guard cell protoplasts

derived from the slac1 mutant. Only very

weak background whole-cell membrane

currents and patch-clamp seal currents were

observed (81). Thus, SLAC1 most likely

corresponds to the long-sought-after anion

channel in the guard cell that is crucial for

ABA-mediated stomatal closure.

A final reason that the study of the slac1

plants was particularly important was that

it provided strong evidence that the R-typeand the S-type anion currents are produced

by different channel proteins. The R-type

anion current, which is transient rather than

sustained, is not affected in the slac1 mu-

tants (81), implying that the S- and R-type

anion channels are unlikely due to post-

translational modification of a single poly-

peptide, which is consistent with previous

suggestion based on physiological studies

(12, 83). AtALMT12, a homolog of the

Table 1. Dissociation constants of representative receptors in the presence and absenceof PP2Cs. The dissociation constants (extrapolation of ligand affinity to achieve half oc-cupancy of the receptor sites) were obtained by using receptors produced in Escherichiacoli and calculated by isothermal titration calorimetry (ITC) or surface plasmon resonance(SPR). In the presence of PP2Cs (ABI1, ABI2, HAB1), the four PYR/PYL/RCAR recep-tors display substantially higher hormone affinity. N/A, not applicable.

Receptors K d (µM) K d in the presence ofPP2C (nM)

Techniques for K d measurements

PYL9/RCAR1 0.7 64 (ABI2) ITC (41)

PYL5/RCAR8 1.1 38 (HAB1) ITC (39 )

PYR1/RCAR11 N/A 125 (0.8 HAB1)* ITC (32 )

PYL8/RCAR3 1.0 18 (0.25 ABI1)* ITC (54 )

PYL1/RCAR12 52 and 340 N/A ITC, SPR (53 )

*Values estimated from IC50 using the indicated relative ratios of PP2C to the receptor.


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aluminum-activated malate transporter, fulfills the physiochemicalcharacteristics of the R-type an-ion channel (84, 85). These chan-nels were first studied in the rootsand are thought to play a role in

releasing malate to chelate alumi-num in the rhizosphere (86 , 87 ).In mutants lacking the functionalAtALMT12, guard cells displayedreduced sensitivity to closingstimuli, such as the transition oflight to darkness, high concen-trations of CO2, and ABA. Thecharacteristic voltage-dependentR-type current was reduced by~40% in the mutant protoplastscompared with those preparedfrom WT plants. The gating of theR-channel is sensitive to malate.

This malate-sensitivity was alsoobserved for AtALMT12 when itwas heterologously expressed andcharacterized in oocytes (85), sug-gesting that this property may beinherent to the transporter itself,unless Xenopus has conservedthe same regulatory machinery. Itis currently not known how ABAregulates AtALMT12.

Guided by the structural stud-ies on the anion channel TehA, theSLAC1 homolog from the bac-terium Haemophilus influenzae,

SLAC1 would be a symmetricaltrimer composed of quasisym-metrical subunits, each having 10transmembrane helices arranged in pairsto form a central five-helix transmembrane

pore (88). The pore is a relatively uniform passage of 5 Å in diameter lined with largelyhydrophobic residues, except for a constric-tion that is gated by an conserved phenylala-nine residue (Phe450). SLAC1 does not seemto have discrete anion binding sites in thechannel, compared, for example, with thoseof the CLC family of channels, which havediscrete ion binding sites with high field

strength. The ion selectivity of SLAC1 isthought to be largely a function of the ener-getic cost of ion dehydration and thus repre-sents a unique pore structure for anion chan-nels. Despite the overall hydrophobicity ofthe ion-conducting tube, the electrostatic po-tential on the pore surface is polarized, andin particular, the electropositive nature of itscytoplasmic surface is thought to contributeto anion efflux.

When the channel is heterologously ex-

pressed in the Xenopus oocyte system, theactivity of SLAC1 was detected only whencoexpressed with any of the following threekinases: OST1 (89), CPK23, or CPK21(90). The major site of phosphorylation byOST1 is Ser 120 in the N-terminal cytosolicdomain of SLAC1 (4, 89, 91). This N-ter-minal cytosolic domain of SLAC1 is phos-

phorylated by the CPKs at other unspecifiedmotifs. There are several other SLAC1 sitesthat are phosphorylated in vitro by OST1,

and whether they have in vivo relevanceis not clear (4). In Xenopus, the activatedSLAC1 displays higher permeability to

NO3 – compared with Cl – and malate (89).

The mutant slac1 can be complemented bythe ectopic expression of either SLAH1 orSLAH3 driven by the guard cell–specific

promoter of SLAC1. However, SLAH1 doesnot contain any extended N- or C-terminalcytosolic domains that could constitutethe targets of phosphorylation. Thus, the

molecular consequences of phosphorylation on the overallSLAC1 structure are not im-mediately obvious.

The protein phosphatasesABI1, ABI2 (89, 90), and

PP2CA (91) block SLAC1-mediated anion efflux in the Xenopus expression system.The other homologous pro-tein phosphatases, such asHAB1 and HAB2, were lesseffective (90), at least in the

Xenopus system. Neither theWT catalytic activity of the ki-nase OST1 (89) nor that of the

phosphatase AtPP2CA (91)is required for these proteinsto interact. An inactive formof AtPP2CA blocked phos-

phorylation of SLAC1 by WTOST1 (91), indicating that theactivity of OST1 can be inhib-ited by physical entrapmentin addition to dephosphoryla-tion by the PP2Cs. ABI1 andABI2 interact with CPK21and CPK23, which was detect-ed with bimolecular fluores-cence complementation whentagged forms of these partnerkinase-phosphatase pairs werecoexpressed in the Xenopus oocytes (90) or in mesophyll

cells (92). Although CPK23can phosphorylate SLAC1 inheterologous systems, such

as Xenopus oocytes, and although the an-ion current is reduced (by 70%) in guardcell protoplasts derived from the knockoutcpk23, its functional importance, if any, in

planta is not clear (90). Despite the reducedanion current, no stoma phenotype wasnoted in the cpk23 knockout mutant in thesestudies (90). The opposite phenotypes ofreduced and increased stomatal apertures,respectively, were observed by others in theknockout mutant and in plants overexpress-

ing AtCPK23 (93). CPK21 functions as anegative regulator of abiotic stress responses

because the cpk21 knockout is more toler-ant, rather than having the expected height-ened sensitivity, to prolonged osmotic stressas compared with WT plants (94). Theseapparently contradictory results suggest thatin the guard cell, there may be other targetsof these CPKs missing in the oocyte assaysin which only single targets were tested.There are also two other calcium-dependent

Table 2. Receptor affinity to ABA depends on the presence of proteinphosphatases and their relative ratios. The IC50 values indicate theconcentration of ABA required to cause 50% inhibition of the PP2Cactivity by the receptor. The ratio of PP2C to receptor has a pro-nounced impact on IC50, which is commensurate with the amount ofinput PP2C. Because of this, the PP2Cs have been regarded by someas coreceptors of ABA. Note that PYR1 with 0.6 HAB1 yielded an IC50

of 390 nM, whereas a value of 125 nM was obtained in Table 1 using0.8 HAB1. These variable results for the same combination of PP2Cand receptors could be due to the different experimental conditions.

Receptors Ratio PP2C:receptor IC50 (nM) Reference

PYL9/RCAR1 0.25 ABI1

0.50 ABI2

0.25 ABI2

35

95

60

(54 )

(54 )

(54 )

PYL5/RCAR8 0.60 HAB1

2.00 ABI2

2.00 ABI1

35

115

123

(39 )

(39 )

(39 )

PYR1/RCAR11 0.60 HAB1 0.60 ABI2

2.00 ABI1

390* 360

330

(39

) (39 )

(39 )

PYL8/RCAR3 0.25 ABI1

0.50 ABI1

2.00 ABI1

0.25 ABI2

2.00 ABI2

0.6 HAB1

18*

23

75

30

118

135

(54 )

(54 )

(39 )

(54 )

(39 )

(39 )

PYL4/RCAR10 2.00 ABI1 272 (39 )

2.00 ABI2 110 (39 )

0.60 HAB1 188 (39 )

*Values can be compared to those in Table 1.


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kinases, CPK3 and CPK6, which have been

implicated in the regulation of anion chan-

nels; however, it is not known whether these

kinases directly phosphorylate SLAC1 aswell, or whether they modify anion channel

activity through an indirect effect (95).

Slow anion conductance, particularly

permeability to NO3 – , is not completely

abolished in the slac1 mutant. For example,

the slac1 mutant can still close the stomates

in response to the transition from light to

dark (80, 81), which requires anion efflux.

This fueled the motivation to trawl the Ara-

bidopsis genome for other anion channels

behind the residual activities in the guard

cell. Ectopically expressed SLAH3 can

functionally restore the mutant phenotypes

of slac1-2, but unlike the original reporton the tissue-specific expression pattern of

the SLAC family (80), later investigations

revealed that SLAH3 itself is expressed in

the guard cell at ~50% of the amount of

SLAC1, as estimated by quantitative PCR

(92). In addition, the abundance of the

SLAH3 transcript in guard cell protoplasts

increased ~twofold in the slac1 background

compared with its abundance in WT plants,

suggesting that these two anion channels

can compensate for each other by feedback.

Patch-clamp measurements of slah3 guard

cells revealed reduced current in nitrate-

based medium, suggesting that SLAH3 isthe likely channel responsible for the re-

sidual anion activities in slac1; however, the

slah3 mutant has no growth phenotype (92).

The SLAH3 activity was also slowly deac-

tivated by negative membrane potentials,

reminiscent of the characteristics of the

S-type anion channel. Like SLAC1, SLAH3

was also phosphorylated at the N-termi-

nal cytosolic segment by CPK21, which

was blocked in the Xenopus experimental

Fig. 4. Current model of the ABA signaling pathway in the guard cell. Inthe absence of ABA, the activities of the three kinases CPK21, CPK23,and OST1 are muted by the upstream PP2Cs (ABI1, ABI2). Light acti-vates H+-ATPases (for example, OST2), which in turn drive secondarytransporters, such as K+ influx channels (probably consisting of KAT1, aheterotetrameric complex, with its closest homolog KAT2). The bindingof ABA to the receptor leads to retention of the PP2Cs, thereby liberat-ing the kinases to phosphorylate the downstream targets. The OST1phosphorylates and inhibits the inward-rectifying K+ channels to pre-vent entry of K+ into the guard cell necessary for stomatal opening (A).This same kinase, however, phosphorylates and activates the NADPH

oxidase AtrbohF to generate the second messenger H2O2, which islinked to Ca2+ release. OST1 and CPK21 or CPK23 phosphorylate andactivate the S-type anion channel SLAC1. OST1 also integrates theCO2 stimulus, but the intermediates (marked as ?) in this pathway havenot been determined. CPK21, but not OST1, phosphorylates and ac-tivates SLAH3 in Xenpous oocytes. Ca2+ inhibits the proton pumpingactivity (for example, OST2), probably through the action of anotherCa2+-dependent kinase PKS5 (128 ). GORK is the major K+-outwardrectifying channel that is sensitive to cytosolic alkalinization and expelsK+ needed for stomatal closing (B) (97 ). Upward arrows denote stimula-tion of activity; downward arrows indicate inhibition of activity.

CDPK SnRK2SnRK2 CDPK CDPK CDPK CDPK

(CPK23?) (OST1) (CPK23?) (CPK21?)(OST1)

Ca2+

Stomata open Stomata closed

SLAH3

(Anion channel opens

anions exit cells)

KAT1 and

KAT2

KAT1

and KAT2

(K + influx

channel closed)

(K + channel

open)

OST2OST2

(H+-ATPase

inactive)

SLAC1

(Anion channel opens

anions exit cells)

?

? ?

GORK1

(K + efflux

channel opens)

(PKS5)

O COOHOH

(CPK21?)

K + H+

CO2

(AtrbohF)

Cytosol

H2O

2

Anions

(H+-ATPase,

H+ ions exported)

C R E D I T : Y . H A M M O N D / S C I E N C E S I G N A L I N G


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system by coexpression of ABI1 and ABI2.

However, there are also some important dif-

ferences between these homologous anion

channels. Unlike SLAC1, SLAH3 is not

phosphorylated by OST1. Compared with

SLAC1, SLAH3 is twice as permeable to

NO3 –

, and this anion has been proposed to be a physiological activator of this channel

(92). In contrast, SLAC1 might be activated

by bicarbonate (96 ), which is blocked in the

knockout mutant ost1. Whether these appar-

ent differences are physiologically relevant

or the consequences of different experi-

mental approaches needs more exploration.

The fact that the activities of SLAC1 and

SLAH3 are regulated by OST1 or CPKs or

both is consistent with the Ca2+-dependent

and -independent nature of the anion ef-

flux critical for stomatal closure. However,

questions remain concerning how these

CPKs fit into the early steps of ABA sig-naling in planta. Like the ABA-dependent

transcriptional pathway (68), the posttrans-

lational regulation of SLAH3 that presum-

ably contributes to stomatal closure has also

been reconstructed in vitro (92). Binding of

ABA to the receptor RCAR1 (also known

as PYL9) blocks ABI1 phosphatase activity,

freeing CPK21 to phosphorylate SLAH3

(more correctly, its N-terminal cytosolic

domain, which was used in the assay to

represent the physiological endpoint) (Fig.

4) (92). In parallel, it is expected that the

depolarization of the plasma membrane

evoked by SLAC1 and SLAH3 would leadto cytosolic alkalinization and activation

by a pH-sensitive pathway activating the

outward-rectifying K + current, which has

been identified as GORK (97 ) (Fig. 4). K +

efflux, therefore, has a twin role: to restore

the charge unbalance due to expulsion of

the anions by SLAC1 and SLAH3, and to

relieve guard cell turgor pressure requisite

for stomatal closure.

OST1, an Integrator of ABA andCO2 SignalsThe work on the functional relation between

SLAC1 and OST1 in guard cell signalinghas parlayed into fresh insight into how

ABA signaling is integrated with the re-

sponse to CO2, the other signal besides H2O

with direct relevance to accumulation of

biomass and climate change (96 ). Plants re-

spond to increased CO2 [800 parts per mil-

lion (ppm), compared with ambient CO2 of

~350 ppm] by closing the stomates, which

requires carbonic anhydrase activity (98),

but the early signaling events have not been

entirely clear (99). CO2 is thought to dif-

fuse passively across the plasma membrane

during photosynthesis, but pharmacologi-

cal studies and reverse genetic studies have

suggested that certain aquaporins present in

the plasma membrane and chloroplast enve-

lope might actively transport CO2, at leastin the mesophyll of tobacco (100 – 102). The

identification of the slac1 mutant brings a

genetic proof that the guard cell itself is

equipped with CO2 sensors and signal trans-

duction pathways. In fact, increased CO2

activates anion currents in the guard cells

(98). High and low bicarbonate concentra-

tions that promoted either stomatal closing

and opening, respectively, had also been

observed decades ago (103). Guard cells of

the slac1 mutant are insensitive to several

environmental stimuli, including changes

in CO2 concentration (4, 80), and guard

cell–derived protoplasts of slac1 plants donot produce anion currents when exposed

to high concentrations of CO2 and carbonic

acid (CO2/HCO3 – ; a mixture of CO2 and car-

bonic acid was used in these experiments);

HCO3 – is condensed from CO2 and water, a

reaction catalyzed by the CO2-binding pro-

teins carbonic anhydrases (96 ). These stud-

ies also suggested that HCO3 – , more than

CO2, might be the intracellular activator

of anion channels (96 ). It appears that one

of the consequences of CO2/HCO3 – is the

priming or enhancement of the Ca2+ sensi-

tivity of SLAC1. SLAC1 is phosphorylated

by OST1, and mutant ost1 plants exhibited anormal stomatal opening response to low at-

mospheric CO2 (60). Thus, it was surprising

to find that, compared with WT guard cell

protoplasts, the anion currents from those

of ost1 were also not triggered by increased

CO2/HCO3 – , and stomatal closing was im-

paired. In contrast, the stomatal opening

response to bicarbonate was normal, albeit

slower, in the quadruple mutant for the ABA

receptors pyr1, pyl1, pyl2, and pyl4 (96 ).

Together, these results suggest that OST1 is

a convergent point for both ABA and CO2 in

the stomatal closure pathways.

Concluding Remarks andFuture ProspectsWe have come a long way since the first

biochemical identification of ABA-bind-

ing proteins in the plasmalemma of the V.

faba guard cell (28). The discovery of the

cytosolic ABA receptors, characterized by

the presence of the START domain, has

led to elucidation of the early steps in the

ABA signaling pathway. The accessibility

of ABA to this family of inside receptors

is probably partially modulated by ATP-

binding cassette transporters (104, 105),

which is reminiscent of the carrier-medi-

ated ABA uptake reported for Commelina

(16 , 17 ). A parsimonious ABA signaling

pathway, as defined by reconstitution invitro, is composed of a soluble ABA re-

ceptor (PYR1), a PP2C (ABI1), a SnRK2

(OST1), and a transcription activator b-ZIP

(ABF2) that binds ABA-regulated promot-

ers (68). Phosphorylation of a fragment of

ABF2 by OST1 in this in vitro reconstitu-

tion was shown to be ABA-dependent (68).

Likewise, a similar minimal pathway was

reconstructed for the ABA response in the

guard cell. Because SLAH3 is functionally

equivalent to SLAC1 and the slac1 mutant

stomata are insensitive to a battery of clos-

ing signals (80), the prototypical members

in the stomatal-closing pathway consist ofRCAR1 (PYL9) (the cytosolic ABA recep-

tor), AB11 and ABI2 (the negative regulato-

ry PP2Cs), OST1 and possibly CPK23 and

CPK21 (the positively regulating kinases),

and SLAC1 and SLAH3 (as the S-type an-

ion channels initiating the depolarization of

the plasma membrane prerequisite to sto-

matal closure) (92). Because the receptors,

PP2Cs, CPKs, and SnRK2s are all encoded

by large gene families, tremendous combi-

natorial possibilities are possible, enabling

plants to finely modulate the intensities of

the output. Just one example of this inher-

ent flexibility is the combination of apo-receptor and PP2C, which affects the bind-

ing constants to ABA (39, 54) (Table 1).

Immunoprecipitation of ABI1 (tagged

with yellow fluorescent protein) from plant

extracts recovered a large number of solu-

ble receptors (9 of 14) (67 ). Other proteins

that coprecipitated with ABI1 included the

REGULATORY PARTICLE NONATPASE

10 (RPN10), a subunit of the 19S regula-

tory complex of the 26S proteasome (106 ).

Whether this indicates that some of these

signaling components might be regulated

by protein stability in the guard cell is not

known (67 ). However, in germinating seeds,the b-ZIP transcription regulator ABI5 ac-

cumulates in the mutant rpn10 (106 , 107 ).

Additional proteins that coprecipitated with

ABI1 included the sucrose-phosphate syn-

thase 1F and the ribosomal protein PRL12B.

The H+-ATPase, OST2 (108), whose activ-

ity is suppressed by ABA before stomatal

closing, was also recovered along with the

receptors in the ABI1 immunoprecipita-

tions. AHA2, which shares overlapping


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functions with OST2 (109), was not recov-

ered, however. The proton pumps are usu-

ally considered to be the endpoints of sig-

naling pathways; thus, the direct association

of OST2 with the ABA receptor complex

hints at the possibility that the pathway is

much more complex in composition in the plant context. How the functions of these

other “accessory” proteins are integrated

into the “core” ABA signaling components

established in vitro remains to be investi-

gated. Besides PYR/PYL/RCAR, there are

also membrane-associated candidate re-

ceptors—GTG1, GTG2 (110), and ABAR

(111, 112)—although some have been un-

able to reproduce the binding of ABA to

ABAR (113).

It is still too early to pronounce wheth-

er GTG1 and GTG2 might fit the profile

of the outside ABA perception site. Also,

where or how the GTG1 and GTG2 relateto the PYR/PYL/RCAR-mediated pathway

or whether they represent part of parallel

and independent signaling cascades remain

fascinating questions. Structural studies

of PYR1 bound to both S-(+)-ABA and

R-(–)-ABA stereoisomers showed that the

differences in the chirality of both isomers

are accommodated within the soluble re-

ceptors by the rotation of the ABA ring by

~180° (50). Neither ABAR nor GTG1 or

GTG2 can bind the nonnatural R-(–)-ABA

isomer (110, 114); Nonetheless, the R-(–)-

ABA isomer induces long-term responses,

such as seed germination (115). SLAC1is phosphorylated by OST1 and the Ca2+-

dependent CPK21, at least when assayed

in the Xenopus oocyte system. Reverse

genetics and electrophysiological studies

have also implicated CPK3 and CPK6 in

the regulation of the S-type anion efflux

in response to ABA (95). The precise rela-

tion between these two Ca2+-dependent ki-

nases and S-type anion transporters is not

yet clear, but does reinforce the importance

of Ca2+ in “priming” or accentuating the re-

sponse to ABA (43). The calcium-binding

protein NpSCS exerts a suppressing effect

on all SnRK2s tested in vitro, and this in-hibition is calcium-dependent (116 ). If so,

this suggests that SLAC1 may be regulated

by two mutually exclusive phosphorylation

pathways in guard cells.

ABA also seems to play developmen-

tal roles other than in stress signaling and

drought protection. Studies carried out in

tomato and Arabidopsis suggest that ABA

is required to limit ethylene production

during the course of normal plant growth

(117 , 118). The dose-dependent effect of

ABA is evident in roots, where elongation

in Arabidopsis is stimulated by exogenous

ABA at 0.1 µM and is inhibited when the

hormone is applied at concentrations above

1.0 µM (119). Suppressing ABA production

by mutations or in transgenic plants resultsin developmental defects, such as altered

organization of the mesophyll and disrupted

stomatal morphogenesis (120, 121). Plants

in unstressed conditions contain a function-

ally relevant basal amount of ABA. Careful

liquid chromatography–mass spectrometry

measurements of ABA content in 4-week-

old Arabidopsis seedlings detected between

10 to 40 nM of the hormone (122). How-

ever, ABA is unequally distributed in vari-

ous cells in plants. Using an ABA-sensitive

promoter driving the expression of a lucifer-

ase gene, the hormone is more concentrated

in guard cells and in the root tips, with anestimated detectable threshold of 0.3 µM

(123). In V. faba and on a per-guard-cell

basis, ABA in the concentration range of 0.7

to 1.6 fg of ABA (equivalent to ~0.7 to 1.6

µM) has been calculated (29, 124, 125). In

vitro, the IC50 of PP2C activity by ABA act-

ing through several members of the PYR1/

PYL1/RCAR family occurs in the nanomo-

lar range (39, 41, 54). Thus, the amounts of

ABA required to inhibit PP2Cs and activate

the signaling pathway are near the basal

concentrations of ABA in guard cells. Be-

cause the synthetic ABA agonist, pyrabac-

tin, can bind PYL2 in two orientations byan induced fit mechanism to produce either

a productive or nonproductive conforma-

tion, it has been suggested that there might

be naturally existing antagonists of ABA

receptors in plants that could lock the ABA

receptors in nonproductive orientations,

perhaps as a safety mechanism against aber-

rant ABA signaling (45, 126 ). The structural

insight gained from PYL2 complexed with

either ABA or pyrabactin offers a tangible

possibility to embark on rational design of

chemical modulators of drought resilience

for crops.

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Joshi-saha2011_A Brand New START_Abscisic Acid Perception and Transduction in the Guard Cell