Joshi-Saha2011 - A Brand New START - Abscisic Acid Perception and Transduction in The Guard Cell

` A Brand New START: Abscisic Acid Perception and Transduction in the
Guard Cell
Archana Joshi-Saha, Christiane Valon and Jeffrey Leung (29 November 2011)
Science Signaling 4 (201), re4. [DOI: 10.1126/scisignal.2002164]
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REVIEW
PLANT BIOLOGY
A Brand New START: Abscisic Acid

Perception and Transduction in the
Guard Cell
Archana Joshi-Saha,1 Christiane Valon,1 Jeffrey Leung1*
Introduction
Land plants, being rooted in place, must
sense and adapt to their incessantly uctuating surroundings. At one time or another, we
have all noticed a plant neglected in a shady
corner exhibiting heliotropism: leaning out
and orienting its leaves perpendicularly toward the Suns rays to maximize photosynthesis. In contrast to laboratory settings,
plants in heterogeneous eld 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 quality and intensity, uctuations in temperature,
humidity, CO2, and possibly uneven water
distribution in the soil. Carbon dioxide and
water are among the most important ingredients contributing to plant biomass [6CO2
+ 6H2O + light sugar + 6O2]; in return,
plants recycle CO2 and H2O, with O2 release
Institut des Sciences du Vgtal, Centre National de la Recherche Scientique, Unit Propre de Recherche 2355, 1 Avenue de la Terrasse,
Btiment 23, 91198 Gif-sur-Yvette, France.
1
*Corresponding author. E-mail, leung@isv.

cnrs-gif.fr
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 possible almost exclusively through stomatal
pores (Fig. 1A). In most plants, these pores
are anked 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, respectively (Fig. 1B). Sunlight stimulates stomatal
opening to allow diffusion of atmospheric
CO2 to photosynthetic tissues. This, however, exacts a trade-off in water loss through
transpiration, which will compromise growth
(2). Stomates also close in response to elevated CO2, the cause of global climate
warming, and to high concentrations of pollutants, 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
plants conicting 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
A Retrospective on Abscisic Acid

Signaling in the Guard CellThe
Circuitry of Ion Fluxes
Water decit stimulates the synthesis and,
to a much lesser extent (~5%), release from
storage of the hormone ABA to promote stomatal closure. The early physiological intricacies of ABA signaling in guard cells were
teased out largely by pharmacological and
biophysical approaches that provided the
rst sketch of the circuitry (Fig. 1B). These
studies showed that an early detectable event
triggered by ABA is the production of reactive oxygen species [(ROS), sometimes also
known as oxidative burst], which stimulates
Ca2+ release from internal stores and inux
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 (610). At the
same time, Ca2+ also prevents membrane
hyperpolarization by inhibiting H+adenosine triphosphatases [(ATPases), proton
pumps coupled to ATP hydrolysis] required
to drive stomatal opening (11). Two distinct
types of anion efux currents are detectable
in the guard cell, designated as slow (S) or
rapid (R) (12, 13). The S-type current is carried by a range of anions that include NO3,
Cl, and malate (14, 15). It was proposed
that the S-type, and not the R-type, current
was responsible for ABA-mediated stomatal
www.SCIENCESIGNALING.org
29 November 2011 Vol 4 Issue 201 re4
The soluble receptors of abscisic acid (ABA) have been identied in Arabidopsis
thaliana. The 14 proteins in this family, bearing the double name of PYRABACTIN
RESISTANCE/PYRABACTIN-LIKE (PYR/PYL) or REGULATORY COMPONENTS
OF ABA RECEPTOR (RCAR) (collectively referred to as PYR/PYL/RCAR), contain between 150 and 200 amino acids with homology to the steroidogenic acute
regulatory-related lipid transfer (START) protein. Structural studies of these receptors have provided rich insights into the early mechanisms of ABA signaling.
The binding of ABA to PYR/PYL/RCAR triggers the pathway by inducing structural changes in the receptors that allows them to sequester members of the
clade A negative regulating protein phosphatase 2Cs (PP2Cs). This liberates the
class III ABA-activated Snf1-related kinases (SnRK2s) to phosphorylate various
targets. In guard cells, a specic SnRK2, OPEN STOMATA 1 (OST), stimulates
H2O2 production by NADPH oxidase respiratory burst oxidase protein F and inhibits potassium ion inux by the inward-rectifying channel KAT1. OST1, the kinase CPK23, the calcium-dependent kinase CPK21, and the counteracting PP2Cs
modulate the slow anion channel SLAC1, a pathway that contributes to stomatal
responses to diverse stimuli, including ABA and carbon dioxide. A minimal ABA
response pathway that leads to activation of the SLAC1 homolog, SLAH3, and
presumably stomatal closure has been reconstituted in vitro. The identication
of the soluble receptors and core components of the ABA signaling pathway provides promising targets for crop design with higher resilience to water decit
while maintaining biomass.
sugars. Depending on the nature of the input stimuli, coordinated ionic uxes across
membranous compartments of the guard
cell will be assured by teams of different
channels and transporters. The electrical
signals generated by ion uxes across the
plasma membrane are then converted by
the cell into chemical messages to shape the
nal physiological output (in this case, the
binary decision of stomatal opening or closing). 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 factors. 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 to
other plant tissues or organs that respond to
this hormone.
REVIEW
A
B
H+
Caa2+
Light / Low CO2
Hyperpolarization
ABA
Fusicoccin
High humidity
H2O2
Low CO2
Light
K+
AH2O2
ABA
K+
Low humidity
High CO2
Darkness
pH
Depolarization
CREDITS: (A) JEFFREY LEUNG; (B) Y. HAMMOND/SCIENCE SIGNALING
Fig. 1. Biophysics of stomatal movement. (A) One-week-old Arabidopsis rosette leaf is shown with an image of a single stomate.
The microscopic pores contoured by the two anking guard cells
dene a stomatal opening. The uorescent round structures are
chloroplasts. (B) Stomatal opening (left) and closing (right). Increasing turgor pressure inside the cells causes the two cells to swell
and bow out from each other, resulting in the opening of the pore.
Stomatal opening requires hyperpolarization of the plasma membrane and entry of K+. ABA accumulates in response to drought
and fosters stomatal closing. The earliest detectable signal is the
presence of reactive oxygen species (H2O2) and then a transient
closure. However, it was not clear at the time

whether the two types of anion currents occurred through distinct channels or through
modication of the same channel. ABA also
induces the net efux 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 signaling branch modulates K+ efux 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 closure 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 cytosolic receptors. Furthermore, the guard cells
of Commelina communis have substantial
carrier-mediated uptake of ABA (16, 17),
which could deliver externally applied ABA
increase in Ca2+. A second signaling intermediate is sensitive to pH

and stimulates K+ efux through K+-outward rectifying channels. Because 90% of the volume of the guard cell is the vacuole, the efux
of ions must rst cross the vacuolar membrane (pale green oblong
structure), then the plasma membrane, and nally move into the
apoplastic space. For simplicity, the left cell shows the plasma membrane proteins that are active during stomatal opening, and the right
cell shows the plasma membrane proteins that are active during stomatal closure. Red, H+-ATPase; yellow, K+ inward-rectifying channel;
light blue, Ca2+ permeant channel; dark blue, anion channels; light
green, K+ outward-rectifying channel.
to intracellular reception sites. The requirement for an extracellular receptor was suggested by the ability of ABA at high pH,
when it is charged and can no longer cross
the 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 -amylase synthesis by gibberellic acid,
whereas microinjecting up to 250 M ABA
was ineffective (20). A cell surfacelocalized receptor was also concordant with K+
uxes (21) and reporter gene expression in
either Arabidopsis or rice cell cultures that
were stimulated by ABA coupled to carriers that could not penetrate membranes (21,
22). Immunolocalization of presumptive extracellular 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 applied 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 comparatively less effective in regulating stomatal

aperture at higher versus lower pH, the latter
of which favors uptake by passive diffusion
of the protonated form of ABA, providing
indirect evidence for an intracellular receptor (17, 19, 25, 26). The presence of an intracellular 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 the
Core Signaling Complex: Soluble
Receptor, Protein Phosphatase 2C,
and the Kinase SnRK2
To dissect the underlying mechanisms by
which ABA rapidly causes stomatal closing, 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
rst report more than 25 years ago of binding proteins in the plasmalemma of V. faba
guard cells (28, 29). After many candidates
that have been described as false starts (30),
ABA
those with properties matching

the required physiological and
Br
Br
molecular proles were identied in 2009. This momentous
OH
discovery, celebrated as one of
O
O OH
O=S=O
O=S=O
the top 10 discoveries of the
NH
NH
year (31), represents an awakening in our understanding of the
N
initial ABA signaling events.
This soluble receptor family
has 14 members in Arabidopsis Fig. 2. ABA and pyrabactin used in classical and chemithaliana, and this high degree cal genetic screens, respectively, to identify signaling
of functional redundancy may components. (A) Chemical structure of ABA, highlighting
have cloaked their identity from its active carboxyl group that directly binds to a conserved
being revealed by standard ge- Lys residue (Lys86 in PYL1) deep in the pocket of the renetic screens (at least for the ceptors (except PYL13). (B) Structures of the synthetic
loss-of-function category of mu- chemical pyrabactin (left) and its analog apyrabactin
tations). However, the discovery (right). The pyradyl nitrogen (arrow) is important for the
that the synthetic compound, pyrabactin agonist effect, because apyrabactin is inactive.
4-bromo-N-[pyridin-2-yl methyl]naphthalene-1-sulfonamide, known as 1), all reduced the interaction between the
pyrabactin (Fig. 2), partially mimicked receptor and PP2C. Independently, Ma et al.
the inhibitory effects of ABA on seed ger- isolated a protein named REGULATORY
mination, early seedling growth, and gene COMPONENT OF ABA RECEPTOR 1
expression program (32), presumably by (RCAR1), as a partner of ABI1 and ABI2
binding and modifying the activities of sev- (41). The mutation G168D in ABI2 (abi2-1)
eral of the soluble ABA receptors simulta- or the equivalent mutation G180D in ABI1
neously, allowed Park et al. to side-step the (abi1-1) also abolished its interactions with
functional redundancy barrier. By selecting RCAR1 (also known as PYL9).
for mutagenized seeds that germinated in
The crystal structures of several recepthe presence of inhibitory concentrations tors were rapidly accomplished, and ne
of pyrabactin, followed by map-based clon- structural details were obtained in quick sucing of one such locus named PYRABACTIN cession for (i) ABA-bound, (ii) pyrabactinRESISTANCE 1 (PYR1), they succeeded in bound, (iii) and ligand-free (PYR1, PYL1,
isolating a gene encoding a homolog to the PYL2) apo-receptors, as well as (iv) the
steroidogenic acute regulatory lipid trans- tertiary complex ABA-PYL/RCAR-PP2C.
fer (START) proteins. These proteins are These structural studies have uncovered
characterized by a structural scaffold that a wealth of mechanistic insights into the
can accommodate a large spectrum of hy- early events of ABA signaling (4250). The
drophobic ligands, such as lipids, antibiot- receptor protein has a central cradle that is
ics, and hormones (33). Importantly, PYR1 formed by the alignment of seven-stranded
interacted in a yeast two-hybrid screen, in antiparallel sheets wrapped around by a
an ABA-dependent manner, with several long helix from the C-terminal end of the
phosphatases of the protein phosphatase 2C protein. The bottom of the cradle is created
(PP2C) family (namely, ABI1, ABI2, and by two other helices situated between the
HAB1) that had previously been established rst and second sheets. The ABA molecule
as key negative regulators of the ABA sig- is held inside the cavity by a combination
naling cascade (3440). Moreover, the proof nonpolar and polar interactions. Among
tein-protein interaction pattern established the charged interactions, the carboxyl
by the yeast two-hybrid system related to group of the ABA is plunged deep within
the phenotypes of the respective mutant the pocket, and it is in direct contact with
plants. For example, the loss-of-function Lys59 of PYR1 (or Lys86 of PYL1 and Lys64
mutations Pro88 to Ser88 (P88S) and Ser152 of PYL2) (Fig. 3, A, C, and D). This lysine
to Leu152 (S152L) in PYR1 that confer the is conserved in the gene family, with the exreceptors property of pyrabactin resistance ception of PYL13 in which this residue is
and, conversely, the gain-of-function Gly168 occupied by a glutamine. The access to the
to Asp168 (G168D) mutation in ABI2, which ABA molecule from outside the receptor is
dened this negative regulator and confers controlled by two important structures: The
ABA resistance in the mutant plants (abi2- rst 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 members (51). This closing of the gate is further
secured by the positioning of the latch and
the extension of an -helical loop (recoil
region) (50). This recoil region encompasses 13 amino acids (Met147 to Phe159 in PYR,
which align with Val177 to Phe189 in PYL1 in
Fig. 3) that, after ABA binding, coil into the
C-terminal helix of the receptor.
In the absence of ABA, the receptor
(PYR as the model) exists as an asymmetric dimer (50) with ~10 deviation from a
twofold (180) symmetry. These monomeric 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 compact structure with a biconcave disc shape
resembling a red blood cell. Coimmunoprecipitation assays conrmed the existence
of dimer in vivo both with and without exogenous ABA. It seems that ABA binding
causes the dimer to dissociate into monomers 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 ) PYR1 and
at 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 synthetic compound could theoretically antagonize activation of PYL2 by ABA (45, 49).
In the productive ABA receptorpyrabactin
congurations [derived from the structural
studies of PYL1-pyrabactin (45), PYR1pyrabactin, and PYL1-pyrabactin-ABI1 (45,
49)], the orientation of the bound pyrabactin
provides the necessary van der Waals interactions to induce gate closure. In contrast,
in the nonproductive mode [deduced from
the PYL2-pyrabactin structure (45, 49)], the
CREDIT: Y. HAMMOND/SCIENCE SIGNALING
REVIEW
REVIEW
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 median 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 (Table 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 pathways 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 deep
within the cavity of the receptor. However,
Trp300 of ABI1 (or Trp385 of HAB1), sometimes 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 simultaneously through a combination of watermediated and hydrophobic interactions,
respectively (Fig. 3). Mutational analysis
showed that this tryptophan is not essential
for phosphatase activity, but only its afnity with the receptor and, as a consequence,
ABA-dependent inhibition of ABI1 (53) or
HAB1 (55) is affected when this residue
is mutated. Conformational changes in the
receptor induced by its interaction with this
key tryptophan residue facilitate the fastening of the receptors gate and latch into the
closed conguration. Whether this could
provide a structural rationale for the more
than 10-fold increase in ABA binding afnity observed for the PYL-PP2C complexes as
compared with the apo-receptors still needs
to be conrmed (39, 41, 44, 51, 53, 54).
Several research groups have independently, and by different experimental approaches, identied an ABA-activated and
calcium-independent kinase in wheat (56),
the broad bean V. faba (57, 58), and its ortholog 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 puried 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 Arabidopsis, known variously as OPEN STOMATA
1 (OST1), SRK2E, and SnRK2.6 (59, 60),
also blocked the typical stomatal closing response to ABA and to progressive drought.
Yoshida et al. demonstrated a direct interaction between ABI1 and OST1 by the yeast
two-hybrid approach. Further, they delineated 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 known
elementary ABA responses (6365). Several 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 dephosphorylated the ABA-stimulated OST1 recovered
from cell extracts (65, 66). Also, relative to
WT plants, the ABA-activated kinase activity 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 regulated 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 Arabidopsis protein extracts. The composition of
the copuried proteins did not change regardless 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 of
the same signalosome simultaneously, but
different combinations of the constituents
could exist in different tissues.
Regulation of Ion Transport Across
the Plasma Membrane by the ABA
Core Signaling Complex
Relative to its closest homologs SnRK2.2
and SnRK2.3, mutations in the OST1 locus
have the most negative impact on guard cell
relative orientation of the pyrabactin along

the length of the molecule is ipped by 180
in PYL2. The pyrabactin in this inverted orientation can no longer supply the necessary
van der Waals forces to maintain gate closure. Thus, the binding of the ligand (ABA
or pyrabactin) within the cavity is not itself
sufcient to trigger the downstream pathway. 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 conguration is needed 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 moiety
of the complex, suggesting that the receptor is a rigid structure. An important insight
informed by the tertiary complex is that
amino acid Ser112 of PYL1, or the equivalent
Ser89 of PYL2, make contact with Glu142 and
Gly180 of ABI1 (Fig. 3). Thus, the Ser112 or
Ser89 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 aspartic
acid, would permit escape from repression
by receptor binding during ABA signaling,
which would explain the dominant or constitutive nature of these mutations. Again,
the structural data with these soluble receptors and the PP2Cs t those from the genetic
analysis of the mutants and molecular properties of their proteins.
The dissociation constants (Kd) of four
representative receptors (expressed as recombinant proteins in bacteria) are unexpectedly high, near or in the micromolar
range. However, their afnity for ABA increases to the nanomolar range when compatible PP2Cs are present (Table 1). This
increase in receptor afnity for ABA in the
presence of PP2C was also reected by in
vitro assays of the inhibition of protein phosphatase 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 efciency of ABAmediated inhibition of phosphatase activity
in vitro was generally higher for ABI1 than
REVIEW
response to environmental stress (59, 60,
69, 70). OST1 may thus be the key guard
cell kinase regulating a large roster of targets. A substantial fraction of the transcriptome 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 corABA receptor
PYL1
Recoil
ABA
Lys
Trp300
ABA
Latch
Latch
Gly180
Ser112
gate
Apo-gate
Glu
Ser
Gly
Gate
Trp
Glu142
Catalytic
center
Recoil
Latch
PYL1
ABI1

pyr1-9
Gate

pyr1-3

pyr1-8
pyr1-5
Latch

(

pyr1-6
CREDITS: (A) Y. HAMMOND/SCIENCE SIGNALING; (B) MODIFIED WITH PERMISSION FROM NATURE 462, 609614 (2009)
Clade A PP2C
ABI1
Apo-latch

ABA-bound
gate
5HFRLO
pyr1-2 1-4

Fig. 3. The mechanistic basis of ABA-mediated inhibition of PP2C

activity. (A) Ribbon models of superimposed apo-PYL2 (gray) and
ABA-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-bound
gate. (B) The PYL1-ABA-ABI1 tertiary complex [modied with permission from (53)]. The Trp lock of ABI1 is shown as yellow spacell.
(Right) Close-up view of the intermolecular interactions that explain
receptor sequestration of PP2C upon ABA binding. (C) A generalized scheme of the receptor-ABA-PP2C complex highlighting the essential serine residue (Ser112 in PYL1) in the receptor that tethers the
PP2Cs by interacting with a Glu (Glu142 in ABI1) and a Gly residue
(Gly180 in ABI1). A conserved Trp in the PP2Cs (Trp300 in ABI1) interacts with the ABA through a water molecule (blue dot). The carboxyl
group of ABA is in contact with a Lys residue (Lys86 in PYL1) deep in

DEL

the pocket. N, N terminus. (D) In PYL1, amino acids participating in

ABA binding are underlined in black (86, 171, 116 to 121, and 143 to
149). The START homology spans from amino acids 50 to 206. Red
dots denote amino acids that dock onto the catalytic regions of ABI1.
Residues underlined in blue denote -helical structures (amino
acids 34 to 47, 69 to 77, 82 to 84, and 183 to 208); orange underlines
indicate strands (57 to 67, 89 to 93, 105 to 110, 117 to 122, 135
to 137, and 148 to 175); and the red underline denotes the helical
turn at amino acids 128 to 131 (127). Various mutant alleles of the
PYR1 locus have been transposed onto the equivalent amino acids
of PYL1. (E) ABI1; blue residues represent the catalytic region. Yellow dots mark the amino acids that, when mutated, decreased PYL1
binding. Orange overlines indicate E142, G180 (abi1-1), and W300
(Trp lock), which dene the entrance to the ABI1 catalytic center in
the crystal structure. Mutations around W300 show an eightamino
acid insert specic to plant PP2Cs (42).
REVIEW
Table 1. Dissociation constants of representative receptors in the presence and absence
of PP2Cs. The dissociation constants (extrapolation of ligand afnity to achieve half occupancy of the receptor sites) were obtained by using receptors produced in Escherichia
coli 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 receptors display substantially higher hormone afnity. N/A, not applicable.
Receptors
Kd (M)
Kd in the presence of
PP2C (nM)
Techniques for Kd
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.
ABA-mediated ROS production in the ost1

mutant guard cell (60).
The target of OST1 that has attracted most
of the attention is involved in the regulation
of anion efux critical for ABA-mediated
stomatal closure. Anion efux is controlled
by a balance between phosphorylation and
dephosphorylation events (79). Two groups
independently converged on the locus
SLOW ANION CHANNELASSOCIATED
1 (SLAC1) as the gene encoding the most
likely S-type anion channel involved in stomatal closure (80, 81) (Fig. 4). Both groups
used impaired stomatal closure in response
to either high CO2 (80) or hypersensitivity to damage of photosynthetic tissues by
ozone (O3) (81) as the phenotypic criterion
for 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 consequence of perturbed ionic homeostasis (80).
There are three related SLAC1 homologs
(SLAH) in the Arabidopsis genome. Reverse transcriptase polymerase chain reaction (RT-PCR) coupled to histochemical
analysis of the -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 specific conditions used for this analysis (80). Despite the differences in their tissue-specic
rectly, a ~80amino acid protein fragment)

to represent the transcriptional output of
this minimal pathway (68).
OST1 also modies membrane transport properties in the guard cells by phosphorylating and inactivating one of the major potassium inward-rectifying channels,
KAT1, which was shown in vitro and in
the Xenopus oocyte heterologous expression system (Fig. 4) (74). The ensuing reduction in K+ inux is consistent with the
known electrophysiological effect of ABA
on modifying guard cell membrane transport (Fig. 1). ABA stimulates the production of H2O2 (Fig. 1) through plasma membranelocalized NADPH oxidases, which
are encoded by 10 genes in the Arabidopsis
genome. Although H2O2 is similar in structure 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 M)long enough to
oxidize its effector molecules, but not so
long as to cause cellular damage. Hence, to
curb rampant H2O2 cytotoxicity, the activities 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) identied mutations in two
genes encoding NADPH oxidase catalytic
subunits, AtrbohD and AtrbohF (respiratory burst oxidase homolog D and F), that
abolished ABA-induced stomatal closure,
ABA-mediated promotion of ROS production, and ABA-induced increase in cytosolic Ca2+. In vitro, OST1 phosphorylates
AtrbohF, but not AtrbohD (Fig. 4) (78),
which is also consistent with the lack of
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 cellspecic promoter complements
the phenotypes of CO2 insensitivity and accumulation of organic and inorganic ions of
slac1-2 (80). The slac1 mutant is phenotypically 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 rst is that they provide genetic evidence that the CO2, O3, low humidity, and
ABA perception pathways are interconnected and that SLAC1 has an important role in
integrating these environmental and endogenous signals. Two of the transferred DNA
insertion 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 0.2 l/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 important
is that, in contrast to WT plants, the characteristic slow and sustained anion current,
which is weakly voltage-dependent, is barely 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 nal reason that the study of the slac1
plants was particularly important was that
it provided strong evidence that the R-type
and 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 mutants (81), implying that the S- and R-type
anion channels are unlikely due to posttranslational modication of a single polypeptide, which is consistent with previous
suggestion based on physiological studies
(12, 83). AtALMT12, a homolog of the
REVIEW
aluminum-activated malate trans- Table 2. Receptor afnity to ABA depends on the presence of protein molecular consequences of
porter, fullls the physiochemical phosphatases and their relative ratios. The IC50 values indicate the phosphorylation on the overall
characteristics of the R-type an- concentration of ABA required to cause 50% inhibition of the PP2C SLAC1 structure are not imion channel (84, 85). These chan- activity by the receptor. The ratio of PP2C to receptor has a pro- mediately obvious.
nels were rst studied in the roots nounced impact on IC50, which is commensurate with the amount of
The protein phosphatases
and are thought to play a role in input PP2C. Because of this, the PP2Cs have been regarded by some ABI1, ABI2 (89, 90), and
releasing malate to chelate alumi- as coreceptors of ABA. Note that PYR1 with 0.6 HAB1 yielded an IC50 PP2CA (91) block SLAC1of 390 nM, whereas a value of 125 nM was obtained in Table 1 using
num in the rhizosphere (86, 87). 0.8 HAB1. These variable results for the same combination of PP2C mediated anion efux in the
In mutants lacking the functional and receptors could be due to the different experimental conditions.
Xenopus expression system.
AtALMT12, guard cells displayed
The other homologous proreduced sensitivity to closing
tein phosphatases, such as
Receptors
Ratio PP2C:receptor IC50 (nM)
Reference
stimuli, such as the transition of
HAB1 and HAB2, were less
light to darkness, high concen- PYL9/RCAR1
effective (90), at least in the
0.25 ABI1
35
(54)
trations of CO2, and ABA. The
Xenopus system. Neither the
0.50 ABI2
95
(54)
characteristic voltage-dependent
WT catalytic activity of the ki0.25 ABI2
60
(54)
R-type current was reduced by
nase OST1 (89) nor that of the
~40% in the mutant protoplasts PYL5/RCAR8
phosphatase AtPP2CA (91)
0.60 HAB1
35
(39)
compared with those prepared
is required for these proteins
2.00 ABI2
115
(39)
from WT plants. The gating of the
to interact. An inactive form
2.00 ABI1
123
(39)
R-channel is sensitive to malate.
of AtPP2CA blocked phos0.60 HAB1
390*
(39)
This malate-sensitivity was also PYR1/RCAR11
phorylation of SLAC1 by WT
observed for AtALMT12 when it
OST1 (91), indicating that the
0.60 ABI2
360
(39)
was heterologously expressed and
activity of OST1 can be inhib2.00 ABI1
330
(39)
characterized in oocytes (85), sugited by physical entrapment
0.25 ABI1
18*
(54)
gesting that this property may be PYL8/RCAR3
in addition to dephosphoryla0.50 ABI1
23
(54)
inherent to the transporter itself,
tion by the PP2Cs. ABI1 and
unless Xenopus has conserved
ABI2 interact with CPK21
2.00 ABI1
75
(39)
the same regulatory machinery. It
and CPK23, which was detect0.25 ABI2
30
(54)
is currently not known how ABA
ed with bimolecular uores2.00 ABI2
118
(39)
regulates AtALMT12.
cence complementation when
0.6 HAB1
135
(39)
Guided by the structural studtagged forms of these partner
ies on the anion channel TehA, the PYL4/RCAR10
kinase-phosphatase pairs were
2.00 ABI1
272
(39)
SLAC1 homolog from the baccoexpressed in the Xenopus
2.00 ABI2
110
(39)
terium Haemophilus inuenzae,
oocytes (90) or in mesophyll
0.60 HAB1
188
(39)
SLAC1 would be a symmetrical
cells (92). Although CPK23
trimer composed of quasisym- *Values can be compared to those in Table 1.
can phosphorylate SLAC1 in
metrical subunits, each having 10
heterologous systems, such
transmembrane helices arranged in pairs pressed in the Xenopus oocyte system, the as Xenopus oocytes, and although the anto form a central ve-helix transmembrane activity of SLAC1 was detected only when ion current is reduced (by 70%) in guard
pore (88). The pore is a relatively uniform coexpressed with any of the following three cell protoplasts derived from the knockout
passage of 5 in diameter lined with largely kinases: OST1 (89), CPK23, or CPK21 cpk23, its functional importance, if any, in
hydrophobic residues, except for a constric- (90). The major site of phosphorylation by planta is not clear (90). Despite the reduced
tion that is gated by an conserved phenylala- OST1 is Ser120 in the N-terminal cytosolic anion current, no stoma phenotype was
nine residue (Phe450). SLAC1 does not seem domain of SLAC1 (4, 89, 91). This N-ter- noted in the cpk23 knockout mutant in these
to have discrete anion binding sites in the minal cytosolic domain of SLAC1 is phos- studies (90). The opposite phenotypes of
channel, compared, for example, with those phorylated by the CPKs at other unspecied reduced and increased stomatal apertures,
of the CLC family of channels, which have motifs. There are several other SLAC1 sites respectively, were observed by others in the
discrete ion binding sites with high eld that are phosphorylated in vitro by OST1, knockout mutant and in plants overexpressstrength. The ion selectivity of SLAC1 is and whether they have in vivo relevance ing AtCPK23 (93). CPK21 functions as a
thought to be largely a function of the ener- is not clear (4). In Xenopus, the activated negative regulator of abiotic stress responses
getic cost of ion dehydration and thus repre- SLAC1 displays higher permeability to because the cpk21 knockout is more tolersents a unique pore structure for anion chan- NO3 compared with Cl and malate (89). ant, rather than having the expected heightnels. Despite the overall hydrophobicity of The mutant slac1 can be complemented by ened sensitivity, to prolonged osmotic stress
the ion-conducting tube, the electrostatic po- the ectopic expression of either SLAH1 or as compared with WT plants (94). These
tential on the pore surface is polarized, and SLAH3 driven by the guard cellspecic apparently contradictory results suggest that
in particular, the electropositive nature of its promoter of SLAC1. However, SLAH1 does in the guard cell, there may be other targets
cytoplasmic surface is thought to contribute not contain any extended N- or C-terminal of these CPKs missing in the oocyte assays
to anion efux.
cytosolic domains that could constitute in which only single targets were tested.
When the channel is heterologously ex- the targets of phosphorylation. Thus, the There are also two other calcium-dependent
REVIEW
Stomata open
Stomata closed
OH
COOH
CO2
Ca2+
?
CDPK
CDPK
SnRK2
(CPK21?)
(CPK23?)
(OST1)
CDPK
SnRK2
(CPK23?)
(OST1)
H2O2
CDPK
CDPK
(CPK21?)
(PKS5)
H+
Cytosol
Membra
Mem
brane
bra
ne
KAT1
and KAT2
(K+ channel
open)
OST2
(H+-ATPase,
H+ ions exported)
SLAC1
KAT1 and
KAT2
(K+ inux
channel closed)
(Anion channel opens

anions exit cells)
CREDIT: Y. HAMMOND/SCIENCE SIGNALING
Fig. 4. Current model of the ABA signaling pathway in the guard cell. In
the absence of ABA, the activities of the three kinases CPK21, CPK23,
and OST1 are muted by the upstream PP2Cs (ABI1, ABI2). Light activates H+-ATPases (for example, OST2), which in turn drive secondary
transporters, such as K+ inux channels (probably consisting of KAT1, a
heterotetrameric complex, with its closest homolog KAT2). The binding
of ABA to the receptor leads to retention of the PP2Cs, thereby liberating the kinases to phosphorylate the downstream targets. The OST1
phosphorylates and inhibits the inward-rectifying K+ channels to prevent entry of K+ into the guard cell necessary for stomatal opening (A).
This same kinase, however, phosphorylates and activates the NADPH
kinases, CPK3 and CPK6, which have been

implicated in the regulation of anion channels; however, it is not known whether these
kinases directly phosphorylate SLAC1 as
well, 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 efux.
This fueled the motivation to trawl the Arabidopsis genome for other anion channels
SLAH3
OST2
GORK1
(AtrbohF)
(Anion channel opens

anions exit cells)
(H+-ATPase
inactive)
(K+ eux
channel opens)
oxidase AtrbohF to generate the second messenger H2O2, which is

linked to Ca2+ release. OST1 and CPK21 or CPK23 phosphorylate and
activate the S-type anion channel SLAC1. OST1 also integrates the
CO2 stimulus, but the intermediates (marked as ?) in this pathway have
not been determined. CPK21, but not OST1, phosphorylates and activates SLAH3 in Xenpous oocytes. Ca2+ inhibits the proton pumping
activity (for example, OST2), probably through the action of another
Ca2+-dependent kinase PKS5 (128). GORK is the major K+-outward
rectifying channel that is sensitive to cytosolic alkalinization and expels
K+ needed for stomatal closing (B) (97). Upward arrows denote stimulation of activity; downward arrows indicate inhibition of activity.
behind the residual activities in the guard

cell. Ectopically expressed SLAH3 can
functionally restore the mutant phenotypes
of slac1-2, but unlike the original report
on the tissue-specic 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 nitratebased medium, suggesting that SLAH3 is
the likely channel responsible for the residual anion activities in slac1; however, the
slah3 mutant has no growth phenotype (92).
The SLAH3 activity was also slowly deactivated by negative membrane potentials,
reminiscent of the characteristics of the
S-type anion channel. Like SLAC1, SLAH3
was also phosphorylated at the N-terminal cytosolic segment by CPK21, which
was blocked in the Xenopus experimental
Anions
K+
REVIEW
OST1, an Integrator of ABA and

CO2 Signals
The work on the functional relation between
SLAC1 and OST1 in guard cell signaling
has parlayed into fresh insight into how
ABA signaling is integrated with the response to CO2, the other signal besides H2O
with direct relevance to accumulation of
biomass and climate change (96). Plants respond to increased CO2 [800 parts per million (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 diffuse passively across the plasma membrane
during photosynthesis, but pharmacological studies and reverse genetic studies have
suggested that certain aquaporins present in
the plasma membrane and chloroplast envelope might actively transport CO2, at least
in the mesophyll of tobacco (100102). The
identication of the slac1 mutant brings a
genetic proof that the guard cell itself is
equipped with CO2 sensors and signal transduction pathways. In fact, increased CO2
activates anion currents in the guard cells
(98). High and low bicarbonate concentrations 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
cellderived protoplasts of slac1 plants do
not produce anion currents when exposed
to high concentrations of CO2 and carbonic
acid (CO2/HCO3; a mixture of CO2 and carbonic acid was used in these experiments);
HCO3 is condensed from CO2 and water, a
reaction catalyzed by the CO2-binding proteins carbonic anhydrases (96). These studies 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+ sensitivity of SLAC1. SLAC1 is phosphorylated
by OST1, and mutant ost1 plants exhibited a
normal stomatal opening response to low atmospheric CO2 (60). Thus, it was surprising
to nd 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 impaired. 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 and
Future Prospects
We have come a long way since the rst
biochemical identication of ABA-binding 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 ATPbinding cassette transporters (104, 105),
which is reminiscent of the carrier-mediated ABA uptake reported for Commelina
(16, 17). A parsimonious ABA signaling
pathway, as dened by reconstitution in
vitro, is composed of a soluble ABA receptor (PYR1), a PP2C (ABI1), a SnRK2
(OST1), and a transcription activator b-ZIP
(ABF2) that binds ABA-regulated promoters (68). Phosphorylation of a fragment of
ABF2 by OST1 in this in vitro reconstitution 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 closing signals (80), the prototypical members
in the stomatal-closing pathway consist of
RCAR1 (PYL9) (the cytosolic ABA receptor), AB11 and ABI2 (the negative regulatory PP2Cs), OST1 and possibly CPK23 and
CPK21 (the positively regulating kinases),
and SLAC1 and SLAH3 (as the S-type anion channels initiating the depolarization of
the plasma membrane prerequisite to stomatal closure) (92). Because the receptors,
PP2Cs, CPKs, and SnRK2s are all encoded
by large gene families, tremendous combinatorial possibilities are possible, enabling
plants to nely modulate the intensities of
the output. Just one example of this inherent exibility is the combination of aporeceptor and PP2C, which affects the binding constants to ABA (39, 54) (Table 1).
Immunoprecipitation of ABI1 (tagged
with yellow uorescent protein) from plant
extracts recovered a large number of soluble receptors (9 of 14) (67). Other proteins
that coprecipitated with ABI1 included the
REGULATORY PARTICLE NONATPASE
10 (RPN10), a subunit of the 19S regulatory 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 accumulates in the mutant rpn10 (106, 107).
Additional proteins that coprecipitated with
ABI1 included the sucrose-phosphate synthase 1F and the ribosomal protein PRL12B.
The H+-ATPase, OST2 (108), whose activity is suppressed by ABA before stomatal
closing, was also recovered along with the
receptors in the ABI1 immunoprecipitations. AHA2, which shares overlapping
system by coexpression of ABI1 and ABI2.

However, there are also some important differences 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 apparent differences are physiologically relevant
or the consequences of different experimental 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 efux critical for stomatal closure. However,
questions remain concerning how these
CPKs t into the early steps of ABA signaling in planta. Like the ABA-dependent
transcriptional pathway (68), the posttranslational regulation of SLAH3 that presumably 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 lead
to cytosolic alkalinization and activation
by a pH-sensitive pathway activating the
outward-rectifying K+ current, which has
been identied as GORK (97) (Fig. 4). K+
efux, 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.
REVIEW
(117, 118). The dose-dependent effect of
ABA is evident in roots, where elongation
in Arabidopsis is stimulated by exogenous
ABA at 0.1 M and is inhibited when the
hormone is applied at concentrations above
1.0 M (119). Suppressing ABA production
by mutations or in transgenic plants results
in developmental defects, such as altered
organization of the mesophyll and disrupted
stomatal morphogenesis (120, 121). Plants
in unstressed conditions contain a functionally relevant basal amount of ABA. Careful
liquid chromatographymass spectrometry
measurements of ABA content in 4-weekold Arabidopsis seedlings detected between
10 to 40 nM of the hormone (122). However, ABA is unequally distributed in various cells in plants. Using an ABA-sensitive
promoter driving the expression of a luciferase gene, the hormone is more concentrated
in guard cells and in the root tips, with an
estimated detectable threshold of 0.3 M
(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
M) has been calculated (29, 124, 125). In
vitro, the IC50 of PP2C activity by ABA acting through several members of the PYR1/
PYL1/RCAR family occurs in the nanomolar 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. Because the synthetic ABA agonist, pyrabactin, can bind PYL2 in two orientations by
an induced t mechanism to produce either
a productive or nonproductive conformation, 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 aberrant 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.
References and Notes
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52715276 (2008).
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A Brand New START: Abscisic Acid

*Corresponding author. E-mail, leung@isv.

as a photosynthetic by-product (1). Because

A Retrospective on Abscisic Acid

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Light / Low CO2

CREDITS: (A) JEFFREY LEUNG; (B) Y. HAMMOND/SCIENCE SIGNALING

closure. However, it was not clear at the time

increase in Ca2+. A second signaling intermediate is sensitive to pH

melina, extracellular ABA was comparatively less effective in regulating stomatal

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those with properties matching

signature amino acid motif SGLPA; A, Ala),

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CREDIT: Y. HAMMOND/SCIENCE SIGNALING

transiently in wild-type (WT) Vicia guard

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relative orientation of the pyrabactin along

b-ZIP transcription factors (71), which are

components consist of PYR1, ABI1, and

     

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Fig. 3. The mechanistic basis of ABA-mediated inhibition of PP2C

       

   

       

the pocket. N, N terminus. (D) In PYL1, amino acids participating in

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125 (0.8 HAB1)*

ITC, SPR (53)

ABA-mediated ROS production in the ost1

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rectly, a ~80amino acid protein fragment)

expression patterns, at least two of these are

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(Anion channel opens

CREDIT: Y. HAMMOND/SCIENCE SIGNALING

kinases, CPK3 and CPK6, which have been

(Anion channel opens

oxidase AtrbohF to generate the second messenger H2O2, which is

behind the residual activities in the guard

can compensate for each other by feedback.

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OST1, an Integrator of ABA and

of ABA to this family of inside receptors

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system by coexpression of ABI1 and ABI2.

1. A. M. Hetherington, F. I. Woodward, The role of

work: Advances in understanding abscisic acid,

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