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Guard Cell
Archana Joshi-Saha, Christiane Valon and Jeffrey Leung (29 November 2011)
Science Signaling 4 (201), re4. [DOI: 10.1126/scisignal.2002164]
The following resources related to this article are available online at http://stke.sciencemag.org.
This information is current as of 1 October 2012.
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PLANT BIOLOGY
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
www.SCIENCESIGNALING.org
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.
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A
B
H+
Caa2+
Hyperpolarization
ABA
Fusicoccin
High humidity
H2O2
Low CO2
Light
K+
AH2O2
ABA
K+
Low humidity
High CO2
Darkness
pH
Depolarization
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
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-
www.SCIENCESIGNALING.org
ABA
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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
www.SCIENCESIGNALING.org
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
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
DEL
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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
ITC (32)
PYL8/RCAR3
1.0
18 (0.25 ABI1)*
ITC (54)
PYL1/RCAR12
52 and 340
N/A
*Values estimated from IC50 using the indicated relative ratios of PP2C to the receptor.
www.SCIENCESIGNALING.org
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www.SCIENCESIGNALING.org
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)
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
SLAH3
OST2
GORK1
(AtrbohF)
(H+-ATPase
inactive)
(K+ eux
channel opens)
www.SCIENCESIGNALING.org
Anions
K+
REVIEW
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
www.SCIENCESIGNALING.org
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
www.SCIENCESIGNALING.org
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
10
functions with OST2 (109), was not recovered, however. The proton pumps are usually considered to be the endpoints of signaling 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 investigated. Besides PYR/PYL/RCAR, there are
also membrane-associated candidate receptorsGTG1, GTG2 (110), and ABAR
(111, 112)although some have been unable to reproduce the binding of ABA to
ABAR (113).
It is still too early to pronounce whether GTG1 and GTG2 might t the prole
of the outside ABA perception site. Also,
where or how the GTG1 and GTG2 relate
to 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 receptors 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). SLAC1
is 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 efux
in response to ABA (95). The precise relation between these two Ca2+-dependent kinases and S-type anion transporters is not
yet clear, but does reinforce the importance
of Ca2+ in priming or accentuating the response to ABA (43). The calcium-binding
protein NpSCS exerts a suppressing effect
on all SnRK2s tested in vitro, and this inhibition 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 developmental 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
REVIEW
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