Molecular Plant • Volume 6 • Number 2 • Pages 528–538 • March 2013 ReseaRch aRticle
ABA Signaling in Guard Cells Entails a Dynamic
Protein–Protein Interaction Relay from the
PYL-RCAR Family Receptors to Ion Channels
Sung Chul Leea,b,1,2, Chae Woo Limb,2, Wenzhi Lana,c,2, Kai Hea and Sheng Luana,d,1
a Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
b School of Biological Sciences (BK21 Program), Chung-Ang University, Seoul, 156–756, Korea
c Plant Molecular Biology Institute and College of Life Sciences, Nanjing University, China
d WCU Program, Chonnam National University, Gwangju, Korea
ABSTRACT Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trig-
ger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward
anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA
signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we
reconstituted the ABA signaling pathway as a protein–protein interaction relay from the PYL/RCAR-type receptors, to
the PP2C–SnRK2 phosphatase–kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited
PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate
the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular
fluorescence complementation assays were used to verify the interactions among the components in the pathway. These
biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The
SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the
presence of different combinations of signaling components. Further study using transgenic plants overexpressing one
of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.
Key words: abscisic acid; ABA receptor; protein kinase; protein phosphatase; SLAC1.
1 To whom correspondence should be addressed S.C.L.: E-mail sclee1972@
cau.ac.kr, tel. 82–2–820–5207, fax 82–2–825–5206 S.L.: E-mail sluan@berke-
ley.edu, tel. (510) 642–6306, fax (510) 642–4995.
2 These authors contributed equally to this work.
© The Author 2012. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPB and
IPPE, SIBS, CAS.
doi:10.1093/mp/sss078, Advance Access publication 30 August 2012
Received 28 June 2012; accepted 11 July 2012
Plants close their stomata to conserve water in response to
a water-deficit condition. Plant hormone abscisic acid (ABA)
plays a key role in the adaptation to water-deficit conditions
through regulatory pathways that control gene expression
and stomatal closure (Luan, 2002; Zhu, 2002; Wasilewska
et al., 2008). The levels of ABA in plant tissues increase
under drought conditions, and ABA down-regulates guard
cell turgor pressure and thus triggers stomatal closure by
modifying activities of a number of ion channels (Schroeder
et al., 2001). The ion channels targeted by ABA signaling
include K+ channels and anion channels, which control ionic
fluxes across the plasma membrane and tonoplast, thereby
adjusting guard cell turgor pressure (Schroeder et al.,
1987; Schroeder and Hagiwara, 1989; Lemtiri-Chlieh and
MacRobbie, 1994; Negi et al., 2008; Vahisalu et al., 2008). The
process starts with an increasing level of ABA activating anion
efflux through anion channels thus inducing depolarization
of guard cell plasma membrane. Depolarization drives K+
efflux through outward-rectifying K+ channels leading to
water efflux, which reduces guard cell volume and leads to
stomatal closure (Ache et al., 2000; Li et al., 2000; Ward et al.,
To initiate ABA signaling, ABA receptors must exist in
plant cells. Previous studies have identified several types
of putative receptors that may mediate ABA functions
(McCourt and Creelman, 2008; Cutler et al., 2010). Most
recently, a family of START domain proteins, known as
Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel 529
PYR/PYLs-RCARs, were shown to function as ABA recep-
tors (Fujii et al., 2009; Ma et al., 2009; Melcher et al., 2009;
Miyazono et al., 2009; Park et al., 2009). These ABA recep-
tors interact with and inhibit the activity of group A pro-
tein phosphatase 2C (PP2C) (Ma et al., 2009; Santiago et al.,
2009b; Szostkiewicz et al., 2010).
Genetic studies have identified several PP2C genes that
are required for ABA signaling in Arabidopsis. Of these
members, a group A PP2C, including ABI1, ABI2, HAB1,
HAB2, AGH1, and PP2CA, generally function as nega-
tive regulators of the ABA response (Merlot et al., 2001;
Nishimura et al., 2004; Saez et al., 2004; Kuhn et al., 2006;
Rubio et al., 2009). If PP2Cs function as negative regulators
of the ABA signaling pathways, protein kinases are expected
to act as positive regulators in the same pathways (Mustilli
et al., 2002; Yoshida et al., 2006a; Fujii et al., 2007). OST1 is
an Arabidopsis SnRK2-type protein kinase named SnRK2.6.
Several other members in this family, such as SnRK2.2 and
SnRK2.3, are also shown to function in ABA response (Belin
et al., 2006; Yoshida et al., 2006a; Chae et al., 2007; Fujii
et al., 2007). On the contrary to A-type PP2Cs, these three
kinases are positive regulators of ABA signaling during seed
development, germination, and in response to water stress
(Fujii et al., 2007; Fujii and Zhu, 2009; Fujita et al., 2009;
Nakashima et al., 2009).
Downstream from the receptors, PP2Cs, and SnRKs are ion
channels that control stomatal movements (Fujii et al., 2009;
Geiger et al., 2009; Lee et al., 2009). Two groups identified
the gene encoding the guard cell slow anion channel, named
SLAC1, involved in stomatal regulation in Arabidopsis (Negi
et al., 2008; Vahisalu et al., 2008). SLAC1 is required for sto-
matal closure induced by high CO2 and ABA (Vahisalu et al.,
2008) indicating that the SLAC1 protein may represent a criti-
cal component of guard cell anion channels that are respon-
sible for stomatal closure induced by ABA or other signals.
Indeed, recent studies have shown that SLAC1 serves as a
substrate for and is activated by SnRK2.6/OST1 (Geiger et al.,
2009; Lee et al., 2009). Furthermore, OST1 activity against the
SLAC1 protein is inhibited by PP2Cs (Geiger et al., 2009; lee
et al., 2009).
In the present study, we addressed this question using the
Xenopus oocyte expression system to assess SLAC1 activity and
its regulation by ABA receptors (PYL-RCAR), kinases (SnRK2),
and phosphatases (PP2C) in the ABA signaling pathway. The
interactions among the ABA receptor, phosphatases, kinases,
and the ion channel proteins was also examined using an
electrophysiological method as well as yeast two-hybrid and
bimolecular fluorescence complementation (BiFC) assay. Our
data provide evidence for a series of physical protein–pro-
tein interactions starting from ABA receptors that interact
with and inhibit PP2CA. PP2CA regulates kinase activity by
directly interacting with the OST1/SnRK2.6 kinase, which, in
turn, interacts with and phosphorylates the SLAC1 channel
RESuLTS And dISCuSSIOn
Members of the PYL/RCAR Family Physically Interact
with A-Type PP2Cs and Form a Complex network
Previous studies have shown that PYL/RCAR proteins interact
with PP2C A-type family members and inhibit their activities
(Ma et al., 2009; Park et al., 2009). We have shown previ-
ously that PP2CA, an A-type PP2C, interacts with and inhib-
its SnRK2.6/OST1 activity thereby inhibiting the SLAC1 ion
channel. To examine whether PP2CA mediates ABA signaling
through PYL/RCAR-type ABA receptors and to further address
the specificity and overlap of the functions of different fam-
ily members in the PYL/RCAR family, we conducted a com-
prehensive interaction analysis of PP2CA and all PYL/RCAR
We first used the yeast two-hybrid system to screen for
protein–protein interactions between PP2CA and all PYL/
RCAR proteins. Figure 1 shows that PP2CA interacted with a
majority of PYL/RCAR members except RCAR11, 12, and 14
without ABA. In previous reports, ABA is shown to promote
interactions between some PYL members and PP2Cs (Ma
et al., 2009; Santiago et al., 2009a); thus, it is possible that
interactions between PP2CA and RCAR11, 12, and 14 would
occur in the presence of ABA. Therefore, we added 10 µM
ABA to the selection media, which enhanced the interactions
between PP2CA with both RCAR12 and RCAR14, indicating
that PP2CA interacts with these two RCAR members in an
ABA-dependent manner. It is also possible that endogenous
ABA from yeast might be sufficient to promote the RCARs–
PP2CA interaction, because several fungi produce ABA (hirai
et al., 2000; Nambara and Marion-Poll, 2005).
We then used the BiFC procedure in plant cells to con-
firm the protein–protein interactions identified in the yeast
two-hybrid assays. As shown in Figure 1B, co-expression of
PP2CA and RCAR2 in epidermal cells of Nicotiana benthami-
ana generated yellow fluorescence protein (YFP) signals only
in the nucleus identified by the arrows, indicating an inter-
action between the PP2CA and RCAR2. This result supports
the yeast two-hybrid assay for a physical interaction between
PP2CA and RCAR2 proteins. It also suggests that the PP2CA
and RCAR proteins are both targeted to the nucleus, where
they regulate gene expression (Yoshida et al., 2006b).
ABA Receptor RCAR2 Relieves PP2CA Inhibition of
If an interaction between PYL/RCAR and PP2CA mediates
ABA-dependent regulation of channels such as SLAC1,
inhibition of SLAC1 by PP2CA, as reported previously (lee
et al., 2009), should be relieved by the presence of the PYL/
RCAR proteins. We tested the functional relationship between
RCAR2, PP2CA, and the SLAC1 ion channel in the Xenopus
oocyte system by co-expressing different combinations
of the proteins (Figure 2). We first expressed the SLAC1
channel with RCAR2, and found that SLAC1 activity was not
530 Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel
changed significantly (Figure 2B). The SLAC1 channel was
partially inhibited when co-expressed with the PP2CA. We
then co-expressed the SLAC1 channel with both RCAR2 and
PP2CA. The current recorded from these oocytes was larger
than that recorded from oocytes expressing the SLAC1 ion
channel with PP2CA, suggesting that RCAR2 counteracted
the effect of PP2CA (Figure 2A). As an ABA receptor, RCAR2
interaction with PP2CA may be affected by presence of ABA
(Ma et al., 2009; Santiago et al., 2009b). We tested whether
SLAC1 channel activity in the oocyte was affected by ABA.
Figure 2B shows that the currents generated by the oocytes
co-expressing SLAC1, RCAR2, and PP2CA were not significantly
changed by ABA. In addition, currents were unchanged by
ABA when we used RCAR6, which is another RCAR member,
instead of RCAR2. These results suggest that inhibition of
SLAC1 channel activity by PP2CA can be restored partially by
Structural studies of PYL/RCARs revealed two models that
can account for the function of ABA in the ABA–receptor
complex. One model suggests that binding of ABA to RCAR
creates a recognition site for PP2C on the surface of RCAR
to form a tertiary complex (Melcher et al., 2009; Miyazono
et al., 2009; Yin et al., 2009). The other model suggests that
PP2Cs stabilize the RCAR–ABA complex and promote bind-
ing affinity between RCAR and ABA (Ma et al., 2009; Melcher
et al., 2009; Miyazono et al., 2009; Yin et al., 2009). These
models may apply to specific cases of PYL/RCAR interactions
with PP2Cs, and our results here suggest that ABA does not
change the interaction of RCAR2–PP2CA, which is consistent
with the yeast two-hybrid result in Figure 1.
RCAR2 Inhibits PP2CA Activity and Recovers OST1
Kinase Activity towards the Channel Protein Substrates
According to previous studies, PYL/RCAR proteins inhibit the
type-A sub-family of plant PP2Cs in the ABA signaling path-
way (Ma et al., 2009; Park et al., 2009). In addition, RCAR
family genes are associated with activating SnRK2-type
kinases (Park et al., 2009). Several studies have shown that
PYL/RCAR-mediated activation of SnRK2 kinases results from
inhibition of PP2Cs (Fujii et al., 2009; Umezawa et al., 2009;
Vlad et al., 2009). Our previous study also showed that PP2CA
interacts with OST1 and inhibits its kinase activity towards
its substrates such as SLAC1 (Lee et al., 2009). It is becom-
ing clear that RCAR2 may relieve SLAC1 inhibition by PP2CA
by activating SnRK2.6/OST1, which is normally inhibited by
PP2CA. To confirm this, we first tested whether RCAR2 would
inhibit PP2CA activity using an in vitro protein phosphatase
assay. When expressed and purified from E. coli, phosphatase
activity corresponding to full-length PP2CA was indeed inhib-
ited by RCAR2 (Figure 3A). RCAR2 inhibited PP2CA activity
with a half maximal inhibitory concentration (IC50) value of
After finding that RCAR2 inhibits PP2CA activity, we
determined whether RCAR2 affects OST1 kinase activity, which
is normally inhibited by PP2CA using an in vitro kinase assay
(Figure 3B). Based on our previous study on OST1 (Lee et al.,
2009), this kinase displays autophosphorylation activity. We
observed auto-kinase activity of OST1 without including any
other substrate in the assay and this activity was abolished by
adding PP2CA (Figure 3B). Interestingly, RCAR2 did not appear
to affect OST1 inhibition by PP2CA, despite the observation
that RCAR2 interacted with and inhibited PP2CA activity.
When the OST1 substrate SLAC1 N-terminus was present, OST1
phosphorylation of SLAC1 was also abolished by the presence
of PP2CA (Figure 3B). However, OST1 partially phosphorylated
SLAC1 by addition of RCAR2 to the reaction containing PP2CA,
OST1, and the SLAC1 N-terminus. In other words, RCAR2 did
not seem to affect PP2CA inhibition of OST1 auto-kinase
activity but partially alleviated the PP2CA inhibition of
SLAC1 phosphorylation by OST1. The proportion of SLAC1
phosphorylation restored by RCAR2 was rather low, suggesting
Figure 1. Physical Interactions among PP2CA and RCARs.
(A) Yeast two-hybrid assay of interactions between PP2CA and RCARs.
Growth on the selection medium (SC-ALTH) was used as an indicator
of interaction (left row) and with 10 µM ABA (middle row). Growth in
SC-LT was used as control (right row).
(B) Bimolecular fluorescence complementation (BiFC) assay of interac-
tions among PP2CA and RCAR2. The fluorescence indicates interaction
between the indicated partner proteins. The images were obtained
from the YFP channel or bright field or a merged picture of the two.
PP2CA-35S-SPYCE(M) co-expressed with RCAR2-35S-SPYNE(R)173.
Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel 531
that the physical interaction between RCAR2 and PP2CA may be
important to SLAC1 phosphorylation. On the contrary, PP2CA
inhibition of OST1 activity against SLAC1 was very strong and
PP2CA may use OST1 as a substrate for its phosphatase activity.
The question is whether PP2CA inactivates the kinase by
dephosphorylating OST1, or by a simple physical interaction
with the kinase without an enzyme–substrate relationship.
Earlier studies seem to support both possibilities (Lee et al.,
2009; Umezawa et al., 2009; Vlad et al., 2009). Using a simple in
vitro kinase assay, we determined whether PP2CA phosphatase
activity is important in regulation of OST1 auto-kinase activity
and activity against the SLAC1 N-terminus. Figure 3C shows
that inclusion of PP2CA and its phosphatase-dead form, which
is a PP2CA mutant protein with two amino acid mutations
that eliminates PP2C activity, effectively abolished both
auto-kinase activity (lanes 2 and 3) and activity towards the
SLAC1 N-terminal domain (lanes 7 and 8). However, when
we added PP2CA or its dead form after the kinase reaction,
OST1 autophosphorylation was not altered by PP2CA (lanes
4 and 5), indicating that PP2CA does not dephosphorylate
phosphorylation by OST1, adding PP2CA after the kinase
assay slightly reduced the level of SLAC1 phosphorylation
(lane 9). Such a reduction seemed to be related to PP2CA
phosphatase activity, because the phosphatase-dead form of
PP2CA showed less of an effect on SLAC1 phosphorylation
level (lane 10). Thus, PP2CA physically interacts with SLAC1
(Lee et al., 2009) and can dephosphorylate SLAC1 to a certain
extent. Nevertheless, inhibition of OST1 activity by PP2CA
does not seem to be associated with OST1 dephosphorylation
in this in vitro assay system. These results suggest that PP2CA
may interact directly and inhibit OST1 kinase activity. Taken
together, our data suggest that the RCAR2 protein physically
interacts with PP2CA, which may break the inhibitory bondage
between PP2CA and OST1 leading to activation of OST1 kinase.
The nature of ‘inhibitory bondage’ of PP2CA and OST1 may
include both physical interaction and enzyme modification.
Dynamic protein–protein interactions may determine the
localization and function of signaling components in the ABA
OST1. In the case of SLAC1
Figure 2. RCARs Regulate PP2CA Inhibition on
(A) RCAR2 recovers SLAC1 activity inhibited
by PP2CA. (Left) Typical whole-cell current
traces recorded from the oocytes injected
with water (Control) or the oocytes injected
with cRNA of RCAR2, SLAC1, SLAC1+PP2CA,
or SLAC1+PP2CA+RCAR2. The current was
recorded by the voltage steps of 50 to −150 mV
(in 15-mV decrements, 7.5-s duration) with a
1.45-s prepulse to 0 mV. Dotted lines represent
zero current level. (Right) The current–volt-
age relationship was deduced from the record-
ings of the control oocytes and the oocytes
expressing RCAR2, SLAC1, SLAC1+PP2CA, and
(B) Exogenous ABA does not change SLAC1
activity regulated by PP2CA+RACR2 or by
PP2CA+RCAR6. The relative value was calcu-
lated as summarized currents at –150 mV gen-
erated by various combinations/summarized
currents at –150 mV generated by SLAC1. Mean
current (± SE) at −150 mV recorded from the
oocytes injected with various combinations of
cRNA including SLAC1 alone, SLAC1+RCAR2,
532 Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel
Previous studies (Geiger et al., 2009; Lee et al., 2009) and
results in this report have demonstrated the importance of
physical interactions among the signaling components dur-
ing ABA regulation of ion channel activity. Each component
often interacts with multiple protein partners at the same
time, and yet these components function in different plant
cell compartments. How do these dynamic interactions
among partner proteins change the subcellular location and
potential function of various components? We tried to pro-
vide some clues for this question using multiple protein–pro-
tein interaction assays to dissect the relationships. First, we
tested whether RCAR2 inhibits the OST1–PP2CA interaction
in a multi-BiFC assay among RCAR2, PP2CA, and OST1. The
interaction between RCAR2 and PP2CA or PP2CA and OST1
generated CFP and YFP signals in the nucleus, respectively
(Figure 4A), indicating that RCAR2 may not have a major
effect on the interaction between PP2CA and OST1.
In the case of PP2CA–OST1–SLAC1 interactions, it was
interesting to observe both nucleus and plasma membrane
localization of the fluorescence, depending on the partner
(Figure 4B). While PP2CA and OST1 formed a complex in the
nucleus, OST1 and SLAC1 interacted at the cell surface where
SLAC1 is located. Both complexes formed simultaneously,
indicating multiple subcellular locations for the OST1 protein.
Along the same line, when OST1 and PP2CA each paired
with SLAC1, both the phosphatase and the kinase were
localized to the plasma membrane, forming a complex with
the SLAC1 channel (Figure 4C). These results show that the
direct interaction of PP2CA with OST1 localized in the nucleus
but that the PP2CA–SLAC1–OST1 complex may be localized
to the plasma membrane. It has become clear that multiple
locations of individual signaling components can be achieved
by interaction with different partner proteins and that such
interactions can take place simultaneously in plant cells.
Overexpression of RCAR2 Confers Transgenic Plants
with ABA Hypersensitivity and drought Tolerance
The presence of RCAR2 antagonized PP2CA inhibition of
SnRK2 in the kinase and SLAC1 activation assays. To deter-
mine whether RCAR2 overexpression in planta would alter
ABA signaling and sensitivity, we generated transgenic plants
overexpressing the coding sequence of the RCAR2 gene
under the control of a strong constitutive 35S promoter.
RCAR2 expression level was undetectable in wild-type plants
under the conditions used in this study. The overexpressing
lines showed significant transcripts levels (Figure 5A). The
RCAR2 transgenic lines and wild-type plants showed signifi-
cantly different phenotypic growth (Figure 5B). The rosettes
of the transgenic lines had slightly smaller stature under nor-
mal growth conditions without significant changes in flower-
ing or seed production.
When we analyzed the ABA responses, the transgenic lines
were ABA-hypersensitive during seed germination and early
seedling growth (Figure 6). Germination rates of untreated
seeds did not differ between wild-type and transgenic lines.
On medium containing 0.5 µM ABA, 90% of the wild-type
seeds germinated within 3 d, whereas the seeds from trans-
genic lines took 5–6 d to reach the same rate. Treatment
Figure 3. In Vitro Dephosphorylation and Phosphorylation Assay with
OST1, SLAC1, PP2CA, and RCAR2.
(A) In vitro dephosphorylation assay of PP2CA with RCAR2. Increasing
amounts of RCAR2 were added to a phosphatase reaction with 10 ng
PP2CA. RCAR2 inhibits the protein phosphatase activity of PP2CA.
(B) RCAR2 affects SLAC1 phosphorylation via OST1 with PP2CA.
(C) PP2CA and PP2CA null mutant inhibits OST1 auto-kinase activity
and activity against SLAC1. Following incubation for 30 min at 30°C,
PP2CA or PP2CA null mutant was added and incubated for 30 min at
30°C in lane 4, 5, 9, or 10. The contents of the kinase assays are shown
at the top of the autoradiography picture. The 32P-labeled protein
bands are indicated by arrows and names of the proteins at the left
side. The presence and absence of the proteins are indicated by +/–
Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel 533
with 1.0 µM ABA significantly inhibited seed germination
in both wild-type and transgenic seeds. However, germina-
tion was again clearly more inhibited in the transgenic seeds
(Figure 6A). In addition, the transgenic lines were also more
sensitive to lower levels of ABA including 0.3 and 0.5 µM at
the seed germination stage (Figure 6B). These results show
that RCAR2 overexpression increases ABA sensitivity in the
We also examined the drought response of adult plants in
the wild-type and transgenic lines (Figure 7). When grown
under a well-watered condition, wild-type and transgenic
plants did not show any significant phenotypic differences.
However, after drought treatment by withholding watering,
the transgenic lines exhibited enhanced drought tolerance
compared to that in wild-type plants (Figure 7A). The
transgenic plants became less wilted as compared to wild-
type plants (Figure 7A), suggesting a decreased transpiration
rate in transgenic plants. We determined the transpiration
rate by measuring water loss from detached rosette leaves
(Figure 7B). The fresh-weight loss of leaf tissues was greater
in wild-type plants compared to that in the transgenic lines
To determine whether lower levels of transpiration rate
resulted from ABA hypersensitivity in transgenic plants, we
performed stomatal assay and found that the transgenic
plants showed smaller stomatal apertures after plants
were treated with ABA. Stomatal apertures in RCAR2-1 and
RCAR2-2 were reduced to 66% and 58%, respectively, upon
treatment with 10 µM ABA. In wild-type plants, the stomatal
aperture was reduced to 77% (Figure 7C). These results indi-
cate that RCAR2 overexpression increases ABA sensitivity of
Figure 5. Construction of RCAR2-overexpressing Transgenic Plants.
(A) RT–PCR analysis of RCAR2 gene expression from wild-type (Col-0)
and transgenic lines.
(B) Growth characteristics of the Arabidopsis transgenic plants overex-
pressing RCAR2. The plant phenotypes of 2-week-old wild-type (Col-0)
and transgenic lines.
Figure 4. Multicolor BiFC Assay among RCAR2,
PP2CA, OST1, and SLAC1.
(A) Simultaneous visualization of RCAR2/PP2CA
(cyan) and OST1/PP2CA (yellow);
(B) PP2CA/OST1 (cyan) and SLAC1/OST1 (yellow);
(C) PP2CA/SLAC1 (cyan) and OST1/SLAC1 (yellow).
The overlay of the cyan and yellow channels and
the bright field image are depicted below the
534 Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel
guard cells, leading to reduced water loss under drought
conditions. The ABA responses in RCAR2 transgenic plants
were completely sensitive in seeds and vegetative tissues,
indicating that RCAR2 may function as a positive regulator
of ABA. Interestingly, these results are in agreement with
the phenotypes of the triple hab1-1 abi1-2 pp2ca-1 mutant
(Rubio et al., 2009). These results again support the same
principle: RCAR2 function serves to remove the brake to
ABA signaling imposed by PP2CA. Taken together, these
observations are consistent with earlier studies (Santiago
et al., 2009b; Umezawa et al., 2009; Nishimura et al., 2010)
and with the biochemical and electrophysiological work
performed in this study: PYR/PYL/RCAR-type ABA receptors
activate ABA responses by inhibiting PP2C A-type phos-
phatases, leading to activation of SnRK2-type kinases to
phosphorylate downstream targets including SLAC1, which
controls guard cell turgor and stomatal aperture.
Previous studies (Geiger et al., 2009; Lee et al., 2009) sug-
gested that PP2C-type phosphatases lie upstream of the
SnRK2-type kinase that, in turn, interact with and activate the
SLAC1 channel to control the ABA response in guard cells. In
this study, we added the ABA receptors to this regulation and
reconstituted the complete pathway that leads ABA signal
to ion channel regulation and stomatal closure as proposed
in Figure 8. Under normal conditions, PP2CA inhibits SLAC1
channel activity by inactivation of OST1. When ABA levels
are increased by environmental cues and PYL/RCAR levels
are also increased, more ABA may bind to PYL/RCAR, induc-
ing the formation of additional PYL/RCAR–PP2C complex
and thus breaking the PP2C–OST1 complex to release active
OST1 kinase. The OST1 kinase interacts with and activates the
SLAC1 ion channel to mediate efflux of anions and decrease
of turgor pressure.
Several questions remain unresolved in this model.
First, it is still not clear how ABA triggers this signaling
pathway. In studies to date, PYL/RCAR proteins directly
associate with PP2Cs without ABA. It will be important to
determine the condition and concentration dependence
of ABA that modulates interaction of PYL/RCAR–PP2C.
Second, the mechanism of OST1 inactivation by PP2Cs
remains undetermined. Several reports have suggested
that phosphatase activity is required for inactivation of
SnRK2-type kinases (Umezawa et al., 2009; Vlad et al., 2009).
Results from our studies suggest that both phosphatase
activity and a physical interaction are important for PP2CA
function in ABA signaling. Phosphatase activity is required
for dephosphorylating and inactivating the SLAC1 ion
channel (Figures 2 and 3). On the other hand, physical
interactions may play a more important role in the inhibition
of OST1 kinase activity. However, it is difficult to explain
how physical interaction alone could effectively inactivate
Considering the diversity of the PYL/RCAR family, which
consists of 14 members and at least two subfamilies, func-
tional redundancy and specificity are complex. Furthermore,
group A PP2Cs and SnRK2-type kinases are also composed
of multiple members. How these different phosphatases
and kinases combine with PYL/RCAR proteins in a particular
cell type or at a certain developmental stage of plants will
provide a challenging area of research for years to come.
Figure 6. Enhanced Tolerance of the
RCAR2-ox Transgenic Lines to Abscisic Acid
(A) Seed germination of wild-type and trans-
genic lines exposed to ABA. Seeds were ger-
minated on MS agar plates containing 0, 0.5,
or 1.0 µM ABA. Data are the means ± standard
deviation from three independent experi-
ments each evaluating 100 seeds.
(B) Phenotype of wild-type and transgenic
plants exposed to ABA. Photographs show
plants after exposure to 0, 0.3, and 0.5 µM
ABA for 7 d.
Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel 535
Yeast Two-Hybrid Analysis
Each construct was built by cDNA fragments amplified by PCR
and cloned into the pGBKT7 and pGADGH vectors. The lithium
acetate method was used to introduce BD and AD plasmids
into yeast strain AH109 (Ito et al., 1983). Yeast two-hybrid
assays were performed as before (Li et al., 2006; Lee et al.,
2007). Transformants were selected in SC-Leucine-Tryptophan
media and transferred on the interaction selection media
(SC-Adenine-Histidine-Leucine-Tryptophan) to score growth
as an indicator of protein–protein interaction. For serial dilu-
tion assay, exponentially grown yeast cells were harvested
and adjusted to OD600 = 0.5 with sterilized double-distilled
water and diluted to 1/10, 1/100, and 1/1000. Yeast cells,
2 µl, were spotted onto SC-Leucine-Tryptophan media and
SC-Adenine-Histidine-Leucine-Tryptophan media with or
without 10 µM ABA.
Bimolecular Fluorescence Complementation
To generate the BiFC constructs, RCAR2, SLAC1, OST1, and
PP2CA full-length cDNA with no stop codon were sub-
cloned via SpeI/SalI into 35S-SPYNE(R)173, 35S-SCYNE(R)173,
35S-VYNE, 35S-SPYCE(M), and 35S-SCYCE(M) vectors (Waadt
et al., 2008). For transient expression, the Agrobacterium
tumefaciens strain GV3101 carrying each construct was used
together with the p19 strain for infiltration of 5-week-old
Nicotiana benthamiana leaves. For microscopic analyses, leaf
discs were cut 4 d after infiltration. The lower epidermis cells
were analyzed by confocal microscopy (model Zeiss 510 UV/
Vis Meta) operated with LSM Image Browser software.
Expression and Purification of GST-Fusion Proteins in
Escherichia coli and Kinase Assay
To produce GST fusion proteins in E. coli, RCAR2, OST1, PP2CA,
and the N-terminus of SLAC1 were cloned into pGEX4T-1
vector. All GST fusion constructs were transformed into E. coli
strain BL21 (DE3) cells. Protein expression and purification of
GST fusion protein were performed as described earlier (li
et al., 2006).
For the kinase assays, the buffer contained 20 mM Tris
HCl (pH 7.5), 2.5 mM MnCl2, 2.5 mM MgCl2, 1 mM CaCl2,
and 1 mM DTT. Total volume of 40 µl included 7.5 µCi of
[γ-32P]ATP and the protein combinations (1 µg of the each
protein) indicated in the figure legends. Following incuba-
tion for 60 min at 0°C, the reaction was stopped by add-
ing 12.5 µl of 5X Laemmli buffer; 20 µl of the mixture was
then separated by SDS/PAGE using a 10% (w/v) acrylamide
gel. The gel was dried and 32P was detected by autoradiog-
raphy using a Typhoon 8600 imager (Molecular Dynamics,
Protein phosphatase activities of PP2CA were performed
by using ProFluor Ser/Thr PPase assay kit (Promega, WI, USA)
according to the manufacturer’s protocol as described ear-
lier (Santiago et al., 2009b). Briefly, phosphatase assays were
performed in a 100-µl reaction volume containing 25 mM
Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, and 10 ng PP2CA
Figure 7. Enhanced Tolerance of the RCAR2-ox Transgenic Lines to
(A) Growth of wild-type and transgenic plants after dehydration for
(B) Stomatal movements in RCAR2-ox transgenic lines are hypersen-
sitive to ABA. Stomatal apertures were measured under the micro-
scope in wild-type and RCAR2-ox transgenic lines. Data are the
means ± standard errors.
(C) Water loss from leaves of wild-type and transgenic plants at various
times after detachment of leaves.
536 Lee et al. • ABA Signal Relay from ABA Receptor to Ion Channel
The genes used in the electrophysiological assay were cloned
into the pGEMHE oocyte expression vector. The preparation
and determination of cRNA concentration prepared by the
mMESSAGE mMACHINE T7 RNA transcription kit were per-
formed as previously described (Li et al., 2006; Lee et al.,
2007). Freshly isolated Xenopus oocytes were injected with
23 nl of cRNA and used for voltage-clamp experiments 2 d
after injection. Two-electrode voltage-clamp recordings were
performed to measure SLAC1 currents. The pipette solution
contained 3 M KCl. To test SLAC1 currents, we first used a
modified ND96 solution in which NaCl was replaced by CsCl
as the bath solution (containing 96 mM CsCl, 1 mM MgCl2,
1.8 mM CaCl2, 2 mM KCl, 5 mM HEPES, pH 7.5 adjusted with
NaOH). The current was recorded by the voltage steps of 50 to
−150 mV (in 15-mV decrements, 7.5-s duration) with a 1.45-s
prepulse to 0 mV. The summarized data of the SLAC1 currents
were generated from the pooled currents at 1.6 s of each
voltage-clamp episode. Data are presented as representative
recordings or as mean ± SE of n observations with three rep-
etitions, in which n is the number of samples. Statistical com-
parisons were made using either Student’s paired or unpaired
t-tests as appropriate, and differences were considered to be
significant at p < 0.05.
Plant Expression Vector Construction and Arabidopsis
To induce constitutive expression of the RCAR2 gene under
the control of the CaMV 35S promoter, the binary vector
pBIN35S was used to generate a plasmid for Arabidopsis
transformation. The full-length RCAR2 cDNA sequence
cloned into pBIN35S. The recombinant plasmids were verified
by sequencing. The binary plasmids were introduced into the
Agrobacterium tumefaciens strain GV3101 via electropora-
tion. Arabidopsis transformation with the RCAR2 gene was
carried out using the floral dipping method (Clough and
Bent, 1998). For selection of RCAR2-ox transgenic lines, seeds
harvested from the putative transformed plants were plated
on MS agar plates containing 50 µg ml–1 kanamycin.
Assays of drought Tolerance
Three-week-old seedlings from the wild-type and RCAR2-ox
transgenic lines were randomly planted in a tray contain-
ing soil mix (peat moss, perlite, and vermiculite, 9:1:1).
Dehydration stress was imposed plants by withholding water-
ing. To determine the drought tolerance in a quantitative
manner, leaves were detached from each plant and placed
in Petri dishes. The dishes were kept in a growth chamber
with 40% relative humidity, and the loss of fresh weight was
determined at the indicated times.
For stomatal aperture bioassays, four rosette leaves from
4-week-old plants were detached and floated in stomatal
opening solution (SOS: 50 mM KCl and 10 mM MES-KOH,
pH 6.15, 10 mM CaCl2) in the light as described by Cheong
et al. (2007). After 2.5 h, buffer was replaced with SOS con-
taining ABA of various concentrations. After 2.5-h further
incubation, 60 stomata were measured in each individual
sample, and each experiment was performed in triplicate.
This work is supported by a grant from the Biogreen21 Program
(PJ008222) Rural Development Administration and the Research
Foundation of Korea (NFR) grant funded by the Korea gov-
ernment (No. 2011–0007600) (to S.C.L.), and the US National
Science Foundation and Korean WCU Program of National
Research Foundation (to S.L.). No conflict of interest declared.
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