Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dδ to transduce hydrogen peroxide signals in the Arabidopsis response to stress.

Abstract

Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases (GAPCs) interact with the plasma membrane-associated phospholipase D (PLDδ) to transduce the ROS hydrogen peroxide (H(2)O(2)) signal in Arabidopsis thaliana. Genetic ablation of PLDδ impeded stomatal response to abscisic acid (ABA) and H(2)O(2), placing PLDδ downstream of H(2)O(2) in mediating ABA-induced stomatal closure. To determine the molecular link between H(2)O(2) and PLDδ, GAPC1 and GAPC2 were identified to bind to PLDδ, and the interaction was demonstrated by coprecipitation using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence complementation. H(2)O(2) promoted the GAPC-PLDδ interaction and PLDδ activity. Knockout of GAPCs decreased ABA- and H(2)O(2)-induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDδ rendered plants less responsive to water deficits than the wild type. The results indicate that the H(2)O(2)-promoted interaction of GAPC and PLDδ may provide a direct connection between membrane lipid-based signaling, energy metabolism and growth control in the plant response to ROS and water stress.

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Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases
Interact with Phospholipase Dd to Transduce Hydrogen
Peroxide Signals in the Arabidopsis Response to Stress
C W
Liang Guo,a,bShivakumar P. Devaiah,a,b,1Rama Narasimhan,a,b,2Xiangqing Pan,a,b,3Yanyan Zhang,a,b,4
Wenhua Zhang,cand Xuemin Wanga,b,5
aDepartment of Biology, University of Missouri, St. Louis, Missouri 63121
bDonald Danforth Plant Science Center, St. Louis, Missouri 63132
cCollege of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University,
Nanjing 210095, People’s Republic of China
Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators
in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases
(GAPCs) interact with the plasma membrane–associated phospholipase D (PLDd) to transduce the ROS hydrogen peroxide
(H2O2) signal in Arabidopsis thaliana. Genetic ablation of PLDd impeded stomatal response to abscisic acid (ABA) and H2O2,
placing PLDd downstream of H2O2in mediating ABA-induced stomatal closure. To determine the molecular link between
H2O2and PLDd, GAPC1 and GAPC2 were identified to bind to PLDd, and the interaction was demonstrated by coprecipitation
using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence
complementation. H2O2promoted the GAPC–PLDd interaction and PLDd activity. Knockout of GAPCs decreased ABA- and
H2O2-induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDd rendered plants less responsive
to water deficits than the wild type. The results indicate that the H2O2-promoted interaction of GAPC and PLDd may provide
a direct connection between membrane lipid–based signaling, energy metabolism and growth control in the plant response
to ROS and water stress.
INTRODUCTION
Reactive oxygen species (ROS) are produced in plants in re-
sponse to a wide variety of stresses, including drought, UV
irradiation, high light, wounding, ozone, low and high temper-
atures, and pathogens (Desikan et al., 2001; Apel and Hirt, 2004;
Suzuki et al., 2012). ROS were originally viewed as by-products
of metabolic pathways, and a high concentration of ROS is toxic
to the cells (Apel and Hirt, 2004; Quan et al., 2008; Finkel, 2011).
It has now been well documented that ROS are generated as
signals that alter various cellular and physiological processes in
plant growth and development (Desikan et al., 2001; Apel and
Hirt, 2004; Gechevet al., 2006; Shao et al., 2008). Hydrogen
peroxide (H2O2) is the major and most stable species of ROS
and plays a signaling role in plant response to stresses, such as
mediating abscisic acid (ABA)–regulated stomatal closure (Pei
et al., 2000; Zhang et al., 2001). H2O2is thought to affect target
protein activities through modification of thiol groups of Cys
residues (Hancock et al., 2005). However, it is unclear how such
oxidative modification affects a signaling cascade that leads to
alteration of cellular function and plant stress responses.
Recent studies indicate that phospholipase D (PLD) and its
product phosphatidic acid (PA) play a role in ROS-mediated
signaling (Sang et al., 2001; Yamaguchi et al., 2004; Zhang et al.,
2009; Lanteri et al., 2011). The Arabidopsis thaliana genome
contains 12 PLDs, PLDa(3), b(2), g(3), d, e, and z(2), and these
PLDs exhibit distinguishable biochemical properties and cellular
functions. Knockout (KO) of PLDa1 decreases the production of
ROS, and addition of PA induces recovery of ROS levels in the
PLDa1 mutant (Sang et al., 2001). PA interacts with NADPH
oxidase and increases its activity and ROS production (Zhang
et al., 2009). PLD and PA are also implicated in promoting the
elicitor-induced generation of ROS in suspension rice (Oryza
sativa) and tomato (Solanum lycopersicum) cells (Yamaguchi
et al., 2004; Lanteri et al., 2011). On the other hand, H2O2-
induced activation of PLD enhances elicitor-induced biosynthesis
of phytoalexins in rice cells (Yamaguchi et al., 2004). Plasma
membrane–associated PLDd is activated by H2O2, and ablation of
it renders Arabidopsis cells more sensitive to H2O2-promoted
programmed cell death than the wild type (Wang and Wang,
2001; Zhang et al., 2003, 2005; Wang et al., 2006). These results
1Current address: Arkansas Biosciences Institute, Arkansas State
University, State University, AR 72467.
2Current address: Department of Crop Physiology, University of Agri-
cultural Sciences, Bangalore 560065, India.
3Current address: Solae LLC, 4300 Duncan Avenue, St. Louis, MO
63110.
4Current address: College of Journalism and Food Science, Shanghai
Business School, Shanghai 200235, China.
5Address correspondence to swang@danforthcenter.org.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Xuemin Wang (swang@
danforthcenter.org).
CSome figures in this article are displayed in color online but in black and
white in the print edition.
WOnline version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.111.094946
The Plant Cell, Vol. 24: 2200–2212, May 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

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suggest that whereas PLDa1 promotes the ROS production,
PLDd mediates plant responses to ROS. However, it is unknown
how H2O2activates PLDd and whether PLDd is involved in me-
diating the H2O2effect in the ABA signaling.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cata-
lyzes the conversion of glyceraldehyde-3-phosphate to 1,3-
bisphosphoglycerate in the glycolytic pathway, thus functioning
to produce energy and supply intermediates for cellular metab-
olism (Plaxton, 1996). The Arabidopsis genome contains seven
phosphorylating GAPDHs, five of which are located in plastids,
whereas GAPC1 and GAPC2 are in the cytosol (Rius et al., 2008;
Muñoz-Bertomeu et al., 2010). GAPDHs have been implicated in
embryo development, pollen development, root growth, and ABA
signal transduction (Rius et al., 2006, 2008; Muñoz-Bertomeu
et al., 2009, 2010, 2011). The catalytic Cys residues of GAPDH
can be oxidized by oxidants such as H2O2, leading to fully or
partially reversible inactivation of GAPDH (Hancock et al.,
2005; Hara et al., 2005; Holtgrefe et al., 2008). GAPC1 has
been suggested to be a H2O2target potentially involved in
mediating ROS response in Arabidopsis (Hancock et al., 2005;
Holtgrefe et al., 2008). Here, we show that GAPC1 and GAPC2
bind to PLDd, that H2O2promotes the GAPC interaction with
PLDd, and that the interaction mediates plant response to ABA
and water deficits.
RESULTS
Ablation of PLDd Compromises ABA- and H2O2-Induced
Stomatal Closure, but Not ABA-Promoted H2O2Production
To determine if PLDd is activated by ABA, we isolated PLDa1
PLDd double KO plda1 pldd (see Supplemental Figure 1 online)
and assayed the PLD activity in response to ABA in wild-type,
plda1, pldd, and plda1 pldd using 1-palmitoyl-2-{12-[(7-nitro-2-
1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phos-
phocholine (NBD-PC)–labeled protoplasts (Figure 1A). The PLDa1
KO mutant was used because PLDa1 was reported to be re-
sponsible for a majority of PA produced in response to ABA
(Zhang et al., 2009). PA production was increased twofold after
wild-type protoplasts were incubated with ABA for 20 min (Figure
1A). The ABA-induced PA production in plda1 and pldd was ;62
and 28% lower, respectively, than in the wild type. No significant
PA increase was observed in response to ABA in PLDa1 PLDd
double KO cells (Figure 1A). The results indicate that in addition to
PLDa1, PLDd is also activated by ABA and that PLDa1 and PLDd
together account for virtually all ABA-induced PLD activity, with
PLDa1 providing twice as much PA as PLDd in response to ABA
in Arabidopsis.
To determine the role of PLDd in ABA response, we in-
vestigated whether the loss of PLDd alters ABA-promoted sto-
matal closure and H2O2production in guard cells. pldd leaf peels
exhibited decreased sensitivity to ABA-promoted stomatal clo-
sure (Figure 1B), a response similar to plda1 (Zhang et al., 2004;
Zhang et al., 2009). H2O2has been shown to induce stomatal
closure in plda1 (Zhang et al., 2009). However, H2O2failed to
induce stomatal closure in pldd (Figure 1B). Introduction of PLDd
driven by its own promoter into pldd restored the phenotype for
both ABA- and H2O2-induced stomatal closure, indicating that
loss of PLDd is responsible for the ABA and H2O2response
phenotype (Figure 1B). In addition, unlike plda1, which decreased
ABA-promoted H2O2production (Zhang et al., 2009), KO of PLDd
did not affect the ABA-induced H2O2production. The basal level
of ROS in pldd and wild-type cells were also similar, as revealed
by the fluorescent dye 29,79-dichlorofluorescin diacetate (H2DCF-
DA) intensity (Figures 1C and 1D). These results indicate that
PLDd is not required for ABA-induced H2O2production but is
involved in stomatal response to ABA and H2O2. The data
suggest that PLDd acts downstream of H2O2in signaling ABA-
induced stomatal closure.
Direct Interaction between GAPC and PLDd
To determine how PLDd is involved in the H2O2response, we
incubated purified PLDd with H2O2and the treatment had no
impact on enzyme activity (Zhang et al., 2003). The transcript
level of PLDd was not increased after ABA treatment for 40 min
(see Supplemental Figure 2 online). These data indicate that the
ABA-induced activation of PLDd in the early phase is not me-
diated by increased PLDd expression or the direct effect of H2O2
on PLDd. To test whether a protein is involved in the H2O2ac-
tivation of PLDd, we investigated the potential interaction of
PLDd with GAPC, because GAPC was reported as a direct
target of H2O2in Arabidopsis (Hancock et al., 2005; Holtgrefe
et al., 2008). His-tagged GAPC1 was expressed in Escherichia
coli and incubated with microsomal proteins from Arabidopsis
leaves, and immunoblotting with PLDd antibodies detected PLDd
in the GAPC1 coprecipitate (see Supplemental Figure 3 online).
To verify the interaction, we purified His-tagged GAPC1 and
GAPC2 proteins expressed in E. coli (see Supplemental Figure
4A online) and used them for reciprocal pulldown with gluta-
thione S-transferase (GST)-PLDd. GAPC2 pulled down PLDd.
PLDd also pulled down GAPC1, as indicated by immunoblotting
with anti-His or anti-GST antibodies (Figure 2A). In addition, the
association of GAPCs and PLDd was increased in the presence
of H2O2but decreased in the presence of the reducing reagent
DTT (Figure 2A). To further validate the interaction, we coex-
pressed GAPC and PLDd in yeast (see Supplemental Figure
4B online) and grew the yeast cells with or without H2O2.
GAPC1 and GAPC2 were detected in the complex with PLDd
when PLDd was immunoprecipitated with FLAG antibody.
PLDd also associated with GAPC1 or GAPC2 when GAPCs
were immunoprecipitated with cMyc antibody. The presence
of H2O2promoted the interaction between GAPC and PLDd
(Figure 2B). These results indicate that the GAPC–PLDd in-
teraction is enhanced in an oxidative but weakened in a re-
ducing environment.
To quantify the interaction between GAPC1 and PLDd, we
used surface plasmon resonance (SPR) to determine the binding
kinetics. Purified GAPC1 was immobilized on an nitrilotriacetic
acid (NTA) chip followed by injection of purified GST or GST-
PLDd. The representative sensorgram showed an increase in
response unit (RU) when GST-PLDd, but not GST, was injected,
indicating that PLDd interacts with GAPC1 (Figure 2C). When
H2O2-treated GAPC1 was used, the GAPC1–PLDd interaction
was enhanced as RU was higher than when GAPC1 was not
GAPC and PLD in Reactive Oxygen Species and Stress Signaling2201

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incubated with H2O2(Figure 2C). H2O2-treated or untreated
GAPC1 displayed comparable association rate constants (Ka=
8.19 3 104M21s21versus 8.33 3 104M21s21). However, the
dissociation rate constant was lower when GAPC1 was exposed
to H2O2(Kd= 5.52 3 1024s21versus 3.23 3 1023s21). The
maximum specific binding is 1564 RU for H2O2-treated GAPC1
and 286 RU for GAPC1 without H2O2treatment (Figure 2C). The
equilibrium binding constant KDis 6.62 3 1029M for GAPC1–
PLDd interaction in the presence of H2O2and 3.94 3 1028M for
GAPC1–PLDd interaction without H2O2. The results indicate that
the GAPC1–PLDd interaction is significantly enhanced by H2O2
and that H2O2stabilizes the interaction by decreasing dissoci-
ation between GAPC1 and PLDd.
To visualize the GAPC–PLDd interaction in plant cells, we
used bimolecular fluorescence complementation (BiFC) that brings
together two yellow fluorescent protein (YFP) fragments fused
to two interacting proteins (Walter et al., 2004). GAPC1 or
GAPC2 was fused to the N terminus of YFP (GAPC1-YFPNor
GAPC2-YFPN), and PLDd was fused to the C terminus of YFP
(PLDd-YFPC). These constructs were cointroduced into tobacco
leaves. No fluorescence was observed when empty vectors
YFPNand YFPCwere cotransformed or when GAPC-YFPNand
PLDd-YFPCwere transformed separately (see Supplemental
Figure 5 online). In the positive controls, bZIP63-YFPNand bZIP63-
YFPC, the transcription factor, formed dimers and brought
YFPNand YFPCtogether to generate fluorescence in the nu-
cleus (see Supplemental Figure 5 online). GAPC1-YFPNor
GAPC2-YFPNcoexpressed with PLDd-YFPCproduced fluo-
rescence in the cell, indicating that both GAPCs interacted with
PLDd (Figure 2D).
Figure 1. Decreased Response of pldd Plants to H2O2and ABA.
(A) ABA-induced PA production in leaf protoplasts of plda1, pldd, plda1 pldd, PLDd-complementation (COM), and the wild type (WT). Values are
means 6 SE (n = 3).
(B) Stomatal closure induced by 25 µM ABA or 100 µM H2O2. Values are means 6 SE (n = 50).
(C) Representative image of ROS production in guard cells, visualized by fluorescent dye. +ABA, epidermal peels were loaded with H2DCF-DA for 10
min followed by addition of 25 µM ABA for 5 min; –ABA, no ABA added. Bars = 50 µm.
(D) Quantification of ROS production based on fluorescence intensity (mean pixel intensity). Values are means 6 SE (n = 50). Columns with different
letters are significantly different from each other (ANOVA, P < 0.05).
[See online article for color version of this figure.]
2202 The Plant Cell

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GAPCs Promote the Activity of PLDd under
Oxidative Conditions
To determine the function of GAPC interaction with PLDd, we
first tested the sensitivity of GAPC1 and GAPC2 purified from E.
coli to H2O2. H2O2inhibited GAPC activity in a dose-dependent
manner, and virtually all GAPC1 or GAPC2 activity was inhibited
at 500 µM H2O2(Figure 3A). When different concentrations of
DTT were added to GAPCs first, followed by addition of 500 µM
H2O2, the loss of GAPC activity was small, showing that H2O2
oxidation of GAPCs can be protected by DTT reduction (see
Supplemental Figure 6A online). After incubation with 500 µM
H2O2, partial GAPC activity could be recovered by addition of
DTT (see Supplemental Figure 6B online).
Purified PLDd was then incubated GAPCs with or without H2O2
to determine the effect of H2O2and GAPC on PLDd activity.
Without GAPC, addition of 100 µM H2O2did not affect PLDd
activity (Figure 3B), verifying that H2O2has no direct effect on PLDd
activity. Incubation of PLDd with GAPC1 and GAPC2 increased
PLDd activity by 34 and 11%, respectively (Figure 3B). However,
pretreatment of GAPC1 and GAPC2 with 100 µM H2O2increased
PLDd activity by 82.1 and 58.9%, respectively (Figure 3B). The
data indicate that H2O2inactivates GAPC but promotes the GAPC
binding to PLDd, and the binding increases PLDd activity.
GAPC Mediates the H2O2Activation of PLDd in the Cell
To evaluate whether GAPC affects the activity of PLDd in living
cells, we compared PLD activity in GAPC-KO, PLDd-KO, and
wild-type protoplasts as affected by H2O2. Two homozygous
T-DNA insertion KO lines of Arabidopsis were isolated for
GAPC1 (gapc1-1, CS328689; gapc1-2, SALK_129091) and for
GAPC2 (gapc2-1, SALK_016539; gapc2-2, SALK_070902) (see
Supplemental Figure 7 online). The GAPC1 transcript was lost in
Figure 2. Interaction of GAPC with PLDd.
(A) Immunoblotting of proteins after coprecipitation using E. coli–expressed GST-PLDd and His-GAPC1/2, as affected by H2O2(100 mM) and DTT (100
mM). i, Coprecipitation of His-GAPC1 with GST-PLDd. GAPC1, immunoblotting of GAPC1 using anti-His antibody for the precipitates; PLDd, the
starting GST-PLDd used for precipitation. ii, Coprecipitation of GST-PLDd with His-GAPC2. PLDd, immunoblotting of PLDd using anti-GST antibody for
the precipitates. GAPC2, the starting His-GAPC2 used for precipitation. DTT was added before the addition of H2O2when both were applied.
(B) Immunoblotting of coprecipitated GAPC and PLDd that were coexpressed in yeast grown in the presence or absence of added H2O2(20 mM). i and ii,
Reciprocal pulldown of PLDd and GAPC1 and GAPC2, respectively. PLDd was fused with a FLAG tag and GAPC1or GAPC2 with a cMyc tag. GAPC1 or
GAPC2 band indicates immunoblotting with cMyc antibody against the sample precipitated with FLAG antibody–conjugated agarose beads. PLDd
band indicates immunoblotting with FLAG antibody against the sample precipitated with cMyc antibody for GAPC1 or GAPC2.
(C) Quantitative SPR analysis of PLDd binding to GAPC1. GAPC1 (no H2O2treatment or pretreated with 100 µM H2O2) was first immobilized on the NTA
chip followed by injection of GST or GST-PLDd.
(D) Representative confocal images of BiFC. Green color represents YFP fluorescence, indicating interaction of GAPC with PLDd. PLDd-YFPCwas
cotransformed with GAPC1-YFPNor GAPC2-YFPNinto tobacco leaves by infiltration. Bars = 50 µm.
GAPC and PLD in Reactive Oxygen Species and Stress Signaling2203

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two GAPC1-KO lines, and GAPC2 transcript was also absent in
two GAPC2-KO lines, suggesting that all four GAPC T-DNA
lines are null mutants (Figure 4A). We then generated two double
KO lines (gapc1-1 gapc2-1 and gapc1-1 gapc2-2) by crossing
the single mutants. Two lines of triple KO mutants (gapc1-1
gapc2-1 pldd and gapc1-1 gapc2-2 pldd) were also isolated
by crossing the GAPC double KO with pldd. NAD-dependent
GAPDH activity was determined in the single and double KO
lines of GAPC. The GAPDH activity in leaves was decreased
by 21% (gapc1-1), 25% (gapc1-2), 23% (gapc2-1), and 21%
(gapc2-2) for GAPC single mutants (Figure 4B). GAPC double
KO plants gapc1-1 gapc2-1 and gapc1-1 gapc2-2 had ;45%
decrease in GAPDH activity (Figure 4B). The results indicate that
GAPC1 and GAPC2 contribute almost equally to the activity,
and together they account for nearly half of NAD-dependent
GAPDH activity in Arabidopsis leaves.
To determine if KO of both GAPCs affects PLD activation by
H2O2, protoplasts of wild-type, pldd, and GAPC double mutants
were labeled with NBD-PC and treated with H2O2. We first ex-
amined how GAPDH activity in protoplasts responded to H2O2.
Protoplasts from GAPC double KOs had significantly lower
GAPDH activity than the wild type or pldd (Figure 4C). H2O2
treatments for 20 min had no significant effect on GAPDH ac-
tivity in the GAPC double KO but decreased GAPDH activity in
the wild type and pldd by 15%. Significant decreases in GAPDH
activity occurred in all genotypes after 40 min of H2O2treatments
(Figure 4C). The results indicate that H2O2inhibits GAPDH activity
in the cell and also could mean that the loss of the GAPDH activity
in the early phase (20 min) results primarily from H2O2inhibition
of GAPCs.
Without addition of H2O2, the PLD activity, as measured by
the formation of PA, in gapc1-1 gapc2-1 and gapc1-1 gapc2-2
was comparable to that of the wild type (Figure 4D). The H2O2
treatment increased PA production nearly twofold after 40 min in
the wild type, whereas it increased PA production only 30% in
pldd. The gapc1 gapc2 double KOs and gapc1 gapc2 pldd triple
KOs exhibited similar attenuated PA increase as pldd in re-
sponse to H2O2(Figure 4D). The results indicate that PLDd is the
main PLD responsible for the H2O2activation of PLD and that
GAPCs mediate the H2O2-induced increase of PLDd activity.
GAPCs Are Involved in ABA-Induced PA Production
To characterize the effect of GAPC and PLDd on PA production
in response to ABA, we measured the PA levels and composi-
tion in 4-week-old Arabidopsis leaves treated with ABA up to
20 min. PA level was induced by ABA in the wild type and
reached a plateau at 10 min after ABA treatment. The total PA
level was increased in pldd, gapc1-1 gapc2-1, and gapc1-1
gapc2-2 leaves after ABA treatment (Figure 5A). However, the
amount of PA was significantly lower in pldd, gapc1-1 gapc2-1,
and gapc1-1 gapc2-2 than in the wild type at 10 and 20 min after
ABA treatment (Figure 5A).
The molecular species of PA in response to ABA at 10 min
were analyzed for the wild type, pldd, gapc1-1 gapc2-1, and
gapc1-1 gapc2-2. In wild-type Arabidopsis leaves, 34:2 (16:0/
18:2), 34:3 (16:0/18:3), 36:4 (mainly 18:2/18:2), 36:5 (18:2/18:3),
and 36:6 (18:3/18:3) are the most abundant PA species (Zhang
et al., 2009). The levels of major PA species, including 34:1,
34:2, 34:3, 36:2, 36:4, and 36:5 PA, were significantly decreased
in pldd, and the major overall decrease of total PA level was due
to the decrease in 34:2, 34:3, 36:4, and 36:5 PA (Figure 5B).
Similarly, the levels of PA species 34:2, 34:3, 36:2 and 36:4 PA
were significantly reduced in gapc1-1 gapc2-1 and gapc1-1
gapc2-2 compared with the wild type after 10 min of ABA
treatment (Figure 5B). The PA acyl combinations affected by
Figure 3. Oxidized GAPC Promotes PLDd Activity.
(A) H2O2inhibition of GAPC1 and GAPC2 activities.
(B) GAPC promotion of PLDd activity under oxidative conditions. Equal
molar ratios of PLDd and GAPC proteins were used. PLDd activity was
assayed in the presence of GAPC1 (i) or GAPC2 (ii) under different
conditions as indicated; 100 mM DTT or 100 mM H2O2was used as in-
dicated. Values are means 6 SE (n = 3). Different letters indicate signifi-
cant differences (ANOVA, P < 0.05).
2204The Plant Cell

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PLDd and GAPC expression are the molecular species typically
derived from hydrolysis of extraplastidic phospholipids (Welti
et al., 2002), consistent with the extraplastidic location of these
enzymes. The results show that the ablation of either PLDd or
GAPCs decreases the ABA-induced PA production. The atten-
uation of ABA-induced activation of PLDd in GAPC double KOs
is consistent with the results that GAPCs are required for the
activation of PLDd activity (Figure 4D).
Loss of GAPCs or PLDd Renders Plants Less Responsive to
Water Deficits
To determine if GAPC–PLDd interaction is involved in the
process of mediating plant response to ROS, we measured
stomatal closure in response to ABA and H2O2in leaves de-
ficient in both GAPCs or GAPC and PLDd. Stomata of gapc1-1
gapc2-1 and gapc1-1 gapc2-2 were less sensitive to ABA or
H2O2, as indicated by greater stomatal aperture in these mutants
than that of the wild type after the treatment of ABA or H2O2
(Figure 6A). Two triple mutants (gapc1-1 gapc2-1 pldd and
gapc1-1 gapc2-2 pldd) were also less sensitive to ABA- and
H2O2-promoted stomatal closure (Figure 6A).
To determine how the effect of GAPCs and PLDd on ABA and
H2O2signaling impacts plant response to water deficits, we
evaluated the effect of GAPCs and PLDd KOs on Arabidopsis
plants grown under three field water capacity (FC) conditions:
100% FC for well-watered control, and 60 and 30% FC for mild
and acute drought stress, respectively (see Supplemental Figure
Figure 4. H2O2Effects on GAPC and PLDd Activities.
(A) RT-PCR detection of GAPC1 and GAPC2 expression in the leaves of wild-type (WT) and mutant plants. 18S rRNA was a control confirming the
synthesis of cDNA.
(B) GAPDH activity in the total protein extracted from the leaves of wild-type and mutant plants.
(C) GAPDH activity using protein extracted from protoplasts after 1 mM H2O2treatment.
(D) H2O2-promoted PA production in protoplasts. Values are means 6 SE (n = 3). Different letters mark significant differences from each other (ANOVA,
P < 0.05).
GAPC and PLD in Reactive Oxygen Species and Stress Signaling 2205

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8 online). Under well-watered conditions, pldd, gapc1-1 gapc2-1,
and gapc1-1 gapc2-2 did not show significant difference from
the wild type in cumulative water transpired and photosynthetic
rate, but gapc1-1 gapc2-1 and gapc1-1 gapc2-2 had higher
stomatal conductance than the wild type (Figure 6B). At 60%
FC, pldd, gapc1-1 gapc2-1, and gapc1-1 gapc2-2 displayed
higher stomatal conductance, higher cumulative water transpira-
tion, and higher photosynthetic rate than wild-type plants (Figure
6B). At the severe water deficit (30% FC), stomatal conductance
was very low in all genotypes, but pldd, gapc1-1 gapc2-1, and
gapc1-1 gapc2-2 mutant lines still exhibited the tendency to
have more cumulative water transpiration than the wild type
(Figure 6B).
As the FCs decreased, wild-type, pldd, gapc1-1 gapc2-1, and
gapc1-1 gapc2-2 mutants accumulated less biomass, as plant
growth was inhibited in response to water deficits. pldd, gapc1-1
gapc2-1, and gapc1-1 gapc2-2 accumulated more biomass
than the wild type under both mild and acute drought con-
ditions. At 60% FC, the three mutants accumulated ;30%
more dry matter than the wild type. The greater biomass in
the mutants than the wild type was consistent with higher
stomatal conductance and photosynthetic rate. The de-
creased drought inhibition of plant growth in the mutants
suggests that the loss of PLDd or GAPCs renders plants
less responsive to adjusting growth under water deficits.
However, the mutants lost much more water and had lower
instant water use efficiency (WUE) than the wild type (Figure
7A). When they were grown in separate pots without main-
taining FC or watering, the PLDd and GAPC mutants wilted
faster than the wild type (Figure 7B), consistent with the
measurements that PLDd- and GAPC-deficient plants lost
more water.
Figure 5. PA Content of GAPC and PLDd Mutant Leaves in Response to ABA.
(A) Total PA content of leaves harvested at different times after spraying with ABA (100 µM). WT, the wild type.
(B) PA molecular species in leaves of the wild type and mutants treated with ABA for 10 min. Values are means 6 SE (n = 5). Asterisks indicate significant
difference from the wild type at the same time point of ABA treatment (P < 0.05, t test).
2206 The Plant Cell

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DISCUSSION
This study demonstrates that PLDd plays a role in mediating
ABA-induced stomatal closure, but it acts in a distinctively dif-
ferent step from PLDa1 in the ABA signaling pathway (Figure 8).
PLDa1 promotes NADPH oxidase activity and H2O2production
(Zhang et al., 2009), whereas PLDd mediates H2O2response but
not H2O2production. Both PLDa1 and PLDd are activated in
response to ABA to generate PA. This raises the question of
whether PA generated by PLDa1 and PLDd targets the same or
different proteins. Our analyses of PLDd- and PLDa1-deficient
mutants show that PLDa1 produces twice as much PA as does
PLDd in response to ABA and that PLDd is the main PLD re-
sponsible for H2O2-stimulated PA production. Also, temporal
comparisons of PA formation in these mutants indicate that
PLDa1 is activated earlier than PLDd. In addition, PLDa1 and
PLDd have different subcellular locations and different substrate
selectivities with PLDa1 and PLDd preferring PC and phos-
phatidylethanolamine, respectively (Figure 8; Wang et al., 2006).
It is conceivable that the different magnitude, timing, and loca-
tion of PA production as affected by PLDa1 and PLDd will
Figure 6. Response of GAPC and PLDd Mutants to ABA and Water
Deficits.
(A) Changes in stomatal aperture after ABA (25 mM) or H2O2(100 mM)
treatment. Values are means 6 SE (n = 50). Different letters mark signif-
icant differences from each other (ANOVA, P < 0.05). WT, the wild type.
(B) Stomatal conductance, cumulative water transpiration, photosyn-
thesis, and dry weight. Asterisks mark significant difference from the wild
type under the same growth condition. Values are means 6 SE (n = 16).
Figure 7. Increased Water Loss in GAPC-KO and PLDd-KO Arabidopsis
Plants.
(A) Instant WUE of wild-type (WT) and mutant plants under 100 and 60%
FC. Arabidopsis seedlings were transplanted to pots and maintained at
100% FC and 60% FC. Instant WUE was calculated as the ratio of the
photosynthetic rate to stomatal conductance; measurements were taken
after the first 4 d after the onset of required stress. Asterisks indicate
significant difference from the wild type. Values are means 6 SE (n = 16;
*P < 0.05, t test).
(B) Increased dehydration of GAPC-KO and PLDd-KO plants when FC
was not maintained. Plants (25 d old) were fully watered and then left
unwatered for 16 d when the photograph was taken. D1 and D2 are
GAPC1 and 2 double KOs gapc1-1 gapc2-1 and gapc1-1 gapc2-2, re-
spectively. T1 and T2 are GAPC1, GAPC2, and PLDd triple KOs gapc1-1
gapc2-1 pldd and gapc1-1 gapc2-2 pldd, respectively.
[See online article for color version of this figure.]
GAPC and PLD in Reactive Oxygen Species and Stress Signaling 2207

Page 9

impact their product PA interaction with target proteins. PA has
been shown to bind to ABA INSENSITIVE1, NADPH oxidase,
and sphingosine kinase. These proteins are involved in the ABA-
mediated stomatal closure and targets of PLDa1 (Figure 8)
(Zhang et al., 2004; Zhang et al., 2009; Guo et al., 2011). In
addition, mitogen-activated protein kinases (MAPKs), which are
involved in various cellular processes, such as H2O2-induced
cell death and ABA-promoted stomatal closure (Zhang et al.,
2003; Zhang et al., 2006; Jammes et al., 2009; Yu et al., 2010),
have been implicated as targets of PA. PLDd-KO cells had
a decreased MAPK activity in response to H2O2(Zhang et al.,
2003); thus, MAPKs could be targets regulated by PA involved in
PLDd-mediated stomatal closure.
The analyses of GAPC and PLDd interaction further augment
the role of PLDd and PA in mediating ROS response. This study
documented the direct interaction between PLDd and GAPCs
qualitatively and quantitatively using different approaches. H2O2
inhibits GAPC activity by oxidizing the catalytic Cys residues in
the enzyme (Hancock et al., 2005). Our results indicate that H2O2
promotes the GAPC interaction with PLDd by decreasing the
dissociation of the GAPC-PLDd binding. KOs of GAPCs atten-
uated the ABA- or H2O2-promoted production of PA in the cell,
providing in vivo support for the role of GAPCs in the H2O2
activation of PLDd. It may be noted that the level of H2O2used in
this study is within physiological range reported for Arabidopsis
leaves, in which H2O2levels varied from 60 µM to more than 5 mM
under different stress conditions or different assays (Karpinski
et al., 1999; Veljovic-Jovanovic et al., 2001; Queval et al., 2008).
In our study, GAPC activity in vitro was significantly inhibited at
50 mM H2O2and almost completely lost at 500 mM H2O2. When
H2O2was applied to protoplasts, we used 1 mM H2O2to ensure
the oxidation of GAPCs because plant cells have a high capacity
to degrade H2O2by several scavenging enzymes.
Plants deficient in GAPCs or PLDd were less sensitive to ABA-
promoted stomatal closure and had higher transpirational water
loss than the wild type under drought stress. Without either
GAPC or PLDd, plants are less responsive to drought-induced
growth inhibition. These results indicate that GAPC–PLDd in-
teraction mediates ROS signaling and increases plant respon-
siveness to water deficits. Under the controlled water deficits
with specific FCs maintained, the GAPC- or PLDd-deficient
plants actually accumulated more biomass than the wild type.
The data are consistent with the observation that GAPC- or
PLDd-deficient plants have higher stomatal conductance and
a higher rate of photosynthesis than the wild type, probably
due to more opened stomata to allow more CO2uptake and
increased nutrient transport than the wild type. However, the
increase in biomass accumulation was at the expense of in-
creased water use. Indeed, without maintaining a specific soil
water level, the GAPC- or PLDd-deficient plants wilted faster
than the wild type when plants were withheld water. Earlier
studies showed that KO of PLDd decreased plant tolerance to
severe stresses, such as freezing, UV irradiation, and salt tol-
erance in Arabidopsis (Katagiri et al., 2001; Zhang et al., 2003;
Li et al., 2004; Bargmann et al., 2009). Decreasing growth under
water deficits is one of the key strategies for plants to cope with
stress and survival. The results indicate that the loss of GAPC
or PLDd compromises plant ability to sense the water stress
and to adjust cellular and physiological response accordingly.
The glycolytic enzyme GAPDH occurs in both the cytosol and
plastids, and the specific contributions of the two glycolytic
pathways to plant metabolism and growth are not well defined
(Plaxton, 1996; Muñoz-Bertomeu et al., 2009). Recent studies
show that KO of both plastid-localized GAPCps causes severe
development and growth defects, including arrested root de-
velopment, dwarfism, and male sterility in Arabidopsis (Muñoz-
Bertomeu et al., 2009, 2010). Genetic ablation of another glycolytic
enzyme, phosphoglycerate mutase, also indicates a critical role
of glycolysis in stomatal movement, vegetative growth, and
pollen production in Arabidopsis (Zhao and Assmann, 2011). By
Figure 8. A Proposed Model for the Role of PLD/PA in Regulating ROS Production and Response under Water Deficits.
This model depicts only the known targets of PLD/PA in ABA-mediated stomatal closure; other ABA regulators are not included in this model. GAPCox
refers to oxidized, catalytically inactive GAPC that interacts with PLDd and promotes PLDd activity. GAPCred refers to reduced, active GAPC that
converts glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-bisPG) with NADH production. PLDa1 uses preferably phosphatidyl-
choline (PC), whereas PLDd prefers phosphatidylethanolamine (PE) as substrate. Solid arrows indicate established links, and dashed arrows denote
putative links. PM, plasma membrane.
2208The Plant Cell

Page 10

comparison, our study reveals that the KO of both cytosolic
GAPCs results in no overt growth defects under normal condi-
tion in Arabidopsis. Instead, the GAPC-deficient plants exhibited
less growth inhibition than the wild type under drought under
the controlled drought conditions. These results suggest that
GAPCs are required for plant growth responsiveness to drought,
and we propose that the H2O2-promoted interaction of GAPCs
with PLDd is involved in the stress signaling leading to growth
inhibition (Figure 8). An alternative hypothesis could be that the
decrease in GAPC would alter the flux through carbon metab-
olism and affect the growth phenotype without GAPC binding
to PLDd. If so, one would expect that under drought stress,
the increased H2O2in plants would inhibit GAPCs, leading to
growth inhibition. But this is not the case because GAPC-KO
plants display less growth inhibition than the wild type. In addi-
tion, GAPC-KO mutants share a similar phenotype as PLDd-KO;
ablation of either GAPCs or PLDd renders plants less responsive
to water deficits, and the drought-induced growth inhibition re-
quires the presence of both PLDd and GAPCs. Thus, our results
are consistent with the proposition that the interaction between
GAPCs and PLDd is involved in mediating H2O2signals in plant
response to water deficits. However, further studies are needed to
understand the requirement for and mechanism of the GAPC–
PLDd interaction in modulating plant growth under stress and the
metabolic role of GAPCs in plant growth and stress responses.
The identification of GAPC interaction with PLDd unveils
a regulatory function of the carbon metabolic enzymes GAPCs
in plants and potentially a molecular node linking stress sig-
naling and metabolic alterations. Some classical metabolic en-
zymes can have crucial regulatory roles in the cell. For example,
hexokinase has been found in the nucleus, where it forms
a protein complex mediating glucose signaling in yeast and
plant (Ahuatzi et al., 2004; Cho et al., 2006). In animal cells,
GAPDH is involved in nonmetabolic processes, including gene
transcription, DNA replication, nuclear tRNA export, and DNA
repair, and these studies indicate that GAPDH has direct re-
lationship to the pathology of various diseases (Sirover, 1997;
Hara et al., 2005; Bae et al., 2006; Harada et al., 2007). Oxidized
GAPDH is thought to be translocated to the nucleus to regulate
gene expression to initiate apoptotic cell death (Hara et al.,
2005; Bae et al., 2006). This study shows that the cytosolic
GAPCs interact with the plasma membrane–bound PLDd and
the interaction is promoted by ROS. We propose that the
GAPC–PLDd interaction in response to ROS provides a molec-
ular link between stress signaling and the alteration of cellular
metabolism and growth (Figure 8). Further investigations on the
specificity, mechanism, and downstream targets of these in-
teractions will provide mechanistic insights to how plants adjust
metabolism and growth in response to different stresses.
METHODS
Isolation of KOs and pldd Complementation
Arabidopsis thaliana (Columbia-0) wild-type and T-DNA insertion lines were
obtained from the ABRC at Ohio State University. plda1 (SALK_053785)
was isolated and confirmed previously (Zhang et al., 2004). The homozy-
gous line of pldd (SALK_023247) was confirmed by PCR. The primers for
PCR screening are listed in Supplemental Table 1 online. Four T-DNA lines
(gapc1, CS328689, SALK_129091; gapc2, SALK_016539 and SALK_
070902) were screened, and the homozygous lines were verified by PCR
(see Supplemental Figure 6 online). The open reading frame of GAPC1 and
GAPC2 shares 89.7% identity, while the 39 untranslated regions of both
genes are not conserved. Thus, primers in the 39 untranslated region of
GAPC1 and GAPC2 were used to distinguish the GAPC1 and GAPC2
transcripts. To complement pldd, a genomic sequence including the pro-
moter of PLDd was cloned (primers listed in see Supplemental Table 1
online) and inserted into binary vector PEC291 for transformations of pldd.
Plant Growth Conditions and Physiological Analysis
Plants were grown in soil in a growth chamber with cool white light of
160 µmol m22s21under 12-h-light/12-h-dark and 23°C/19°C cycles.
Drought stress was created by a gravimetric approach (Sheshshayee
et al., 2005; Peters et al., 2010). Ten-day-old Arabidopsis seedlings were
transplanted to pots containing soil saturated to maximum field capacity
(100% FC). Soil saturation was achieved by adding a known amount of
water based on weight of soil and water holding capacity. The pots were
covered with thick polyethylene sheets to prevent evaporation. One set of
plants was maintained at 100% FC (control), and the other two sets were
maintained at 60% (mild stress) and 30% (acute stress). The pots were
weighed every day, and the difference in weight in subsequent days was
corrected by adding water to maintain specific FCs. The amount of water
added over the experimental period was summed up to give the cu-
mulative water transpired.Stomatal conductance and photosynthetic rate
were recorded on fully expanded leaf using a portable gas exchange
system (LICOR6400-XT; LiCOR Biosciences). Instant WUE was calcu-
lated as ratio of photosynthetic rate to stomatal conductance. Mea-
surements were taken on the first 4 d after the onset of drought stress. At
the end of the stress, the shoots were harvested, dried, and weighed.
Stomatal Aperture Measurements
Stomatal aperture was measured using epidermal peels according to
a described procedure (Zhang et al., 2004). Briefly, epidermal peels were
floated inincubation buffer(10mMKCl,0.2mMCaCl2,0.1mMEGTA, and
10 mM MES-KOH, pH 6.15) for 2.5 h under cool white light at 23°C to
induce stomatal opening. ABA or H2O2was applied separately to epi-
dermal peels, which were incubated for 2.5 h under cool white light at
23°C to induce stomatal closure. Stomata were imaged under a micro-
scope with a digital camera and analyzed with ImageJ software (NIH).
ROS Detection
The endogenous ROS levels in guard cells were detected using epidermal
peels treated with the dye H2DCF-DA (Sigma-Aldrich) (Zhang et al., 2009).
Epidermal peels were floated in incubation buffer for 2 h and then loaded
with 50 µM H2DCF-DA (50 mM stock in DMSO) for 10 min, followed by
20 min washing in incubation buffer. The 25 µM ABA was added for
desired time of treatment. Epidermal peels were observed with a confocal
microscope (Zeiss LSM 510) (green fluorescence: excitation of 488 nm
and emission of 525 nm).
GAPC Cloning, Protein Purification, and Activity Assay
The cDNAs ofGAPC1 and GAPC2 were amplifiedand ligated to pET-28a-
c(+) vector to produce GAPC1 and GAPC2 with six His residues at the
N terminus. The recombinant plasmids were transformed into Escherichia
coli BL21(DE3)pLysS. Induction and purification of protein was as de-
scribed (Guo et al., 2011). Purified proteins were dialyzed in tris-buffered
saline buffer with DTT overnight. Dialyzed proteins were centrifuged at
12,000g for 20 min, and protein concentration was determined using the
Bradford protein assay. Purified proteins were analyzed by 10% SDS-
GAPC and PLD in Reactive Oxygen Species and Stress Signaling2209

Page 11

PAGE, followed by Coomassie Brilliant Blue staining. The prepared pro-
teins were used for activity assay or kept in 50% glycerol at 280°C. NAD-
dependent GAPDH activity assay was done using purified bacterially
expressed GAPC (2 to 5 mg) or total protein (25 to 50 mg) extracted from
Arabidopsis leaves with modification according to the method described
previously (Rius et al., 2008).
Protein Coprecipitation and Coimmunoprecipitation Assays
GST-PLDdconstructandexpressionofPLDdweredescribedpreviously(Qin
et al., 2002). To pull down GAPC, GST-PLDd–bound beads (;15 µg purified
proteins) were incubated with total protein extracted from E. coli expressing
GAPC1or GAPC2 at 4°C for 3 hwith gentle rotation(Zhao and Wang, 2004).
To pull down PLDd, GAPC-bound agarose beads (;10 µg purified proteins)
were incubated with total protein extracted from E. coli expressing GST-
PLDd at 4°C for 3 h with gentle rotation. The beads were collected and
washed three times and subjected to 10% SDS-PAGE followed by im-
munoblotting. To coexpress PLDd and GAPC in yeast for coimmunopre-
cipitation, PLDd and GAPC1 or GAPC2 were cloned into pESC-HIS vector
and transformed into YPH yeast strain (Stratagene). PLDd and GAPC1 or
GAPC2 were coexpressed in yeast after induction by addition of galactose,
andtheyeasts were grownovernight at 30°C. Then,20mMH2O2wasadded
when oxidativecondition was required. Primers used for cloning are listed in
Supplemental Table 1 online. Total protein was extracted from harvested
yeast and used for coimmunoprecipitation analysis.
SPR Analysis
SPR binding assays were performed as described with some mod-
ifications (Guo et al., 2011). The purified proteins were dialyzed in the
running buffer (0.01 M HEPES, 0.15 M NaCl, and 50 µM EDTA, pH 7.4)
overnight at 4°C and then the proteins were centrifuged at 13,000g to
remove insoluble protein before use. For each experiment, the running
buffer with 500 µM NiCl2wasinjected to saturate theNTA chip with nickel.
His-tagged GAPC1 protein (200 nM) was immobilized on a Biacore
Sensor Chip NTA via Ni2+/NTA chelation. PLDd-GAPC1 interaction was
monitored as GST-PLDd (200 nM) was injected in sequence over the
surface of the sensor chip. The purified GST protein was used as control.
During the evaluation, the sensorgrams from the beginning of association
to the end of dissociation for each interaction were analyzed and plotted
by SigmaPlot 10.0 (Systat Software, Inc.). Kinetic constants including
Bmax, association (kon), and dissociation rate (koff) were analyzed using
BIAevaluation software (GE Healthcare).
BiFC
The BiFC vectors were constructed, described, and provided by Walter
et al. (2004). GAPC1 or GAPC2 cDNA was cloned into pSPYNE vector
(GAPC-YFPN), and PLDd cDNA was cloned into pSPYCE vector (PLDd-
YFPC). The constructs were transformed into C58C1 Agrobacterium
tumefaciens strain and grown to stationary phase. Bacterial cells were
collected and resuspended in solution containing 10 mM MES, pH 5.7,
10 mM MgCl2, and 150 mg mL21acetosyringone. Three-week-old Ni-
cotiana benthamiana leaves were infiltrated with the mixed bacteria (GAPC-
YFPNandPLDd-YFPN)solutions(Voinnetetal.,2003).YFPfluorescencewas
examined in tobacco leaves using a Zeiss LSM 510 confocal microscope,
witha 488-nmexcitationmirror anda505-to 530-nm filter to recordimages.
Assaying PLD Activity
For in vivo PLD activity assay, protoplasts prepared from leaves of 4-week-
old plants were incubated in 0.5 mg/mL NBD-PC for 80 min on ice (Zhang
et al., 2004). To determine PLD activity using fluorescent lipids, as affected
byABAtreatmentat differenttimepointsinvivo, 100µMABA wasadded to
the NBD-PC–labeled protoplasts, and 100-mL aliquots (1.5 3 105for each
assay) were transferred to a new tube at the end of each treatment. Then,
0.4 mL hot isopropanol (75°C) was added, and the mixture was incubated
for 10 min at 75°C to inactivate PLD. Lipids extraction, separation, and
quantification were done according to the procedure as described (Zhang
etal.,2004).TotesttheeffectofGAPConPLDd,PLDdactivitywasassayed
using dipalmitoylglycero-3-phospho-[methyl-3H]choline as substrate ac-
cording to the procedure described previously (Qin et al., 2002).
Electrospray Ionization–Tandem Mass Spectrometry Analysis of
Lipid Molecular Species
LipidswereextractedandPAanalyzedbyelectrosprayionization–tandem
mass spectrometry (Xiao et al., 2010). Expanded leaves of 4- to 5-week-
old plants were sprayed with 100 µM ABA with 0.01% Triton X-100. The
leaves were excised and immersed in 3 mL of isopropanol with 0.01%
butylatedhydroxytoluene(preheatedto75°C)immediatelyaftersampling.
The experiment was repeated three times with five replicates of each
treatment each time.
Statistical Analysis
Experimental values represent mean values and standard errors. n rep-
resents the number of independent samples. P values were calculated
with Student’s t test (two-tailed) using Microsoft Excel or analysis of
variance (ANOVA).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: PLDa1, At3g15730; PLDd, At4g35790; GAPC1, At3g04120;
GAPC2, At1g13440; and UBQ10, At4g05320.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Confirmation of Homozygous T-DNA In-
sertion PLD Mutants by PCR.
Supplemental Figure 2. Expression Level of PLDd in Response to
ABA.
Supplemental Figure 3. PLDd-GAPC Association as Identified by
GAPC1 Coprecipitation of PLDd from Microsomal Proteins of Arabi-
dopsis Leaves.
Supplemental Figure 4. Purification and Immunoblotting of PLDd and
GAPCs Produced in E. coli and Yeast.
Supplemental Figure 5. Negative and Positive Control for BiFC.
Supplemental Figure 6. DTT Protection of GAPC Activity.
Supplemental Figure 7. Isolation of GAPC T-DNA Homozygous
Lines.
Supplemental Figure 8. Growth Phenotype of the Wild Type and
GAPC and PLDd Mutants under Control and Drought Conditions.
Supplemental Table 1. Primers Used in This Study.
ACKNOWLEDGMENTS
We thank Jörg Kudla for kindly providing the BiFC vectors, Mary Roth for
technical assistance, and Ruth Welti for critical reading of the article. This
2210 The Plant Cell

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work was supported by National Science Foundation Grant IOS-
0818740, by U.S. Department of Energy Grant DE-SC0001295, and by
U.S. Department of Agriculture Grant 2007-35318-18393.
AUTHOR CONTRIBUTIONS
L.G. and X.W. designed the research. L.G. performed most experiments.
Y.Z. and W.Z. identified pldd stomatal phenotype. X.P. and S.P.D.
performed the interaction and GAPDH activity assays. R.N. performed
the physiological study in Figure 6B. L.G. and X.W. analyzed the data and
wrote the article.
Received December 19, 2011; revised April 10, 2012; accepted April 25,
2012; published May 15, 2012.
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Supplemental Figure 1. Confirmation of homozygous T-DNA insertion PLD mutants by PCR.
PCR was conducted using genomic DNA extracted from plant leaves with a pair of gene
specific primers (PLDα1RP+PLDα1LP for PLDα1 and PLDδRP+PLDδLP for PLDδ) or a
combination of a T-DNA left border primer (LBa1) and gene specific primers (PLDα1RP and
PLDδRP). The presence of a T-DNA band and lack of a PLDα1 or PLDδ band indicate that
pldδ and pldα1pldδ are homozygous T-DNA mutants. The primers used for PCR are listed in
Table S1.
Supplemental Data. Guo et al. (2012). Plant Cell 10.1105/tpc.111.094946
1

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Supplemental Figure 2. Expression level of PLDδ in response to ABA.
RNA was extracted from leaves sprayed with 100 μM ABA with 0.01% Triton X-100. PLDδ
transcript level was measured by real-time PCR normalized to UBQ10. The ABA response
gene ABI1 was used as a positive control. The experiment was repeated three times with
similar results. Values are means ± SE (n = 3) for one representative experiment. The
primers for real-time PCR are listed in Table S1.
Supplemental Data. Guo et al. (2012). Plant Cell 10.1105/tpc.111.094946
2