Cooperative function of PLDδ and PLDα1 in abscisic acid-induced stomatal closure in Arabidopsis.

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K+ channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Stomatal pores are formed by pairs of guard cells and mediate transpiration and carbon dioxide uptake. Abscisic acid (ABA) synthesized in plants subjected to drought stress induces stomatal closure in order to suppress water loss in plants (Assmann and Shimazaki, 1999;Schroeder et al., 2001).
Phospholipase D (PLD) activity increases in response to hyperosmotic stress and dehydration in Craterostigma plantagineum Munnik et al., 2000) and Arabidopsis (Arabidopsis thaliana; Katagiri et al., 2001). PLDs hydrolyze phospholipids, phosphatidylcholine (PC), and phosphatidylethanolamine (PE), releasing phosphatidic acid (PA) and their head group in the plasma membrane. The released PA is thought to function as a signal molecule in cellular signaling.
The Arabidopsis genome has 12 PLD genes that are classified into six subfamilies, a, b, g, d, «, and z (Wang, 2005). Pharmacological studies examining the effect of a PLD inhibitor (1-butanol [1-BuOH]) and exogenous application of PA in Vicia faba have suggested that ABA signaling is mediated by PLDs (Jacob et al., 1999). Previous studies have reported that PLDa1 positively regulates ABA-induced stomatal closure  and the inhibition of stomatal opening by ABA (Mishra et al., 2006). Furthermore, PLDa1 activity is reported to be modulated by a G protein, GPA1, in the process of inhibition of stomatal opening by ABA Mishra et al., 2006). However, a plda1 loss-of-function mutation alone did not inhibit ABAinduced stomata closure (Siegel et al., 2009), which suggests that other PLDs are involved in ABA signaling in guard cells.
PLDd is involved in responses to drought and salinity stress (Katagiri et al., 2001) and cold stress (Li et al., 2004). PLDa1 and PLDd are required for tolerance to high salinity and hyperosmotic stress (Bargmann et al., 2009). Therefore, we hypothesized that PLDd functions cooperatively with PLDa1 in the ABA signal pathway in guard cells.
In this study, we investigated the roles of PLDa1 and PLDd in (1) ABA-induced stomatal closure, (2) ABA-induced production of ROS and NO, cytosolic pH alkalization, and cytosolic free Ca 2+ elevation, and (3) ABA inhibition of inward-rectifying K + (K + in ) channel currents and stomatal opening. We report that PLDa1 and PLDd have overlapping functions in ABAinduced stomatal closure and different functions in the ABA inhibition of light-induced stomatal opening.

Expression of PLDa1 and PLDd in Guard Cells
We examined the accumulation of transcripts of PLDa1 (At3g15370) and PLDd (At4g35790) in isolated guard cell protoplasts (GCPs) using reverse transcription (RT)-PCR. POTASSIUM CHANNEL IN ARABI-DOPSIS THALIANA1 (KAT1) was used as a specific GCP marker (Leonhardt et al., 2004). As shown in Figure 1A, the transcripts of PLDa1 and PLDd were detected in both GCPs and mesophyll cell protoplasts (MCPs). The PLDa1 transcription level in GCPs appeared to be lower than that in MCPs, whereas the PLDd transcription level in GCPs was slightly higher than that in MCPs (Fig. 1A).

ABA-Induced Stomatal Closure in pld Mutants
We isolated mutants with T-DNA inserted in PLDa1 (SALK_063785) and PLDd (KAZUSA T-DNA tag line) loci (Fig. 1B). A double mutant, plda1 pldd, was generated by crossing the single mutants. Disruption of the genes was assessed by RT-PCR with total RNA isolated from whole leaves. The transcripts of PLDa1 were not detected in the plda1 and plda1 pldd mutants, and the transcripts of PLDd were not detected in the pldd and plda1 pldd mutants (Fig. 1C).
Utilizing these loss-of-function mutants, we examined the involvement of PLDa1 and PLDd in ABA-induced stomatal closure. Application of 1 mM ABA induced stomatal closure in the wild type (P , 10 24 ) and both pld single mutants (P , 10 23 for plda1, P , 10 23 for pldd) Figure 1. PLDa1 and PLDd expression and the effect of mutation of PLDs in ABA-induced stomatal closure and PA production. A, RT-PCR analysis of PLDa1 and PLDd gene expression in wild-type GCPs and MCPs. ACTIN2 was used as a positive control for the RT-PCR. KAT1 was used as a control of GCP gene expression. B, The positions of T-DNA insertions in plda1 and pldd mutants. Boxes represent exons. C, RT-PCR analysis of PLDa1 and PLDd gene expression in the wild type (WT) and plda1, pldd, and plda1 pldd mutants. ACTIN2 was used as a positive control for the RT-PCR. D, ABA-induced stomatal closure in the wild type and plda1, pldd, and plda1 pldd mutants. Averages from three independent experiments (60 stomata per bar) are shown. E, Drought-induced PA accumulation in rosette leaves of the wild type and pld mutants. F, ABA-induced PA accumulation in epidermal tissues of the wild type and pld mutants. Radioactivity was normalized to the control values. Averages from six independent experiments are shown. Error bars represent SE. compared with the solvent control ( Fig. 1D) but not in the double mutant (P = 0.096). On the other hand, the application of 10 mM ABA slightly induced stomatal closure in the double mutant (Fig. 1D), indicating greatly reduced ABA sensitivity of double mutant stomata.
Drought induced an accumulation of PA in wild-type rosette leaves (Fig. 1E). The drought-induced PA accumulation in rosette leaves was suppressed in the plda1 pldd double mutant but not in either single mutant (Fig.  1E). ABA-induced PA accumulation in wild-type rosette leaves was not detected (data not shown), in agreement with previous results (Katagiri et al., 2001). On the other hand, in epidermal tissues, 50 mM ABA induced an accumulation of PA in the wild type (P , 0.05; Fig. 1F). ABA also induced a similar accumulation of PA in the pldd single mutant (P , 0.03), which was not significantly different from the accumulation induced in the wild type (P = 0.593; Fig. 1F). ABA did not induce PA accumulation in either the plda1 single mutant (P = 0.222) or the plda1 pldd double mutant (P = 0.163; Fig.  1F). Moreover, suppression of PA production by the double mutation was stronger than that of the plda1 single mutation (P , 0.04). These results suggest that PLDa1 and PLDd are involved in PA production induced by ABA signaling in guard cells. To test whether the expression of PLDa1 or PLDd complements the stomatal phenotype of the double mutant, we generated the plda1 pldd mutants transformed with PLDa1 or PLDd. PLDa1 and PLDd transcripts were detected in the respective complement lines ( Fig. 2A). The transformed plants showed a restored stomatal response to ABA (Fig. 2B), suggesting that mutations of PLDa1 and PLDd are responsible for the ABA-insensitive phenotype observed in the plda1 pldd mutants.

PA-Induced Stomata Closure in pld Mutants
To confirm the function of PLDs in ABA signaling in guard cells, we examined the effects of a PLD inhibitor, 1-BuOH, on ABA-induced stomatal closure. ABA at 10 mM closed stomata of plda1 and pldd at a level comparable to the level in the wild type in the absence of 1-BuOH, whereas 1-BuOH at 50 mM suppressed the ABA-induced stomatal closure (Fig. 3A), which suggests that activation of PLDs is involved in ABAinduced stomatal closure.
PLDs hydrolyze phospholipids, releasing PA. The released PA mediates ABA signaling, leading to stomatal closure (Jacob et al., 1999;Mishra et al., 2006;Zhang et al., 2009). We examined exogenous PAinduced stomatal closure in the plda1 and pldd mutants. PA at 10 and 50 mM induced stomatal closure in the wild type and the pld mutants (Fig. 3B), suggesting that downstream of PA production in the ABA signal cascade is intact in the pld mutants.
Exogenous PA induced ROS accumulation and cytosolic alkalization in guard cells in the wild type and the plda1 pldd mutant (Supplemental Fig. S2, A and C) but did not affect NO accumulation in guard cells in either the wild type or the double mutant (Supplemental Fig. S2B).

ABA-Induced Cytosolic Ca 2+ Oscillations in plda1 pldd Guard Cells
We monitored [Ca 2+ ] cyt in guard cells using a Ca 2+ -sensing fluorescent protein, Yellow Cameleon 3.6 (YC3.6). When the wild-type guard cells were treated with 10 mM ABA, 83% of the guard cells showed

ABA Inhibition of K + in Channel Currents in Guard Cells
In the absence of ABA, K + in channel currents in GCPs were not significantly different between the wild type and the double mutant (P = 0.70). However, in the presence of ABA, K + in channel currents were reduced in the wild type (P , 10 23 ; Fig. 6, A and B) but not in the double mutant (P = 0.24; Fig. 6, C and D). These results indicate that PLDa1 and PLDd are involved in the inhibition of the K + in channel by ABA signaling.

Inhibition of Stomatal Opening by ABA
One of ABA's many roles is to inhibit light-induced stomatal opening (Shimazaki et al., 2007), and PLDa1 is reported to be involved in the ABA inhibition of light-induced stomata opening (Mishra et al., 2006). ABA at 1 mM inhibited stomatal opening in the wild type (P , 0.02) and the pldd single mutant (P , 0.01) but did not inhibit it in the plda1 single mutant (P = 0.76) or the plda1 pldd double mutant (P = 0.15; Fig. 7). On the other hand, 10 mM ABA slightly inhibited stomatal opening in the plda1 single mutant and the plda1 pldd double mutant. These results suggest that PLDd functions differently from PLDa1 in the inhibition of light-induced stomatal opening.

Inhibition of Seed Germination by ABA
The inhibitory effect of ABA on seed germination was slightly reduced in both of the single mutants and strongly reduced in the double mutant (Fig. 8). These results suggest that PLDa1 and PLDd cooperatively function not only in stomatal closure but also in seed germination. In contrast, root growth was inhibited by ABA in a dose-dependent manner in the wild type and the pld mutants (data not shown). Hence, PLDa1 and PLDd do not appear to be involved in all ABA signaling in Arabidopsis. . Effects of the PLD inhibitor 1-BuOH on ABA-induced stomata closure and PA in the wild type (WT) and pld mutants. A, Rosette leaves of the wild type and pld mutants were treated with 10 mM ABA and with or without 50 mM 1-BuOH. 1-BuOH was applied to the assay solution before ABA treatment. B, PA induced stomatal closure in pld mutants. Rosette leaves of the wild type and plda1, pldd, and plda1 pldd mutants were treated with 10 mM ABA, 10 mM PA, and 50 mM PA. Averages from three independent experiments (60 stomata per bar) are shown. Error bars represent SE.

Effects of ABA on Transcription of PLDd
Rosette leaves were placed in stomatal assay solution with and without 50 mM ABA under the light condition for 2 h. Total RNA was isolated from the leaves, and transcript levels of PLDa1, PLDd, and RESPONSIVE TO DEHYDRATION B (RD29B), a positive control for the ABA response (Yamaguchi-Shinozaki and Shinozaki, 1993), were measured by quantitative real-time PCR. The amounts of PLDa1 transcripts in the untreated and treated leaves were not significant different (P = 0.13; Fig.  9, left panel), but the amount of PLDd transcript in the ABA-treated leaves was three times higher than that in the untreated leaves (P , 0.002; Fig. 9, middle panel). RD29B transcripts were remarkably increased by ABA ( Fig. 9, right panel), as expected. These results indicate that transcription of PLDa1 is constitutive and that transcription of PLDd is ABA inducible. They also suggest that PLDd functions in ABA signaling by regulating gene transcription.

Cooperative Function of PLDa1 and PLDd in ABA Signaling in Arabidopsis Guard Cells
In the plda1 single mutant, most studies have reported that ABA-induced stomatal closure is impaired (Zhang et al., , 2009Mishra et al., 2006), Figure 4. Effects of ABA (50 mM) on ROS production, NO production, and cytosolic alkalization in the wild type (WT) and pld mutants. A, Representative gray-scale DCF fluorescence images (top panel) and ROS production as shown by DCF fluorescence in the wild type, plda1, pldd, and plda1 pldd (bottom panel). B, Representative grayscale DAF-2 images (top panel) and NO production as shown by DAF-2 fluorescence (bottom panel). C, Representative BCECF images (top panel) and cytosolic alkalization as shown by BCECF fluorescence (bottom panel). In each graph, fluorescence intensity was normalized to the control values. Bars indicate averages of three independent experiments (60 guard cells per bar). Error bars represent SE. although one study found no strong ABA-insensitive phenotype (Siegel et al., 2009), in agreement with our research here.
Our results show that the plda1 pldd double mutation disrupted ABA-induced PA production and ABAinduced stomatal closure but that the single mutations did not, suggesting that not only PLDa1 but also PLDd positively regulate ABA-induced stomatal closure. The disruption by the double mutation was complemented by the expression of PLDa1 or PLDd by the 35S promoter, suggesting that PLDa1 and PLDd cooperatively function in ABA signaling in guard cells.
A PLD inhibitor, 1-BuOH, inhibited ABA-induced stomatal closure in the plda1 and pldd single mutants as it did in the wild type (Fig. 3A), suggesting that other PLDs are involved in ABA-induced stomatal closure in each pld mutant. Moreover, ABA-induced stomatal closure in the plda1 pldd double mutant was not completely inhibited (Fig. 1D), but it was completely inhibited by the application of 1-BuOH (Fig.  3A). This implies that other PLDs are involved in ABA signaling in guard cells. ABA induces ROS production in guard cells, resulting in stomatal closure (Pei et al., 2000;Murata et al., 2001). The ROS production is mediated by NADPH oxidases, encoded by RESPIRATORY BURST OXI-DASE HOMOLOG D (AtrbohD) and AtrbohF genes (Kwak et al., 2003). OPEN STOMATA1 kinase has been reported to activate AtrbohF via phosphorylation in ABA signaling (Sirichandra et al., 2009), and PA has also been reported to activate AtrbohF via binding (Zhang et al., 2009), suggesting that ABA-induced ROS production occurs downstream of PA production in ABA signaling in guard cells.
In this study, ABA-induced ROS production in guard cells was partially suppressed in the single mutants and completely suppressed in the double mutant (Fig. 4A),  Involvement of PLDd in ABA Signaling in Guard Cells suggesting that both PLDa1 and PLDd are involved in ABA-induced ROS production and that PLDa1 and PLDd cooperatively function upstream of ABAinduced ROS production in guard cells. Like ROS production, NO production and cytosolic alkalization are also accompanied by ABA-induced stomatal closure (Irving et al., 1992;Desikan et al., 2002;Suhita et al., 2004;García-Mata and Lamattina, 2007;Gonugunta et al., 2008;Islam et al., 2010a). ABA-induced NO production and cytosolic alkalization were also impaired in the plda1 pldd double mutant (Fig. 4, B and C), suggesting that PLDa1 and PLDd positively regulate NO production and cytosolic alkalization in ABA signaling.
PLDs hydrolyze phospholipids, releasing PA as a second messenger. Our findings that PA induces ROS production (Supplemental Fig. S2A) and cytosolic alkalization (Supplemental Fig. S2C) confirm that PLDa1 and PLDd function upstream of ROS production and cytosolic alkalization in ABA signaling.
In our study, PA treatment did not evoke NO production in guard cells (Supplemental Fig. S2B). Previous reports have shown that NO production in guard cells is dependent on ABA-induced H 2 O 2 (Bright et al., 2006), suggesting that NO production is downstream of ROS production. However, another report has shown that NO induces PA production (Distéfano et al., 2008). The protein phosphatase 2C abi1 mutation impaired ABA-induced ROS production (Murata et al., 2001) but not ABA-induced NO production (Desikan et al., 2002). Moreover, Lozano-Juste and León (2010) have proposed a NO-independent regulatory mechanism of ABA-induced stomatal closure. Taken together, these results indicate that PA is closely involved in ROS production and cytosolic alkalization in ABA signaling and that the roles of NO production in ABA signaling remain to be investigated.
[Ca 2+ ] cyt oscillation/elevation is known to occur during ABA-induced stomatal closure  and is closely related with ROS production in guard cells (Pei et al., 2000;Islam et al., 2010aIslam et al., , 2010b. Activation of PLDa1 and PLDd requires Ca 2+ , since these PLDs contain a conserved C2 domain that participates in Ca 2+ /phospholipid binding , suggesting that PLD activities are affected by [Ca 2+ ] cyt elevation in guard cells. PA production and [Ca 2+ ] cyt oscillation/ elevation may occur not only in tandem but also in parallel in ABA signaling in guard cells. In this study, ABA induced [Ca 2+ ] cyt oscillation but not ROS production in the plda1 pldd double mutant (Fig. 5). These results contradict the current ABA-signaling model, in which Ca 2+ -permeable cation channels in the plasma membrane are activated by H 2 O 2 (Pei et al., 2000;Kwak et al., 2003). However, long-term Ca 2+ programmed stomatal closure requires stimulus-specific calcium oscillations; that is, certain specific Ca 2+ signatures inhibit stomatal reopening after Ca 2+ (reactive) stomatal closure . Therefore, the H 2 O 2 -independent [Ca 2+ ] cyt oscillations in the plda1 pldd double mutant may have failed to induce stomatal closure and may be attributed to a malfunction of [Ca 2+ ] cyt homeostasis due to the double mutation. Furthermore, spatiotemporal modulation of ROS production and/or differences of pattern of [Ca 2+ ] cyt elevation are important in guard cell ABA signaling Jannat et al., 2011aJannat et al., , 2011b. Spatiotemporal analysis of ROS production and [Ca 2+ ] cyt elevation should resolve this discrepancy.  . Real-time PCR analysis of PLDa1, PLDd, and RD29B gene expression in the wild type. Total RNA was isolated from leaves treated with 50 mM ABA and from untreated leaves, as was done in the stomatal assay procedure. Transcript levels were normalized to the expression of ACTIN2 in the control. Three independent experiments were done. Error bars represent SE.

Inhibition of K + in Channel Currents by ABA in GCPs
Three second messengers, H 2 O 2 , NO, and sphingoshine-1-phosphate, have been reported to inhibit K + in channel currents of GCPs in V. faba (Zhang et al., 2001;Sokolovski et al., 2005) and of Arabidopsis GCPs (Coursol et al., 2003). Exogenous PA was also reported to inhibit K + in channel currents of V. faba GCPs (Jacob et al., 1999). Our findings that exogenous PA inhibited K + in channel currents in Arabidopsis GCPs (Supplemental Fig. S3) and that ABA strongly inhibited K + in channel currents of wild-type GCPs but not of the plda1 pldd GCPs (Fig. 6) suggest that, in ABA signaling, PLDa1 and PLDd are also involved in the inhibition of K + in channel currents.

PLDa1 and PLDd Differentially Function in ABA Signaling in Guard Cells
In this study, we observed several differences of phenotype between the plda1 and pldd single mutants. In the case of ABA-induced stomatal closure, the plda1 single mutation partially impaired stomatal closure induced by 1 mM ABA but the pldd single mutation did not (Fig. 1D). ROS production by ABA in the plda1 single mutant was smaller than that in the pldd single mutant, even though the difference was not significant (Fig. 4A). Similarly, ABA-induced PA production in the plda1 single mutant was less than that in the pldd single mutant (Fig. 1F). Moreover, the plda1 single mutation weakened the ABA inhibition of light-induced stomatal opening but the pldd single mutation did not (Fig. 7). Together, these results suggest that PLDa1 and PLDd have somewhat different roles in ABA signaling.
PLDa1 is located in the cytosol and plasma membrane and prefers PC to PE as a substrate (Fan et al., 1999;Li et al., 2009), whereas PLDd is mainly located at the plasma membrane and prefers PE to PC (Gardiner et al., 2001;Qin et al., 2002;Li et al., 2009). These differences in phenotype between the plda1 and pldd mutants thus may be due to differences in the location of PA production and the molecular species of the produced PA between PLDa1 and PLDd.
Moreover, PLDa1 activity is regulated by the binding of PLDa1 with the G protein a-subunit GPA1 at a DRY motif , while PLDd potentially interacts with G protein because it contains a DRY motif and a hydrophobic motif, which are highly conserved in G protein-binding proteins. Hence, the difference in affinity for G protein may also be responsible for the phenotype differences between the plda1 and pldd mutants.
The PLDa1 gene is constitutively expressed even under drought and saline conditions, whereas the PLDd gene is inductively expressed by dehydration and salinity (Katagiri et al., 2001). Antisense suppression of PLDa1 increases PLDd gene expression (Mane et al., 2007). Our study shows that expression of the PLDa1 gene was constitutive regardless of ABA treatment and that expression of the PLDd gene was ABA inducible (Fig. 9). This suggests that PLDd activity is regulated at the transcriptional level in the stomatal response to ABA. Thus, the plda1 single mutant phenotype is susceptible to a change in PLDd expression that is influenced by growth conditions. As a result, we could see both ABAsensitive and ABA-hyposensitive phenotypes in the plda1 single mutant. By contrast, PLDa1 activity may be posttranslationally regulated by other factors, such as GPA1. In other words, PLDa1 may mainly function under moderate environmental stress conditions and PLDd may cooperatively work with PLDa1 under severe environmental stress conditions.

Other Physiological Functions of PLDa1 and PLDd in Response to ABA
In this study, the inhibitory effect of ABA on seed germination was slightly reduced in both of the single mutants and strongly reduced in the double mutant (Fig. 8), in agreement with the result of Katagiri et al. (2005) that an accumulation of PA facilitates the inhibition of seed germination by ABA in Arabidopsis. However, in Oryza sativa, PLDb1 mutation mitigates the inhibition of germination by ABA (Li et al., 2007). It is unknown whether the mutation also reduces PA production.
In this study, root growth was inhibited by ABA in a dose-dependent manner in the wild type and the pld mutants (data not shown), while a reduction of PA production due to the plda1 pldd double mutation increased the sensitivity to hyperosmotic and salt stress in Arabidopsis roots (Bargmann et al., 2009). In roots, PLDa1 and PLDd appear to be involved in the responses to salinity and hyperosmolarity but not in the response to ABA.

CONCLUSION
Our results show that PLDa1 and PLDd cooperatively function upstream of the production of ROS and NO and the cytosolic alkalization in ABA signaling of Arabidopsis guard cells.

Plant Materials, Growth, and Transformation
Arabidopsis (Arabidopsis thaliana) wild type (Columbia-0) as well as plda1, pldd, and plda1 pldd mutants were grown in a growth chamber at 22°C and 60% humidity with a 16-h light period with 80 mmol m 22 s 21 photon flux density and 8 h of dark. Water containing 0.1% Hyponex was applied two to three times in 1 week on the plant growth tray. [Ca 2+ ] cyt in guard cells was measured using a Ca 2+ -sensing fluorescent protein, YC3.6 (Nagai et al., 2004;Mori et al., 2006). To obtain YC3.6-expressing plants, wild-type and plda1 pldd double mutant plants were crossed with a Columbia-0 plant that had previously been transformed with YC3.6.
For a germination test, 100 seeds of the same age were sown on germination medium agar plates (Katagiri et al., 2001) supplemented with 1% (w/v) Suc. Germination was defined as the emergence of the radicle.

Measurement of Stomatal Aperture
Stomatal apertures were measured as described previously . Briefly, excised rosette leaves were floated on an assay solution containing 5 mM KCl, 50 mM CaCl 2 , and 10 mM MES-Tris, pH 6.15, for 2 h in the light to induce stomatal opening followed by the addition of ABA or PA or H 2 O 2 . After a 2-h incubation, the leaves were shredded in a commercial blender for 30 s, and the remaining epidermal tissues were collected using nylon mesh. For stomatal opening, excised rosette leaves were floated on the assay solution for 2 h in the dark to induce stomatal closure. These leaves were transferred in the light for 3 h with ABA. The leaves were shredded for 30 s, and the remaining epidermis was collected. For each sample, 20 stomatal apertures were measured.

Isolation of MCPs and GCPs
MCPs and GCPs were enzymatically isolated from 4-week-old Arabidopsis plants as described previously (Leonhardt et al., 2004).
Quantitative real-time PCR was performed with an Mx3000P QPCR System (Agilent Technologies) using SYBR Green (Brilliant II QPCR Master Mix; Stratagene) to monitor double-stranded DNA synthesis. A standard curve was constructed for each gene using gene fragments that had been previously amplified and quantified. The levels of gene transcript were normalized to that of ACTIN2 and expressed relative to the amounts observed under control conditions.

Generation of Complement Transgenic Plants in plda pldd
PLDa1 was amplified by PCR from Arabidopsis leaf cDNA using primers 5#-CACCATGGCGCAGCATCTGTTGCACGGG-3# and 5#-AAAGGTTGTAA-GGATTGGAGGCAGGTAG-3#, which were based on GenBank accession At3g15370. Similarly, PLDd was amplified using primers 5#-CACCATGGCG-GAGAAAGTATCGGAGGACG-3# and 5#-AAACGTGGTTAAAGTGTCAG-GAAGAGCC-3#, which were based on At4g35790. These primer sequences were modified to allow insertion in Gateway binary vectors. Amplified DNA fragments were cloned into pENTR/D-TOPO vector (Invitrogen). The final sequence was confirmed by sequencing with an ABI310 sequencer (ABI). The resulting entry clones were introduced into Gateway binary vector pGWB2 (Nakagawa et al., 2007) following the manufacturer's instructions. The plda1 pldd double mutant was transformed using a floral dip procedure (Clough and Bent, 1998), and transformed plants were confirmed to be carrying the transgene by growth on kanamycin-and hygromycin-containing Murashige and Skoog agar medium.

Measurement of ROS and NO Production
ROS production in guard cells was analyzed using H 2 DCF-DA . The epidermal peels were incubated for 3 h in the assay solution containing 50 mM KCl, 50 mM CaCl 2 , and 10 mM MES-Tris (pH 6.15), and then 50 mM H 2 DCF-DA was added to the sample. The epidermal tissues were incubated for 30 min at room temperature, and then the excess dye was washed out with the solution. Collected tissues were again incubated with solution and 50 mM ABA or 50 mM PA for 20 min in the dark condition. The image was captured using a fluorescence microscope (Bio Zero BZ-8000; KEYENCE), and the pixel intensity of the fluorescence in guard cells was measured using ImageJ 1.42q (National Institutes of Health). For ABA-and PA-induced NO detection in guard cells, 10 mM 4,5-diaminofluorescein-2 diacetate was added instead of 50 mM H 2 DCF-DA .

Measurement of Cytosolic pH
Cytosolic pH elevation in guard cells was analyzed using BCECF-AM (Islam et al., 2010a). The epidermal peels were incubated for 3 h in an assay solution containing 50 mM KCl, 50 mM CaCl 2 , and 10 mM MES-Tris (pH 6.5), and then 20 mM BCECF-AM was added to the sample. The epidermal tissues were incubated for 30 min at room temperature, and then the excess dye was washed out with the assay solution. Collected tissues were incubated in the solution with 50 mM ABA or 50 mM PA for 20 min in the dark. The images were obtained and analyzed as described above.

Measurement of [Ca 2+ ] cyt Oscillations
Four-to 6-week-old wild-type and plda1 pldd plants expressing YC3.6 were used for the measurement of guard cell [Ca 2+ ] cyt oscillations as described previously (Islam et al., 2010a(Islam et al., , 2010bHossain et al., 2011). The abaxial side of an excised leaf was gently mounted on a glass slide with a medical adhesive (stock no. 7730; Hollister) followed by removal of the adaxial epidermis and the mesophyll tissue with a razor blade in order to keep the lower epidermis intact on the slide. The remaining abaxial epidermis was incubated in a solution containing 5 mM KCl, 50 mM CaCl 2 , and 10 mM MES-Tris (pH 6.15) under light for 2 h at 22°C to promote stomatal opening. Turgid guard cells were used to measure [Ca 2+ ] cyt oscillations. Guard cells were treated with 10 mM ABA using a peristaltic pump at 5 min after monitoring. For dual-emission ratio imaging of YC3.6, we used a 440AF21 excitation filter, a 445DRLP dichroic mirror, a 480DF30 emission filter for cyan fluorescent protein (CFP), and a 535DF25 emission filter for yellow fluorescent protein (YFP). The CFP and YFP fluorescence intensities of guard cells were imaged and analyzed using the W-View system and AQUA COSMOS software (Hamamatsu Photonics). CFP and YFP fluorescence were simultaneously monitored following simultaneous excitation of CFP and YFP.

Whole-Cell Patch-Clamp Recording of K + in Channel Currents
Arabidopsis GCPs were enzymatically isolated from rosette leaves of 4-to 6-week-old plants as described previously (Munemasa et al., 2007). Whole-cell currents were measured using a patch-clamp amplifier (model CEZ-2200; Nihon Kohden). Data were analyzed with pCLAMP 8.2 software (Molecular Devices). The pipette solution contained 30 mM KCl, 70 mM K-Glu, 2 mM MgCl 2 , 3.35 mM CaCl 2 , 6.7 mM EGTA, and 10 mM HEPES adjusted to pH 7.1 with Tris, and the bath solution contained 30 mM KCl, 2 mM MgCl 2 , 40 mM CaCl 2 , and 10 mM MES titrated to pH 5.5 with Tris (Saito et al., 2008). Osmolarity of the pipette solution and the bath solution was adjusted with Dsorbitol to 500 and 485 mmol kg 21 , respectively. In order to examine the effect of ABA, GCPs were treated with 10 mM ABA for 2 h before recordings.

P Labeling of Phospholipids of Arabidopsis Leaf Discs and Epidermis
Phospholipids were labeled with 32 P as described previously (Katagiri et al., 2001). Leaf discs with a diameter of 3 mm and epidermal tissues were prepared from 3-to 4-week-old Arabidopsis plants. The discs and epidermal peels were incubated in MES-KOH buffer (pH 5.6) supplemented with 3.7 MBq mL 21 [ 32 P]orthophosphoric acid for 12 h. For the control, 32 P-labeled leaf discs were transferred into the MES-KOH buffer without [ 32 P]orthophosphoric acid. For dehydration treatment, dry 32 P-labeled leaf discs were incubated in an Eppendorf tube for 2 h. For ABA treatment, 32 P-labeled leaf discs were transferred into MES-KOH buffer supplemented with 50 mM ABA and then incubated for 2 h. The 32 P-labeled epidermal tissues were collected by centrifugation and washed with MES-KOH buffer. The corrected epidermal tissues were incubated in MES-KOH buffer in the absence or presence of 50 mM ABA for 2 h. After each treatment, an equal volume of MES-KOH buffer supplemented with 0.75% (v/v) 1-BuOH was added to each sample. After a 10-min incubation, the reaction was stopped by the addition of 10 mL of 60% (w/v) HClO 4 . The mixture was incubated in liquid nitrogen for 1 min, and then lipids were extracted from leaf discs and epidermal tissues (Katagiri et al., 2001).

Statistical Analysis
The significance of differences between mean values of stomatal aperture and root growth were assessed by Student's t test and two-factor factorial ANOVA. The frequency of [Ca 2+ ] cyt oscillations was assessed by x 2 test. Differences were considered significant at P , 0.05.

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Effects of exogenous H 2 O 2 (100 mM) on stomatal aperture in the wild type (WT) and pld mutants.
Supplemental Figure S2. Effects of exogenous PA (50 mM) on the production of ROS and NO and cytosolic alkalization in guard cells of the wild type (WT) and pld mutants.
Supplemental Figure S3. Effects of PA (50 mM) on K + in channel currents of guard cells of the wild type.