H

H. cell ABA signaling. AnMPK12-YFPfusion construct rescued the ABA-insensitive stomatal response phenotype ofmpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H2O2treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence thatMPK9andMPK12function downstream of ROS to regulate guard cell ABA signaling positively. Keywords:abscisic acid, anion channels, protein kinase, reactive oxygen species, stomata The phytohormone abscisic acid (ABA) regulates diverse cellular processes and transduces environmental signals to protect plants from abiotic stresses (1,2). Among the identified molecular elements working in ABA signaling are protein kinases and phosphatases that play a central role in regulating the signaling network (3). It is noteworthy that only a few recessive mutations inArabidopsisprotein kinase and phosphatase genes have been identified that act as positive regulators of guard cell ABA signaling. These genes include Ca2+-dependent protein kinases (CPK3,CPK6,CPK4,CPK11), a protein phosphatase 2A regulatory subunit A (RCN1), a receptor-like protein kinase (RPK1), a Ser/Thr protein kinase (OST1), and MAPK cascade genes (MKK1,MPK6) (48). Reactive oxygen species (ROS) were shown previously to induce increases in cytosolic Ca2+and stomatal closure (9). ROS activate hyperpolarization-activated plasma membrane PNZ5 Ca2+-permeable channels in guard cells ofViciaandArabidopsis(10,11). ABA also increases H2O2levels inViciaguard cells in advance of stomatal closure (12). The endogenous source of guard cell ROS has been explored through a combined molecular genetic and functional genomics approach, which revealed that the 2 2 guard cell-expressed AtrbohF and AtrbohD NADPH oxidases, among the 10 NADPH oxidases in theArabidopsisgenome, PNZ5 are responsible for ABA-induced ROS production and subsequent ABA signaling in guard cells (13). ABA was shown to induce MAPK activation in barley aleurone layers (14), and a possible MAPK activity was observed inViciaguard cell protoplasts (15). Furthermore, a study with pea epidermal peels showed that the MAPKK inhibitor PD98059 inhibits ABA-induced stomatal closure and expression of an ABA-inducible dehydrin gene (16,17). Despite these studies indicating that MAPK cascades function in ABA signaling, it remains to be established which specific MAPKs, MAPKKs, and MAPKK kinases (MAPKKKs) form a complete cascade to mediate ABA signaling in guard cells. The large number of genes in the plant MAPK, MAPKK, and MAPKKK families (18) potentially confers a high level of genetic redundancy within signal transduction mechanisms, thereby hampering conventional genetics. Nevertheless, complete MAPK cascades that function in plant innate immunity and stomatal development have been established (1921). Interestingly, all these identified MAPK cascades PNZ5 shareMPK6and/orMPK3. Here, we show that 2 other members of the MPK family,MPK9andMPK12, are PNZ5 preferentially expressed in PNZ5 guard cells, share functional redundancy, and function as positive regulators downstream of ROS in guard cell ABA signaling. == Results == == Guard Cell-Preferential Expression ofMPK9andMPK12. == To test whether MAPK cascades function inArabidopsisguard cell ABA signaling, we first examined ABA-induced stomatal movements in the presence and absence of the MAPKK inhibitor PD98059. Both ABA- and H2O2-induced stomatal closure inArabidopsiswere significantly inhibited by PD98059, indicating that MAPK cascades function downstream of ROS in ABA signaling inArabidopsisguard cells (Fig. S1). We then asked whether any genes encoding MAPK cascade components are specifically expressed in guard cells, a finding that could lead to identification of MAPK cascades required for guard cell ABA signaling. For this purpose, we analyzed ATH1 microarray-derived data in which guard cell and mesophyll cell RNA had been compared (22). This analysis revealed that 2 MAPK genes,MPK9andMPK12, are highly and preferentially expressed in guard cells relative to mesophyll cells (Fig. 1A), suggesting hWNT5A thatMPK9andMPK12might function specifically in guard cell signal transduction and/or development. == Fig. 1. == Expression analyses reveal.