MAP4K1 and MAP4K2 regulate ABA-induced and Ca2+-mediated stomatal closure in Arabidopsis

Abstract Abscisic acid (ABA)-induced stomatal closure limits water loss from plants under drought stress. To investigate signaling pathways involved in stomatal closure, we performed a phosphoproteomic analysis of ABA-treated Arabidopsis guard cell protoplasts (GCPs). From this analysis, we discovered that MITOGEN-ACTIVATED PROTEIN 4 KINASE 1 (MAP4K1) is phosphorylated in response to ABA. Subsequent biochemical assays showed that Ser-479 of MAP4K1 is directly phosphorylated by SRK2E (OST1/SnRK2.6), a protein kinase that plays a central role in ABA-induced responses. Mutational analyses of MAP4K1 , as well as closely related MAP4K2 , revealed that both kinases positively regulate ABA-induced stomatal closure, and that Ser-479 of MAP4K1 was required for this phenotype. In map4k1map4k 2, stomatal closure was induced by exogenous Ca 2+ but not H 2 O 2 . Furthermore, electrophysiology experiments showed that MAP4K1/2 are required for ABA-dependent activation of Ca 2+ -permeable channels in GCPs. Together, our results indicate that SnRK2 and MAP4K1/2 function as a signaling module to regulate Ca 2+ -mediated stomatal closure.