OPEN STOMATA 1 activates SLAC1 anion channel primarily through CPK15 in ABA-induced stomatal closure in <i>Arabidopsis</i>

The Ca²⁺-independent OST1 and Ca²⁺-dependent protein kinases CPKs both activate the anion channel SLAC1 during ABA-induced stomatal closure pathway. However, the mechanism by which OST1 regulates SLAC1 activation and its relationship with CPKs remain unclear. Here, we identify that OST1 primarily activates SLAC1 in this process through CPK15. Mutation of <i>CPK15</i> significantly impairs ABA-induced stomatal closure and increases drought sensitivity. OST1 interacts with CPK15 and phosphorylates it at T103, which is essential for ABA-induced stomatal closure. Moreover, CPK15 can phosphorylate eight sites in the N terminus of SLAC1 to activate its anion currents in oocytes. Expression of <i>SLAC1^8D</i> (a phosphomimetic form) in oocytes constitutively activates anion channel activity and effectively restores the impaired ABA-induced stomatal closure of <i>cpk15-1</i> but not by <i>SLAC1^8A</i> (a phospho-dead form). Furthermore, activated CPK15 by OST1 and Ca²⁺ enhances its activity toward SLAC1, and mutations of both OST1 and CPK15 have additive effect on ABA-induced stomatal closure, suggesting that CPK15 activates SLAC1 through both direct and indirect mechanisms. These findings demonstrate the key role of CPK15 in ABA-induced stomatal closure, revealing a connection between OST1 and CPKs in both Ca²⁺-independent and Ca²⁺-dependent pathways in ABA-induced stomatal closure.