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

Xin Shen, Kaili Yin, Zhiyu Wang, Zhiwei Zhang, Mengqing Liu, Sheng Luo, Shaowu Xue, and Honghong Hu

PNAS; November 20 2025; 122 (47) e2518134122; https://doi.org/10.1073/pnas.2518134122

Significance

Abscisic acid (ABA) activates Ca²⁺ signaling to promote stomatal closure. The Ca²⁺-independent kinase OST1 and the Ca²⁺-dependent protein kinases CPKs directly phosphorylate and activate SLAC1 and SLAC1 homologues (SLAHs). However, the priming of Ca²⁺ signaling and the relationship between these pathways remain unclear. Here, we demonstrate that the Ca²⁺-dependent protein kinase CPK15 is essential for ABA-induced stomatal closure, with OST1 primarily activating SLAC1 anion channel activity through CPK15. OST1 and Ca²⁺ work synergistically to enhance CPK15 activity. These findings reveal a mechanism where OST1 connects Ca²⁺-independent and Ca²⁺-dependent pathways via CPK15 to regulate ABA-induced stomatal movement.

Abstract

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 CPK15 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 SLAC1^8D (a phosphomimetic form) in oocytes constitutively activates anion channel activity and effectively restores the impaired ABA-induced stomatal closure of cpk15-1 but not by SLAC1^8A (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.

See: https://www.pnas.org/doi/10.1073/pnas.2518134122

Figure 1:

OST1 interacts with CPK15. (A) The interaction of OST1 with CPKs by split-LUC assay. N-terminal LUC (nLUC) fused to N terminus of OST1 and C-terminal LUC (cLUC) fused to C-terminus of CPKs were coexpressed in N. benthamiana leaf epidermis. (B) Quantitative analysis of the luminescence intensity in (A). Data shown are mean ± SEM (n = 3 independent biological replicates). Different letters above the error bars indicate the significant differences at P < 0.05 by One-way ANOVA with Tukey’s test. (C) Coimmunoprecipitation of OST1 with CPK15 in Arabidopsis protoplasts coexpressing 35S–OST1–YFP and 35S–CPK15–FLAG, or 35S–YFP and 35S–CPK15–FLAG. Protein extracts were immunoprecipitated with GFP-beads. The input and coimmunoprecipitated proteins were detected with anti-GFP and anti-FLAG antibodies as indicated. IP, immunoprecipitation. Experiments were repeated three times.