CPKs are involved in Ca ²⁺ signaling encoding by enhancing OST1-initiated Ca ²⁺ influx for ABA-induced stomatal closure in Arabidopsis

Both Ca 2+ -independent kinase OPEN STOMATA 1 (OST1) and Ca 2+ -DEPENDENT PROTEIN KINASEs (CPKs) play important roles in ABA-induced stomatal closure. We recently reported that OST1-mediated phosphorylation and activation of Ca 2+ channels constituted mainly with CYCLIC NUCLEOTIDE-GATED CHANNEL 5 (CNGC5), 6, 9, and 12 (CNGC5/6/9/12) are required for ABA-induced stomatal closure in Arabidopsis. However, Ca 2+ -dependent protein kinases and underlying mechanisms that are involved in this Ca 2+ signaling pathway are still largely unknown. In this study, we identified CPK3, 8, and 10 (CPK3/8/10) as Ca 2+ -dependent CNGC-activating kinases with CPK3 as the main one, and a conserved serine site at CNGCs’ C termini is revealed to be the main CPK3-target sites, differing from OST-target sites at CNGCs’ N termini. Double S-to-D (2D) and S-to-A (2A) point mutations at OST1- and CPK3-target sites respectively coactivate and cosuppress CNGCs, but individual S-to-D activation is impaired by S-to-A mutation at the other site. Abscisic acid (ABA)-induced stomatal closure and Ca 2+ oscillations are impaired in Arabidopsis triple mutant cpk3/8/10 , but are rescued fully by the CNGC6’s 2D variant, largely by mixed A/D variant, and not by 2A variant. These results demonstrate that the cytosolic Ca 2+ elevation derived from OST1-CNGC modules-mediated external Ca 2+ influx activates CPK3 via Ca 2+ binding, the Ca 2+ -bound CPK3 evokes more massive external Ca 2+ influx through enhancing the activity of CNGCs by phosphorylation, and CPK3-evoked Ca 2+ influx is required for the encoding of ABA-induced cytosolic Ca 2+ signaling in Arabidopsis guard cells.