Roles of CPKs in ethylene-induced Arabidopsis stomatal closure and their crosstalk with H₂O₂ and NO signalling

Calcium-dependent protein kinases (CPKs) play crucial roles in plant guard cell signal transduction. Ethylene is known to induce stomatal closure, with the hydrogen peroxide (H₂O₂)-nitric oxide (NO) signalling module being pivotal to this process. However, the specific roles of CPKs in this process and their interactions with H₂O₂ and NO remain unclear. In this study, we screened Arabidopsis mutants of nine CPKs and found that in the loss-of-function mutants for CPK3, CPK4, CPK6, CPK11, CPK21, and CPK33, exogenous ethylene failed to induce stomatal closure, indicating that these CPKs act as positive regulators in ethylene-induced stomatal closure. Mutants' stomatal responses to H₂O₂ and NO treatment and changes of endogenous H₂O₂ and NO levels in guard cells upon ethylene treatment indicated that CPK3, CPK4, CPK11, and CPK33 function upstream of the H₂O₂-NO module, while CPK6 and CPK21 act downstream. Furthermore, NADPH oxidases play critical roles in ethylene-induced H₂O₂ production. We identified the interactions of CPK3, CPK4, and CPK11 with AtRBOHF, and CPK4 and CPK11 with AtRBOHD using four different assays, and exogenous ethylene enhanced these interactions. These results suggest that CPK3, CPK4, and CPK11 may mediate ethylene-induced H₂O₂ formation in guard cells through their interactions with AtRBOHD/F. Additionally, exogenous ethylene significantly upregulates the expression of CPK3, CPK4, CPK6, CPK11 and CPK21, providing a potential mechanism by which ethylene modulates CPKs. Our findings not only establish the role of CPKs in ethylene guard cell signalling but also offer insights into the mechanism by which ethylene activates NADPH oxidases to initiate H₂O₂ production.