Plants regulate stomatal mobility to limit water loss and improve pathogen resistance. Ethyl vinyl ketone (evk) is referred to as a reactive electrophilic substance (RES). In this paper, we found that evk can mediate stomatal closure and that evk-induced stomatal closure by increasing guard cell K⁺ efflux. To investigate the role of eATP, and H₂O₂ in evk-regulated K⁺ efflux, we used Arabidopsis wild-type (WT), mutant lines of mrp4, mrp5, dorn1.3 and rbohd/f. Non-invasive micro-test technology (NMT) data showed that evk-induced K⁺ efflux was diminished in mrp4, rbohd/f, and dorn1.3 mutant, which means eATP and H₂O₂ work upstream of evk-induced K⁺ efflux. According to the eATP content assay, evk stimulated eATP production mainly by MRP4. In mrp4 and mrp5 mutant groups and the ABC transporter inhibitor glibenclamide (Gli)-pretreated group, evk-regulated stomatal closure and eATP buildup were diminished, especially in the mrp4 group. According to qRT-PCR and eATP concentration results, evk regulates both relative gene expressions of MRP4/5 and eATP concentration in rbohd/f and WT group. According to the confocal data, evk-induced H₂O₂ production was lower in mrp4, mrp5 mutants, which implied that eATP works upstream of H₂O₂. Moreover, NADPH-dependent H₂O₂ burst is regulated by DORN1. A yeast two-hybrid assay, firefly luciferase complementation imaging assay, bimolecular fluorescence complementation assay, and pulldown assay showed that the interaction between DORN1 and RBOHF can be realized, which means DORN1 may control H₂O₂ burst by regulating RBOHF through interaction. This study reveals that evk-induced stomatal closure requires MRP4-dependent eATP accumulation and subsequent H₂O₂ accumulation to regulate K⁺ efflux.

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evk’s identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Here, we show that evk treatment increased H ₂ O ₂ and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H ₂ O ₂ signaling functioned upstream of Ca ²⁺ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca ²⁺ -ATPASE, ISOFORM 8)–CML8 (CALMODULIN-LIKE 8) interaction promoted Ca ²⁺ efflux to return Ca ²⁺ levels to the resting state. Evk treatment led to the antagonism of salicylic acid (SA) and jasmonic acid (JA). CALMODULIN 5 (CAM5) positively regulates WRKY53 expression, and CAM5 and WRKY53 positively regulate SA-related gene expression. These proteins physically interact and form a complex that is unlocked by Ca ²⁺ to release WRKY53. An electrophoretic mobility shift assay and dual-luciferase reporter assay demonstrated that WRKY53 and TGA5 cooperate to enhance the expression of the defense gene PATHOGENESIS-REALTED 1 ( PR1 ) and that WRKY53 enhances the binding of TGA5 to the PR1 promoter. This paper proposes a framework that evk, as a RES substance, can achieve plant’s ‘REScue’ through complete defense response.