Ethyl Vinyl Ketone Activates K Efflux to Regulate Stomatal Closure by MRP4-Dependent eATP Accumulation Working Upstream of HO Burst in Arabidopsis.

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 HO in evk-regulated K efflux, we used Arabidopsis wild-type (WT), mutant lines of , , and . Non-invasive micro-test technology (NMT) data showed that evk-induced K efflux was diminished in , , mutant, which means eATP and HO work upstream of evk-induced K efflux. According to the eATP content assay, evk stimulated eATP production mainly by MRP4. In and mutant groups and the ABC transporter inhibitor glibenclamide (Gli)-pretreated group, evk-regulated stomatal closure and eATP buildup were diminished, especially in the group. According to qRT-PCR and eATP concentration results, evk regulates both relative gene expressions of and eATP concentration in and WT group. According to the confocal data, evk-induced HO production was lower in , mutants, which implied that eATP works upstream of HO. Moreover, NADPH-dependent HO 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 HO burst by regulating RBOHF through interaction. This study reveals that evk-induced stomatal closure requires MRP4-dependent eATP accumulation and subsequent HO accumulation to regulate K efflux.