Ethyl Vinyl Ketone Activates K+ Efflux to Regulate Stomatal Closure by MRP4-Dependent eATP Accumulation Working Upstream of H2O2 Burst in Arabidopsis

Abstract

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 <i>mrp4</i>, <i>mrp5</i>, <i>dorn1.3</i> and <i>rbohd/f</i>. Non-invasive micro-test technology (NMT) data showed that evk-induced K⁺ efflux was diminished in <i>mrp4</i>, <i>rbohd/f</i>, <i>and dorn1.3</i> 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 <i>mrp4</i> and <i>mrp5</i> mutant groups and the ABC transporter inhibitor glibenclamide (Gli)-pretreated group, evk-regulated stomatal closure and eATP buildup were diminished, especially in the <i>mrp4</i> group. According to qRT-PCR and eATP concentration results, evk regulates both relative gene expressions of <i>MRP4/5</i> and eATP concentration in <i>rbohd/f</i> and WT group. According to the confocal data, evk-induced H₂O₂ production was lower in <i>mrp4</i>, <i>mrp5</i> 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.