Find in Library

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.