Ethyl vinyl ketone activates oxidative and calcium burst and CML8-ACA8 participates in calcium recovery in Arabidopsis leaves.

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. Surface plasmon resonance (SPR) binding assay shows that evk indeed bind to RBOHD. Here, we show that evk treatment increased H2 O2 and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H2 O2 signaling functioned upstream of Ca2+ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca2+ -ATPASE, ISOFORM 8)-CML8 (CALMODULIN-LIKE 8) interaction promoted Ca2+ efflux to return Ca2+ levels to the resting state.