Exogenous OPDA-induced stomatal closure is mediated by hydrogen peroxide and Ca2+ in Arabidopsis thaliana

12-oxo-phytodienoic acid (OPDA), a biosynthetic precursor of jasmonic acid (JA), also functions as a signaling molecule and has been implicated in stomatal regulation. However, the molecular mechanisms underlying OPDA-induced stomatal closure remain largely unclear. Here, we demonstrate that exogenous OPDA induces stomatal closure without strict dependence on canonical JA or abscisic acid (ABA) biosynthesis and signaling pathways, as evidenced by analyses of multiple JA- and ABA-related mutants in Arabidopsis thaliana . Exogenous OPDA treatment also promoted hydrogen peroxide (H 2 O 2 ) accumulation in guard cells. Notably, the NADPH oxidase respiratory burst oxidase homolog protein F (RbohF), but not RbohD, was required for OPDA-induced reactive oxygen species (ROS) production and stomatal closure. Furthermore, disruption of extracellular calcium ion (Ca 2+ ) availability or influx significantly impaired OPDA-induced stomatal closure, indicating that Ca 2+ influx is an essential component of this response. In addition, the OPDA-binding protein RESISTANT TO CORONATINE 4 (ROC4) was required for OPDA-triggered H 2 O 2 accumulation and stomatal closure, suggesting that ROC4 functions upstream of ROS production during OPDA-induced stomatal closure. Transcriptomic analysis of OPDA-treated OPDA reductase 3 ( OPR3 ) and CORONATINE INSENSITIVE 1 ( COI1 ) double mutant opr3/coi1-30 ice-blended epidermal fragments containing guard cells identified a limited set of OPDA-responsive genes associated with transcriptional regulation, redox homeostasis, detoxification, and stress-related signaling pathways. Collectively, these results indicate that exogenous OPDA promotes stomatal closure through a signaling module involving ROC4-dependent activation of RbohF, ROS production, extracellular Ca 2+ influx, and demonstrate that OPDA-mediated stomatal regulation can occur without strict dependence on canonical JA and ABA pathways under the tested conditions.