Uncovering molecular mechanisms involved in microbial volatile compounds-induced stomatal closure in Arabidopsis thaliana

Microbial volatile compounds (mVCs) may cause stomatal closure to limit pathogen invasion as part of plant innate immune response. However, the mechanisms of mVC-induced stomatal closure remain unclear. In this study, we co-cultured Enterobacter aerogenes with Arabidopsis (Arabidopsis thaliana) seedlings without direct contact to initiate stomatal closure. Experiments using the reactive oxygen species (ROS)-sensitive fluorescent dye, H 2 DCF-DA, showed that mVCs from E. aerogenes enhanced ROS production in guard cells of wild-type plants. The involvement of ROS in stomatal closure was then demonstrated in an ROS production mutant ( rbohD ). In addition, we identified two stages of signal transduction during E. aerogenes VC-induced stomatal closure by comparing the response of wild-type Arabidopsis with a panel of mutants. In the early stage (3 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and receptor-like kinase THESEUS1 mutant ( the1-1 ) but not in rbohD , plant hormone-related mutants ( nced3 , erf4 , jar1-1 ), or MAPK kinase mutants ( mkk1 and mkk3 ). However, in the late stage (24 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and rbohD but not in nced3 , erf4 , jar1-1 , the1-1 , mkk1 or mkk3 . Taken together, our results suggest that E. aerogenes mVC-induced plant immune responses modulate stomatal closure in Arabidopsis by a multi-phase mechanism.