Coronatine Inhibits Stomatal Closure through Guard Cell-Specific Inhibition of NADPH Oxidase-Dependent ROS Production

Abstract

Microbes trigger stomatal closure through microbe-associated molecular patterns (MAMPs). The bacterial pathogen <i>Pseudomonas syringae</i> pv. <i>tomato</i> (<i>Pst</i>) synthesizes the polyketide toxin coronatine, which inhibits stomatal closure by MAMPs and by the hormone abscisic acid (ABA). The mechanism by which coronatine, a jasmonic acid-isoleucine analog, achieves this effect is not completely clear. Reactive oxygen species (ROS) are essential second messengers in stomatal immunity, therefore we investigated the possible effect of coronatine on their production. We found that coronatine inhibits NADPH oxidase-dependent ROS production induced by ABA, and by the flagellin-derived peptide flg22. This toxin also inhibited NADPH oxidase-dependent stomatal closure induced by darkness, however, it failed to prevent stomatal closure by exogenously applied H₂O₂ or by salicylic acid, which induces ROS production through peroxidases. Contrary to what was observed on stomata, coronatine did not affect the oxidative burst induced by flg22 in leaf disks. Additionally, we observed that in NADPH oxidase mutants <i>atrbohd</i> and <i>atrbohd/f</i>, as well as in guard cell ABA responsive but flg22 insensitive mutants <i>mpk3, mpk6, npr1-3</i>, and <i>lecrk-VI.2-1</i>, the inhibition of ABA stomatal responses by both coronatine and the NADPH oxidase inhibitor diphenylene iodonium was markedly reduced. Interestingly, coronatine still impaired ABA-induced ROS synthesis in <i>mpk3, mpk6, npr1-3</i>, and <i>lecrk-VI.2-1</i>, suggesting a possible feedback regulation of ROS on other guard cell ABA signaling elements in these mutants. Altogether our results show that inhibition of NADPH oxidase-dependent ROS synthesis in guard cells plays an important role during endophytic colonization by <i>Pst</i> through stomata.