Abscisic Acid Increase Hydrogen Peroxide in Mitochondria to Facilitate Stomatal Closure

Abstract

Abscisic acid (ABA) drives stomatal closure to minimize water loss due to transpiration in response to drought. We examined the subcellular location of ABA increased accumulation of reactive oxygen species (ROS) in guard cells that drive stomatal closure. ABA-dependent increases in fluorescence of the generic ROS sensor, dichlorofluorescein (DCF), were observed in mitochondria, chloroplasts, cytosol, and nuclei. The ABA response in all these locations were lost in an ABA-insensitive quintuple receptor mutant. The ABA-increased fluorescence in mitochondria of both DCF and an H2O2-selective probe, Peroxy Orange 1 (PO1), colocalized with Mitotracker Red. ABA treatment of guard cells transformed with the genetically-encoded H2O2 reporter targeted to the cytoplasm (roGFP2-Orp1), or mitochondria (mt-roGFP2-Orp1), revealed H2O2 increases. Consistent with mitochondrial ROS changes functioning in stomatal closure, we found that guard cells of a mutant with mitochondrial defects, abo6, have elevated ABA-induced ROS in mitochondria and enhanced stomatal closure. These effects were phenocopied with rotenone, which increased mitochondrial ROS. In contrast, the mitochondrially targeted antioxidant, MitoQ, dampened ABA effects on mitochondrial ROS accumulation and stomatal closure in Col-0 and reversed the guard cell closure phenotype of the abo6 mutant. ABA-induced ROS accumulation in guard cell mitochondria was lost in mutants in genes encoding Respiratory Burst Oxidase Homolog (RBOH) enzymes and reduced by treatment with the RBOH inhibitor VAS2870, consistent with RBOH machinery acting in ABA-increased ROS in guard cell mitochondria. These results demonstrate that ABA elevates H2O2 accumulation in guard cell mitochondria to promote stomatal closure. One sentence summaryGenetically encoded biosensors and chemical probes revealed ABA-dependent increases in hydrogen peroxide, a reactive oxygen species with signaling activity, in guard cell cytoplasm and mitochondria.