Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity

Hydrogen sulfide (H₂S) is a gaseous signaling molecule involved in numerous physiological processes in plants, including gas exchange with the environment through the regulation of stomatal pore width. Guard cells are pairs of specialized epidermal cells that delimit stomatal pores and have a higher mitochondrial density and metabolic activity than their neighboring cells. However, there is no clear evidence on the role of mitochondrial activity in stomatal closure induction. In this work, we showed that the mitochondrial-targeted H2S donor AP39 induces stomatal closure in a dose-dependent manner. Experiments using inhibitors of the mitochondrial electron transport chain (mETC) or insertional mutants in cytochrome c (CYTc) indicated that the activity of mitochondrial CYTc and/or complex IV are required for AP39-dependent stomatal closure. By using fluorescent probes and genetically-encoded biosensors we reported that AP39 hyperpolarized the mitochondrial inner potential (ψm) and increased cytosolic ATP, cytosolic hydrogen peroxide levels, and oxidation of the glutathione pool in guard cells. These findings showed that mitochondrial-targeted H2S donors induce stomatal closure, modulate guard cell mitochondrial ETC activity, the cytosolic energetic and oxidative status, pointing to an interplay between mitochondrial H2S, mitochondrial activity, and stomatal closure.