Hydrogen Sulfide Increases Production of NADPH Oxidase-Dependent Hydrogen Peroxide and Phospholipase D-Derived Phosphatidic Acid in Guard Cell Signaling.

Hydrogen sulfide (HS) is an important gaseous signaling molecule in plants that participates in stress responses and development. l-Cys desulfhydrase 1, one of the enzymatic sources of HS in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of HS in stomatal closure and the interplay between HS and other second messengers of the guard cell signaling network, such as hydrogen peroxide (HO) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for HS-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that HS stimulates HO production in Arabidopsis guard cells. Additionally, we observed an interplay between HS and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for HS-induced stomatal closure, and most of the HS-dependent HO production required the activity of PLDα1. Finally, we showed that HS induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of HS in the signaling network that controls stomatal closure, and suggest that HS regulates NADPH oxidase and PLD activity in guard cells.