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

Hydrogen sulfide (H 2 S) 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 H 2 S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H 2 S in stomatal closure and the interplay between H 2 S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H 2 O 2 ) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis ( Arabidopsis thaliana ). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H 2 S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H 2 S stimulates H 2 O 2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H 2 S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H 2 S-induced stomatal closure, and most of the H 2 S-dependent H 2 O 2 production required the activity of PLDα1. Finally, we showed that H 2 S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H 2 S in the signaling network that controls stomatal closure, and suggest that H 2 S regulates NADPH oxidase and PLD activity in guard cells.