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

Abstract

Hydrogen sulfide (H₂S) 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 H₂S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H₂S in stomatal closure and the interplay between H₂S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H₂O₂) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (<i>Arabidopsis thaliana</i>). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H₂S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H₂S stimulates H₂O₂ production in Arabidopsis guard cells. Additionally, we observed an interplay between H₂S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H₂S-induced stomatal closure, and most of the H₂S-dependent H₂O₂ production required the activity of PLDα1. Finally, we showed that H₂S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H₂S in the signaling network that controls stomatal closure, and suggest that H₂S regulates NADPH oxidase and PLD activity in guard cells.