Abstract
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.