Phospholipase Dδ and H2S increase the production of NADPH oxidase-dependent H2O2 to respond to osmotic stress-induced stomatal closure in Arabidopsis thaliana

Osmotic stress is one of the main stresses that seriously affects the survival of plants, destroying normal cell activities, and potentially leading to plant death. Phospholipase D (PLD), a major lipid hydrolase, hydrolyzes membrane phospholipids to produce phosphatidic acid (PA) and responds to many abiotic stresses. Hydrogen sulfide (H2S) emerges as the third gaseous signaling molecule involved in the complex network of signaling events. Hydrogen peroxide (H2O2) plays a crucial role as a signaling molecule in plant development and growth, and responds to various abiotic and biotic stresses. In this study, the functions and the relationship of PLDδ, H2S, and H2O2 in osmotic stress-induced stomatal closure were explored. By using the seedlings of ecotype (WT), PLDδ-deficient mutant (pldδ), l-cysteine desulfhydrase (LCD)-deficient mutant (lcd), and pldδlcd double mutant, atrbohD, and atrbohF mutant as materials, and the stomatal aperture were analyzed. The relative water loss of pldδ, lcd, and pldδlcd was higher than that of WT. Exogenous PA and NaHS could partially alleviate the leaf wilting and yellowing phenotypes of pldδ, lcd, and pldδlcd under osmotic stress, but the mutants could not be restored to the same phenotype as WT. The fluorescence intensity of H2O2 in guard cells of pldδ, lcd, and pldδlcd was lower than that of WT, indicating that PLDδ and LCD were involved in the production of H2O2 in guard cells. Exogenous application of H2O2 to WT, pldδ, lcd, and pldδlcd significantly induced stomatal closure under osmotic stress. Exogenous NaHS induced stomatal closure of WT, but could not induce stomatal closure of atrbohD and atrbohF under osmotic stress. These results suggest that the accumulation of H2O2 was essential to induce stomatal closure under osmotic stress, and PLDδ and LCD acted upstream of H2O2.