Osmotic stress is one of the main stresses seriously affects the growth and development of plants. Hydrogen sulfide (H2S) emerges as the third gaseous signal molecule to involve in the complex network of signaling events. Phospholipase D delta (PLD delta), as signal enzyme, responds to many biotic or abiotic stress responses. In this study, the functions and the relationship of PLD delta and H2S in stomatal closure induced by osmotic stress were explored. Using the seedlings of ecotype (WT), PLD delta deficient mutant (pld delta), L-cysteine desulfhydrase (LCD) deficient mutant (lcd) and pld delta lcd double mutant as materials, the Realtime quantitative PCR (RT-qPCR) and the stomatal aperture were analyzed. Osmotic stress induced the expressions of PLD delta and LCD. The H2S content and the activities of PLD and LCD ascended in WT under osmotic stress. The phenotypes of pld delta, lcd and pld delta lcd were more sensitive to osmotic stress than WT. Compared with pld delta, the stomatal of lcd showed lower sensitivity to osmotic stress, and the stomatal aperture of pld delta lcd was similar to that of lcd. Simultaneous application of PA and NaHS resulted in tighter closure of stomatal than application of either PA or NaHS alone. These results suggested that osmotic stress-triggered stomatal closure requires PLD delta and H2S in A. thaliana. LCD acted downstream of PLD delta to regulate the stomatal closure induced by osmotic stress. (c) 2020 Elsevier Inc. All rights reserved.