The role of phytosphingosine-1-phosphate (Phyto-S1P) and its relationships with cytosolic pH and hydrogen peroxide (H2O2) during stomatal closure by darkness in broad bean

We examined the role of phytosphingosine-1-phosphate (Phyto-S1P) and its relationships with cytosolic pH and hydrogen peroxide (H2O2) during stomatal closure by darkness using broad bean as experimental material. Darkness caused cytoplasmic alkalization, induced H2O2 generation, and led to stomatal closure. DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS) inhibited these effects of darkness. Exogenous Phyto-S1P promoted cytoplasmic alkalization, H2O2 synthesis, and caused stomatal closure. Butyric acid largely suppressed cytoplasmic alkalization and prevented stomatal closure by Phyto-S1P. The H2O2 modulators ascorbic acid (ASA), catalase (CAT), and diphenyleneiodonium (DPI) obviously restricted Phyto-S1P-promoted H2O2 synthesis and stomatal closure. These results suggest that Phyto-S1P was involved in darkness-induced stomatal closure by causing cytoplasmic alkalization and H2O2 synthesis. Furthermore, darkness-induced H2O2 synthesis was inhibited by butyric acid, suggesting that cytoplasmic alkalization caused by Phyto-S1P was probably necessary for H2O2 synthesis. The rise in cytosolic pH promoted by Phyto-S1P became noticeable at 6 min and reached a maximum at 18 min, while H2O2 levels sharply increased at 8 min and peaked at 22 min, and butyric acid prevented Phyto-S1P-promoted H2O2 synthesis, which confirmed the conclusion that Phyto-S1P-induced cytosolic alkalization acts upstream of the production of H2O2 during stomatal closure by darkness. We suggest that Phyto-S1P is involved in stomatal closure by darkness through cytoplasmic alkalization and H2O2 synthesis, and Phyto-S1P-promoted cytoplasmic alkalization acts upstream of H2O2 synthesis in the process.