Sphingosine-1-phosphate （S1P） mediates darkness-induced stomatal closure through raising cytosol pH and hydrogen peroxide （HO） levels in guard cells in Vicia faba

The role and signaling of sphingosine-1-phosphate (S1P) during darkness-induced stomatal closure were examined in Vicia faba. Darkness substantially raised S1P and hydrogen peroxide (H 2 O 2 ) levels and closed stomata. These darkness effects were significantly suppressed by DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS), two inhibitors of long-chain base kinases. Exogenous S1P led to stomatal closure and H 2 O 2 production, and the effects of S1P were largely prevented by the H 2 O 2 modulators ascorbic acid, catalase, and diphenyleneiodonium. These results indicated that S1P mediated darkness-induced stomatal closure by triggering H 2 O 2 production. In addition, DL-threo-DHS and DMS significantly suppressed both darkness-induced cytosolic alkalization in guard cells and stomatal closure. Exogenous S1P caused cytosolic alkalization and stomatal closure, which could be largely abolished by butyric acid. These results demonstrated that S1P syn-thesis was necessary for cytosolic alkalization during stomatal closure caused by darkness. Furthermore, together with the data described above, inhibition of darkness-induced H 2 O 2 production by butyric acid revealed that S1P synthesis-induced cytosolic alkalization was a prerequisite for H 2 O 2 production during stomatal closure caused by darkness, a conclusion supported by the facts that the pH increase caused by exogenous S1P had a shorter lag and peaked faster than H 2 O 2 levels and that butyric acid prevented exogenous S1P-induced H 2 O 2 production. Altogether, our data suggested that darkness induced S1P synthesis, causing cytosolic alkalization and subsequent H 2 O 2 production, finally leading to stomatal closure.