Ma, Y., She, X. and Yang, S. 2013. Cytosolic alkalization-mediated H₂O₂and NO production are involved in darkness-induced stomatal closure inVicia faba. Can. J. Plant Sci. 93: 119-130. Darkness raised cytosolic pH, hydrogen peroxide (H₂O₂) and nitric oxide (NO) levels in guard cells while inducing Vicia faba stomatal closure. These darkness effects were prevented by weak acid butyric acid, H₂O₂ modulators ascorbic acid (ASA), catalase (CAT), diphenyleneiodonium (DPI) and NO modulators 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), N^G-nitro-L-arg-methyl ester (L-NAME) respectively. The data suggest that cytosolic alkalization, H₂O₂ and NO all participate in darkness-induced stomatal closure. During darkness treatment, pH rise became noticeable at 10 min and peaked at 25 min, while H₂O₂ and NO production increased significantly at 20 min and reached their maximums at 40 min. The H₂O₂ and NO levels were increased by methylamine in light and decreased by butyric acid in darkness. The results show that cytosolic alkalization induces H₂O₂ and NO production. ASA, CAT and DPI suppressed NO production by methylamine, c-PTIO and L-NAME prevented H₂O₂ generation by methylamine. Calcium chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) restricted darkness-induced alkalization, H₂O₂ and NO production and stomatal closure. We suggest that cytosolic alkalization is necessary for H₂O₂ and NO production during darkness-induced stomatal closure. H₂O₂ mediates NO synthesis by alkalization, and vice versa. Calcium may act upstream of cytosolic alkalization, H₂O₂ and NO production, besides its known action downstream of H₂O₂ and NO.