Abstract: In the present studies, the effects and mechanisms of natural and synthetic auxin IAA, NAA, 2,4-D, cytokinin ZT, KT, 6-BA on dark- and ABA-induced stomatal closure were investigated by means of stomatal bioassay and using laser-scanning confocal microscopy. Isolated epidermal strips of Vicia faba were incubated with IAA (10 mmol L^-1), NAA (10 mmol L^-1), 2,4-D (10 mmol L^-1), ZT (0.1 mmol L^-1), KT (0.2 mmol L^-1), 6-BA (0.2 mmol L^-1), NO scavenger c-PTIO (200 mmol L^-1), Hb (100 mmol L^-1), NOS inhibitor L-NAME (25 mmol L^-1), H₂O₂ scavenger AsA (100 mmol L^-1), CAT (100 U mL^-1), inhibitor of H₂O₂-generating enzyme NADPH oxidase DPI (10 mmol L^-1) for 3 h, in darkness or in light in the presence of ABA (1 mmol L^-1), respectively. The results showed that auxin, cytokinin, as well as c-PTIO, Hb, L-NAME, AsA, CAT, and DPI, reversed dark- and ABA-induced stomatal closure significantly. Epidermal strips treated with auxin and cytokinin were loaded with NO-fluorescent dye DAF-2DA or H₂O₂-fluorescent dye H₂DCF-DA. The results indicated that darkness and ABA could induce an intense DAF-2DA or H₂DCF-DA fluorescence in guard cells. However, dark- and ABA-induced DAF-2DA and H₂DCF-DA fluorescence were largely prevented by auxin and cytokinin tested. Similarly, the treatments of c-PTIO, Hb, L-NAME and AsA, CAT, DPI also substantially suppressed dark- and ABA-induced DAF-2DA and H₂DCF-DA fluorescence, respectively. These results provide the evidence that auxin and cytokinin tested lessen assuredly NO and H₂O₂ levels induced by dark and ABA in guard cells. Considering synthetic auxin, cytokinin NAA, 2,4-D, KT, 6-BA and natural IAA, ZT were used in the present work and IAA, ZT are representative of endogenous auxin and cytokinin respectively, the effects of auxin, cytokinin tested on dark- and ABA-induced stomatal closure and NO, H₂O₂ level can be attributed to an universal effect of auxin or cytokinin.

Key words: Auxin, Cytokinin, Nitric oxide, Hydrogen peroxide, Dark, ABA, Stomatal closure