Abstract: The stomatal aperture，guard cell H2O2 levels and gene expressions were studied pharmacologically in the abaxial epidermis of detached apple（Malus × domenstica Borkh.‘Fuji’）leaves with laser scanning confocal microscopy and qRT-PCR techniques to investigate the role and possible regulatory mechanisms of microtubule skeleton and PP1/PP2A protein phosphatase in the inhibitory effect of 5-aminolevulinic acid（ALA）on abscisic acid（ABA）-induced stomatal closure. It was observed that vinblastine（VBT），a microtubule disassemble agent，depressed stomatal opening induced by ALA，while taxol，a microtubule stabilizer，undermined stomatal closure induced by ABA. This suggests that ALA inhibits ABA-induced stomatal closure depending on the microtubule polymerization of the guard cells. Exogenous ALA reversed the down-regulation of the expressions of tubulin coding genes including TUB1，MAP65-1 and MAP65-3 induced by ABA，suggesting that ALA promoted stomatal opening by promoting microtubule polymerization in guard cells. On the other hand，okadaic acid（OA），an inhibitor of PP1/PP2A protein phosphatase caused both microtubule depolymerization and stomatal closure，indicating that microtubule polymerization and stomatal opening of the guard cells were positively regulated by PP1/PP2A. Furthermore，OA promoted H2O2 accumulation in guard cells，which caused microtubule depolymerization and stomatal closure. This suggests that PP1/PP2A acts at the upstream of H2O2 generation. The above results indicate that ALA may promote PP1/PP2A protein phosphatase activity in apple leaf guard cells，and then inhibit ABA-induced H2O2 production，which up-regulates the expression of tubulin genes and promotes microtubule polymerization of stomatal guard cells，suppressing ABA-induced the stomatal closure. Taken together，we revealed a possible mechanism for ALA-improved photosynthesis in plants，providing a theoretical basis for the application of ALA in agricultural production.

Key words: apple, ABA, ALA, H2O2, microtubule, PP1/PP2A protein phosphatase, stomata