MdDGK3-like as a negative regulator participates in ALA-induced PP2AC to promote stomatal opening in apple leaves

5-Aminolevulinic acid (ALA) can inhibit abscisic acid (ABA)-induced stomatal closure. However, the molecular mechanism is unclear. In this study, we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple (Malus domestica Borkh. cv. ‘Fuji’) leaves. With the promoter of MdPP2AC as bait, a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid (Y1H) from the cDNA library of the epidermis of apple leaves treated by exogenous ALA. Additional to binding the promoter of MdPP2AC, MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter, while ALA significantly eliminated the role of MdDGK3-like. In tobacco leaves, MdDGK3-like was localized in the nucleus of stomatal guard cells. Therefore, MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure. To further identify MdDGK3-like functions, several transiently transgenic apple leaves (including overexpression and interference) were established. The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca²⁺ and H₂O₂ and decreasing flavonol levels in the guard cells. Conversely, MdDGK3-like (ⅰ) led the stomatal opening with lower levels of Ca²⁺ and H₂O₂ but higher flavonols. Based on these, we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity, which improved the stomatal aperture. Since it was the first time that MdDGK3-like was showed to act as a transcription factor, the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.