MdPP2AC interacting with MdCLC to mediate ALA-induced salt tolerance in apple

5-Aminolevulinic acid (ALA) is a plant growth regulator that enhances salt tolerance, yet the underlying mechanisms remain unclear. In this study, we investigated the function and molecular mechanism of the Protein Phosphatase 2A (PP2A) catalytic subunit MdPP2AC in ALA-induced salt tolerance in detached leaves, calli, and rooted plantlets of 'Gala' apple (Malus × domestica). Results showed that pretreatment with ALA before NaCl stress significantly alleviated NaCl-induced damage, enhanced antioxidant enzyme activities, and improved chloride (Cl^-) interception in the roots with less transport to the aboveground. Further analysis revealed that the PP2A activity and MdPP2AC expression were induced by NaCl and further by ALA. Functional studies showed that overexpressing (OE)-MdPP2AC enhanced salt tolerance, whereas RNA interference (RNAi)-MdPP2AC or application of cantharidin (CT, a specific inhibitor of PP2A activity) compromised salt tolerance, but exogenous ALA mitigated CT-aggravated salt injury. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), luciferase complementation imaging (LCI), sequence truncation and site-directed mutagenesis assays confirmed that MdPP2AC can interact with the chloride channel protein MdCLC-c2, and the C-terminal Glu⁶⁸⁴ residue of MdCLC-c2 is indispensable for the molecular interaction and Cl^- transport activation. Function identification with Δgef1 mutant yeast showed that MdCLC-c2 transformation promoted intracellular Cl^- accumulation as well as cell salt tolerance, and the effects were further promoted by MdPP2AC transformation and exogenous ALA. Collectively, we propose that MdPP2AC mediates ALA-induced salt tolerance in apple by interacting with MdCLC-c2, which promotes Cl^- sequestration into root vacuoles with less transport to the aboveground, thus maintaining Cl^- homeostasis at the cellular and plant levels. These findings reveal a novel mechanism whereby PP2AC modulates chloride channel function to maintain ion homeostasis within the ALA signaling pathway, providing critical insights into ALA-mediated apple salt tolerance.