Functional characterization of MhbZIP62 in regulating drought tolerance in apple

MhbZIP62-MhbZIP23 module has a positive regulatory effect on tobacco and apple calli under drought stress. Drought stress seriously restricts the efficient cultivation and sustainable development of the apple industry. In this study, 19 apple MhbZIP genes were identified from transcriptome datasets, followed by systematic analyses of their physicochemical characteristics, promoter cis-acting elements, and stress-responsive expression profiles. Bioinformatic analysis revealed that most encoded proteins are hydrophilic and unstable, and their promoter regions contain abundant cis-elements responsive to diverse abiotic stresses and phytohormones. Transcriptional assays showed that MhbZIP family genes are markedly upregulated upon PEG 6000, mannitol and ABA treatments, with MhbZIP62 displaying the most prominent inducible expression. Functional assays in transgenic tobacco and apple calli verified that ectopic overexpression of MhbZIP62 substantially improves drought tolerance, elevates endogenous ABA levels, and stimulates antioxidant enzyme activities. Consistently, VIGS-mediated knockdown of MhbZIP62 compromises drought-associated physiological traits, whereas exogenous ABA supplementation partially restores the defective phenotype. Yeast two-hybrid and subsequent biochemical assays validated physical protein-protein interaction between MhbZIP62 and MhbZIP23. In apple calli, co-overexpression of the two genes synergistically strengthens drought resistance relative to single-gene overexpression. Furthermore, transient silencing of MhbZIP23 in apple seedlings leads to aggravated leaf wilting, enlarged stomatal aperture, suppressed antioxidant enzyme activities, and reduced accumulation of proline and chlorophyll, ultimately impairing drought adaptability. Collectively, this work characterizes the drought-resistant functions of MhbZIP62 and MhbZIP23 as well as their synergistic regulation via protein interaction, and phenotypic and physiological data imply potential involvement of ABA-dependent pathways in their drought regulatory cascade, which lays a theoretical foundation for drought-resistant molecular breeding and agronomic improvement in apple.