CCCH zinc-finger proteins constitute a unique class of transcription factors that play vital roles in mediating plant tolerance to biotic and abiotic stresses and regulating various physiological and developmental processes. This study systematically identified and characterized the apple (Malus domestica) CCCH (MdC3H) gene family, aiming to elucidate its evolutionary patterns, functional characteristics, and regulatory mechanisms under drought stress. Genome-wide analysis revealed 85 MdC3H genes, which were unevenly distributed across chromosomes and exhibited significant differences in physiochemical properties, suggesting functional divergence. Phylogenetic analysis classified these genes into 9 subfamilies with distinct conservation. Collinearity analysis indicated a close evolutionary relationship between apple and Malus sieversii, with 150 collinear gene pairs identified, highlighting the conservation of the C3H gene family during speciation. Cis-acting element prediction in promoter regions uncovered abundant stress-responsive elements (e.g., ABRE, DRE, MYB), implying the potential of MdC3H genes in coordinating environmental signals. Functional verification demonstrated that MdC3H49, a key member of the family, is localized in the nucleus and possesses transcriptional activation activity. Overexpression of MdC3H49 in Arabidopsis and apple calli significantly enhanced drought tolerance, characterized by reduced malondialdehyde (MDA) content, relative electrical conductivity, and increased proline accumulation. Mechanistic studies revealed that MdC3H49 directly regulates the expression of MdP5CS, a core gene in proline biosynthesis, thereby strengthening the cellular antioxidant capacity and mitigating drought-induced damage. Collectively, this study establishes MdC3H49 as a critical regulator in apple drought stress response, providing valuable insights into the molecular mechanisms underlying abiotic stress tolerance in perennial plants and laying a foundation for genetic improvement of drought resistance in apple breeding.
Zhang et al. (2026) studied this question.