Genome-Wide Identification of the Ca2+-ATPase Gene Family and Functional Analysis of MdACA39 in Resistance to Alternaria alternata in Malus domestica

The calcium ion-transporting ATPase (Ca2+-ATPase) gene family maintains plant intracellular Ca2+ homeostasis and regulates growth, development and stress immunity; however, its functions remain poorly characterized in Malus domestica. Here, we performed a genome-wide identification of apple Ca2+-ATPase genes and obtained 45 members, which were classified into MdACA (39) and MdECA (6) subfamilies and unevenly distributed on 14 chromosomes. Phylogenetic analysis of Ca2+-ATPase genes from Malus domestica, Arabidopsis thaliana, and Oryza sativa classified these proteins into five subgroups. The ACA and ECA subfamilies were highly conserved across species, whereas Group D was apple-specific. Collinearity and Ka/Ks analyses indicated that segmental duplication and purifying selection dominated the evolution of apple Ca2+-ATPase genes. Promoter cis-element prediction uncovered numerous regulatory elements related to phytohormone signaling, growth, development and stress defense. Codon usage bias analysis indicated that AUG (methionine) was the dominant codon. Tissue expression profiles showed differential expression of apple Ca2+-ATPase genes in various organs. Quantitative real-time PCR (qRT-PCR) assays demonstrated widespread responses of Ca2+-ATPase genes to Alternaria alternata infection, exogenous CaCl2, salicylic acid (SA) and methyl jasmonate (MeJA), among which MdACA39 was strongly induced under all treatments. Subcellular localization verified that MdACA39 resides on the plasma membrane. Moreover, transient overexpression of MdACA39 significantly enhanced apple resistance to A. alternata, likely due to the activation of SA, MeJA and Ca2+ signaling-mediated immune pathways, the induction of disease resistance-related genes, and elevated antioxidant enzyme activity. Collectively, this study systematically characterizes the apple Ca2+-ATPase family and identifies MdACA39 as a key regulator of fungal resistance, providing valuable gene resources for dissecting Ca2+ signaling-mediated disease resistance in apple.