Genome-wide identification of α-ʟ-arabinofuranosidase and β-ᴅ-xylosidase genes in apple and functional characterization of <i>MdAF3</i> in fruit softening

Abstract α-ʟ-Arabinofuranosidase (α-AFase) and β-ᴅ-xylosidase (β-Xyl) are cell wall-modifying enzymes that often exhibit overlapping substrate specificity and are involved in the coordinated disassembly of pectin and hemicellulose networks during fruit softening. A genome-wide analysis of this functionally linked gene family in apple is lacking. Here, we identified 13 α-AFase/β-Xyl genes in the apple genome. Phylogenetic, structural, and promoter analyses revealed their evolutionary relationships and potential for complex regulation. Expression profiling via reverse transcription quantitative polymerase chain reaction (RT-qPCR) demonstrated that three genes were detectable in flesh tissue of ‘Oregon Spur II’ fruit during ambient storage, with MdAF3 showing the strongest induction. Functional characterization through transient transformation indicated that MdAF3, a cell wall-localized protein, positively regulates fruit softening by modulating the activities of multiple cell wall-degrading enzymes and accelerating the solubilization and degradation of cell wall polysaccharides. Our work provides an integrated analysis of the α-AFase/β-Xyl family in apple and establishes MdAF3 as a key regulator of fruit texture, offering a potential target for improving postharvest quality in apple.