The phosphate-responsive protein MdEXO enhances postharvest firmness in apple by modulating brassinosteroid biosynthesis and cell wall metabolism

Abstract Fruit softening is a critical determinant of shelf life and quality of apples ( Malus domestica ). While the degradation of cell wall components is a well-established driver of this process, the specific involvement of brassinosteroids (BRs) in postharvest apple softening and how their upstream regulators influence cell wall integrity perception remain unclear. In this study, transcriptomic analysis identified EXORDIUM ( MdEXO ) as a gene whose expression declined during storage. Real-time quantitative PCR (RT–qPCR) assays and in situ hybridization confirmed this downregulation and revealed a positive correlation between MdEXO expression level and fruit firmness. Further characterization showed that the MdEXO protein was phosphate-responsive and localized to the cell wall. Functional studies demonstrated that overexpression of MdEXO significantly enhanced fruit firmness, increased protopectin and cellulose content, and reduced soluble pectin levels. These changes were accompanied by the downregulation of key cell wall degradation-related genes, including polygalacturonase 1 ( MdPG1 ), pectate lyase 5 ( MdPL5 ), and β-galactosidase 9 ( Mdβ-Gal9 ). Additionally, MdEXO overexpression increased endogenous BR levels by upregulating the BR biosynthetic gene DWARF 4 ( MdDWF4 ). On the contrary, treatment with brassinazole (BRZ), the BR biosynthesis inhibitor attenuated the firmness-promoting effect of MdEXO overexpression. Furthermore, VIGS-mediated transient silencing of MdEXO in apple fruits produced the opposite phenotypic effects compared to the overexpression lines. These results demonstrate that MdEXO delays apple fruit softening by modulating BR biosynthesis, offering new insights into the hormonal regulation of postharvest fruit quality.