Apple transcription factor MdDof43 promotes fruit softening by regulating cell wall-modifying genes Mdβ-Gal2 and Mdα-AF3

Apple (Malus × domestica Borkh.) fruit firmness is a crucial determinant of fruit quality and postharvest storage capacity. The DNA-binding one-finger (Dof) transcription factor family has been demonstrated to play a significant role in regulating fruit ripening and softening; however, the genome-wide role of Dofs in determining apple firmness remains unclear. Here, we examined the expression patterns of 50 MdDofs in two apple varieties, ‘Fuji’ and ‘Cripp's Pink’, known for their distinct fruit firmness and cell wall components during fruit development and ripening. We also clarified the function and regulatory network of the MdDof43 in regulating apple fruit firmness. Functional validation indicated that MdDof43 enhanced the water-soluble pectin content, increased neutral sugar loss, decreased hemicellulose content and fruit firmness in transgenic apple, calli and tomato. RNA sequencing analysis revealed that MdDof43 overexpression induced the expression levels of many genes involved in the cell wall degradation in apple calli. DNA affinity purification and sequencing (DAP seq) demonstrated that MdDof43 directly binds to the promoters of Mdβ-Gal2 and Mdα-AF3, two genes encoding pectin side-chain regulating enzymes. In vitro assays confirmed that MdDof43 positively regulates the expression of Mdβ-Gal2 and Mdα-AF3, facilitating the degradation of cell wall components and promoting fruit softening. These findings provide insights into the regulatory mechanisms of cell wall structures that contribute to apple fruit firmness, which has practical significance for fruit quality in breeding.