MdEXPA8 integrates transcriptional regulation and cell wall remodeling to modulate fruit softening in apple

Fruit softening is a critical determinant of apple fruit texture and postharvest quality; however, the transcriptional mechanisms controlling cell wall remodeling during this process remain largely unknown. Previous studies have shown that an A/T allelic variation at nucleotide position 517 of the transcription factor MdNAC5 is significantly associated with natural variation in fruit firmness. Based on this finding, the present study integrated ChIP-seq and transcriptome data from MdNAC5 transgenic materials to identify an expansin gene, MdEXPA8 , involved in cell wall remodeling. Yeast one-hybrid, dual-luciferase reporter, and electrophoretic mobility shift assays demonstrated that MdEXPA8 is a direct downstream target of MdNAC5, and that the MdNAC5 A allele exhibits a significantly stronger transcriptional activation of MdEXPA8 than MdNAC5 T . Consistently, the contents of cell wall components were significantly lower in MdNAC5 A transgenic lines than in MdNAC5 T lines. Further homologous and heterologous expression analyses in apple and tomato revealed that overexpression of MdEXPA8 markedly accelerated cell wall disassembly, reduced fruit firmness, and promoted fruit softening. Spatiotemporal expression analysis showed that MdEXPA8 expression gradually increased during fruit development and ripening, was significantly negatively correlated with fruit firmness, and was higher in cultivars exhibiting faster softening. Collectively, these results demonstrate that allelic variation in MdNAC5 regulates apple fruit softening through allele-dependent transcriptional activation of MdEXPA8 , providing an important candidate gene for improving fruit texture.