A natural variant in MdNAC25 promoter contributes to fruit harvest date divergence in apple

Fruit ripening is a quantitative trait governed by multiple genes, influencing fruit quality and commercial values. However, the genetic mechanisms underlying this process remain poorly understood. Here, we elucidate the role of MdNAC25 in regulating apple fruit ripening. We identified allele-specific expression (ASE) of MdNAC25 in various apple cultivars, driven by a 4-bp deletion in its promoter. This deletion created a key regulatory element, ACS2-TAAAATAT, which enhanced MdNAC25 promoter activity in tobacco leaf promoter-reporter assays. Additionally, a long non-coding RNA (lncRNA-nac25) located upstream of MdNAC25 also exhibited ASE and was positively correlated with MdNAC25 expression. The ACS2-TAAAATAT element, positioned between MdNAC25 and lncRNA-nac25, functioned as a bidirectional enhancer, regulating both genes. Overexpression and silencing experiments demonstrated that lncRNA-nac25 enhanced MdNAC25 expression. Genetic association studies in 154 apple accessions linked the MdNAC25 allele with the 4-bp deletion to earlier fruit ripening. This allele was significantly more frequent in cultivated apples than in wild species, indicating artificial selection. Furthermore, overexpression of MdNAC25 in non-ripening (nor) mutant tomatoes restored ripening, reinforcing its functional significance. Comparative genomic analysis revealed conservation of the ACS2-TAAAATAT element across Rosaceae fruit trees, including peach, pear, plum, and apricot, underscoring MdNAC25 as a key regulator of fruit ripening across species.