Upregulation of <i>PECTATE LYASE5</i> by a NAC transcription factor promotes fruit softening in apple

Flesh firmness is a critical breeding trait that determines consumer selection, shelf life, and transportation. The genetic basis controlling firmness in apple (Malus × domestica Borkh.) remains to be fully elucidated. We aimed to decipher genetic variance for firmness at harvest and develop potential molecular markers for marker-assisted breeding. Maturity firmness for 439 F1 hybrids from a cross of "Cripps Pink" and "Fuji" was determined in 2016 and 2017. The phenotype segregated extensively, with a Gaussian distribution. In a combined bulked segregant analysis (BSA) and RNA-sequencing analysis, 84 differentially expressed genes were screened from the 10 quantitative trait loci regions. Interestingly, next-generation re-sequencing analysis revealed a Harbinger-like transposon element insertion upstream of the candidate gene PECTATE LYASE5 (MdPL5); the genotype was associated with flesh firmness at harvest. The presence of this transposon repressed MdPL5 expression and was closely linked to the extra-hard phenotype. MdPL5 was demonstrated to promote softening in apples and tomatoes. Subsequently, using the MdPL5 promoter as bait, MdNAC1-L was identified as a transcription activator that positively regulates ripening and softening in the developing fruit. We also demonstrated that MdNAC1-L could induce the up-regulation of MdPL5, MdPG1, and the ethylene-related genes MdACS1 and MdACO1. Our findings provide insight into TE-related genetic variation and the PL-mediated regulatory network for the firmness of apple fruit.