PpNAC transcription factors regulate <i>ENDOPOLYGALACTURONASE</i> alleles and control peach fruit texture and stone adhesion

Fruit texture variation in peach (Prunus persica L. Batsch), especially softening accompanied by the melting phase, directly affects marketability and storage. Two tandem endopolygalacturonase (endoPG) genes at the F-M locus, PGF and PGM, determine the melting or nonmelting trait, which is associated with the freestone or clingstone trait. In this study, freestone melting flesh (FMF), clingstone melting flesh (CMF), and clingstone nonmelting flesh (CNMF) cultivars were sampled across various developmental and postharvest stages. PGF-PGM haplotypes were identified using molecular markers, and their contributions to the FMF/CMF/CNMF phenotypes were investigated. In FMF peach, the FMF phenotypes were related to elevated PGF expression with spatiotemporal differences between the flesh near the stone at early developmental stages and the whole flesh at the postharvest stage. PpNAC74 and PpNAC87, specifically expressed in the flesh near skin or stone, cooperatively activated PGF transcription in FMF. Two PGM alleles, PGMSer and PGMPhe, exhibited functional divergence with increased expression of the former dominantly conferring the CMF phenotype, and the recessive PGMPhe determining the CNMF phenotype. PpNAC1-PpNAC72-PpNAC100 synergistically activated PGMSer transcription during ethylene-induced melting in CMF. Transient overexpression or silencing of these PpNACs induced or repressed the expression of target endoPGs and accelerated or delayed softening, respectively. Whole-genome bisulfite sequencing suggested that DNA methylation mediates the expression of key softening-related genes and thus participates in the regulation of the flesh melting and freestone traits. Our findings elucidate the genetic and molecular regulatory network diversification that determines flesh texture and stone adhesion in peach fruit.