Tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of the PpPGF to regulate peach softening during fruit ripening.

Peach fruit rapidly soften after harvest, a significant challenge for producers and marketers as it results in rotting fruit and significantly reduces shelf life. In this study, we identified two tandem genes, PpNAC1 and PpNAC5, within the sr (slow ripening) locus. Phylogenetic analysis showed that NAC1 and NAC5 are highly conserved in dicots and that PpNAC1 is the orthologous gene of Non-ripening (NOR) in tomato. PpNAC1 and PpNAC5 were highly expressed in peach fruit, with their transcript levels up-regulated at the onset of ripening. Yeast two-hybrid and bimolecular fluorescence complementation assays showed PpNAC1 interacting with PpNAC5 and this interaction occurs with the tomato and apple orthologues. Transient gene silencing experiments showed that PpNAC1 and PpNAC5 positively regulate peach fruit softening. Yeast one-hybrid and dual luciferase assays and LUC bioluminescence imaging proved that PpNAC1 and PpNAC5 directly bind to the PpPGF promoter and activate its transcription. Co-expression of PpNAC1 and PpNAC5 showed higher levels of PpPGF activation than expression of PpNAC1 or PpNAC5 alone. In summary, our findings demonstrate that the tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of PpPGF to regulate fruit softening during peach fruit ripening.