PpNAP4 and ethylene act in a regulatory loop to modulate peach fruit ripening and softening.

Ripening significantly influences fruit quality and commercial value. Peaches (Prunus persica), a climacteric fruit, exhibit increased ethylene biosynthesis and decreased fruit firmness during ripening. NAC-like proteins activated by AP3/P1 (NAP) proteins are a subfamily of NAC transcription factors, and certain NAPs have been shown to intervene in fruit ripening. Here, we revealed that one NAP member PpNAP4, along with ethylene, positively regulated peach ripening and softening. Positive regulation of fruit ripening by PpNAP4 was demonstrated by overexpressing PpNAP4 in both peaches and tomatoes, resulting in enhanced fruit ripening through targeted modulation of specific ethylene biosynthesis and cell wall degradation-related genes. Further investigation revealed that PpNAP4 targets and upregulates key ethylene biosynthesis genes PpACS1, PpACO1 and PpEIN2, which is the core component of ethylene signaling. PpNAP4 positively modulates fruit softening by binding to and activating the promoters of cell wall degradation-related genes PpPL1 and PpPL15. Additionally, expression of PpPL1 and PpPL15 was directly affected by ethylene, with further investigation revealing that their promoters were clearly induced by ethylene. Our findings demonstrated a synergistic role played by the interaction between PpNAP4 and PpNAP6, enhancing the expression of PpACS1, PpACO1, PpPL1, PpPL15 and PpEIN2, thereby contributing to fruit ripening and softening. Overall, our study revealed the intricate mechanisms responsible for PpNAP4, PpNAP6, and ethylene roles during peach fruit ripening, highlighting a regulatory loop in which PpNAP4 and ethylene mutually enhance each other during the ripening process. These enhancements further contribute to peach fruit softening by upregulating specific cell wall degradation-related genes.