Transcription factor PpNAC1 and DNA demethylase PpDML1 synergistically regulate peach fruit ripening

Fruit ripening is accompanied by dramatic changes in color, texture, and flavor and is regulated by transcription factors (TFs) and epigenetic factors. However, the detailed regulatory mechanism remains unclear. Gene expression patterns suggest that PpNAC1 (NAM/ATAF1/2/CUC) TF plays a major role in peach (Prunus persica) fruit ripening. DNA affinity purification (DAP)-seq combined with transactivation tests demonstrated that PpNAC1 can directly activate the expression of multiple ripening-related genes, including ACC synthase1 (PpACS1) and ACC oxidase1 (PpACO1) involved in ethylene biosynthesis, pectinesterase1 (PpPME1), pectate lyase1 (PpPL1), and polygalacturonase1 (PpPG1) related to cell wall modification, and lipase1 (PpLIP1), fatty acid desaturase (PpFAD3-1), and alcohol acyltransferase1 (PpAAT1) involved in volatiles synthesis. Overexpression of PpNAC1 in the tomato (Solanum lycopersicum) nor (nonripening) mutant restored fruit ripening, and its transient overexpression in peach fruit induced target gene expression, supporting a positive role of PpNAC1 in fruit ripening. The enhanced transcript levels of PpNAC1 and its target genes were associated with decreases in their promoter mCG methylation during ripening. Declining DNA methylation was negatively associated with increased transcripts of DNA demethylase1 (PpDML1), whose promoter is recognized and activated by PpNAC1. We propose that decreased methylation of the promoter region of PpNAC1 leads to a subsequent decrease in DNA methylation levels and enhanced transcription of ripening-related genes. These results indicate that positive feedback between PpNAC1 and PpDML1 plays an important role in directly regulating expression of multiple genes required for peach ripening and quality formation. Transcription factor PpNAC1 and DNA demethylase PpDML1 form a feedback loop to synergistically regulate ethylene synthesis, softening, and flavor formation in peach fruit.