Transcription Factor NAC And DNA Methylation Synergistically Regulate Volatile Formation During Peach Fruit Ripening

Aroma is an important fruit quality trait.Peach is a bulk fruit species,whose character aroma is contributed by ester and lactone volatiles derived from fatty acid pathway.Although important volatiles of peach fruit and synthesis related genes such as Pp AAT1 have been identified,the regulatory mechanism of volatile formation during peach fruit ripening remains incomplete.Transcription factor NAC and DNA methylation are involved in the regulation of fruit ripening,however,it remains unclear whether these two regulators play roles in the formation of volatiles in peach fruit.In this research,peach fruit ‘Hujingmilu’(Prunus persica L.Batsch cv Hujingmilu)were used as materials.Dynamic changes in metabolome,transcriptome and DNA methylome during fruit ripening were investigated.To validate regulatory role of NAC and DNA methylation in peach fruit volatile formation,electrophoretic mobility shift assay,transient overexpression and tomato fruit transformation were applied.The main results are as follows:Volatiles accumulated during peach fruit ripening,involving hexyl acetate and γ-decalactone.Ethylene treatment increased contents of esters and lactones,while 1-MCP and low temperature treatment significantly reduced volatile contents in peach fruit.These results indicated a regulation network of volatile formation during peach fruit ripening.The increased expression level of Pp NAC1,which is considered as a candidate gene to regulate volatile formation,was observed during peach fruit ripening.DAP-Seq for transcription factor Pp NAC1 analysis identified 9238 binding sites,among which 2245 annotated genes were located in the promoter region.By analyzing the expression data of these genes,combined with the WGCNA analysis of volatiles content,and integrated the results of EMSA and tobacco dual-luciferase report assay,it was found that Pp FAD3-1,Pp LIP1 and Pp AAT1 which related to fatty acid-derived volatiles biosynthesis were target genes of Pp NAC1.Moreover,the regulatory role of Pp NAC1 in volatile biosynthesis was confirmed via homologous transient overexpression in peach fruit and heterologous transgenic technology in tomato nor mutants.These results indicated that transcription NAC plays an essential role in the synthesis of aroma volatiles during peach fruit ripening.Based on results from tobacco dual-luciferase report assay,EMSA,Ch IP-Seq and Ch IP-q PCR,transcription factor Pp NAC1 activated Pp DML1 expression via binding to Pp DML1 promoter.Tomato transcription factor NAC-NOR also activated Sl DML2 expression by directly binding to its promoter.Overexpressing Pp NAC1 in peach fruit significantly induced the expression of Pp DML1.The knockout of NAC-NOR significantly decreased the expression of Sl DML2,accompanied by an increase in DNA methylation in tomato fruit.Therefore,Pp DML1 and Sl DML2 which encode DNA demethylase were target genes of transcription factor Pp NAC1 and NAC-NOR,respectively.DNA methylation inhibitor 5-Aza treatment induced the expression of structural genes related to volatile formation,involving Pp FAD3-1,Pp LIP1 and Pp AAT1 in peach fruit.Moreover,expression of transcription factor Pp NAC1 was also activated after treatment.Knock-out of Sl DML2 in model fruit tomato increased DNA methylation level,and expression levels of structural genes related to volatile biosynthesis were reduced.Notably,the expression of transcription factor NAC-NOR was inhibited in sldml2 mutants,accompanied by the increased DNA methylation in the promoter region.These results indicated that DNA methylation reduced fruit volatile production through inhibiting expression of transcription factor NAC and structural genes associated with volatile synthesis during fruit ripening.In summary,transcription factors Pp NAC1 and NAC-NOR could activate the expression of DNA demethylation enzymes Pp DML1 and Sl DML2,and structural genes related to volatile biosynthesis.Meanwhile,Pp DML1 and Sl DML2 activate the transcription of the above transcription factors and structural genes through DNA demethylation.Therefore,the transcription factor NAC and DNA methylation synergistically regulate the volatiles formation during fruit ripening.