Transcription Factor PpNAC1 And Epigenetic Modification Are Involved In The Synthesis Of Volatiles In Peach Fruits By Regulating The Expression Of PpFAD3-1

Aroma is an important quality indicator of fruit,and the material basis of its formation is volatiles.Fatty acids are important precursors for the biosynthesis of fruit volatiles,such as the unsaturated fatty acid linolenic acid(18:3)involved in the synthesis of(E)-2-hexenal and(E)-2-hexenyl acetate.However,the regulatory pathway for the formation of volatiles in the linolenic acid pathway remains unclear.In this paper,peach fruit(Prunus persica L.Batsch cv Hujingmilu)is used as the main material to identify the structural genes and the transcription factors involved in the biosynthesis of linolenic acid.Then clarify their roles in the biosynthesis of fruit volatiles,and further carry out molecular mechanism research at the level of epigenetic modification.The main results are as follows:1、w-3 fatty acid desaturase(w-3 FAD)PpFAD3-1 is involved in the formation of linolenic acid and volatiles in peach fruit.Exogenous addition of linolenic acid significantly promoted the accumulation of volatiles such as(E)-2-hexenal and(E)-2-hexenyl acetate,which increased by approximately 1.4 and 6.0 times,respectively.Gene expression results showed that PpFAD3-1 was a member of w-3FADs with the highest transcript abundance during peach fruit ripening.PpFAD3-1also had a significant positive correlation with the content of linolenic acid and downstream volatiles.Overexpression of PpFAD3-1 significantly increased the linolenic acid content of transgenic tomato fruits,while reducing the content of linoleic acid(18:2),and was accompanied by a significant decrease in the content of hexanal,a volatile derived from linoleic acid,which resulted in elevated ratios of linolenic acid/linoleic acid(18:3/18:2)and(E)-2-hexenal/hexanal.2、The transcription factor PpNAC1 can bind to the promoter of PpFAD3-1 and activate its transcription.By using the RNA-Seq results of peach fruit ripening,23 transcription factors that were positively correlated with the expression of PpFAD3-1were screened.The tobacco dual-luciferase experiment proved that the transcription factor PpNAC1 could activate the promoter activity of PpFAD3-1,and its activation effect reached 20 times.EMSA experiments showed that PpNAC1 could directly bind to the NAC binding site on the PpFAD3-1 promoter in vitro.Ch IP-Seq results showed that PpNAC1 could bind to the promoter of PpFAD3-1 in vivo.The expression of PpNAC1 and PpFAD3-1 in different varieties of peach fruit had a significant positive correlation.The above results indicated that PpNAC1 in peach fruit can transcriptionally activate the expression of PpFAD3-1.3、Transgenic overexpression of the transcription factor PpNAC1 promotes the accumulation of linolenic acid and its derived volatiles in fruit.Overexpression of PpNAC1 significantly upregulated the expression of Sl FAD3,increased the linolenic acid content,and elevated the ratio of linolenic acid/linoleic acid in transgenic tomato fruit.Different from the transgenic fruit that overexpressed the structural gene PpFAD3-1,the overexpression of the transcription factor PpNAC1 significantly increased the expression of Sl LOXC,Sl HPL1,and Sl ADH2,which related to the synthesis of volatiles in tomato fruit.Thereby increasing the contents of 18:3 pathway volatiles such as(E)-2-hexenal and(Z)-3-hexenol.The above results showed that the transcription factor PpNAC1 not only affected the expression of genes involved in the linolenic acid synthesis pathway but also changed the expression of genes that can synthesize volatiles,thereby promoting the synthesis of fruit volatiles by providing the precursor substance linolenic acid.4、The expression of the structural gene PpFAD3-1 and transcription factor PpNAC1 was accompanied by changes in epigenetic modification during peach fruit ripening.The epigenetic analysis of genes for the synthesis of volatiles in the linolenic acid pathway of peach fruit was carried out by using the Fruit ENCODE database.It was found that the chromatin openness of PpFAD3-1 and PpNAC1 increased as the fruit ripened,accompanied by a significant increase in gene expression level.The trimethylation(H3K4me3)of H27K4me3,a histone activated by corresponding gene expression,increased as the fruit ripened,while the expression inhibition of H27K4me3 decreased.In addition,the DNA methylation level of the PpFAD3-1 promoter region showed a decreasing trend during fruit ripening.These results suggest that the increased expression levels of transcription factor PpNAC1 and structural gene PpFAD3-1,which are related to aromatics synthesis in the linolenic acid pathway,are related to epigenetic modifications such as histone methylation and DNA methylation.