DNA Methylation Regulation Mechanism Of Furanone Biosynthesis,a Characteristic Volatile Compound In Strawberry Fruit

Characteristic volatile compounds in cultivated strawberry(Fragaria ×ananassa)fruit are the main factors to attract customers,which are highly correlated with consumer preference and overall satisfaction for strawberry.Furanone is a characteristic aroma in strawberry,and has a significant impact on fruit flavor.Research on the regulation mechanism of fruit aroma compounds can provide theoretical basis for improving fruit flavor quality.Recently,increasing evidence suggests that DNA methylation plays a crucial role in fruit ripening in horticultural crops.However,the role of DNA methylation in regulating specific traits,such as furanone biosynthesis,remains unclear.In this paper,aromatic ‘Yuexin’ strawberry fruit is used as material.Focusing on the topic of ‘Regulation and synthesis of furanone,a characteristic aromatic compound in strawberry fruit’,the regulation mechanism of Rd DM pathway and DNA demethylation pathway on the key gene Fa QR in furanone synthesis was explored.The main results are as follows:1.Fa OR3 and its DNA methylation modification play a vital role in furanone synthesis.Four Fa QRs homologs commonly exist in cultivated strawberry,and one of them,Fa QR3 contributes to approximately 50% of Fa QRs transcripts,indicating a major role of Fa QR3 in furanone biosynthesis.The detection of DNA methylation showed that m CG and m CHG of M2 section in Fa QR3 promoter decreased significantly during fruit ripening.DNA methylation of M2 region in Fa QR3 promoter is negatively correlated with Fa QR3 expression and furanone content.2.The change of DNA methylation can significantly affect strawberry fruit ripening.Postharvest ‘Yuexin’ strawberry fruits at T stage were placed at 10 ℃ and20 ℃,and treatment at 20 ℃ could promote fruit ripening and demethylation of M2 region in Fa QR3 promoter,thus promoting Fa QR3 expression and furanone accumulation.DNA methyltransferase inhibitor 5-Azacytidine accelerates the loss of m CG and m CHG in the M2 region of Fa QR3 promoter,thereby promoting Fa QR3 expression and furanone accumulation,and accelerating fruit ripening.3.Fa QR3 transcription can be driven by the M2 region in Fa QR3 promoter.The M2 region in Fa QR3 promoter where DNA methylation lost during fruit ripening is enriched with many cis-acting elements,including the binding elements of MYBs,ERFs,WRKYs and other TFs.Among them,21 TFs have regulatory effects on Fa QR3 promoters.The absence of M2 region reduces the transcriptional activation effect of Fa RAV#4,Fa MYB115 and Fa WRKY2 for Fa QR3 promoter,indicating the M2 region in Fa QR3 promoter is the critical reigon driving Fa QR3 transcription.4.24-nt si RNAs were enriched in M2 region of Fa QR3 promoter and were reduced during fruit ripening.The transient overexpression and RNAi of AGO4,a key gene in the Rd DM pathway,significantly promoted and decreased the DNA methylation level of M2 region in the Fa QR3 promoter,respectively,suggesting that methylation of Fa QR3 promoter could be mediated by Rd DM pathway.5.DNA demethylation is involved in furanone accumulation.Transient RNAi of strawberry demethylation gene Fa DMLs can inhibit the loss of DNA methylation level of M2 region in Fa QR3 promoter,thereby inhibiting Fa QR3 expression and furanone biosynthesis,suggesting that DNA demethylation is also involved in furanone accumulation.Rd DM and Fa DMLs mediated DNA demethylation pathway antagonize each other,and balance and regulate the DNA methylation level of M2 region in Fa QR3 promoter,thus affecting furanone synthesis and accumulation.Taken together,Fa QR3 is a key gene in the biosynthesis of furanone,a characteristic volatile component in strawberry fruit.DNA methylation of Fa QR3 promoter is negatively correlated with furanone accumulation.Rd DM and DNA demethylation pathway can co-regulate DNA methylation level of Fa QR3 promoter,thereby affecting the Fa QR3 expression and regulating furanone accumulation.