Hypermethylation in the promoter regions of flavonoid pathway genes is associated with skin color fading during ‘Daihong’ apple fruit development

Abstract

Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar ‘Daihong’ (DH) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1, MdCHS, MdANS, MdUFGT, MdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5′ and 3′ flanking regions of genes and transposable elements (TEs), and in TE bodies in all CG, CHG and CHH contexts, especially the mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1, MdROS1, and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHS, MdCHI, MdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHS, MdCHI, MdANR, and MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin. Treatment with the DNA demethylation agent 5-aza-2′-deoxycytidine verified the negative correlation between DNA methylation and gene expression within the flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during DH apple development.

Acknowledgements

This work was supported by the Natural Science Foundation of Shandong Province (ZR2023MC169), the Taishan Scholar Foundation of Shandong Province (tstp20221134), the Agricultural Variety Improvement Project of Shandong Province (2023LZGCQY007; 2021LZGC024; 2022LZGC010), the China Agriculture Research System Foundation (CARS-27), and Science and Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta (2022SZX34).

Author contributions

J.X. wrote the manuscript. J.L.Y. revised the manuscript. J.X., Y.Z., and J.L.Y. designed the experiments. L.X., P.Z., J.X., S.J., X.S., C.D., H.J., and X.X. performed the experiments and analyzed the data. All the authors approved the final manuscript.

Data availability

The transcriptome and methylome data in this study have been deposited in the Sequence Read Archive (SRA) database in NCBI, with bioProject accession numbers PRJNA1000056 and PRJNA1000046, respectively.

Conflict of interest

The authors declare that there is no conflict of interest associated with this work.

Supplementary data

Supplementary data is available at Horticulture Research online.

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