MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple.

Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four continuous-generation bud sport mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin color traits remains elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in 'Golden Delicious' fruits, 'Gala' calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in 'Red Fuji' apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.

Whole-genome DNA methylation patterns and complex associations with gene expression associated with anthocyanin biosynthesis in apple fruit skin.

MAIN CONCLUSION: DNA methylation of anthocyanin biosynthesis-related genes and MYB/bHLH transcription factors was associated with apple fruit skin color revealed by whole-genome bisulfite sequencing. DNA methylation is a common feature of epigenetic regulation and is associated with various biological processes. Anthocyanins are among the secondary metabolites that contribute to fruit colour, which is a key appearance and nutrition quality attribute of apple fruit. Although few studies reported that DNA methylation in the promoter of MYB transcription factor was associated with fruit skin color, there is a general lack of understanding of the dynamics of global and genic DNA methylation in apple fruit. Here, whole-genome bisulfite sequencing was carried out in fruit skin of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four-generation bud sport mutants, including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4) at color break stage. Correlation and linear-regression analysis between DNA methylation level and anthocyanin content, as well as the transcription levels of genes related to anthocyanin biosynthesis were carried out. The results showed that the number of differentially methylated regions (DMRs) and differentially methylated genes (DMGs) was considerably increased from G1 to G4 versus the number observed in G0. The mCHH context was dominant in apple, but the levels of mCG and mCHG of DMGs were significantly higher than that of the mCHH. Genetic variation of bud sport mutants from 'Red Delicious' was associated with differential DNA methylation. Additionally, hypomethylation of mCG and mCHG contexts in flavonoid biosynthesis pathway genes (PAL, 4CL, CYP98A, PER, CCoAOMT, CHS, and F3'H), mCHG context in MYB10 at upstream, led to transcriptional activation and was conductive to anthocyanin accumulation. However, hypermethylation of mCG context in bHLH74 at upstream led to transcriptional inhibition, inhibiting anthocyanin accumulation.