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The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

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Key message

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.


Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

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Chalone synthase


Flavonol synthase


Dihydroflavonol reductase


Cinnamate 4-hydroxylase


Anthocyanin synthase


UDP-glucose:flavonoid 3- glucosyltransferase


ERF-associated amphiphilic repression


Green fluorescent protein


Glutathione S-transferase






Murashige and Skoog


No tryptophan


No leucine


No histidine


No adenine


Activation domain


Binding domain


Coding DNA sequence


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This work was supported by the National Natural Science Foundation of China (31572091) and National Key Research and Development Project of China (2016YFC0501505).

Author contributions

XC and HX: conceived and designed the experiments. HX and NW: performed the experiments. HX: analyzed the data. JL, CQ, YW, SJ, NL, ZZ: contributed reagents/materials/analysis tools. HX and XC: wrote the paper.

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Correspondence to Xuesen Chen.

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Xu, H., Wang, N., Liu, J. et al. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol Biol 94, 149–165 (2017). https://doi.org/10.1007/s11103-017-0601-0

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  • DOI: https://doi.org/10.1007/s11103-017-0601-0