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

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.

Abstract

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.

     

Overexpression of the transcription factor MdbHLH33 increases cold tolerance of transgenic apple callus

As cold stress greatly affects plant growth and development, understanding the mechanisms underlying cold tolerance in plants is important. In this study, we analyzed the expression levels of apple (Malus domestica) MdbHLH33 and MdCBF1–5 by semi-quantitative PCR after exposure to 4 °C for different amounts of time and generated evolutionary trees for MdbHLH33 and the MdCBFs. Overexpressing MdbHLH33 pro-GUS in ‘Orin’ callus, indicated that transgenic callus had higher GUS activity and was more deeply stained at 4 °C than at 25 °C. Subcellular localization showed that MdbHLH33 was located in the nucleus. Overexpressing MdbHLH33 in ‘Orin’ callus increased the expression level of MdCBF2, MdCOR15A-1, and MdCOR15A-2, and resulted in increased cold tolerance. EMSA and Chip-PCR analysis showed that MdbHLH33 could bind the LTR cis-acting element found in the MdCBF2 promoter. Overexpressing MdCBF2 in ‘Orin’ callus indicated that MdCBF2 could also increase the expression level of MdCOR15A-1 and MdCOR15A-2 and improve cold tolerance; we also found that transgenic callus overexpressing MdCBF2 had reduced MdCBF1 and MdCBF5 expression and increased MdCBF3 and MdCBF4 expression. Overall, these results show that MdbHLH33 can regulate the expression of MdCBF2 and improve the cold tolerance of transgenic callus.     

Jasmonate induces biosynthesis of anthocyanin and proanthocyanidin in apple by mediating the JAZ1–TRB1–MYB9 complex

Jasmonate (JA) induces the biosynthesis of anthocyanin and proanthocyanidin. MdMYB9 is essential for modulating the accumulation of both anthocyanin and proanthocyanidin in apple, but the molecular mechanism for induction of anthocyanin and proanthocyanidin biosynthesis by JA is unclear. In this study, we discovered an apple telomere-binding protein (MdTRB1) to be the interacting protein of MdMYB9. A series of biological assays showed that MdTRB1 acted as a positive modulator of anthocyanin and proanthocyanidin accumulation, and is dependent on MdMYB9. MdTRB1 interacted with MdMYB9 and enhanced the activation activity of MdMYB9 to its downstream genes. In addition, we found that the JA signaling repressor MdJAZ1 interacted with MdTRB1 and interfered with the interaction between MdTRB1 and MdMYB9, therefore negatively modulating MdTRB1-promoted biosynthesis of anthocyanin and proanthocyanidin. These results show that the JAZ1–TRB1–MYB9 module dynamically modulates JA-mediated accumulation of anthocyanin and proanthocyanidin. Taken together, our data further expand the functional study of TRB1 and provide insights for further studies of the modulation of anthocyanin and proanthocyanidin biosynthesis by JA.