title: The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.
abstract: 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.
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Xu
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Jingxuan
authors.initials: J
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Changzhi
authors.initials: C
authors.lastname: Qu
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Shenghui
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Shandong institute of pomology, Tai-An, Shandong, China.
authors.forename: Ninglin
authors.initials: N
authors.lastname: Lu
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Deyun
authors.initials: D
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China.
authors.forename: Zongying
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China. chanyetixi@163.com.
authors.forename: Xuesen
authors.initials: X
authors.lastname: Chen
mesh_terms: D000872:Anthocyanins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005638:Fruit; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D055786:Gene Knockout Techniques; D027845:Malus / Q000235:genetics / Q000378:metabolism*; D020081:Phyllachorales; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000872:Anthocyanins; D010940:Plant Proteins; D014157:Transcription Factors
keywords: Anthocyanin; EAR suppression sequence; First-filial generation; Homodimers; Malus sieversii f. neidzwetzkyana; MdMYB16; MdbHLH33