The R2R3-MYB, bHLH, WD40, and related transcription factors in flavonoid biosynthesis

Lei Zhao, Liping Gao, Hongxue Wang, Xiaotian Chen, Yunsheng Wang, Hua Yang, Chaoling Wei, Xiaochun Wan, Tao Xia

R2R3-MYB, bHLH, and WD40 proteins have been shown to control multiple enzymatic steps in the biosynthetic pathway responsible for the production of flavonoids, important secondary metabolites in Camellia sinensis. Few related transcription factor genes have been documented. The presence of R2R3-MYB, bHLH, and WD40 were statistically and bioinformatically analyzed on 127,094 C. sinensis transcriptome unigenes, resulting in identification of 73, 49, and 134 genes, respectively. C. sinensis phylogenetic trees were constructed for R2R3-MYB and bHLH proteins using previous Arabidopsis data and further divided into 27 subgroups (Sg) and 32 subfamilies. Motifs in some R2R3-MYB subgroups were redefined. Furthermore, Sg26 and Sg27 were expanded compared to Arabidopsis data, and bHLH proteins in C. sinensis were grouped into nine subfamilies. According to the functional annotation of Arabidopsis, flavonoid biosynthesis in C. sinensis was predicted to include R2R3-MYB genes in Sg4 (6), Sg5 (2), and Sg7 (1), as well as bHLH genes in subfamily 2 (2) and subfamily 24 (5). The wide evolutionary gap prevented phylogenetic analysis of WD40s; however, a single gene, CsWD40-1, was observed to share 80.4 % sequence homogeny with AtTTG1. Analysis of CsMYB4-1, CsMYB4-2, CsMYB4-3, CsMYB4-4, CsMYB5-1, and CsMYB5-2 revealed the interaction motif [DE]Lx2[RK]x3Lx6Lx3R, potentially contributing to the specificity of the bHLH partner in the stable MYB-bHLH complex. Full-length end-to-end polymerase chain reaction (PCR) and quantitative reverse transcriptase (qRT)-PCR were used to validate selected genes and generate relative expression ratio profiles in C. sinensis leaves by developmental stage and treatment conditions, including hormone and wound treatments. Potential target binding sites were predicted.