AUTHOR=Huang Wenjun , Lv Haiyan , Wang Ying 

TITLE=Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum

JOURNAL=Frontiers in Plant Science

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.01274

DOI=10.3389/fpls.2017.01274

ISSN=1664-462X

ABSTRACT=<p><italic>Epimedium</italic> species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs) and anthocyanins, predominantly contributing to the color diversity of <italic>Epimedium</italic> flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF) as well as bHLH TF and WD40 protein at the transcriptional level. <italic>MYB</italic> TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from <italic>Epimedium sagittatum</italic>, but a <italic>R2R3-MYB</italic> TF involved in regulating both these two pathways has not been functionally characterized to date in <italic>Epimedium</italic> plants. In this study, we report the functional characterization of <italic>EsMYB9</italic>, a <italic>R2R3-MYB</italic> TF previously isolated from <italic>E. sagittatum</italic>. The previous study indicated that <italic>EsMYB9</italic> belongs to a small subfamily of <italic>R2R3-MYB</italic> TFs containing grape <italic>VvMYB5a</italic> and <italic>VvMYB5b</italic> TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of <italic>EsMYB9</italic> in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of <italic>EsMYB9</italic> contributes to the autoactivation activity, and <italic>EsMYB9</italic> interacts with <italic>EsTT8</italic> or <italic>AtTT8 bHLH</italic> regulator. Transient reporter assay shows that <italic>EsMYB9</italic> slightly activates the expression of <italic>EsCHS</italic> (chalcone synthase) promoter in transiently transformed leaves of <italic>Nicotiana benthamiana</italic>, but the addition of <italic>AtTT8</italic> or <italic>EsTT8 bHLH</italic> regulator strongly enhances the transcriptional activation of <italic>EsMYB9</italic> against five promoters of the flavonoid pathway genes except <italic>EsFLS</italic> (flavonol synthase). In addition, co-transformation of <italic>EsMYB9</italic> and <italic>EsTT8</italic> in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The potential role of <italic>EsMYB9</italic> in modulating the biosynthesis and accumulation of sucrose-induced anthocyanin and flavonol-derived BCs is also discussed. These findings suggest that <italic>EsMYB9</italic> is a novel <italic>R2R3-MYB</italic> TF, which regulates the flavonoid biosynthetic pathway in <italic>Epimedium</italic>, but distinctly different with the anthocyanin or flavonol-specific <italic>MYB</italic> regulators identified previously in <italic>Epimedium</italic> plants.</p>