AUTHOR=Lim Sun-Hyung , Kim Da-Hye , Kim Jae K. , Lee Jong-Yeol , Ha Sun-Hwa 

TITLE=A Radish Basic Helix-Loop-Helix Transcription Factor, RsTT8 Acts a Positive Regulator for Anthocyanin Biosynthesis

JOURNAL=Frontiers in Plant Science

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.01917

DOI=10.3389/fpls.2017.01917

ISSN=1664-462X

ABSTRACT=<p>The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish (<italic>Raphanus sativus</italic> L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene <italic>RsTT8</italic>. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of <italic>Arabidopsis thaliana</italic> protoplasts harboring the <italic>RsTT8-sGFP</italic> construct. We evaluated anthocyanin biosynthesis and <italic>RsTT8</italic> expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., <italic>RsCHS, RsCHI, RsF3H, RsDFR</italic>, and <italic>RsANS</italic>). Heterologous co-expression of both <italic>RsTT8</italic> and <italic>RsMYB1</italic> in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the <italic>RsCHS</italic> and <italic>RsDFR</italic> promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis <italic>tt8-1</italic> mutant, which lacks red pigmentation in the leaves and seeds, with <italic>RsTT8</italic> restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis.</p>