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

Abstract

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 (<i>Raphanus sativus</i> 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 <i>RsTT8</i>. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of <i>Arabidopsis thaliana</i> protoplasts harboring the <i>RsTT8-sGFP</i> construct. We evaluated anthocyanin biosynthesis and <i>RsTT8</i> 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., <i>RsCHS, RsCHI, RsF3H, RsDFR</i>, and <i>RsANS</i>). Heterologous co-expression of both <i>RsTT8</i> and <i>RsMYB1</i> 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 <i>RsCHS</i> and <i>RsDFR</i> promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis <i>tt8-1</i> mutant, which lacks red pigmentation in the leaves and seeds, with <i>RsTT8</i> 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.