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 (Raphanus sativus 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 RsTT8. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 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., RsCHS, RsCHI, RsF3H, RsDFR, and RsANS). Heterologous co-expression of both RsTT8 and RsMYB1 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 RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 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.
Highlights
Radishes (Raphanus sativus L.) belong to Brassicaceae family and are economically important vegetable crops cultivated for producing seed oil and sprouts, as well as edible taproots
RsTT8 contains several domains that are conserved in flavonoid-related bHLHtype transcription factors (TFs), including an N-terminal MYB-interaction region (MIR) domain, a WD/activation domain (AD) domain, a basic helix-loop-helix domain, and a Cterminal aspartokinase, chorismate mutase, TyrA (ACT)-like domain
The bHLH domain contains nearly 60 amino acids involved in DNA binding; among these residues, 19 amino acid residues are conserved in anthocyanin-related bHLH TFs (Supplementary Figure 2)
Summary
Radishes (Raphanus sativus L.) belong to Brassicaceae family and are economically important vegetable crops cultivated for producing seed oil and sprouts, as well as edible taproots. They are good model crops for deciphering the anthocyanin biosynthesis mechanisms because they have variety of pigmentation pattern and intensity depending on the anthocyanin accumulation in leaves, stems and roots. The anthocyanin and flavonoid biosynthetic pathways have been extensively characterized, and the corresponding structural genes involved in these pathways have been studied in several plant species including Arabidopsis thaliana, Oryza sativa (rice), and Malus domestica (apple) (Grotewold, 2006; Lin-Wang et al, 2010; Lim and Ha, 2013). The structural genes encoding enzymes in the anthocyanin biosynthetic pathway are regulated by transcription factors (TFs) and are expressed synergistically during anthocyanin accumulation (Koes et al, 2005; Grotewold, 2006)
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