Functionally characterizing transcription factors LC from Zea mays and NTAN2 from Nicotiana tabacum and exploring their roles in combinatorial regulation of anthocyanin biosynthesis

Anthocyanins are important plant secondary metabolites and their synthesis is regulated by a combination of basic helix-loop-helix transcription factor (bHLH TF), MYB TF and WD40 protein.;Lc is a key regulator of anthocyanin pathway in maize. Despite possessing a conserved bHLH sequence, Lc is incapable of binding the E-box sequence (CANNTG), a common DNA motif recognized by bHLH domains. We demonstrated that the isolated Lc bHLH domain has the ability to form a homodimer and specifically binds to G-box (CACGTG). The dimerization is affected by a leucine zipper-like (LZL) structure adjacently. Another dimer-forming, ACT-like domain at the C-terminal of Lc suppresses the DNA-binding potential of bHLH, providing a possible explanation for Lc's inability to bind E-box sequence. The dimerization mediated by the bHLH domain of Lc inhibits its interaction with maize RIF1, a chromatin modification factor. Lc can activate a G-box/luciferase-reporter in vivo and both the bHLH and ACT-like domains are necessary for the activation. Our study demonstrating the cooperation of multiple dimer interfaces underscores a unique mechanism of combinatorial transcriptional regulation by the Lc-like bHLH TFs.;Ectopic expression of Lc in tobacco results in increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with Lc in anthocyanin gene activation. We isolated NtAn2, a gene encoding an R2R3-MYB TF that shares high sequence homology with other known flavonoid-related MYB TFs and is expressed specially in developing flowers. Constitutive expression of NtAn2 induces whole plant anthocyanin production in tobacco in which both subsets of early and late flavonoid pathway genes are upregulated. Suppression of NtAn2 by RNAi resulted in a white-flowered phenotype and the inhibition of the late pathway genes. These results suggest NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.;In addition, in order to create a gene expression vector that induces anthocyanin production in various plant species, we generated a chimeric TF (Lc-NtAn2), in which Lc is fused with NtAn2 by a peptide linker. Constitutive expression of the Lc-NtAn2 in Arabidopsis led to anthocyanin accumulation in multiple tissues and upregulation of multiple anthocyanin pathway genes.;Keywords. Basic Helix-loop-helix (bHLH) Transcription Factor, R2R3-MYB Transcription Factor, Chimeric Transcription Factor, Anthocyanin biosynthesis, Transcriptional Regulation...