Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco

Abstract

Tobacco is a commonly used heterologous system for studying combinatorial regulation of the flavonoid biosynthetic pathway by the bHLH-MYB transcription factor (TF) complex in plants. However, little is known about the endogenous tobacco bHLH and MYB TFs involved in the pathway. Ectopic expression in tobacco of heterologous bHLH TF genes, such as maize Lc, leads to increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with the Lc-like bHLH TFs. We isolated a gene (NtAn2) encoding a R2R3 MYB TF from developing tobacco flowers. NtAn2 shares high sequence homology with other known flavonoid-related MYB TFs and is mostly expressed in developing flowers. Constitutive ectopic expression of NtAn2 induces whole-plant anthocyanin production in tobacco and Arabidopsis. In transgenic tobacco and Arabidopsis expressing NtAn2, both subsets of early and late flavonoid pathway genes are up-regulated. Suppression of NtAn2 by RNAi in tobacco resulted in a white-flowered phenotype and the inhibition of the late pathway genes. Yeast two-hybrid assays demonstrated that NtAn2 can interact with five heterologous bHLH TFs known to induce anthocyanin synthesis in other species including maize, perilla, snapdragon and Arabidopsis. Bimolecular fluorescent complementation using split YFP demonstrated that NtAn2 interacts with Lc in tobacco cells and that the complex is localized to nuclei. Transient co-expression of NtAn2 and Lc or Arabidopsis TT8 in tobacco protoplasts activated the promoters of two key flavonoid pathway genes, chalcone synthase and dihydroflavonol reductase. These results suggest that NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.

The Transcription Factor CrWRKY1 Positively Regulates the Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus

“…Reverse transcription (RT)-PCR and qPCR were performed as described by Pattanaik et al (2010). The primers used in qPCR for several pathway genes are listed in Supplemental Table S1.…”

Regulatory switch enforced by basic helix-loop-helix and ACT-domain mediated dimerizations of the maize transcription factor R

“…To test protein-protein interaction in planta, protoplasts were isolated from tobacco-cell suspension cultures (Nicotiana tabacum L. cv. Xanthi b 'Brad'), and electroporation with supercoiled plasmid DNA was performed as previously described (29). The reporter and effector plasmids used in the protoplast assay are described in SI Materials and Methods.…”

Transcriptional regulators of flavonoid biosynthesis and their application to flower color modification in Japanese gentians

“…These transgenic tobacco plants had significantly decreased ANS and DFR transcripts among anthocyanin biosynthetic genes. The NtAN2 transcription factor regulates anthocyanin biosynthesis in tobacco plants (Pattanaik et al 2010). The expression level of NtAN2 did not significantly change between control and transgenic plants, indicating that this native MYB transcription factor was not responsible for the anthocyanin reduction in transgenic tobacco flowers.…”

“…Regulatory genes have been also characterized in several ornamental plant species, including Japanese morning glory (Morita et al 2006), gerbera (Elomaa et al 1998;Elomaa et al 2003), torenia (Nishijima et al 2013), Mimulus aurantiacus (Streisfeld et al 2013), and peach (Uematsu et al 2014). Several reports have appeared on the regulation of floral anthocyanin pigmentation in non-ornamental plants, such as soybean (Takahashi et al 2013), kiwifruit (Fraser et al 2013), and tobacco (Pattanaik et al 2010). Little is known regarding the regulation of the early flavonoid biosynthetic pathway in flowers.…”