Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco
- 1.3k Downloads
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.
KeywordsAnthocyanin bHLH transcription factor Combinatorial transcriptional regulation MYB transcription factor Nicotiana
Quantitative real-time PCR
Yellow fluorescent protein
We thank Dr. G. Collins of the University of Kentucky for the RNAi vector and Dr. E. Grotewold of the Ohio State University of the split YFP vector. We also express our appreciation to Dr. K. Saito for providing the Myc-RP cDNA, Dr. S. Wessler for the Lc cDNA and the John Innes Research Center for the Delila cDNA. This work is supported by a grant from the Kentucky Tobacco Research and Development Center to L.Y.
- Carey CC, Strahle JT, Selinger DA, Chandler VL (2004) Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana. Plant Cell 16:450–464CrossRefPubMedGoogle Scholar
- Gong ZZ, Yamagishi E, Yamazaki M, Saito K (1999a) A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization, heterologous expression in transgenic plants and transactivation in yeast cells. Plant Mol Biol 41:33–44CrossRefPubMedGoogle Scholar
- Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86:7092–7096CrossRefPubMedGoogle Scholar
- Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, Matheis N, Schuster DK, Menasco DJ, Wagoner W, Lightner J, Wagner DR (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15:1689–1703CrossRefPubMedGoogle Scholar
- Nakatsuka T, Haruta KS, Pitaksutheepong C, Abe Y, Kakizaki Y, Yamamoto K, Shimada N, Yamamura S, Nishihara M (2008) Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. Plant Cell Physiol 49:1818–1829CrossRefPubMedGoogle Scholar
- Niu SS, Xu CJ, Zhang WS, Zhang B, Li X, Lin-Wang K, Ferguson IB, Allan AC, Chen KS (2009) Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor. Planta 6:101–112Google Scholar
- Pattanaik S, Xie CH, Kong Q, Shen KA, Yuan L (2006) Directed evolution of plant basic helix–loop–helix transcription factors for the improvement of transactivational properties. Biochim Biophys Acta-Gene Struct Express 1759:308–318Google Scholar
- Shinya T, Galis I, Narisawa T, Sasaki M, Fukuda H, Matsuoka H, Saito M, Matsuoka K (2007) Comprehensive analysis of glucan elicitor-regulated gene expression in tobacco BY-2 cells reveals a novel MYB transcription factor involved in the regulation of phenylpropanoid metabolism. Plant Cell Physiol 48:1404–1413CrossRefPubMedGoogle Scholar
- Tamura K, Dudley J, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599 Google Scholar