, Volume 231, Issue 5, pp 1061–1076 | Cite as

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

  • Sitakanta Pattanaik
  • Que Kong
  • David Zaitlin
  • Joshua R. Werkman
  • Claire H. Xie
  • Barunava Patra
  • Ling YuanEmail author
Original Article


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.


Anthocyanin bHLH transcription factor Combinatorial transcriptional regulation MYB transcription factor Nicotiana 



Anthocyanidin synthase


Chalcone isomerase


Chalcone synthase


Dihydroflavonol 4-reductase


Flavanone 3-hydroxylase


4-Coumarate-CoA ligase


Phenylalanine ammonia-lyase


Quantitative real-time PCR


RNA interference


Transcription factor


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.

Supplementary material

425_2010_1108_MOESM1_ESM.tif (1 mb)
SI Fig. 1a Schematic diagram of the expression vector containing the full-length NtAn2 cDNA expressed under the control of a modified Mirabilis mosaic virus (MMV) full-length transcript promoter and ribulose bisphosphate carboxylase (rbcS) terminator. b Schematic diagram of the RNAi vector containing a part of the NtAn2 cDNA both in sense and anti sense orientation with a Glycine max FAD3 intron expressed under the control of CaMV 35S promoter and rbcS terminator. 35S-T,CaMV35S terminator; NPT II, neomycin phosphotransferase; LB, left T-DNA border; RB, right T-DNA border (TIFF 1069 kb)
425_2010_1108_MOESM2_ESM.tif (7.6 mb)
SI Fig. 2 Relative expression levels of NtAn2 transcripts in different floral parts. CX, calyx; CL corolla limb; CT corolla tube; AN, anther; OV, ovary. The gene expression levels were measured by quantitative PCR (qPCR). The results were analyzed using the comparative Ct method and presented as fold-changes compared with the wild-type control (TIFF 7741 kb)
425_2010_1108_MOESM3_ESM.tif (463 kb)
SI Fig. 3 Tobacco flowers of empty-vector control plant (a) and homozygous transgenic tobacco plants overexpressing the bHLH transcription factors Delila (b), Myc-RP (c) and Lc (d) (Pattanaik et al. 2008; Pattanaik et al. unpublished data) (TIFF 463 kb)
425_2010_1108_MOESM4_ESM.tif (239 kb)
SI Fig. 4 Protein-protein interactions between NtAn2 and Arabidopsis bHLH TFs. a-b. In a yeast two hybrid assay, the NtAn2/GAL4-activation domain fusion (pAD-NtAn2) was co-transformed with fusion constructs of GAL4-DNA-binding domain with MYB-interaction domain (ID) of GL3 or TT8. The transformants were grown in media with double (A) or quadruple (B) selections. 1, pAD-NtAn2+pBD-GL3aa 1-209; 2, pAD-NtAn2+pBD-TT8aa 1-204; 3, pAD+pBD-TT8aa1-204; 4, pAD-NtAn2+pBD (TIFF 239 kb)


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Sitakanta Pattanaik
    • 1
    • 2
  • Que Kong
    • 1
  • David Zaitlin
    • 2
  • Joshua R. Werkman
    • 1
  • Claire H. Xie
    • 1
    • 2
  • Barunava Patra
    • 2
  • Ling Yuan
    • 1
    • 2
    Email author
  1. 1.Department of Plant and Soil SciencesUniversity of KentuckyLexingtonUSA
  2. 2.Kentucky Tobacco Research and Development CenterUniversity of KentuckyLexingtonUSA

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