, 234:363 | Cite as

Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active

  • Yanhong Bai
  • Sitakanta Pattanaik
  • Barunava Patra
  • Joshua R. Werkman
  • Claire H. Xie
  • Ling Yuan
Original Article


The basic helix-loop-helix (bHLH) transcription factors (TFs) comprise one of the largest families of TFs involved in developmental and physiological processes in plants. Here, we describe the functional characterization of two bHLH TFs (NtAn1a and NtAn1b) isolated from tobacco (Nicotiana tabacum) flowers. NtAn1a and NtAn1b originate from two ancestors of tobacco, N. sylvestris and N. tomentosiformis, respectively. NtAn1a and NtAn1b share high sequence similarity with other known flavonoid-related bHLH TFs and are predominantly expressed in flowers. GUS expression driven by the NtAn1a promoter is consistent with NtAn1 transcript profile in tobacco flowers. Both NtAn1a and NtAn1b are transcriptional activators as demonstrated by transactivation assays using yeast cells and tobacco protoplasts. Ectopic expression of NtAn1a or NtAn1b enhances anthocyanin accumulation in tobacco flowers. In transgenic tobacco expressing NtAn1a or NtAn1b, both subsets of early and late flavonoid pathway genes were up-regulated. Yeast two-hybrid assays showed that NtAn1 proteins interact with the previously characterized R2R3-MYB TF, NtAn2. The NtAn1–NtAn2 complex activated the promoters of two key anthocyanin pathway genes, dihydroflavonol reductase and chalcone synthase. The promoter activation is severely repressed by dominant repressive forms of either NtAn1a or NtAn2, created by fusing the SRDX repressor domain to the TFs. Our results show that NtAn1 and NtAn2 act in concert to regulate the anthocyanin pathway in tobacco flowers and NtAn2 up-regulates NtAn1 gene expression.


Anthocyanin bHLH transcription factor Flavonoids Transcriptional regulation MYB transcription factor 



Anthocyanidin synthase


Chalcone isomerase


Chalcone synthase


Dihydroflavonol 4-reductase


Flavanone 3-hydroxylase


4-Coumarate-CoA ligase


Phenylalanine ammonia-lyase


Quantitative real-time PCR


Transcription factor

Supplementary material

425_2011_1407_MOESM1_ESM.tif (260 kb)
Supplementary Figure (TIFF 259 kb)
425_2011_1407_MOESM2_ESM.doc (34 kb)
Supplementary Table (DOC 33 kb)


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Yanhong Bai
    • 1
    • 3
  • Sitakanta Pattanaik
    • 2
    • 3
  • Barunava Patra
    • 2
    • 3
  • Joshua R. Werkman
    • 2
  • Claire H. Xie
    • 2
    • 3
  • Ling Yuan
    • 2
    • 3
  1. 1.College of AgronomyNorthwest A&F UniversityShaanxiPeople’s Republic of China
  2. 2.Department of Plant and Soil SciencesUniversity of KentuckyLexingtonUSA
  3. 3.Kentucky Tobacco Research and Development CenterUniversity of KentuckyLexingtonUSA

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