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

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Abstract

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.

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Abbreviations

ANS:

Anthocyanidin synthase

CHI:

Chalcone isomerase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol 4-reductase

F3H:

Flavanone 3-hydroxylase

4CL:

4-Coumarate-CoA ligase

PAL:

Phenylalanine ammonia-lyase

qPCR:

Quantitative real-time PCR

TF:

Transcription factor

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Acknowledgments

This work is supported by a grant from the Kentucky Tobacco Research and Development Center to L.Y.

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Authors and Affiliations

  1. College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, People’s Republic of China

    Yanhong Bai

  2. Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546, USA

    Sitakanta Pattanaik, Barunava Patra, Joshua R. Werkman, Claire H. Xie & Ling Yuan

  3. Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, 40546, USA

    Yanhong Bai, Sitakanta Pattanaik, Barunava Patra, Claire H. Xie & Ling Yuan

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  1. Yanhong Bai
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  2. Sitakanta Pattanaik
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  4. Joshua R. Werkman
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  5. Claire H. Xie
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Corresponding author

Correspondence to Ling Yuan.

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Y. Bai and S. Pattanaik contributed equally to this work.

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Bai, Y., Pattanaik, S., Patra, B. et al. Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active. Planta 234, 363–375 (2011). https://doi.org/10.1007/s00425-011-1407-y

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