Flavonoid-related basic helix-loop-helix regulators, NtAn1a and.pdfVIP

Planta (2011) 234:363–375 DOI 10.1007/s00425-011-1407-y ORIGINAL ARTICLE 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 Received: 1 March 2011 / Accepted: 28 March 2011 / Published online: 12 April 2011 ? Springer-Verlag 2011 Abstract The basic helix-loop-helix (bHLH) transcrip- tion 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) Xowers. 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 Xavonoid- related bHLH TFs and are predominantly expressed in Xowers. GUS expression driven by the NtAn1a promoter is consistent with NtAn1 transcript proWle in tobacco Xowers. 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 Xowers. In transgenic tobacco expressing NtAn1a or NtAn1b, both subsets of early and late Xavonoid pathway genes were up-regulated. Yeast two-hybrid assays showed that NtAn1 proteins interact with the previously character- ized R2R3-MYB TF, NtAn2. The NtAn1–NtAn2 complex activated the promoters of two key anthocyanin pathway genes, dihydroXavonol 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 Xowers and NtAn2 up-regulates NtAn1 g