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

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.

Molecular genetics of blood‐fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors

“…However, the dual luciferase assay indicates that PpMYB10.1 alone can induce the activation of the transiently expressed gene PpDFR and PpUFGT. Two highly homologous bHLH genes, NtAN1a and NtAN1b, have been reported in tobacco (Bai et al, 2011). We found that PpMYB10.1 alone is unable to induce the activation of the transiently expressed gene PpDFR when the NtAN1-like gene is knocked out using RNA interference (RNAi) technology ( Figure 4A).…”

The Upregulation of NtAN2 Expression at Low Temperature is Required for Anthocyanin Accumulation in Juvenile Leaves of Lc-transgenic Tobacco (Nicotiana tabacum L.)

“…NtAN2 and NtAN1a/b, representing R2R3-MYB and bHLH type of transcription factors, respectively, interacted with each other to regulate anthocyanin biosynthesis in tobacco (Pattanaik et al, 2010;Bai et al, 2011). Besides, NtAN2 could interact with maize Lc by yeast two-hybrid or biomolecule fluorescence complementation assay (Pattanaik et al, 2010).…”

“…It is also a commonly-used heterologous system for studying the function and interaction of genes in plants. Recently, NtAN2 (a R2R3-Myb transcription factor) and NtAN1a/b (bHLH transcription factors) were reported to coordinately regulate anthocyanin biosynthesis in flowers of tobacco plants (Pattanaik et al, 2010;Bai et al, 2011). In this study, Lc-transgenic tobacco lines were generated using a maize anthocyanin regulatory gene Lc under the control of the promoter of AtSPX3 (Duan et al, 2008), which was involved in phosphorus homeostasis in Arabidopsis.…”

Heterologous expression of IbMYB1a by different promoters exhibits different patterns of anthocyanin accumulation in tobacco

“…Among these, co-expression of two classes of TFs, R2R3-MYB and bHLH, is indispensable for the activation of anthocyanin biosynthetic genes. Recently, tobacco bHLH and MYB TFs involved in flavonoid biosynthetic pathway have been reported (Bai et al, 2011;Pattanaik et al, 2010). Tobacco NtAn2 (accession number: FJ472650) gene encoding an R2R3-MYB TF was isolated from developing tobacco flowers (Pattanaik et al, 2010).…”

“…In addition, ectopic expression of VvMYB5b in tobacco leads to the up-regulation of most flavonoid structural genes, including CHS, CHI, F3H, and ANS, resulting in the accumulation of anthocyanidin-and proanthocyanidin-derived compounds in flowers (Deluc et al, 2008). In apple fruit, MdMYB10 controls red flesh coloration, while MdMYB1, MdMYBA, and MdMYB3 are responsible for red skin coloration (Ban et al, 2007;Espley et al, 2007;Vimolmangkang et al, 2013). Furthermore, overexpression of MdMYB3 in transgenic tobacco plants results in transcriptional activation of several flavonoid pathway genes, including CHS, CHI, UFGT, and FLS, these plants exhibit increased pigmentation and accumulate higher levels of anthocyanins and flavonols in flowers, but not in leaves (Vimolmangkang et al, 2013).…”