Wild type Nicotiana benthamiana
is not capable of betalain synthesis. This prevents false-positive results when the RUBY reporter is not activated. However, the induction of betalain synthesis might cause a harmful metabolic imbalance. To prevent this, we decided to develop an additional reporter system based on anthocyanins. These pigments occur naturally in N. benthamiana
leaves. Therefore the metabolism should be better adapted to cope with anthocyanin biosynthesis. We call this newly developed reporter system ANTHOS.
As explained above, anthocyanins are, like betalains, one of the most common plant pigment groups and belong to the flavonoids, a large class of specialized metabolites in plants. Anthocyanins are responsible for the red, purple and blue colors of many flowers and fruits and attract pollinators and seed dispersers . The synthesis of anthocyanins plays a role in the plant’s protection from light and pathogens and is induced under stress conditions.
The regulation of anthocyanin biosynthesis is highly conserved in angiosperms. To activate the anthocyanin production, a so-called MBW complex is required. It consists of a R2R3MYB transcription factor, a bHLH transcription factor and a WD Repeat Protein (WDR) . The bHLH transcription factor and the WDR protein fulfill several functions beyond anthocyanin regulation, while the involved R2R3-MYB transcription factor specifically regulates the production of anthocyanins.
To identify which combination of transcription factors we should express for a functional anthocyanins-based reporter system (ANTHOS), we talked to Dr. Ralf Stracke
, expert on transcription factors at Bielefeld University. In fact, he annotated the R2R3-MYB transcription factor family in 2001 . He recommended using the proteins At
bHLH2 and At
TTG1 of Arabidopsis thaliana
For the cloning of ANTHOS, we used two different approaches. For the first approach, we cloned each of the three transcription factors in one separate open reading frame with its own promoter and terminator. The promoter for each of the three genes is the estrogen inducible lexA promoter. As terminators, the G7 terminator was used for At
MYB75 and At
bHLH2, the TUB9 terminator for At
TTG1. For the second approach, we used one open reading frame for the expression of the transcription factors, separated by 2A peptides, which are also used for the RUBY expression. Like in the first approach, we chose the lexA promoter combined with the G7 terminator. We decided to use these combination of promoter and terminators based on a recent publication from Andreas Andreou
and Nayomi Nakayama
, where they investigated promoter-terminator interactions. We used terminators, which showed a high expression in combination with the lexA promoter.
Andreas Andreou also developed the cloning method Mobius Assembly in Nayomi Nakayama’s lab, which we used for cloning ANTHOS. This method is based on Golden Gate cloning systems, while the backbones are binary plasmids
. These plasmids are suitable for plant transformation. In Mobius Assembly, basic parts like promoters, terminators and coding sequences are first cloned into level 0 vectors. In level 1, several basic parts are combined to form a transcriptional unit (TU). In level 2, TUs are assembled to form a multi-TU construct.