Due to the difficulty of studying biosynthetic pathways in secondary metabolism, researchers have in general isolated few biosynthetic genes and characterized their promoter sequences, except for anthocyanin biosynthesis (Winkel-Shirley, 2001). The molecular characterization of anthocyanin biosynthesis is a successful application of transcriptional regulation to metabolite production; that is, introduction of transcriptional regulator-R and C1 in a heterologous system (Lloyd et al., 1992) and recent successes with activation tagging methods (Borevitz et al., 2000) show the high potential and bright future of these approaches. For example, the pap1 general transcriptional factor gene in anthocyanin biosynthesis was isolated from Arabidopsis based on the visible inspection of highly pigmented plants after activation tagging (Borevitz et al., 2000). Additional successful isolation of ORCA3, a transcriptional factor with a JA-responsive AP2 domain, in indole alkaloid biosynthesis in Catharanthus using activation tagging demonstrated the effectiveness of this approach, while the selection methodology had to be further developed due to the colorless nature of target compounds (Van Der Fits and Memelink, 2000).
ORCA expression is induced by JA. Ectopic expression of ORCA3 in cultured cells from C. roseus increases the expression of the terpenoid indole alkaloid biosynthetic genes TDC, STR, CPR, and D4H but does not regulate the genes encoding G10H and DAT (Fig. 11.3). Transgenic cells that overexpressed ORCA3 accumulated significantly more tryptophan and tryptamine. However, since no terpenoid indole alkaloids were detected, the terpenoid branch of the pathway remains limiting for terpenoid indole alkaloid production. Although ORCA3 plays an important role in regulating terpenoid indole alkaloid biosynthesis, it is not sufficient to regulate the complete pathway. This indicates that other transcription factors are also involved. The use of an enhancer domain of the STR promoter as bait in a yeast-one-hybrid screen resulted in the isolation of CrBPF1, an MYB-like transcription factor (Van Der Fits et al., 2000). CrBPF1 expression is induced by elicitor but not JA. In addition, the STR promoter contains a promoter element that is conserved in plants, called the G-box, which is located adjacent to the JERE element. A yeast-one-hybrid screen using the G-box as bait isolated G-box-binding factors (crGBF) of the basic leucine zipper class and MYC-type bHLH transcription factors (CrMYC) (Chatel et al., 2003). CrGBFs have been shown to repress STR expression (Siberil et al., 2001), whereas this factor is not sufficient to control the overall gene expression for indole alkaloid biosynthesis. As discussed above, regulation of the whole metabolic pathway would be more complicated since the spatial and developmental integration of metabolism are needed.
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