Transcriptional Regulation and Overall Activation
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.