## 3 Transcriptional Regulation of TIA Biosynthesis
roseus_ hairy roots significantly increased the transcripts levels of genes in both tryptophan pathway and seco-iridoid pathway, and also increased several TIAs, especially taberosine. Therefore, ORCA4 is functionally overlapping but divergent with ORCA3 [42]. CrWRKY1, belonging to the group III WRKY superfamily, was identified in _C. roseus_, which can be induced by several phytohormes and preferentially expresses in roots [43]. Studies found CrWRKY1 regulates _TDC_ by binding W box element in _TDC_ promoter. Overexpression of _CrWRKY1_ in _C. roseus_ hairy roots increased several key pathway genes, such as _AS_, _DXS_, _SLS_, _SGD_, especially _TDC_; as well as TF encoding genes, such as _ZCTs_, while it represses the transcriptional activators _ORCA2_, _ORCA3_, and _CrMYC2_. Moreover, the accumulation of serpentine was significantly increased, while catharanthine was decreased. Therefore, CrWRKY1 is an ideal candidate to regulate serpentine branch to produce more ajmalicine and serpentine. CrBPF1 is a kind of MYB transcription factor that binds to BA region in _STR_ promoter. Overexpression _CrBPF1_ in _C. roseus_ hairy roots changed the expression of many pathway genes. As for regulatory genes, overexpressing _CrBPF1_ increased the transcripts of _ORCA3_, _CrMYC1_, _CrMYC2_, _BIS1_, _GBF2_ and _ZCTs_, but little affected the expressions of _ORCA2_ and _CrWRKY1_. Although overexpression of _CrBPF1_ could not obviously increase the accumulation of TIAs and even caused the decrease of serpentine, it could extensively regulate TIA pathway genes and increase the expression of TIA transcriptional repressors, which may be a good direction to the research of TIA biosynthesis [44]. It has been reported that using G-box element in the \(STR\) promoters as bait to screen _C. roseus_ yeast expression library resulted in identification of G-box binding factors CrGBF1 and CrGBF2 [37; 45]. Both CrGBF1 and CrGBF2 act as transcriptional repressors of the \(STR\) via binding to the NR element in \(STR\) promoter, which indicated that GBFs may play an important role in the regulation of the expression of \(STR\) and the accumulation of TIAs. Three Cys2/His2-type zinc finger transcription factors from _C. roseus_, ZCT1, ZCT2 and ZCT3, were isolated through a yeast one-hybrid screening system using an elicitor-responsive DB element in \(TDC\) promoter as bait [46]. All of them repress the activities of \(STR\) and \(TDC\) promoters in trans-activation assays, which may probably be resulted from the existence of a potent repression domain, LxLxL motif in the C-terminal region of ZCTs. In addition, the ZCT proteins can also repress the transcriptional activating activation of the ORCAs, and are found to be induced by yeast extract (YE) and JA. However, there are several differences between ZCT1, ZCT2 and ZCT3. The \(STR\) and \(TDC\) promoters binding sites of ZCT1, ZCT2 are different from one of ZCT3, and the structures of ZCT1, ZCT2 and ZCT3 are also different [46]. Furthermore, the functions of them are partially different ZCT1 and ZCT2 act as repressors of hydroxymethylbutenyl 4-diphosphate synthase (HDS) while ZCT3 has no effect on HDS [47]. A recent study shows that silencing ZCT1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes, which may be resulted from the remained elevated expression level of ZCT3. These results reveal that the ZCTs may play overlapping but distinct functions in TIA biosynthesis [48]. Figure 2: Regulation of TIA biosynthetic pathways by transcription factors in _Catharanthus roseus_. Various structural genes (marked in blue) in TIA biosynthetic pathway are regulated by different transcription factors (TFs). ORCAs are believed to be key regulators that directly bind to the promoters of structural genes involved in TIA biosynthesis. Several TFs such as WRKYs not only control the expression of structural genes but also regulate the expression of other TFs including MYC2, ORCAs, ZCTs, etc.