## 3 Transcriptional Regulation of TIA Biosynthesis
Therefore, the overexpression of _ORCA3_ alone in two types of tissue cultures could not cover all the pathway genes, such as _G10H_, _SGD_ and _DAT_. Another study shows that co-overexpression of _ORCA3_ and _SGD_ in _C. roseus_ hairy roots resulted in a significant increase of many TIAs [33]. Co-overexpression of _G10H_ and _ORCA3_ in _C. roseus_ plant and hairy roots both increased the accumulation of TIAs, indicating that co-overexpression of regulator and critical synthetic gene is an efficient way to increase metabolite production [34]. Besides JA, other ORCA3 inducers are found recently. Feeding artemisinin acid to _C. roseus_ meristematic cells resulted in the increase of the transcript level of _ORCA3_, which indicated that artemisinin acid may be anotherway to induce ORCA3 [35]. Another study shows that inoculation of fungal endophytes in _C. roseus_ could up-regulate the expression of _ORCA3_ and enhance the accumulation of vindoline [36]. The fact that _ORCAs_ can be induced by not only JA but also other elicitor implies the expressions of two _ORCAs_ are regulated by more transcription factors, such as CrMYC2. As a positive regulator for the expressions of _ORCAs_, CrMYC2 was initially isolated through a yeast one-hybrid screening system using a tetramer of G-box from the _STR_ promoter as bait, while it cannot control the active of the _STR_ promoter [37]. CrMYC2 can regulate the expression _ORCA3_ gene via binding to a specific sequence in the jasmonate-responsive element (JRE) from _ORCA3_ promoter [38]. Overexpression or knockdown of _CrMYC2_ significantly changed the expression of _ORCAs_ and also had a strong effect on the accumulation of catharanthine and taberosine, but showed no influence on _STR_ and _TDC_ expression, which indicated that CrMYC2 is critical for the expression of MeJA-responsive _ORCAs_ and the accumulation of alkaloid but it could not regulate such structural genes as _STR_ and _TDC_[39]. A bHLH transcription factor BIS1 from clade IVa isolated from _C. roseus_ could regulate the expression of structural genes that ORCA3 cannot cover [40]. Overexpression of BIS1 in _C. roseus_ cells and hairy roots caused a significant increase in the expression of the seco-iridoid pathway genes up stream of _LAMT_ and the 2-C-methyl-D-erythnitol-4-phosphate (MEP) pathway genes. Meanwhile, loganic acid, secologanin and strictosidine were highly accumulated in the BIS1-overexpressing cells. However, the accumulation of TIAs in transgenic hairy roots was not increased, which may be resulted from the down-regulation of ORCA3 target genes _LAMT_, _SLS_, _TDC_, and _SGD_. Decreased expression of the ORCA3 target genes was not caused by decreased expression of _ORCA2_ or _ORCA3_, nor of any other known _C. roseus_ TF encoding gene previously linked with regulation of the MIA pathway, and thus involves other, yet unknown, regulatory mechanisms. Another bHLH transcription factor BIS2 was identified recently, which is the homolog of BIS1 that can form homo- or heterodimers with BIS1. Same with BIS1, overexpressing _BIS2_ in _C. roseus_ suspension cells could also up-regulate MEP as well as seco-iridoid pathway genes, knockdown of _BIS2_ completely abolished the JA-induced up-regulation of the seco-iridoid pathway genes and the accumulation of TIAs. The expression of _BIS2_ could be regulated by BISs, while the BISs-binding sites of _BIS2_ promoter remain to be determined. BIS1 and BIS2 could both regulate the structural genes that ORCA2 and ORCA3 cannot affect, such as _G10H_[41]. Therefore, BISs together with ORCAs may control the whole upstream pathway of TIA biosynthesis and support sufficient synthetic precursor of TIAs. Two AP2/ERF proteins form a cluster with ORCA3, ORCA4 and ORCA5 were cloned, which are homologous to ORCA3. The expression of _ORCA4_ and _ORCA5_ can be also induced by JA like _ORCA3_. However, unlike _ORCA3_, _ORCA4_ and _ORCA5_ cannot be directly regulated by CrMYC2, they may be regulated by other transcription factors. Moreover, overexpression of _ORCA4_ in _C.