## 4 A Hairy Root Platform for the Production of Bioactive Abietane Diterpenes
### Metabolic Engineering to Enrich the Content of Abietane Diterpenes in S. sclarea Hairy Roots
#### 5.2.5.5. Orchestrating the Expression of Multiple Biosynthetic Genes of the MEP-Pathway by TFs  
Transcription factors (TFs) provide an attractive alternative for modifying a metabolic flux in plants since they are able to activate in a coordinated manner the transcription of multiple biosynthetic pathway genes. It is widely reported that TFs may act alone or in combination with other regulators to activate and/or to inhibit/de-repress gene transcription [76]. A bioinformatic analysis of _A. thaliana_ genes encoding enzymes of the MEP-pathway revealed that the promoter of many of these genes contains the W-box (_TTGAC_), the known binding site of WRKY TFs, and the G-box (_CACGT_), the binding domain of MYC2, a TF known to be regulated by MJ (Figure 4). The WRKY TFs, members of a plant-specific TF family, characterized by a conserved peptide motif WRKYGQK and a zinc finger domain, play different biological roles and have a prominent involvement in controlling the plant response to biotic and abiotic constraints [77; 78]. Among the different WRKY TFs thus far identified, we focused our attention on AtWRK18 and AtWRK40, since we identified in their promoter region multiple conserved methyl jasmonate responsive elements (MJRE), localized at \(-800\) bp from the TSS, and confirmed their transcriptional activation in MJ-elicited Arabidopsis plantlets [42]. The overexpression of At_WRKY18_, At_WRKY40_ genes in _S. sclarea_ HRs positively activated the transcription of different biosynthetic genes of the plastidial MEP-derived pathway. In particular, _S. sclarea_ genes _DXS, DXR, HDS, GPPS, and CPPS were up-regulated by overexpression of At_WRKY18_ and AtMYC2, while At_WRKY40_ preferentially activated the transcription of the _DXS_ and _CPPS_ genes. Targeted metabolic profiling of overexpressing WRKY HR lines revealed that the simultaneous up-regulation of the biosynthetic genes correlated to an increased content of abietane diterpenes compared to control line (a fold-increase >4 and >2 in the At_WRKY40_ and _AtWRK18_ overexpressing lines, respectively), especially in the production of salvi-isone and aethiopinone [42]. This is one of the first published evidence on the involvement of WRKY TFs in the specific metabolic pathway of abietane diterpenes in _S. sclarea_, which was confirmed also for the biosynthesis of tanshinones in _S. militorhyza_[79].  
We also overexpressed in _S. sclarea_ HRs the AtMYC2 TF, which contains a basic helix-loop-helix (bHLH) domain, and, as already discussed, is a master regulator of the JA signaling pathway and also controls secondary metabolism in plants [80; 81; 82] (Figure 4). _AtMYC2_ overexpression was able to enhance the transcript levels of several biosynthetic genes of the MEP-derived pathway [42]. This coordinated up-regulation resulted in a significant accumulation of the total abietane diterpene content (a fold-increase > 5) (Table 2), which caused however, a considerable growth inhibition of the overexpressing HRs [42], due possibly to the multiple roles of MYC2 in JA signal transduction. As mentioned earlier, a possible unexpected modification of the level of the synthesis of phytohormones or chlorophyll derived from the MEP-pathway might also explain partially the growth impairment of the MYC2 overexpressing HR lines. At any rate, the MYC2- or WRKY-dependent increased content of abietane diterpenes was lower than that obtained by MJ or Cor elicitation, suggesting that it is more likely a combinatorial role for different TFs, belonging to the same TF group or to different families, in the transcriptional regulation of biosynthetic genes of JA-mediated metabolic pathways [42].