## 4 A Hairy Root Platform for the Production of Bioactive Abietane Diterpenes
### Elicitation with Methyl jasmonate and Its Analogue Coronatine
sclarea_ HRs with Methyl-jasmonate (MJ) or Cor has been very informative for us to pinpoint biosynthetic genes of MEP-pathway that might limit the accumulation of aithiopinone and other bioactive triterpen in _S. sclarea_ HRs, as reported below. 1.1 MJ-Induced Accumulation of Bioactive Abietane Diterpenes in _S. sclarea_ Is Due to the Transcriptional Regulation of Genes of the MEP-Pathway  
It is well documented that the JA-activated biosynthesis of different terpenoids [47], is due to transcriptional up-regulation of genes belonging to either the MEP-pathway or the MVA-pathway [48; 49; 50]. We found that also in _S. sclarea_ HRs, MJ elicitation was able to enhance the transcriptional levels of several biosynthetic genes of the plastidial MEP-dependent pathway, such as _SsDXS_, _SsDXR_, _SsCMK_, _SsMCS_, _SsHDS_, _SsHPR_, _SsGGPPS_, and _SsCPPS_ genes [51]. The most induced transcript by MJ was _SsDXS_ (60-fold increase), the enzyme acting up-stream the MEP-derived pathway. Since it is well known that this enzymatic reaction is limiting the accumulation of different isoprenoids in many plant species, increasing the DXS activity by metabolic engineering has been successful to enhance the content of terpenoids in different plant species and prokaryotic cells [52]. The _SsGGPPS_ gene encoding the synthase involved in the condensation of IPP and DMAPP to GGPP, the common precursor of many terpen of plastidial origin, was also transcriptionally activated by MJ (20-fold increase) in _S. sclarea_ HRs as well as the _SsCPPS_ gene (60-fold increase). This is indirect  
Figure 4: **A Schematic representation of the MJ or Cor signal perception and transduction mediated by the MYC2 transcription factor, based on the current knowledge available in literature (original figure). A detailed description of MYC2-mediated signaling and transduction in normal conditions (low level of JA-Ile or Cor) (a) or in stress or elicitation conditions (high level of JA-Ile or Cor) (b) is reported in the text. Abbreviations: JA-Ile, jasmonoyl isoleucine; Cor, coronatine; JAZ, jasmonate ZIM domain; NINJA, novel interactor of JAZ; TPL, topless; HDA6, HDA19, histone deacetylase 6, 19; ASK1, Arabidopsis SKP1 (S-phase kinase-associated protein (1) homologue; CULL, CULLIN; E2, ubiquitin-conjugating enzyme; MYC2, bHLH zip transcription factor; RBX, RING-H2 protein; SCF-complex, complex consisting of Skp1, Cullin-1, and F-box protein; Ub, ubiquitin; COII, coronatine insensitive 1; MED25, mediator 25. **  
evidence that abietane-type diterpenes in _S. sclarea_ are produced from the conversion of GGPP via a copalyl-diphosphate (CPP) intermediate, by using this CPP synthase, as widely reported for other plant diterpenoids [53; 54; 55]. This MJ-coordinated expression of multiple genes of the MEP-pathway induced a significant increase of the abietane diterpene content in _S. sclarea_ HRs, as phenotypically visible from the red color of the MJ-elicited HRs (Figure 3). Compared to control HRs (14.50 \(\pm\) 0.31 mg L\({}^{-1}\)), MJ elicitation (100 \(\upmu\)M) for 7 days induced an approximatively 20-fold enhancement in the total abietane diterpene content (304.01 \(\pm\) 2.21 mg L\({}^{-1}\)). A long-term elicitation (4 weeks) with MJ also increased the content of total abietane diterpenoids, but it was associated with a severe HR growth inhibition, which penalized the final yield of total abietane diterpenes (210.74 \(\pm\) 1.26 mg L\({}^{-1}\)) (Figure 5) [51]. This set of data are consistent with those reported in _Salvia millionriraz_ HRs elicited with MJ, which triggered the accumulation of tanshinone and phenolic acids [56], due also to the up-regulation of biosynthetic genes [57]. MJ has been also proved to induce simultaneously the transcription of most of the biosynthetic genes in specific pathways of other plant species, as reported for the TIA (terpenoid-indole-alkaloid) in _C. roseus_[58], for nicotine in _N. tabacum_[59], and for artemisinin in _A. annua_[60].