# Dynamics of Monoterpene Formation in Spike Lavender Plants
## 3 Discussion
### \({}^{13}\)CO\({}_{2}\) Labelling in Transgenic HMGR5 Plants  
Metabolic engineering studies in spike lavelander demonstrated that MEP and MVA pathways may be implicated in the monoterpene biosynthesis. Nevertheless, essential oil yield was always higher in those transgenic spike lavelander plants overexpressing the DXS gene. Previous work using specific inhibitors of both pathways and feeding experiments with [U-\({}^{13}\)C6]-glucose, suggested a minor contribution of the MVA pathway that was increased in transgenic (HMGR5) spike lavelander plants [37]. Results presented here using labelled \({}^{13}\)CO\({}_{2}\) confirmed that in WT and transgenic HMGR spike lavelander plants, the biosynthesis of camphor and cineole happen in a very robust way through the MEP pathway according to the results achieved with NMR and GC/MS. Still, outcomes with HMGR \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & & \multicolumn{2}{c}{**Excess Values (\%)**} & \multicolumn{2}{c}{**Ratio**} \\ \hline - & - & M+2 & M+3 & M+2/M+3 & M+2 \\ \cline{3-6}  & & & & & (M+2) + (M+3) \\ \hline
1,8-cineole & HMGR5 & - & - & - & - \\ \hline - & 96 h & 0.27 & 0.16 & 1.69 & 0.63 \\ - & 96 h & 0.22 & 0.12 & 1.83 & 0.65 \\ - & 168 h & 0.07 & 0.04 & 1.75 & 0.64 \\ - & 240 h & 0.21 & 0.11 & 1.91 & 0.66 \\ - & 240 h & 0.32 & 0.14 & 1.79 & 0.70 \\ - & 240 h & 0.17 & 0.12 & 1.42 & 0.59 \\ - & WT & - & - & - & - \\ - & 96 h & 0.05 & 0.03 & 1.67 & 0.63 \\ - & 264 h & 0.53 & 0.27 & 1.96 & 0.66 \\ - & 264 h & 0.15 & 0.09 & 1.67 & 0.63 \\ \multicolumn{1}{c}{} & 264 h & 0.87 & 0.48 & 1.81 & 0.64 \\ \hline \hline \end{tabular}
\end{table} 
Table 7: GC/MS excess data for M+2 and M+3 isotopomeres in 1,8-cineole extracted with chloroform-d and analyzed by GC/MS from spike lavelander leaves from lines HMGR5 and WT at different points of time after the labelling pulse phase with \({}^{13}\)CO\({}_{2}\). h: hours. oveerexpressing spike laverder plants cast doubt about the fact that the MVA pathway might be involved, even in a small share, on the production of monoterpenes because of the higher content of essential oil in those plants. It is well known that the MVA and MEP pathways are not totally independent as there is some crosstalk between them through common intermediates [11; 39; 53]. Experiments with labelled intermediates [2-\({}^{13}\)C\({}_{1}\)]mevalonolactone or [U-\({}^{13}\)C\({}_{6}\)]glucose in _Catharantlus roseus_ using NMR techniques showed that crosstalk between the MVA and MEP pathways cannot be explained in detail by a simple two compartment model and requires an additional in depth study of complex regulatory mechanisms [19]. For some time, \({}^{13}\)CO\({}_{2}\) has been used several times as a tool for the analysis of quantitative aspects of plant metabolism in whole plants including _Nicotiana tabacum_[45], _Panax ginseng_[39] and _Pentalinon andrieuxii_[53]. This tool allows central and secondary plant metabolic pathways to be elucidated under close to unperturbed _in vivo_ conditions [42]. The detection of specific distributions of multiply labelled isotopologues in two monoterpenes form \({}^{13}\)CO\({}_{2}\)-labelled _L. latifolia_ confirmed the validity of the experimental approach for the present study. HMGR in plants is considered the most important enzyme for regulating the MVA pathway; it is regulated at the levels of transcription, post-transcription, translation and post-translation [36]. As expected, overexpression of HMGR enzyme in tobacco, spike laverder, tomato, _Arabidopsis_ and ginseng has been reported to increase sterol content [36]. In the case of _Artemisia annua_ it was also reported an increase in artemisinin production [54]; in _Salvia mitiorrhizia_ total tanshinone production was enhanced [55]; and in _Panax ginseng_ triterpene saponin production increased [56]. However, spike laverder is the only plant model were an increase of non-mevalonate metabolites is noted after overexpression of the HMGR enzyme [37].