# Dynamics of Monoterpene Formation in Spike Lavender Plants
## 2 Results
### \({}^{13}\)Co\({}_{2}\) Labelling in Transgenic HMGR5 Plants  
WT and HMGR5 plants were labelled with \({}^{13}\)CO\({}_{2}\) for 5 h and harvested at different chase times. The terpen were extracted and analyzed by GC/MS as previously described. Transgenic HMGR5 accumulated more monoterpenes than the WT (Table 5). The percentages of the five main components of the leaf essential oil (the monoterpenes cineole, camphor, \(\alpha\)-pinene, \(\beta\)-pinene and limonene) were considered in relation to the 15 main peaks of each sample and are presented in Table 5. These five molecules accounted for almost 88% of the 15 peaks in the HMGR5 plants and 78% for the WT plants, revealing that apparently the HMGR5 plants had a higher proportion of monoterpenes in their essential oil. Coumarine was also detected in most samples (Table S3), elevating the total percentage of identified compounds to over 94% in the case of HMGR5 plants and 88% for WT plants. The main compounds in all samples were camphor and 1,8-cineole that accounted for a total of 79% in the case of HMGR5 plants and less than 67% in the case of WT plants. These results are in accordance with those previously described by our group, with HMGR overexpressing plants that accumulated significantly more essential oil constituents, mainly monoterpenes and sesquiterpenes, as compared to controls. Enhanced expression of HMGR1S also increased the amount of the end-product sterols, \(\beta\)-sitosterol and stigmasterol, but did not affect the accumulation of carotenoids or chlorophylls [37]. Although all plants were labelled for a pulse time of five hours, low excess \({}^{13}\)C incorporation was achieved, between 0.01% and 0.83% (Table S4). This might be due to a poor watering of the plants that may induce the stomata closing and a photosynthetic reduction. Nevertheless, some of the obtained results can be discussed. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Position Carbon Atoms** & **Chemical Shift [ppm]** & \({}^{13}\)**C Coupling** & \({}^{13}\)**C-\({}^{13}\)C Coupling** \\  & & & **Constant [Hz]** \\ \hline \multicolumn{4}{c}{Camphor} \\ \hline
10 & 9.27 & to C1 & 41.1 \\
9 & 19.15 & to C7 & 37.9 \\
8 & 19.8 & to C5 & 2.5 \\
5 & 27.04 & to C4 to C8 & 32.2 2.5 \\
6 & 29.91 & to C2 & 2.3 \\
4 & 43.04 & to C5 & 32.2 \\
3 & 43.32 & to C2 & 34.3 \\
7 & 46.84 & to C9 & 37.9 \\
1 & 57.76 & to C10 & 41.1 \\
2 & 220.06 & to C6 & 2.3 \\ \hline \multicolumn{4}{c}{1,8-cineole} \\ \hline
3/5 & 22.8 & C3–C4 & 32.9 \\
7 & 27.55 & to C1 & 43.3 \\
9/10 & 28.87 & C10–C8 C9–C3/4 & 41.0 2.2 \\
2/6 & 31.48 & - &? \\
4 & 32.91 & to C3 & 32.9 \\
1 & 69.93 & to C7 & 43.3 \\
8 & 73.76 & to C10 & 41 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: NMR analysis of camphor and 1,8-cineole derived from chloroform-d extraction of spike lavender leaves pulse-labelled with \({}^{13}\)CO\({}_{2}\) for 5.2 h; chase time 192 h.  
The M+2 and M+3 ratios for 1,8-cineole and camphor are displayed in Figures 8A and 9A and Tables 6 and 7, respectively. These ratios were determined for eight transgenic plants and for four wild type plants. Three HMGR5 plants were harvested 96 h after the pulse time, two after 168 h and three after 240 h. The WT plants were harvested after 96 h (one plant) and 264 h (three plants). Only harvested plants showing an excess of \({}^{13}\)C incorporation to 1,8-cineole are included in Figure 8A. Mean results for cineole revealed that HMGR5 and WT plants have very similar M+2/M+3 ratios (1.7 \(\pm\) 0.2 and 1.8 \(\pm\) 0.1, respectively) (Figure 8B). Similar results were observed in the M+2/M+3 ratios for camphor in WT plants (mean ratio value of 1.9 \(\pm\) 0.1; Figure 9B). In contrast, HMGR5 plants showed a high deviation in the M+2/M+3 ratio within the same chase group (mean value for the ratios were 2.0 \(\pm\) 0.4 and 1.8 \(\pm\) 0.4 for 4 and 10 days chase