# Dynamics of Monoterpene Formation in Spike Lavender Plants
## 2 Results
### NMR Data  
The positional distribution of the labels in cineole and camphor (Figure 4) were determined by \({}^{13}\)C NMR spectroscopy (Tables 2 and 3). Notably, NMR signals for camphor and 1,8-cineole could be detected with the crude chloroform extracts of the leaves by comparison with reference data and on the basis of two-dimensional NMR experiments (Tables 2-4). The overall \({}^{13}\)C-enrichments of carbon atoms were similar and in the range of 2-10% for most of the samples. Unfortunately, the \({}^{13}\)CO\({}_{2}\) labelling experiments afforded similar, if not identical, \({}^{13}\)C-enrichments for the various carbon positions in the monoterpenes, as exemplified for cineole (Table S2). However, the generation of multiply \({}^{13}\)C-labelled isotopologues was dependent on the trunk biosynthetic pathways, as outlined below. Both monoterpenes displayed \({}^{13}\)C-coupled satellites due to the presence of multiply \({}^{13}\)C-labelled isotopologues. On the basis of the specific coupling constants and the detection of specific correlation patterns in INADEQUATE and 1,1-ADEQUATE experiments (Figures 5-7), it can be concluded that the isotopologue profiles of both compounds were highly specific. As shown in Tables 2-4 and Figures 5-7, we assigned 10 and 7 \({}^{13}\)C\({}_{2}\)-isotopologues in camphor and cineole, respectively. Moreover, the detection of long-range \({}^{13}\)C-couplings lead to the assignment of \({}^{13}\)C-isotopologues in camphor and cineole, as well (Tables 2-4 and Figures 5-7). In the case of camphor, the ten C atoms from the monoterpene can be undoubtedly distinguished in the spectrum (Table 4). Coupling constants of satellite pairs C1-C10, C9-C7, C8-C5, C3-C2 and C4-C8 can be clearly attributed (see Figures 5 and 6 and Table 4). The signal for carbon atom C5 shows a satellite pair caused by coupling to C4 (32.2 Hz) and an additional coupling to C8 (2.5 Hz), reflected by a splitting of the satellite pair. Only the long-range coupling of the carbonyl-C2 to C6 cannot be resolved in detail. These results were confirmed by the INADEQUATE experiment, which is shown in Figure 6. Clearly, Figure 6 shows all the couplings already described in Figure 5. The long-range coupling of the carbonyl-C2 to C6 cannot be resolved in the INADEQUATE spectrum as well. Regarding cineole, the 10 C atoms were also determined; as expected, satellite pairs C3-C5, C9-C10, and C2-C6 are indistinguishable from each other because of the circular symmetric structure of the monoterpene. Coupling constants of satellite pairs caused by C3-C4, C7-C1, C10-C8, C9-C4, and C10/C9 can be clearly attributed (see Figure 7). A long-range coupling caused by C9 to C3 is also detected. It is possible that the signal of C3/C5 overlays with a \(\alpha\)-pinene signal. Figure 4: Numbering and biosynthetic origin of carbon atoms from camphor and 1,8-cineole synthesized via the MEP pathway. Carbon atoms derived from: DMAPP (red), IPP (blue), Pyruvate (Green), G3P (Black). Figure 5: \({}^{13}\)C NMR camphor signals, chloroform-d leaf essential oil extract from spike lavelander after incorporation of \({}^{13}\)CO\({}_{2}\) (pulse 5.2 h, chase 192 h). * indicate the satellite signals of the carbon atoms under study. Figure 6: INADEQUATE \({}^{13}\)C NMR camphor signals; chloroform-d leaf essential oil extract from spike lavender after incorporation of \({}^{13}\)CO\({}_{2}\) (pulse 5.2 h, chase 192 h. (NMR parameter: pulse program: inadqf, TD1: 300, NS: 128, J (CC): 50 Hz, D1: 2 s), measurement time: 22 h.  
Figure 7: \({}^{13}\)C NMR 1,8-cineole signals; chloroform-d leaf essential oil extract from spike laverader after incorporation of \({}^{13}\)CO\({}_{2}\) (pulse 5.2 h, chase 192 h).