# Dynamics of Monoterpene Formation in Spike Lavender Plants
## 1 Introduction
Due to its high economic interest, the composition and quality of spike lavender oil has been widely studied including chemical composition of essential oil from some Spanish wild populations [26; 27; 28; 29]. All these studies revealed a great intraspecific variability in the chemical composition of oils that can be attributed to several variation sources: genotypic, climatic, geographical and/or seasonal [28; 29; 30; 31; 32; 33]. Modifications of yield and composition of the spike lavender essential oil by genetic engineering may have important scientific and commercial applications. Until now, three genes involved in the first steps of the synthesis of terpen have been overexpressed in spike lavender, the _Arabidopsis_ HMG1, DXS and DXR genes, encoding the respective HMGR1S, DXS and DXR enzymes. As expected, the overexpression of the DXS gene produced a significant increase of the end-product monoterpenes derived from an elevated supply of precursors of the MEP isoprenoid pathway without affecting the chlorophyll and carotenoid content [34]. Surprisingly, the overexpression of the DXR enzyme did not lead to an increase of monoterpenes or photosynthetic pigments [35]. In contrast, the overexpression of the HMG1 gene from _Arabidopsis_, led to an increased yield of essential oil since transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). These transgenic plants also increased its amount of the end-product sterols, \(\beta\)-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not accumulate more carotenoids or chlorophylls as compared to controls. The potential of the mexolonate pathway for enhanced isoprenoid production by upregulation of HMGR and other enzymes of the pathway as well as its interaction with the MEP pathway have been updated  
Figure 2: Scheme for the synthesis of the monoterpenes cineole and camphor. GPP, geranyl diphosphate; 1, cineole synthase; 2, bornyl diphosphate synthase; 3, bornyl diphosphate diphosphate; 4, borneol dehydrogenase. Adapted from [22; 23; 24; 25]. recently [36]. The increased levels of essential oil and sterols observed in the transgenic T0 plants were maintained in the progeny that inherited the HMG1 transgene. Therefore, genetic manipulation of the MVA pathway also increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species [37]. The incorporation of \({}^{13}\)C- or \({}^{14}\)C-labelled tracer compounds into the current metabolism is one of the most powerful tools to determine the metabolites biosynthetic pathways. To study primary and secondary metabolism in plants, general precursors (e.g., glucose) or more specific intermediates can be used as tracers and supplied to plant tissues maintained either in vitro (e.g., plant cell cultures, stem or leave cultures, seedlings) [38; 39] or ex vitro [39]. By studying the presence and distribution of the labelled atoms in the final products, it is possible to reconstruct the metabolic pathways followed [39]. The use of \({}^{13}\)CO\({}_{2}\) as a tracer is a powerful tool, since this strategy represents the possibility of studying metabolic pathways in plants by mimicking, as much as possible, their physiological growth conditions [40; 41; 42]. The labelling period (pulse) is followed by a chase period in which the plants grow under normal conditions and natural \({}^{13}\)CO\({}_{2}\) abundance for several hours/days. During that time, unlabeled products and intermediates are produced, that also are used in the production of the final metabolites. Therefore, complex patterns of [\({}^{12}\)C]- and [\({}^{13}\)C]-isotopologues are expected to be found in the final natural products.