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Increasing knowledge about isoprenoid biosynthesis pathways has provided new tools for aromatic plant breeding using biotechnological approaches. Notably, there are possibilities to modify essential oil profiles and enhance production of valuable monoterpenes. This attains a particular significance in Lavandula latifolia Medik. (spike lavender), one of the most important essential oil crops in Spain. This chapter summarizes work done to improve essential oil yield and quality by engineering: (1) the enzymes controlling regulatory steps of methyl-D-erythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways to increase C5 units employed for monoterpene biosynthesis and (2) the monoterpene synthases to produce changes in the qualitative profile of particular monoterpenes.

The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in developing spike lavender ( Lavandula latifolia Med) was analyzed using specific inhibitors and on the basis of 13 C-labeling experiments. The presence of mevinolin (MEV), an inhibitor of the MVA pathway, at concentrations higher than 0.5 μM significantly reduced plant development, but not the synthesis of chlorophylls and carotenoids. On the other hand, fosmidomycin (FSM), an inhibitor of the MEP pathway, at concentrations higher than 20 μM blocked the synthesis of chlorophyll, carotenoids and essential oils, and significantly reduced stem development. Notably, 1.2 mM MVA could recover the phenotype of MEV-treated plants, including the normal growth and development of roots, and could partially restore the biosynthesis of photosynthetic pigments and, to a lesser extent, of the essential oils in plantlets treated with FSM. Spike lavender shoot apices were also used in 13 C-labeling experiments, where the plantlets were grown in the presence of [U– 13 C 6 ]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C 5 -precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the methylerythritol phosphate (MEP) pathway. However, on the basis of the isotopologue profiles, a minor contribution of the MVA pathway was evident that was increased in transgenic spike lavender plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first enzyme of the MVA pathway. Together, these findings provide evidence for a transport of MVA-derived precursors from the cytosol to the plastids in leaves of spike lavender.

[EN] Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme, This work has been funded by the Regional Government of Valencia (PROMETEO/2009/075, Generalitat Valenciana); the Spanish Ministerio de Educacion y Cultura, FPU fellowship to I. M-P (AP2007/04606); and Fundacion Ramon Areces, fellowship to I. M-P.

Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.