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We analyzed VOC composition of complete inflorescences and single flowers of lavender during the flowering period. Our analyses, focused on the 20 most abundant terpenes, showed that three groups of components could be separated according to their patterns of variation during inflorescence ontogeny. These three groups were associated with three developmental stages: flower in bud, flower in bloom and faded flower. The expression of two terpene synthases (TPS) was followed using qPCR during inflorescence ontogeny. A comparison of these chemical and molecular analyses suggested that VOC production in lavender spike is mainly regulated at the transcriptional level. These results highlighted that lavender could be a model plant for future investigations on terpene biosynthesis and regulation, and could be used to explore the functions of terpene metabolites.
Terpene metabolites are implicated in several ecological and physiological functions. Their basic carbon skeletons are produced by terpene synthases, enzymes that are regulated throughout plant development in response to biotic and abiotic environmental factors.1,2 We tested the influence of flower maturity on terpene composition. First, we extracted terpenes of single flowers (12 flowers per sample) at different maturity stages during the flowering period. No difference in terpene composition was found between flowers of the same maturity stage, wherever their position on the inflorescence (basal, medium or top). Three different terpene compositions could be distinguished in relation to flower maturity stages (unopened flowers, opened flowers and faded flowers). In a second investigation, we monitored weekly the terpene composition of the whole inflorescence over the entire flowering period (from May to July, Fig. 1) and repeated the experiment over three years. The scent of the whole lavender inflorescence followed the same pattern of evolution as individual flowers, though several maturity stages could be found in a spike. The three different groups of components appeared sequentially during the flowering period (group 1 when flowers were mainly all in bud, group 2 when inflorescences were flowering, group 3 when flowers faded). The total amount of terpenes was the highest when a majority of flowers on a spike were opened.
Qualitative and quantitative changes in terpenes during lavender flower development may serve pollination and post-pollination ecological functions as reported in orchids.3 Terpenes of group 2 included linalyl acetate and several sesquiterpenes as major compounds. These could act as attractive molecules for bees, the main generalist pollinator of lavender. In contrast, terpenes of group 1 and group 3 were composed of non-oxygenated (1,8-cineole, ocimene, limonene) and oxygenated monoterpenes (linalool and terpinen-4-ol) respectively and could act as repellents to protect immature flowers and seeds against damaging insects. Linalool and terpinen-4-ol were reported as toxic compounds against insects in other plants.4 Our results emphasize the use of lavender as a suitable model for the functional characterization of terpenes in Lamiaceae during pollination events. In future experiments, the attractiveness of several terpenes of group 2, such as linalyl acetate, could be tested using choice olfactometer experiments. Moreover, the correlation between the synthesis of terpenes belonging to group 2 and the synthesis of nectar could be examined. Finally, experiments including white inflorescence mutants of lavender are planned to evaluate the importance of scent versus color in attracting pollinators.
Two terpene synthases (TPS) previously cloned by Landmann et al.5 were used to assess the potential relationship between volatile terpene accumulation and biosynthetic gene expression during lavender inflorescence ontogeny. LaLIMS (limonene synthase), a gene directly responsible for the biosynthesis of limonene and co-products (all terpenes in group 1) was downregulated as its corresponding terpenes decreased in full bloom inflorescences. In contrast, the expression of LaLINS (linalool synthase), the gene responsible for production of linalool (group 2), was at the highest level in full bloom inflorescences with linalool and linalyl acetate, the main compounds. These comparative results between terpene production and gene expression suggested that VOC production in the lavender spike is mainly regulated at the transcriptional level. Lavender produces more than 75 mono or sesquiterpenes6 and constitutes a suitable model to study the regulation of several TPS under physiological changes, such as flower development. Others TPS will be characterized and we plan to study their expression during inflorescence ontogeny.
Peltate trichomes are the main structures responsible for synthesis and accumulation of terpenes in lavender flowers (Fig. 2). These secretary glands are localized in the depressed sinus of the calyx. Croteau and coworkers7,8 have extensively studied differentiation of peltate trichomes in peppermint. It is assumed from these studies that secretary trichomes are differentiated at an early stage during organ formation and that terpene synthase activity is restricted to the first stage of trichome formation. Scanning electron microscopy observations confirmed that peltate glands were already differentiated in the young calyx of unopened flowers in lavender. It would be particularly interesting to study the function of peltate trichomes when several terpene synthases could be expressed sequentially. In accordance with our results, such a secreting model suggests that terpenes of group 1 are either evaporated through the cuticle or are remetabolized in secretory cells.
An expressed sequence tag database in lavender has been provided recently by Lane et al.9 and a pyrosequencing project is in progress in our laboratory. These two data banks will help to further the characterisation and expression of other TPS involved in terpene synthesis during inflorescence ontogeny. Moreover, these molecular tools could allow identification of genes involved in secondary modifications of terpenes such as acetyltransferases, which use terpene alcohol as a substrate to produce the corresponding acetate esters. These acetyltransferases have been characterized in several plants10 but belong to a large family with a high molecular polymorphism.11 The study of the regulation of acetyltransferase, during flower ontogeny in lavender inflorescences, should be of great interest to characterize the biological role of acetylated terpenes. Lavender oil production and quality may be improved through a better understanding of factors regulating the production of linalyl acetate.
Overall, lavender is a model plant to study terpene synthesis both at the molecular, cellular and ecological level.
The author would like to thank Prof. David Leach for his fruitful advice during the writing of this manuscript.
Addendum to: Guitton Y, Nicolè F, Moja S, Valot N, Legrand S, Jullien F, Legendre F. Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence developmentPhysiol Plant2010138150163 doi: 10.1111/j.1399-3054.2009.01315.x.
Previously published online: www.landesbioscience.com/journals/psb/article/11704