Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway

Chalvin et al. Horticulture Research (2021)8:206 https://doi.org/10.1038/s41438-021-00640-w ARTICLE Horticulture Research www.nature.com/hortres Open Access Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Camille Chalvin1, Stéphanie Drevensek1, Françoise Gilard 1, Caroline Mauve1, Christel Chollet1, Halima Morin1, Edith Nicol2, Eva Héripré3, Lucie Kriegshauser1, Bertrand Gakière1, Michel Dron1, Abdelhafid Bendahmane1 and Adnane Boualem1 ✉ Abstract Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ ionization mass spectrometry imaging (LDI–FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs’ gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-13C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites. Introduction Ambergris, a natural perfume ingredient, is a waxy substance secreted by the digestive tract of male sperm whales and the best-known amber odorant natural ingredient1–3. Ambergris has been particularly used in the perfume industry for its intense fragrance and unequaled fixative properties4. Ambergris properties are mainly due to one of its constitutive components, ambroxide5. The growing demand for ambergris-type odorants prompted the search for synthetic alternative routes to ambroxide. The most commonly used route to ambroxide is the hemisynthesis starting from sclareol, natural occurring terpene6,7. Correspondence: Adnane Boualem () 1 Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), 91405 Orsay, France 2 Molecular Chemistry Laboratory (LCM), UMR 9168, CNRS, Ecole Polytechnique, Institut Polytechnique de Paris, Route de Saclay, 91128 Palaiseau Cedex, France Full list of author information is available at the end of the article These authors contributed equally: Camille Chalvin, Stéphanie Drevensek Sclareol is a natural diterpene originally characterized in clary sage (Salvia sclarea). Salvia is the largest genus of the Lamiaceae family of plants with almost 1000 described species8. It is widely spread around the world, as Salvia species are found in both temperate and subtropical regions. Clary sage is a biennial herbaceous species naturally growing in the Mediterranean basin and Western Asia9,10. Due to its remarkable aromatic properties, this plant is grown in different European countries, particularly in France, Hungary, and Bulgaria, but also in North America and China11,12. Clary sage has been traditionally used as a medicinal plant11 and numerous biological effects including anti-inflammatory, antimicrobial and cytotoxic activity have been associated with its flower extracts13,14. Today, clary sage is mainly exploited for its aromatic properties. Essential oils (EOs) and sclareol extracted from clary sage are mainly used in flavor and fragrance industries. EOs are extracted by steam distillation of fresh inflorescences and sclareol is © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Chalvin et al. Horticulture Research (2021)8:206 then purified from the remaining plant material by solid/ liquid extraction with an organic solvent15,16. EO is used directly as a perfume component for its tenacious, herbaceous, sweaty, and amber odor16, while sclareol is used as starting material for the hemisynthesis of ambroxide17. In Angiosperms, terpenoid production is often localized in specialized secretory structures18–22. The Lamiaceae are generally characterized by the presence of epidermal secretory structures called glandular trichomes (GTs), which secrete various compounds mainly involved in pollinator attraction or defense against herbivores23. Like most members of the Lamiaceae family, clary sage displays two types of GTs: capitate and peltate GTs24,25. This classification is based on their distinct morphology: capitate GTs have a long stalk topped by a small spherical secretory head, while peltate GTs are sessile trichomes with a very short stalk and a large flattened glandular head (Fig. S1). Two subtypes of capitate GTs have been described in clary sage according to their size: small and large capitate GTs25 (Fig. S1). Linalyl acetate, the acetylated derivative of the monoterpene linalool, is a major component of clary sage EO and is generally considered to be produced by clary sage GTs11,25. By contrast, the localization of sclareol production is less clear. A study using a set of microscopy and analytical chemistry approaches highlighted the presence of abundant epicuticular sclareol crystals at the surface of the calyx15. The authors of this study hypothesized that sclareol may be produced by calyx epidermal cells, like other cuticular secretions15. Terpene biosynthesis starts with the condensation of five-carbon building blocks: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), to form linear compounds called prenyl intermediates. Prenyl intermediates are then converted into functional terpenes through a number of chemical modifications, for example, cyclization and hydroxylation2 (...truncated)