Clary Sage (Salvia Sclarea L., Lamiaceae)
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Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae) Jean-Claude Caissard1*, Thomas Olivier2, Claire Delbecque3, Sabine Palle4, Pierre-Philippe Garry3, Arthur Audran3, Nadine Valot1, Sandrine Moja1, Florence Nicole´ 1, Jean-Louis Magnard1, Sylvain Legrand1,5, Sylvie Baudino1, Fre´de´ric Jullien1 1 Laboratoire de Biotechnologies Ve´ge´tales Applique´es aux Plantes Aromatiques et Me´dicinales, Universite´ Jean Monnet, Universite´ de Lyon, Saint-Etienne, France, 2 Laboratoire Hubert Curien, Universite´ Jean Monnet, Universite´ de Lyon, Saint-Etienne, France, 3 Bontoux S.A., Saint-Auban-sur-Ouve`ze, France, 4 Centre de Microscopie Confocale Multiphotonique, Universite´ Jean Monnet, Universite´ de Lyon, Saint-Etienne, France, 5 Laboratoire Stress Abiotiques et Diffe´renciation des Ve´ge´taux Cultive´s, Universite´ Lille Nord de France, Universite´ Lille 1, Villeneuve d’Ascq, France Abstract Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces. Citation: Caissard J-C, Olivier T, Delbecque C, Palle S, Garry P-P, et al. (2012) Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae). PLoS ONE 7(10): e48253. doi:10.1371/journal.pone.0048253 Editor: Joshua L. Heazlewood, Lawrence Berkeley National Laboratory, United States of America Received July 11, 2012; Accepted September 21, 2012; Published October 25, 2012 Copyright: ß 2012 Caissard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is part of the Claryssime Programme (http://www.claryssime.fr/) supervised by CD, and including a PostDoc grant to SL. Claryssime is funded by ‘‘Poˆle de Compe´titivite´ Parfums, Aroˆmes, Senteurs, Saveurs’’, ‘‘Direction Ge´ne´rale de la Compe´titivite´, de l’Industrie et des Services’’ and ‘‘Re´gion Rhoˆne-Alpes’’. Laser microscopy is funded by Universite´ Jean Monnet, CNRS and Leica Microsystem. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: CD, AA and PPG are employees of Bontoux SA. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: caissard@univ-st-etienne.fr Introduction Sclareol can be extracted from inflorescences of Salvia sclarea L., a pluriannual herb from the Lamiaceae family, which is more Ambergris is a biogenic volatile organic compound with a sweet commonly cultivated for its essential oil. The relatively easy earthy aroma and a very high value. It is essentially used as a farming of this herb and its high sclareol yield have encouraged fixative in fragrance chemistry. It is a fatty secretion of the intestine clary sage producers to begin genetic improvement programs and of male sperm whales (Physeter macrocephalus L.) produced after a expand clary sage plantations. However, the sclareol yield is very beak injury with a squid. Details about its maturation process are variable for reasons that are unknown but related to the different unknown and it is assumed that a freshly expelled viscous block of ability of clary sage inflorescences to accumulate sclareol during ambergris floats on the sea, loses some volatiles by evaporation, field production. Differences in sclareol yield are also related to the ages during a long period, and washes ashore as a 10 g to 1 kg extraction process. According to an AFNOR directive (NFT 75– lump. 255) and industrials, essential oil obtained from 1,000 kg of dry Unfortunately, due to high demand and decrease in whale straw contains 20 to 260 g (i.e. 0.002% to 0.026% yield) of sclareol. populations, ambergris has become a scarce product. As a This should be compared to the 1.5% yield, which can be consequence, chemists and perfumers have designed synthetic obtained by manufacturers when they use solid/liquid extraction routes for the industrial production of (+/2)-norlabdane oxide, the (sclareol vs. organic solvent). The huge difference in yields is not most odorant molecule of ambergris. Most strategies of chemical explained. synthesis are based on the hemisynthesis of (+/2)-norlabdane In all members of the Lamiaceae that have been studied, essential oxide from simple natural diterpenes or carotenoid derivatives oil accumulation takes place in subcuticular pockets of secretory such as nerolidol, b-ionone, thujone, manool and sclareol [1–4] trichomes, that are scattered over most of the plant epidermis from and refs therein. This last starting substrate can be transformed via leaves to flower calyces and bracts [5–10]. In clary sage, essential synthetic organic chemistry to sclareolide and then to (+/2)- oil is obtained by steam distillation of fresh inflorescences in full norlabdane oxide by a two-step oxidation process and a reduction bloom and mostly contains mono- and sesquiterpenes [3], [10– step respectively. 13]. Even though clary sage essential oil contains some sclareol, PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48253 Localization of Sclareol in Sage the main site of its accumulation is unknown and cannot be Observation of trichomes and crystal-like structures on assumed to be within or outside the glandular trichomes. The lack calyx epidermis of information on the physiological aspects of sclareol accumula- An ESEM observation of clary sage calyx epidermis showed tion within clary sage hampers efforts to improve both knowledge that it is densely covered by capitate glands and that peltate glands on diterpene biology and agronomic practices. are rare (figure 2A). These two types of glands were described In this work, investigations on sclareol localization were previously by Werker [5], [6] in this species. We further observed undertaken at the cellular level with a combination of comple- that both gland types accumulate essential oil as in some other mentary techniques of microscopy (histochemistry; environmental essential oil-producing members of the Lamiaceae. Our ESEM and scanning electron microscopy, ESEM; two-photon fluorescence light microscopy observations have indeed revealed the existence microscopy, TPF microscopy; and second harmonic generation of a large subcuticular space above the head cells of all mature microscopy, SHG microscopy) and gas chromatography coupled glandular trichomes (figure 2B). A staining of lipid bodies with to mass spectrometry (GC-MS). Nadi [14] showed the presence of terpenes in large intracellular droplets located within the neck cell above the stalk cell of mature Results capitate glands (figure 2C). Tissue localization of sclareol in clary sage During ESEM and light microscopy observations, numerous crystals were seen on the surface of the cuticle (figure 2D–F). These Growers harvest clary sage inflorescences at full bloom stage were present as clumps of precipitate-like (figure 2D) or crystal-like (figure 1A). These contain individual flowers, their peduncles, (figures 2E, F) structures scattered on the entire calyx surface bracts and varying quantities of leaves located above the basal including trichome stems and heads (all types of trichomes) and rosette and attached to the main inflorescence stem. On an pavement cells. These structures differed in length, but seemed to industrial scale, we obtained 100 g of sclareol in 10 kg of essential have a similar shape and really looked like crystals at high oil after steam distillation of 1,000 kg of dry inflorescences (i.e. magnification (figure 2F). They were not evenly distributed over 0.01% yield) but 15 kg of sclareol by direct solid/liquid extraction the calyx surface but were encountered at random in irregular of 1,000 kg of dry straw (i.e. 1.5% yield). These results confirm that clusters. They were present on fresh calyces in natura since the bud it is not possible to extract sclareol efficiently by steam distillation stage, but easier to find in flowers at the fully opened stage. of the essential oil. To know if sclareol is lost during the process or Furthermore, less than one day after harvest, calyces were covered if it is not present in the essential oil in the plant, we wanted to with crystal-like structures. This indicates that precipitation or localize it more precisely. Indeed, sclareol being a diterpene, it is crystallisation also depends on drying. less volatile than monoterpenes and sesquiterpenes, and it could simply not be efficiently steam-distilled. Thus, in our work, we also include observations in planta before and after distillation (see last Microscopical analyses of crystal-like structures paragraph of results). Since pure sclareol spontaneously crystallises at ambient At the laboratory scale, GC-MS analyses of sclareol contents of temperature, we focussed on the crystal-like structures present individual organs revealed that flower calyces were the richest on the surface of the calyx cuticle. Under polarized light, they source of sclareol in the plant. These contained 30 times as much displayed the same light refraction pattern as pure sclareol crystals sclareol per unit of dry weight as leaves did (figure 1B).