par rapport aux populations de référence

Résumé en français 1. Introduction

Le sclaréol est un terpène produit par les organes floraux de la sauge sclarée (Salvia sclarea), en particulier par les calices. Il est utilisé pour l'hémisynthèse de l'ambroxide, un composé très apprécié en parfumerie pour son odeur ambrée et ses propriétés fixatrices augmentant la ténacité des parfums. Le sclaréol utilisé pour produire de l'ambroxide est extrait à partir de sauge sclarée cultivée. En réponse au développement de l'utilisation de l'ambroxide en parfumerie fonctionnelle (détergents, shampooings…), la demande mondiale de sclaréol est actuellement en hausse, stimulant la recherche de moyens permettant d'en accroître la production.

Le projet PISTILL, porté par l'équipe « Développement des fleurs et des carpelles » de l'Institut de Sciences des Plantes de Paris-Saclay, a pour objectif d'augmenter le rendement de la production de sclaréol via l'amélioration génétique ciblée de la sauge sclarée. Le travail présenté dans ce manuscrit visait à compléter les connaissances actuelles sur la biosynthèse du sclaréol et sa régulation chez la sauge sclarée, avec un double objectif: d'une part, orienter le projet PISTILL vers des stratégies prometteuses pour l'augmentation du rendement en sclaréol, et d'autre part, améliorer notre compréhension générale de la production des terpènes chez les plantes. Nous avons entrepris de préciser l'origine cellulaire et métabolique du sclaréol dans les calices de sauge sclarée, et d'identifier les mécanismes de régulation contrôlant son accumulation. Nous avons également commencé à étudier comment la diversité naturelle de la sauge sclarée pourrait être exploitée. Cette étude ouvre des perspectives intéressantes pour la recherche fondamentale sur la production des terpènes chez les plantes, tout en mettant en évidence des pistes prometteuses pour l'amélioration génétique ciblée des performances de la sauge sclarée.

Résultats

Localisation de la biosynthèse du sclaréol

L'épiderme des calices de sauge sclarée est caractérisé par une forte densité de trichomes glandulaires. Chez de nombreuses espèces de plantes, ces structures multicellulaires sont spécialisées dans la production de terpènes [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. La présence de sclaréol a été détectée dans les différents types de trichomes glandulaires de la sauge sclarée (peltés et capités) [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. Cependant, d'autres cellules de l'épiderme du calice pourraient également sécréter du sclaréol [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Dans le premier chapitre de ce manuscrit, nous présentons plusieurs éléments appuyant l'hypothèse selon laquelle le sclaréol est principalement sécrété par les trichomes glandulaires. La répartition spatiale du sclaréol à la surface des calices a été analysée par imagerie par spectrométrie de masse, et les résultats indiquent que le sclaréol est particulièrement abondant au niveau des trichomes glandulaires capités. De plus, des mutants de sauge sclarée dépourvus de ces trichomes (obtenus via une mutagénèse à l'EMS) produisent moins de sclaréol. Dans le futur, l'identification de la mutation (ou des mutations) à l'origine de ce phénotype améliorerait notre compréhension du développement des trichomes glandulaires et de la production du sclaréol chez la sauge sclarée.

Identification de l'origine métabolique du sclaréol par marquage isotopique

La biosynthèse des terpènes débute par la condensation d'un nombre variable d'unités à cinq carbones : l'isopentényl diphosphate (IPP) et son isomère le diméthylallyl diphosphate (DMAPP). Chez les plantes, l'IPP et le DMAPP sont produits par deux voies métaboliques différentes : la voie du mévalonate dans le cytoplasme (voie MVA) et la voie du méthylérythritol-phosphate dans les chloroplastes (voie MEP). L'activité de ces deux voies alimente deux pools d'IPP et de DMAPP physiquement séparés au sein de la cellule. Selon la localisation subcellulaire des terpènes-synthases impliquées, la production d'un terpène donné ne dépend généralement que de l'un de ces deux pools [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF]. Lorsqu'elles sont exprimées de manière transitoire dans des feuilles de tabac (Nicotiana benthamiana), les terpènes-synthases impliquées dans la biosynthèse du sclaréol sont adressées aux chloroplastes [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF], le compartiment où la voie MEP est localisée. Cependant, l'origine métabolique du sclaréol n'avait pas encore été étudiée. Les approches développées dans le deuxième chapitre de ce manuscrit mettent en évidence les contributions respectives des voies MVA et MEP à la biosynthèse de trois terpènes dans les calices de sauge sclarée : le sclaréol, l'acétate de linalyle et le β-caryophyllène. Nous avons réalisé un marquage isotopique en fournissant du glucose-1-13 C à des tiges d'inflorescence coupées. L'analyse des résultats par RMN du 13 C indique que le sclaréol et l'acétate de linalyle proviennent tous deux de la voie MEP, comme la plupart des mono-et diterpènes [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF]. En revanche, le β-caryophyllène semble être d'origine mixte, alors que la plupart des sesquiterpènes proviennent de la voie MVA [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF] ; mais ce résultat nécessite d'être confirmé.

La biosynthèse du sclaréol est-elle régulée par les jasmonates ?

La biosynthèse des terpènes est stimulée en réponse à des signaux développementaux ou environnementaux via l'activation de voies de signalisation spécifiques. Le renforcement de l'activité de ces voies de signalisation représente une autre possibilité d'accroître la production d'un terpène d'intérêt [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF]. Cette stratégie nécessite l'identification préalable des voies de signalisation stimulant la biosynthèse du terpène d'intérêt. Étant donné que la production de nombreux terpènes est activée par les jasmonates [START_REF] Colinas | Combinatorial Transcriptional Control of Plant Specialized Metabolism[END_REF][START_REF] Wasternack | Jasmonates are signals in the biosynthesis of secondary metabolites -Pathways, transcription factors and applied aspects -A brief review[END_REF], le troisième chapitre de ce manuscrit examine l'impact de ces phytohormones sur la production de sclaréol dans les calices de sauge sclarée. Du méthyljasmonate en solution a été fourni à des tiges d'inflorescence coupées ou appliqué sur plante entière par spray. Cependant, les expériences réalisées n'ont pas permis de démontrer une implication des jasmonates dans la régulation de la production de sclaréol. Deux possibilités restent plausibles: soit les jasmonates ne sont pas impliqués dans la régulation de la biosynthèse de sclaréol, soit le protocole expérimental doit encore être optimisé.

Etude de la diversité naturelle de la sauge sclarée

Les conditions environnementales et les polymorphismes génétiques sont responsables de variations de la teneur en terpènes entre différentes populations de la même espèce végétale. La pression de sélection exercée par les conditions de croissance peut conduire à la sélection de modifications génétiques modulant les niveaux de terpènes et rendant la plante plus adaptée à son environnement [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF][START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. La sauge sclarée est présente à l'état sauvage dans divers types d'environnements du bassin méditerranéen et d'Asie occidentale [START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF]. Les populations de sauge sclarée cultivées ne représentent probablement qu'un échantillon de la diversité naturelle de la sauge sclarée en termes de polymorphismes génétiques et de teneur en sclaréol. Le quatrième chapitre de ce manuscrit explore la diversité génétique et phénotypique de populations sauvages croates originaires de trois types d'environnements différents: côte, montagne et plateau. Bien que des polymorphismes génétiques soient détectables dans les gènes de biosynthèse du sclaréol, les populations croates cultivées dans le même environnement ne diffèrent pas significativement en termes de teneur en sclaréol et de date de floraison. Les conditions climatiques croates n'ont apparemment pas entraîné de différenciation génétique affectant la production de sclaréol et la floraison. Cependant, la différence de teneur en sclaréol observée entre les populations de référence « Vatican White » et « Milly » mérite une étude plus approfondie. En effet, l'identification de la cause de cette différence pourrait mener à l'identification de mécanismes de régulation de la production de sclaréol.

Revue de la littérature

Les trichomes glandulaires des plantes sont des structures épidermiques dédiées à la sécrétion de divers métabolites spécialisés. Ces métabolites contribuent à l'adaptation des plantes à leur environnement et beaucoup d'entre eux possèdent des propriétés exploitées par différents secteurs de l'industrie (parfumerie, pharmacie…). L'identification du réseau de gènes contrôlant le développement des trichomes glandulaires permettrait de mieux comprendre comment les plantes produisent ces métabolites spécialisés. Nos connaissances sur ce processus développemental sont encore limitées, mais des gènes contrôlant l'initiation et la morphogenèse du trichome glandulaire ont récemment été identifiés. La revue de la littérature présentée dans ce manuscrit a pour objectif de synthétiser ces découvertes récentes. D'après notre analyse, les facteurs de transcription appartenant aux sous-familles R2R3-MYB et HD-ZIP IV semblent jouer un rôle essentiel dans l'initiation du trichome glandulaire chez Artemisia annua et la tomate. Dans notre revue, nous nous concentrons sur les résultats obtenus chez ces deux espèces et proposons de premiers modèles de régulation génétique intégrant ces données.

Perspectives

Plusieurs stratégies visant à accroître la production de sclaréol chez la sauge sclarée peuvent être envisagées à partir des résultats décrits dans ce manuscrit. Le sclaréol étant dérivé de la voie MEP et principalement produit dans les trichomes glandulaires, l'augmentation de la densité des trichomes glandulaires ou la stimulation de la voie MEP pourraient permettre d'augmenter le contenu en sclaréol. De plus, étant donné que l'acétate de linalyle est également dérivé de la voie MEP et produit dans les trichomes glandulaires, bloquer la biosynthèse de l'acétate de linalyle pourrait augmenter la production de sclaréol en réduisant la compétition pour le substrat. La voie du MEP et la biosynthèse de l'acétate de linalyle pourraient être manipulées au moyen de modifications génétiques ciblant les enzymes impliquées dans ces voies. De même, la densité des trichomes glandulaires pourrait être augmentée via la manipulation des régulateurs de l'initiation des trichomes glandulaires. Par ailleurs, les programmes de sélection de la sauge sclarée pourraient être enrichis par une exploitation efficace de la diversité naturelle de la sauge sclarée. Les populations croates étudiées dans le quatrième chapitre de ce manuscrit représentent une ressource génétique précieuse, car nous avons montré qu'elles portent de nouveaux allèles Introduction 1. Ambergris and sclareol

The search for substitutes to ambergris

Ambergris is a waxy substance secreted by the digestive tract of male sperm whales (Physeter macrocephalus) upon mucosa injuries caused by squid beaks (Figure 1). It is then expelled by the animal as viscous lumps which float on the sea and blocks of 10 g to 1 kg can eventually be found on beaches [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. After several years of aging and exposure to sunlight, air and water, ambergris acquires a musky and sweet earthy smell [START_REF] Barrero | Synthesis of Ambrox® from (-)-sclareol and (+)-cis-abienol[END_REF]. It has been used for centuries in perfume manufacture for its distinct odor and it also possesses fixative properties which increase the tenacity of perfume compositions [START_REF] Barrero | Synthesis of Ambrox® from (-)-sclareol and (+)-cis-abienol[END_REF][START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Ambergris properties are mainly due to one of its constitutive components, ambroxide (Ambrox®), a product of ambrein autoxidation occurring during ambergris aging [START_REF] Barrero | Synthesis of (±)-Ambrox from (E)-Nerolidol and β-Ionone via Allylic Alcohol[END_REF][START_REF] Leffingwell | Biotechnology -Conquests and Challenges in Flavors & Fragrances[END_REF] (Figure 1). The decrease in sperm whale populations increased ambergris scarcity and prompted the search for synthetic alternative routes to ambroxide. Total synthesis protocols have been elaborated, as well as hemisynthesis strategies starting from various naturally occuring terpenes [START_REF] Barrero | Synthesis of Ambrox® from (-)-sclareol and (+)-cis-abienol[END_REF][START_REF] Barrero | Synthesis of (±)-Ambrox from (E)-Nerolidol and β-Ionone via Allylic Alcohol[END_REF]. Sclareol is a natural diterpene which was originally characterized in clary sage (Salvia sclarea). It is present in at least five other species of the genus Salvia [START_REF] Çulhaoglu | Bioactive constituents of Salvia chrysophylla Stapf[END_REF][START_REF] Moridi Farimani | Labdane diterpenoids from Salvia reuterana[END_REF][START_REF] Rapposelli | Relationship Between Soil and Essential Oil Profiles in Salvia desoleana Populations: Preliminary Results[END_REF][START_REF] Park | Sclareol isolated from Salvia officinalis improves facial wrinkles via an antiphotoaging mechanism[END_REF][START_REF] Craft | The Chemotaxonomy of Common Sage (Salvia officinalis) Based on the Volatile Constituents[END_REF][START_REF] Mofidi Tabatabaei | A nor-diterpene from Salvia sahendica leaves[END_REF] and has been reported in species belonging to other plant families, namely Cistus creticus (Cistaceae), Nicotiana glutinosa (Solanaceae), Cleome spinosa (Brassicaceae) [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Mastino | Interpopulation Variability in the Essential Oil Composition of Cistus creticus subsp. eriocephalus from Sardinia[END_REF] and two species of the genus Pseudognaphalium (Asteraceae) [START_REF] Mendoza | Antibacterial activity of 13-episclareol, a labdane-type diterpene isolated from Pseudognaphalium heterotrichium and P. cheiranthifolium (Asteraceae)[END_REF]. Due to its sweet and balsamic scent, sclareol is employed as flavoring in food and tobacco industry and as perfume ingredient in cosmetic industry (Ample Market Research, 2019). Moreover, its labdane-type hydrocarbon skeleton and its hydroxyl groups are particularly attractive for organic synthesis of labdane-derived compounds like ambroxide [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF] (Figure 1). Today, hemisynthesis from sclareol is the most commonly used synthetic route to ambroxide. The optimization of this route is still an object of research and a one-pot synthesis of ambrox starting from sclareol has been recently described [START_REF] Yang | One-pot synthesis of (-)-Ambrox[END_REF]. Sclareol-derived ambroxide is widely used in perfume manufacture as a substitute to ambergris [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF].

Interest in increasing sclareol global production

The sclareol market is mainly driven by the growing demand of cosmetic industry, which already accounts for around 45.8% of the global consumption of sclareol (Ample Market Research, 2019). Sclareol-derived ambroxide has been long employed in the formulation of high-end perfumes. However, it is more and more found among the ingredients of functional products like soap, detergent or beauty care products, where its fixative properties are also particularly desirable (source: Symrise). The large size of functional perfumery market compared to luxury perfumery drives the current increase in sclareol demand. Estimated to be worth 62 million US$ in 2019, the worldwide market for sclareol is expected to grow at a compound annual growth rate of roughly 5.5% over the next five years, and may reach 86 million US$ in 2024 (Ample Market Research, 2019).

Today, sclareol is mainly produced through extraction from cultivated clary sage [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Leffingwell | Biotechnology -Conquests and Challenges in Flavors & Fragrances[END_REF]. Clary sage produces relatively high amounts of sclareol compared to other plants, but the yield is submitted to important variations which remain unexplained [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. The raising global demand for sclareol prompted various initiatives aiming at increasing sclareol production and securing the supply. Research programs have been conducted during the past decade to develop biotechnological production of sclareol using bioengineered yeast or moss cell cultures [START_REF] Schalk | Method for producing sclareol[END_REF][START_REF] Simonsen | Heterologous production of patchoulol, beta-santalene, and sclareol in moss cells[END_REF]. Since growing clary sage and extracting sclareol is a relatively easy and well established process, the genetic improvement of clary sage is another promising strategy which could rapidly enhance sclareol production. In

Clary sage

Botanical description of clary sage

Salvia is the largest genus of the Lamiaceae family of plants with almost 1000 species described [START_REF] Hao | Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea[END_REF][START_REF] Foutami | Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean[END_REF]. It is widely spread around the world, as Salvia species are found in both temperate and subtropical regions [START_REF] Ozdemir | The morphological, anatomical and karyological properties of Salvia sclarea L[END_REF]. Clary sage (Salvia sclarea L.) is a biennial or short-living perennial herbaceous plant species of the genus Salvia [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF]. It is a native species of the Mediterranean basin and Western Asia [START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF] and has become an invasive species in North America, where it is known as European sage [START_REF] Foutami | Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean[END_REF]. The plant generally grows in dry and rocky places [START_REF] Ozdemir | The morphological, anatomical and karyological properties of Salvia sclarea L[END_REF][START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF] (Figure 2), preferably sunny [START_REF] Kumar | Effect of shading and plant density on growth, yield and oil composition of clary sage (Salvia sclarea L.) in north western Himalaya[END_REF]. It develops a rosette which survives the winter and then flowers in June-July [START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF]. White, pink or purple flowers are grouped verticillately in inflorescences of the heterothetic compound raceme type [START_REF] Ozdemir | The morphological, anatomical and karyological properties of Salvia sclarea L[END_REF]. One plant generally develops several floral scapes, each of them measuring between 50 and 150 cm [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF] (Figure 2).

Insect-driven cross-pollination is important for clary sage reproduction, albeit not indispensable. Indeed, in the genus Salvia, the absence of a genetic incompatibility system enables autogamy [START_REF] Claßen-Bockhoff | The staminal lever mechanism in Salvia L. (Lamiaceae): A key innovation for adaptive radiation?[END_REF]. Cross-pollination is mainly performed by carpenter bees (Xylocopa violacea), which are strongly attracted by clary sage flowers [START_REF] Kugler | Zur Bestäubung von Salvia sclarea L. durch Holzbienen (Xylocopa violacea L.). Österreichische Bot[END_REF].

Sclareol biological function in clary sage

The biological function of sclareol in clary sage remains elusive. Sclareol is mainly produced in clary sage flowers, especially calyces [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF] (Figure 3). Calyces are tubular structures formed by the fusion of sepals and are generally considered to play a role in the protection of flower reproductive organs [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF]. Some insects are known to pierce holes in the calyx to access nectar without pollination benefit for the plant, a behavior known as nectar robbing [START_REF] Irwin | Nectar Robbing: Ecological and Evolutionary Perspectives[END_REF]. Sclareol has been shown to form epicuticular crystals at the surface of the calyx [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. These crystals may protect the flower against florivory and nectar robbing, and could also reduce fungal spores adhesion [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF][START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF].

Michel Dron

The biological function of sclareol has been investigated in other plant species [START_REF] Seo | Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis[END_REF][START_REF] Fujimoto | Sclareol Induces Plant Resistance to Root-Knot Nematode Partially Through Ethylene-Dependent Enhancement of Lignin Accumulation[END_REF]. Notably, sclareol has been reported to display antimicrobial activity against different species of plant pathogenic bacteria and fungi [START_REF] Seo | Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis[END_REF][START_REF] Fujimoto | Sclareol Induces Plant Resistance to Root-Knot Nematode Partially Through Ethylene-Dependent Enhancement of Lignin Accumulation[END_REF][START_REF] Chain | FT-IR, FT-Raman, UV-Visible, and NMR spectroscopy and vibrational properties of the labdane-type diterpene 13-epi-sclareol[END_REF]. Exogenous application of sclareol on tobacco, tomato and Arabidopsis thaliana plants was shown to induce resistance to bacterial wilt disease [START_REF] Seo | Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis[END_REF]. In tomato and Arabidopsis thaliana, sclareol could also inhibit the penetration of the root-knot nematode in the roots, thereby conferring resistance to this pathogen [START_REF] Fujimoto | Sclareol Induces Plant Resistance to Root-Knot Nematode Partially Through Ethylene-Dependent Enhancement of Lignin Accumulation[END_REF]. Taken together, these results suggest that sclareol may be involved in the defense of clary sage flowers against pathogenic microorganisms. A: Clary sage calyx. B: Apex of a clary sage inflorescence [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. C: Sclareol quantification in different flower organs of clary sage [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Legends: Br, bract; FB, flower bud; Co, corolla; Ca, calyx; Le, leaf.

Clary sage commercial use

Clary sage has been traditionally used as a medicinal plant for its antispasmodic, carminative and estrogenic properties [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. Notably, essential oil extracted from clary sage has been reported to have numerous biological effects, including anti-inflammatory, antioxidant, antimicrobial and cytotoxic activity (Kuźma et al., 2009b;[START_REF] Sharopov | The essential oil of salvia sclarea L. from Tajikistan[END_REF][START_REF] Kumar | Effect of shading and plant density on growth, yield and oil composition of clary sage (Salvia sclarea L.) in north western Himalaya[END_REF]. The individual contribution of each of its components to essential oil medicinal properties is not known and synergy, antagonism or additive effects may be at play (Kuźma et al., 2009b). In particular, sclareol has been shown to display anti-bacterial and anti-proliferative activity [START_REF] Mendoza | Antibacterial activity of 13-episclareol, a labdane-type diterpene isolated from Pseudognaphalium heterotrichium and P. cheiranthifolium (Asteraceae)[END_REF][START_REF] Sashidhara | Cell growth inhibitory action of an unusual labdane diterpene, 13-epi-sclareol in breast and uterine cancers in vitro[END_REF].

A B C

Today, clary sage is exploited at industrial scale mainly for its aromatic properties. It is commercially cultivated in different European countries, particularly in France, Hungary and Bulgaria, but also in North America and China [START_REF] Lawrence | Production of Clary Sage Oil and Sclareol in North America[END_REF][START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. Clary sage essential oil is used as a flavor for the elaboration of diverse processed food products [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Sharopov | The essential oil of salvia sclarea L. from Tajikistan[END_REF]. Most importantly, essential oil and sclareol extracted from clary sage are employed in perfume manufacture. Essential oil is used directly as a perfume component for its tenacious, herbaceous, sweaty and amber odor [START_REF] Laville | Amphilectane diterpenes from Salvia sclarea: Biosynthetic considerations[END_REF][START_REF] Kumar | Effect of shading and plant density on growth, yield and oil composition of clary sage (Salvia sclarea L.) in north western Himalaya[END_REF], while sclareol is a common starting material for the hemisynthesis of the perfume component ambroxide [START_REF] Yang | One-pot synthesis of (-)-Ambrox[END_REF]. When grown for commercial use in flavor and fragrance industry, clary sage inflorescences are dried and submitted to steam distillation in order to collect essential oil. Sclareol is then obtained from remaining plant material by solid/liquid extraction with an organic solvent [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF][START_REF] Laville | Amphilectane diterpenes from Salvia sclarea: Biosynthetic considerations[END_REF][START_REF] Leffingwell | Biotechnology -Conquests and Challenges in Flavors & Fragrances[END_REF] (Figure 4). In general, essential oil yield is between 0.1% and 0.3% [START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF] and sclareol yield is between 0.5% and 1.5% [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]ITEIPMAI, 7èmes Rencontres du Végétal, 2013).

First advances in clary sage genetics and biotechnology

The identification of genes involved in specific biological processes is greatly facilitated when genomic data are available in the species of interest or in related species. The genome of clary sage is not sequenced yet, but genome sequences of Salvia miltiorrhiza and Salvia splendens have been recently published [START_REF] Xu | Analysis of the Genome Sequence of the Medicinal Plant Salvia miltiorrhiza[END_REF][START_REF] Dong | High-quality assembly of the reference genome for scarlet sage, Salvia splendens, an economically important ornamental plant[END_REF]. The study of clary sage chromosomic material shows that it is a diploid species with a karyotype of 2n=22 [START_REF] Ozdemir | The morphological, anatomical and karyological properties of Salvia sclarea L[END_REF]. Genome size was estimated around 600 Mb (Drevensek et al., unpublished data). Two sets of clary sage transcriptomic data are currently available. The first clary sage transcriptome was obtained from calyces and allowed the identification of putative candidate genes for different steps of terpenoid biosynthesis [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF].

Another transcriptome has been generated from leaves and roots treated with the phytohormone methyljasmonate [START_REF] Hao | Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea[END_REF].

Biotechnological tools enabling the modification of gene expression level are highly valuable for the functional characterization of genes of interest. In many plants, the development of roots named "hairy roots" can be induced using the bacterium Agrobacterium rhizogenes, which is able to transfer human-engineered genetic material into plant genome. Hairy roots can then be separated from aerial parts and cultivated in a bioreactor [START_REF] Ron | Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model[END_REF].

Several studies show that transgenic hairy roots can be generated from clary sage roots (Kuźma et al., 2009a;[START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF][START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF][START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF] (Figure 5). Stable lines of clary sage calli and cell cultures can also be obtained from seeds or stem explants, and were shown to produce sclareol [START_REF] Banthorpe | Accumulation of the anti-fungal diterpene sclareol by cell cultures of Salvia sclarea and Nicotiana glutinosa[END_REF]. Moreover, protocols enabling in vitro regeneration of whole clary sage plants from fragments of somatic tissue are available, starting from immature zygotic embryo cotyledons [START_REF] Liu | Regeneration of Salvia sclarea via organogenesis[END_REF] or shoot tips (Kuźma et al., 2009b). Essential oil composition of clary sage plants regenerated from shoot tips was comparable to plants reproduced from seeds, but the yield was twice lower (Kuźma et al., 2009b). Therefore, the metabolic activity of regenerated clary sage plants may be different compared to plants reproduced from seeds, but further investigation is needed to confirm this hypothesis.

Figure 5: Clary sage hairy root culture [START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF].

Clary sage breeding in France

The Through this method, a clary sage variety with high sclareol yield was obtained: "Scalia".

Unfortunately, this variety displayed a late-flowering phenotype. This phenotype can be detrimental to sclareol yield because late-flowering plants are potentially more impacted by summer drought [START_REF] Otto | Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping[END_REF]. Further recurrent selection cycles led to the development of the "Toscalia" variety, which is characterized by a high sclareol yield without any late-flowering phenotype. A third variety, "Claryssima", is currently being developed using the same approach (source: ITEIPMAI).

Terpene biosynthesis and function in plants

Specialized metabolism and terpenoids

Throughout their lifetime, plants produce a wide variety of chemical compounds which perform different biological functions. Plant compounds have been historically divided into two classes according to their biological role. Metabolites which are essential to sustain normal growth are called primary metabolites. For example, this category comprises structural compounds like cell wall carbohydrates or membrane lipids, and compounds involved in energy metabolism or photosynthesis like organic acids or coenzymes. In addition to primary metabolites, plants produce various other compounds which are not indispensable for plant growth and development, but are involved in plant adaptation to biotic and abiotic environmental factors [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF][START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF][START_REF] Chezem | Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs[END_REF]. These compounds are therefore critical for plant survival in a fluctuating environment and are known as secondary or specialized metabolites. Unlike primary metabolites, which are generally present in all plant lineages, specialized metabolites are highly diverse between plant species. In a single plant species, the amount of specialized metabolites produced can also vary drastically according to environmental conditions.

Plant metabolites can also be classified according to early steps of their biosynthesis pathway. Terpenoids represent the largest class of plant metabolites with at least 50,000 compounds described [START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF]. Some terpenoids play critical roles in plant growth and development. They are therefore considered as primary metabolites, like for example phytohormones of the gibberellin family and photosynthetic pigments of the carotenoid family. However, most plant terpenoids are specialized metabolites involved in diverse biological functions related to plant interaction with its environment [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF].

The biosynthesis of all terpenoids starts with the condensation of the same five-carbon building blocks: isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. The number of five-carbon units condensed defines subclasses among terpenoids: for instance, compounds derived from the condensation of 2, 3 or 4 fivecarbon units belong to monoterpene (10C), sesquiterpene (15C) and diterpene (20C) subclasses, respectively. Sclareol belongs to the diterpene subclass.

The diverse biological functions of terpenes

Numerous terpenes are indispensable for plant cell function and correct plant growth and development. Primary metabolites belonging to the terpenoid family include carotenoids, which are essential for light harvesting during photosynthesis, and phytosterols, which are structural elements of plant plasma membranes (Figure 6). They also include important signaling metabolites, as several classes of important plant hormones are terpenoids:

gibberellins, abscisic acid, strigolactones and brassinosteroids. Moreover, some protein posttranslational modifications are related to terpenoid metabolism: protein prenylation consists in the transfer of a prenyl moiety to a cysteine, and the terpenoid dolichol is involved in protein glycosylation [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF].

Some terpenoids are involved in plant response to abiotic stress, although this is not their most common function. For example, in poplar, isoprene emissions provide protection against thermal stress and ozone-induced oxidative stress [START_REF] Behnke | Transgenic, non-isoprene emitting poplars don't like it hot[END_REF][START_REF] Behnke | RNAi-mediated suppression of isoprene biosynthesis in hybrid poplar impacts ozone tolerance[END_REF]. By contrast, a large number of plant terpenoids are key elements of plant constitutive and induced chemical defense systems against pathogenic microorganisms (fungi, bacteria) and insect herbivores (Figure 6). They can act as direct defense by displaying toxicity to plant pathogens or by acting as herbivore repellents [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. For example, numerous rice diterpenoid phytoalexins such as oryzalexins show antibiotic activity against the fungal pathogen Magnaporthe oryzae [START_REF] Schmelz | Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins[END_REF], and 17-hydroxygeranyllinalool diterpenoid glycosides produced by Nicotiana attenuata work as antifeedants against tobacco hornworm [START_REF] Jassbi | Silencing Geranylgeranyl Diphosphate Synthase in Nicotiana attenuata Dramatically Impairs Resistance to Tobacco Hornworm[END_REF]. Terpenoids can also act as indirect defense via the attraction of natural enemies of plant pathogens, i.e. predators or parasites [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Such indirect action is illustrated by the response of maize roots to feeding by Diabrotica virgifera larvae. Damaged maize roots produce β-caryophyllene, which in turn strongly attracts an entomopathogenic nematode of Diabrotica virgifera larvae [START_REF] Rasmann | Recruitment of entomopathogenic nematodes by insect-damaged maize roots[END_REF].

In addition to defense against pathogenic microorganisms and herbivores, terpenoids play other roles in plant-insect interactions. Notably, volatile scent terpenoids emitted by flowers are critical for pollinator attraction. The monoterpene 1,8-cineole is frequently present in the scent of orchids and strongly attract euglossine bees, a group of bees specifically pollinating orchids [START_REF] Dodson | Biologically active compounds in orchid fragrances[END_REF]. Plant-plant interactions can also be mediated by terpenes. Some terpenoids allow parasitic plants to detect their host, induce defense responses in neighboring plants, or display toxicity to plant competitors [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. For example, momilactone diterpenes produced by rice roots have a negative effect on the growth of barnyard grass, a plant competitor of rice [START_REF] Xu | Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa)[END_REF].

Camille Chalvin

Industrial exploitation of terpenes

Because of their various ecological functions, terpenes display distinct properties and biological activities which are valuable for industrial applications. In particular, a number of plant-derived terpenoids have been used for a long time as scents or flavors, and as active pharmacological ingredients [START_REF] Tetali | Terpenes and isoprenoids: a wealth of compounds for global use[END_REF]. Essential oils, which are mainly composed of terpenoids, are important natural ingredients of flavor and perfume industry, and their components are also individually employed for specific uses. For example, the sesquiterpene nootkatone is the main molecule responsible for the distinct aroma of grapefruit, and the monoterpenes limonene, linalool and 1,8-cineole are common ingredients of lemon or lime flavor [START_REF] Hausch | Flavor Chemistry of Lemon-Lime Carbonated Beverages[END_REF][START_REF] Tetali | Terpenes and isoprenoids: a wealth of compounds for global use[END_REF]. Pharmacologically active molecules are found in all subclasses of terpenes, including monoterpenes and diterpenes (Figure 7). The most famous plant diterpene possessing medicinal properties may be the cancer drug paclitaxel (Taxol®), initially isolated from yew [START_REF] Mafu | Plant diterpenoid metabolism for manufacturing the biopharmaceuticals of tomorrow: prospects and challenges[END_REF]. The diterpene ingenol mebutate from Euphorbia peplus also displays antitumor effects and has been recently approved in clinical trials for skin cancer treatment [START_REF] Mafu | Plant diterpenoid metabolism for manufacturing the biopharmaceuticals of tomorrow: prospects and challenges[END_REF]. In addition to cancer therapy, terpenes are used in many other medicinal applications; for example, the diterpene forskolin is a cardioprotective compound found in the roots of Coleus forskohlii [START_REF] Mafu | Plant diterpenoid metabolism for manufacturing the biopharmaceuticals of tomorrow: prospects and challenges[END_REF], and the sesquiterpene artemisinin produced by Artemisia annua is a reference for the treatment of malaria [START_REF] Tetali | Terpenes and isoprenoids: a wealth of compounds for global use[END_REF].

In addition to scents and medicines, other plant-derived terpenoids are used in food, cosmetic and chemical industries [START_REF] Tetali | Terpenes and isoprenoids: a wealth of compounds for global use[END_REF]. Some terpenoids have important nutritional value, as illustrated by the role of carotenoids which represent an essential source of vitamin A for humans [START_REF] Rodriguez-Concepcion | A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health[END_REF]. Carotenoids are also employed as antioxidants in cosmetic industry, notably in the elaboration of skin care products [START_REF] Sathasivam | A Review of the Biological Activities of Microalgal Carotenoids and Their Potential Use in Healthcare and Cosmetic Industries[END_REF]. Terpenoid properties are useful in other sectors of chemical industry, like for instance polymer chemistry in the case of polyisoprene constituents of natural rubber produced by Hevea brasiliensis. Moreover, due to their low hygroscopy, high energy density, and good fluidity at low temperatures, biotechnologically-produced sesquiterpenes and diterpenes have been suggested as potential renewable alternatives for transportation fuels [START_REF] Tippmann | From flavors and pharmaceuticals to advanced biofuels: production of isoprenoids in Saccharomyces cerevisiae[END_REF].

Terpene biosynthesis

The first step of terpenoid biosynthesis corresponds to the production of the five-carbon building blocks IPP and DMAPP. In plants, IPP and DMAPP are produced by two distinct metabolic pathways: the mevalonate pathway (MVA pathway) and the methylerythritolphosphate pathway (MEP pathway). Then, linear intermediate compounds called prenyl intermediates are formed by condensation of IPP and DMAPP units [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. Prenyl intermediates are finally converted into functional terpenes through a number of molecular modifications, for example cyclization and addition of functional groups like hydroxyl groups. and archae only have the MVA pathway [START_REF] Hemmerlin | A raison d'être for two distinct pathways in the early steps of plant isoprenoid biosynthesis?[END_REF][START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF]. In plants, MVA and MEP pathways are compartmentalized: the MVA pathway is localized in the cytosol, whereas the MEP pathway is localized in plasts. It is now admitted that plants inherited the MEP pathway from a photosynthetic prokaryote through endosymbiosis [START_REF] Vranová | Network Analysis of the MVA and MEP Pathways for Isoprenoid Synthesis[END_REF].

In plants, the MVA pathway involves 6 enzymatic steps [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF] (Figure 8). MEcPP is reduced into 4-hydroxy-3-methylbut-2-enyl-diphosphate (HMBPP) by the HMBPPsynthase (HDS) and HMBPP is finally converted into a mixture of IPP and DMAPP by the HMBPP-reductase (HDR) with a ratio of 5 to 6:1.

Condensation of five-carbon building blocks by prenyltransferases

The second step of terpenoid biosynthesis corresponds to the condensation of IPP and DMAPP units to form prenyl diphosphate intermediates, which are the percursors of all terpenes. These condensation reactions are catalyzed by enzymes called prenyl diphosphate synthases or prenyltransferases. First, an IPP molecule is condensed with a DMAPP molecule to produce a ten-carbon intermediate, and further addition of IPP units leads to the formation of prenyl diphosphate intermediates of various lengths [START_REF] Kharel | Molecular analysis of cis-prenyl chain elongating enzymes[END_REF]. Prenyltransferase activity requires an appropriate ratio of IPP and its more reactive isomer DMAPP. Conversion of IPP into DMAPP is performed by the IPP isomerase (IDI). IPP isomerization is more important in the cytosol than in plastids because the MVA pathway produces only IPP, whereas the MEP pathway produces both five-carbon units [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF].

Prenyltransferases are divided in two classes according to the stereochemistry of double bonds in the product of the condensation reaction: cis-prenyltransferases and transprenyltransferases produce all-cis and all-trans prenyl diphosphate intermediates, respectively [START_REF] Kharel | Molecular analysis of cis-prenyl chain elongating enzymes[END_REF]. Condensation of 2, 3 or 4 five-carbon units in trans orientation leads to the formation of geranyl pyrophosphate (GPP, 10 carbons), (E,E)farnesyl pyrophosphate (FPP, 15 carbons) and geranylgeranyl pyrophosphate (GGPP, 20 carbons), respectively (Figure 9). These trans-prenyl diphosphate intermediates are the main precursors of monoterpene, sesquiterpene and diterpene biosynthesis, respectively. They are more abundant than their all-cis isomers and constitute central metabolic branching points for the biosynthesis of terpenoids involved in primary and specialized metabolism [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. For these reasons, most research studies conducted so far have been focused on trans-prenyltransferases.

GGPP, FPP and GPP are produced by geranylgeranyl pyrophosphate synthases (GGPPsynthases), farnesyl pyrophosphate synthases (FPP-synthases) and geranyl pyrophosphate synthases (GPP-synthases), respectively (Figure 9). Most GGPP and GPP molecules are produced in plasts, whereas FPP synthesis essentially occurs in the cytosol [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF].

GGPP-synthase and FPP-synthase genes are generally present in multiple copies in plant genomes, GGPP-synthase gene families being larger than FPP-synthase gene families [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. In angiosperms, GGPP-synthases and FPP-synthases usually work as homodimers to produce respectively GGPP and FPP as major or unique product [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. By contrast, several GPP-synthases have been shown to work as heterodimers including a large catalytic subunit and a small regulatory subunit, like those described in Antirrhinum majus, Clarkia breweri and Humulus lupulus [START_REF] Tholl | Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases[END_REF][START_REF] Wang | Heterodimeric geranyl(geranyl)diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis[END_REF]. The large subunit is closely related to GGPP-synthases and displays GGPP-synthase activity in vitro, whereas the small subunit shows only limited sequence similarity with GGPP-synthases (around 20%) and does not show any catalytic activity. It is admitted that in vivo, the small subunit binds to the large subunit and modifies its activity to make it produce GPP instead of GGPP [START_REF] Burke | Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate[END_REF][START_REF] Orlova | The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta[END_REF]. Homodimeric and heterotetrameric GPP-synthases have also been characterized [START_REF] Burke | Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer[END_REF][START_REF] Burke | Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate[END_REF][START_REF] Hsiao | A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif[END_REF].

Notably, the first GPP-synthase discovered in plants was identified in the Lamiaceae Mentha piperita and works as a heterotetramer with two large catalytic subunits and two small regulatory subunits similar to subunits forming heterodimeric GPP-synthases [START_REF] Burke | Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer[END_REF][START_REF] Burke | Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate[END_REF]. Some plant species have both heteromeric and homomeric GPP-synthases, for example Catharanthus roseus [START_REF] Rai | Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis[END_REF].

Terpene synthases

In the last step of terpenoid biosynthesis, prenyl diphosphate intermediates undergo various molecular modifications which eventually lead to functional terpenes. Possible modifications include cyclization or cleavage of the hydrocarbon skeleton, and functionalization reactions such as hydroxylation, peroxidation, methylation, acylation or glycosylation [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. This step involves enzymes of the terpene synthase (TPS) superfamily and often requires other types of enzymes, like for example cytochrome P450 monoxygenases [START_REF] Zerbe | Plant diterpene synthases: Exploring modularity and metabolic diversity for bioengineering[END_REF][START_REF] Bathe | Cytochrome P450 enzymes: A driving force of plant diterpene diversity[END_REF].

Plant genomes generally contain numerous putatively functional TPS genes. Only 2 were found in the genome of the moss Physcomitrella patens, but genomes of vascular plants generally have more than 20 TPS genes, up to 113 in Eucalyptus grandis [START_REF] Chen | The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[END_REF].

The large number of TPS genes found in plant genomes, the promiscuous activity of these enzymes and their ability to acquire new catalytic properties by minor structural changes are believed to be responsible for the huge diversity of plant terpenoids [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. According to phylogenetic studies, TPS genes are divided into 8 subfamilies: TPSa to TPSh. TPS enzymes can also be classified in 2 classes according to the mechanism of the enzymatic reaction they catalyze [START_REF] Chen | The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[END_REF]. This enzymatic reaction always involves the formation of a carbocation within the prenyl disphosphate substrate, which opens up the possibility of intramolecular rearrangements or attacks by nucleophilic compounds such as water. Class I TPS enzymes ionize their substrate by removing the diphosphate group, whereas class II TPS do it through a protonation reaction [START_REF] Chen | The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[END_REF]. In vitro, TPS enzymes are sometimes able to use prenyl diphosphate substrates of different lengths, but in vivo their activity is determined by the substrate pool present in the compartment where they are localized.

Given the respective proportions of GPP, FPP and GGPP molecules in plasts and in the cytosol, plastidial TPS enzymes mainly produce monoterpenes and diterpenes, whereas cytosolic TPS enzymes mainly produce sesquiterpenes [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. However, exceptions to this rule have been reported; for instance, plastidial sesquiterpene synthases have been characterized in tomato [START_REF] Sallaud | A novel pathway for sesquiterpene biosynthesis from Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites[END_REF].

Focus on diterpene biosynthesis

Diterpene biosynthesis usually occurs in chloroplasts, where most GGPP precursors are produced [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. The various diterpene biosynthesis pathways are believed to have evolved from gibberellin biosynthesis [START_REF] Peters | Two rings in them all: The labdane-related diterpenoids[END_REF]. A majority of diterpenes, including sclareol, are labdane-type diterpenes. Formation of diterpenes with a hydrocarbon skeleton of the labdane type usually involves the cyclization of GGPP through the consecutive action of a class II diterpene synthase (Figure 10) and a class I diterpene synthase [START_REF] Peters | Two rings in them all: The labdane-related diterpenoids[END_REF].

The obtained bicyclic skeleton can be further modified, for example by the addition of hydroxyl groups mediated by diterpene synthases or cytochrome P450 monoxygenases [START_REF] Zerbe | Plant diterpene synthases: Exploring modularity and metabolic diversity for bioengineering[END_REF][START_REF] Bathe | Cytochrome P450 enzymes: A driving force of plant diterpene diversity[END_REF]. Another cyclization reaction leads to tricyclic abietane diterpenes [START_REF] Peters | Two rings in them all: The labdane-related diterpenoids[END_REF] (Figure 10).

Terpene biosynthesis regulation

Factors affecting terpene content

Specialized metabolite content varies at all scales, between species and populations, but also between individuals and between the different parts of the same plant [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF].

In a given species, populations displaying distinct metabolite profiles are referred to as chemotypes [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Physiological variations, environmental conditions and genetic polymorphisms, along with the interactions of these three factors, are known to determine specialized metabolite content in plants [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF][START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF].

In a single plant, terpenoid production depends on the organ considered, on the developmental stage of this organ, and also on circadian and seasonal cycles [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Important qualitative and quantitative variations are observed between the different organs of the plant. For example, the flowers of Lavandula pinnata produce more monoterpenes and less sesquiterpenes than leaves and stems. These differences are related to the important role played by volatile monoterpenoids in guiding pollinators to the flowers [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF]. In Achillea millefolium, the amount of volatile terpenoids produced by flowers differs according to their developmental stage. The amount of sesquiterpene chamazulene present in the essential oil decreases with flower maturation, while the proportion of the monoterpenes 1,8-cineole and camphor increases [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF].

Diurnal variations of light level impact MEP and MVA pathways in the opposite way: the MEP pathway is stimulated by exposure to light, whereas the MVA pathway is upregulated in the dark [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. Seasonal variations in terpenoid content can also be observed in some species. In sea fennel (Crithmum maritimum), proportions of sabinene and γ-terpinene in the essential oil vary in opposite ways: sabinene is more abundant during the flowering season (between July and October), whereas γ-terpinene predominates during the rest of the year and peaks in April [START_REF] Barroso | Seasonal Variation in the Composition of the Essential Oil of Crithmum maritimum L. Flavour Fragr[END_REF] (Figure 11). [START_REF] Barroso | Seasonal Variation in the Composition of the Essential Oil of Crithmum maritimum L. Flavour Fragr[END_REF].

Environmental conditions impacting specialized metabolite content include climatic factors (temperature, hygrometry), biotic factors (herbivores, microorganisms) and edaphic factors.

Terpenoid production is often tightly related to weather conditions. Monoterpene emission by slash pine (Pinus elliotii) has been shown to increase with temperature [START_REF] Tingey | Influence of light and temperature on monoterpene emission rates from slash pine[END_REF]. Drought has contrasted effects on terpenoid production depending on the species considered: for example, hydric stress enhances terpenoid emissions in Ocimum basilicum, but decreases artemisinin production in Artemisia annua [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF]. Since many terpenoids play a role in plant defense against pathogens, biotic factors significantly influence terpene levels in various plants. For instance, the sesquiterpene capsidiol accumulates in pepper stems upon infection with the oomycete Phytophthora capsici [START_REF] Egea | Capsidiol accumulation in capsicum annuum stems during the hypersensitive reaction to Phytophthora capsici[END_REF]. Finally, soil physical and chemical structure is also considered as an important factor impacting specialized metabolite content in plants. Notably, the proportion of silt and sand as well as potassium oxide content influence the composition of Salvia desoleana essential oil [START_REF] Rapposelli | Relationship Between Soil and Essential Oil Profiles in Salvia desoleana Populations: Preliminary Results[END_REF].

Genetic polymorphisms are responsible for variations in terpenoid content between plants belonging to the same species or to related species. In species of the genus Mentha, the dominant allele C has an important impact on essential oil composition: CC or Cc genotypes are associated with high carvone content, whereas the cc genotype promotes the production of pulegone and menthol [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF]. Genetic variations are shaped by the selective pressure exerted by environmental factors mentioned above. The action of natural selection is illustrated by the study of monoterpene production by Pinus ponderosa trees in the context of the development of bark beetles, which are the most destructive agent of conifer forest worldwide. Directional selection has been shown to take place for trees producing high amounts of limonene, a monoterpene toxic to the beetle [START_REF] Trapp | Defensive resin biosynthesis in conifers[END_REF].

Molecular mechanisms of terpene biosynthesis regulation

Phytohormone signaling pathways and master transcriptional regulators of plant growth and development control terpenoid biosynthesis in response to environmental and developmental signals. In Arabidopsis thaliana, the upregulation of the MEP pathway and carotenoid biosynthesis in light conditions was shown to be performed through the action of phytochrome interacting factors [START_REF] Toledo-Ortiz | Direct regulation of phytoene synthase gene expression and carotenoid biosynthesis by phytochromeinteracting factors[END_REF] and the biosynthesis of floral sesquiterpenes is induced by master regulators of flower maturation in response to jasmonates, auxin and gibberellins [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. Jasmonates stimulate terpenoid biosynthesis in many other plants [START_REF] Goossens | Jasmonates: signal transduction components and their roles in environmental stress responses[END_REF]; for example, they induce artemisinin biosynthesis in Artemisia annua (Chen et al., 2017b).

Genes involved in the same terpene biosynthesis pathway are often organized as gene clusters in plant genomes, an organization which facilitates their transcriptional coregulation [START_REF] Töpfer | The PhytoClust tool for metabolic gene clusters discovery in plant genomes[END_REF]. Positive transcriptional regulators of terpene biosynthesis genes have been identified in several plant species and generally belong to WRKY, AP2/ERF, bHLH or basic leucine zipper (bZIP) transcription factor families [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF]. For example, AaWRKY1, AaERF1, AaERF2, AaORA1 and AabZIP1 transcription factors positively regulate the biosynthesis of the sesquiterpene artemisinin in Artemisia annua [START_REF] Yu | The Jasmonate-Responsive AP2/ERF Transcription Factors AaERF1 and AaERF2 Positively Regulate Artemisinin Biosynthesis in Artemisia annua L[END_REF][START_REF] Lu | AaORA , a trichome-specific AP2/ERF transcription factor of Artemisia annua , is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea[END_REF][START_REF] Han | Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants[END_REF]Zhang et al., 2015a), TcWRKY1 stimulates the production of the diterpene paclitaxel in Taxus chinensis [START_REF] Li | Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis[END_REF] and the bZIP transcription factor OsTGAP1 positively regulates the biosynthesis of diterpene phytoalexins in rice [START_REF] Okada | OsTGAP1, a bZIP transcription factor, coordinately regulates the inductive production of diterpenoid phytoalexins in rice[END_REF].

In plants, MVA and MEP pathways are compartmentalized. Therefore, they can be differentially regulated, and controlled regulatory crosstalks between terpenoid biosynthesis pathways have been reported [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. Notably, terpenoid biosynthesis has been shown to be regulated at the post-translational level by protein prenylation [START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF]. For instance, the production of the MVA-derived sesquiterpene capsidiol in tobacco relies on MEP-dependent protein geranylgeranylation [START_REF] Huchelmann | S-Carvone Suppresses Cellulase-Induced Capsidiol Production in Nicotiana tabacum by Interfering with Protein Isoprenylation[END_REF] and in Catharanthus roseus, MVA-derived farnesylated proteins regulate the expression of genes involved in MEP-dependent monoterpenoid biosynthesis [START_REF] Courdavault | CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathwayin Catharanthus roseus cells[END_REF].

Glandular trichomes

Capitate and peltate glandular trichomes of the Lamiaceae

In Angiosperms, terpenoid production is often localized in specialized secretory structures.

For example, in Hevea brasiliensis, polyisoprene components of natural rubber are produced in specialized ducts known as laticifers [START_REF] Lange | The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity[END_REF]. Lamiaceae are generally characterized by the presence of epidermal secretory structures called glandular trichomes, which produce various compounds mainly involved in pollinator attraction or defense against herbivores [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF]. Terpenoids secreted by glandular trichomes are major components of the essential oil which can be extracted by hydrodistillation. Glandular trichomes of the Lamiaceae are multicellular and composed of 3 parts: a base, a stalk and a gland (or glandular head) (Figure 12). The gland is responsible for the secretion of specialized metabolites, the stalk is the structure bearing the gland, and the base connects the stalk to surrounding epidermal cells [START_REF] Lange | The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity[END_REF]. Both vegetative and reproductive organs of the Lamiaceae harbor glandular trichomes already at early development stages [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF]. [START_REF] Lange | The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity[END_REF] Glandular trichomes of the Lamiaceae show high morphological diversity [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF][START_REF] Gul | Foliar epidermal anatomy of Lamiaceae with special emphasis on their trichomes diversity using scanning electron microscopy[END_REF]. Lamiaceae generally display at least two types of glandular trichomes: capitate glandular trichomes and peltate glandular trichomes [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF]. This classification is based on their distinct morphology: capitate trichomes have a long stalk topped by a small spherical secretory head, while peltate trichomes are sessile trichomes with a very short stalk and a large flattened glandular head. In addition to these morphological differences, capitate and peltate trichomes also differ in the way they secrete chemicals. Both glandular trichome types accumulate secreted chemicals under an elevated cuticle. In peltate glandular trichomes, the cuticle is thick and ruptures only upon contact with an insect or as a consequence of another mechanical stimulation. By contrast, compounds secreted by capitate glandular trichomes are regularly released to the outside; they are thought to be either exuded through the cuticle, or released after spontaneous breakage of the cuticle [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF][START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF][START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF].

Clary sage glandular trichomes

Like in most plants of the Lamiaceae family, both capitate and peltate glandular trichomes are found in clary sage, along with non-glandular trichomes [START_REF] Werker | Glandular hairs and their secretions in the vegetative and reproductive organs of Salvia sclarea and S. dominica[END_REF][START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF] (Figure 13). Several subtypes of capitate trichomes have been described according to their size: small capitate trichomes and large capitate trichomes are the most frequently observed, along with few intermediate trichomes. Small capitate heads measure between 10 and 20 µm, while large capitate heads measure between 50 and 100 µm (Figure 13). By contrast, the morphology of clary sage peltate trichomes is relatively uniform. Peltate glands have a diameter of around 80-90 µm (Figure 13). Capitate and peltate glandular trichomes are present on most aerial organs of clary sage, including leaves (mostly on the abaxial face), floral stems and flower organs [START_REF] Ozdemir | The morphological, anatomical and karyological properties of Salvia sclarea L[END_REF]. Their density is particularly high on flower organs, namely the bract, the corolla and especially the calyx [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. Proportion of each glandular trichome type varies between the different organs. On calyces, large capitate glandular trichomes are more abundant than peltate trichomes, with a ratio of approximately 10:1 [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. According to scanning electron microscopy observations of clary sage calyces at different developmental stages, glandular trichome density was reported to be maximum at bud stage and to gradually decrease during calyx development, probably because of pavement cell expansion [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. Glandular trichomes are not fully expanded at bud stage and glands progressively increase in size during calyx development [START_REF] Ozdemir | The morphological, anatomical and karyological properties of Salvia sclarea L[END_REF][START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF].

Single-gland analyses of glandular trichome content show that capitate and peltate glandular trichomes accumulate the same chemicals, but in different proportions: capitate glands tend to produce more monoterpenes and less sesquiterpenes than peltate glands [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. Quantitative variations of terpene production between capitate and peltate glandular trichomes have also been evidenced in peppermint, another plant of the Lamiaceae family [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF]. Moreover, single-gland analyses performed on clary sage highlight important variations between glands of the same trichome type [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. The proportion of compounds accumulated in glandular trichomes of the same type is different between leaves and floral organs. Notably, floral glandular trichomes accumulate more monoterpenes involved in pollinator attraction like linalool and linalyl acetate. In all organs of clary sage, both capitate and peltate glandular trichomes produce sclareol.

Depending on the organ, sclareol represents in average 60.5-76.6% and 21.2-48.6% of capitate and peltate glandular head content, respectively [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF].

Genetic control of glandular trichome development

See review in the Annex (page 131).

Terpene production by clary sage

Essential oil production in aerial parts

So far, terpene production by clary sage and its regulation have mainly been investigated through the study of essential oil yield and composition. Although clary sage oil chemical composition shows important variations between studies, linalool and its acetylated derivative linalyl acetate are generally reported as being its main constituents [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF][START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Sharopov | The essential oil of salvia sclarea L. from Tajikistan[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF]. The composition of clary sage essential oils produced in different countries of the Mediterranean basin have been compared and chemotypes have been identified according to the proportion of each compound in the essential oil [START_REF] Sharopov | The essential oil of salvia sclarea L. from Tajikistan[END_REF]. Variations observed between clary sage chemotypes can probably be attributed to natural genetic diversity and to the influence of growth conditions. The impact of environmental conditions is illustrated by a study demonstrating that clary sage essential oil production is affected by shade level [START_REF] Kumar | Effect of shading and plant density on growth, yield and oil composition of clary sage (Salvia sclarea L.) in north western Himalaya[END_REF]. Shade level increases when the distance between plants decreases; in the study, it was artificially fixed using green shading nets. Essential oil yield was lower in shade conditions compared to open-sun conditions, and oil composition varied upon shade level: linalool content was highest in open-sun conditions and decreased with shade, whereas the opposite was observed for the sesquiterpene germacrene D [START_REF] Kumar | Effect of shading and plant density on growth, yield and oil composition of clary sage (Salvia sclarea L.) in north western Himalaya[END_REF]. [START_REF] Lawrence | Production of Clary Sage Oil and Sclareol in North America[END_REF].

In a single clary sage plant, essential oil production is submitted to physiological variations.

Notably, it was shown to vary during the day, being the lowest early in the morning, and reaching its maximum during the afternoon [START_REF] Lawrence | Production of Clary Sage Oil and Sclareol in North America[END_REF] (Figure 14). Moreover, essential oil yield depends on inflorescence maturity [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. Being initially low before flower opening, essential oil content of inflorescences gradually increases during inflorescence maturation and peaks at full bloom stage (all flowers open) before sharply declining at full maturity (withered flowers and brown seeds) [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. The proportion of linalyl acetate and other monoterpenes in the essential oil also reaches its maximum at full bloom stage and decreases at full maturity [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. By contrast, the proportion of sclareol and sesquiterpenes in the essential oil does not drop at full maturity [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. The sharper decline of monoterpene content may be due to higher evaporation of monoterpene compounds; indeed, these compounds are generally more volatile than sesquiterpenes and diterpenes [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF]. However, it is important to note that sclareol is poorly extracted by hydrodistillation compared to solidliquid extraction with an organic solvent [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Its concentration in essential oil may not accurately reflect the overall sclareol content of the inflorescence.

Sclareol biosynthesis and secretion

Like all other diterpenes, sclareol is produced from GGPP and this conversion is performed through the action of diterpene synthases. Several enzymes involved in sclareol biosynthesis from GGPP have been cloned and characterized by three different research groups [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF][START_REF] Günnewich | A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate[END_REF]. A class II diterpene synthase was shown to catalyze in vitro the protonation-initiated bicyclization of GGPP along with the capture of a hydroxyl ion at position 8, resulting in a labdane-related intermediate: labda-13en-8-ol diphosphate (LPP) [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF][START_REF] Günnewich | A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate[END_REF] (Figure 15). Besides, a class I diterpene synthase was shown to produce sclareol from LPP in vitro after cleavage of the diphosphate group and an additional hydroxylation at position 13 [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF] (Figure 15). These two enzymes were called labda-13-en-8-ol diphosphate-synthase (LPPS) and sclareol-synthase (SS), respectively [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. When combined in vitro or co-expressed in yeast, they were responsible for the production of sclareol in two steps from GGPP [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF]. Both enzymes possess an N-terminal signal sequence associated with chloroplast localization and were localized in chloroplasts according to transient expression experiments of GFP-fusion constructs in tobacco [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. LPPS and SS are hydroxylating terpene synthases, a function which is quite rare among terpene synthases in angiosperms [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. Hydroxyl groups on the carbon skeleton of terpenes are usually obtained after oxidation by cytochrome P450 monoxygenases [START_REF] Bathe | Cytochrome P450 enzymes: A driving force of plant diterpene diversity[END_REF]. Sclareol is known to accumulate on the epidermis of clary sage calyces [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF], but the identity of cells secreting sclareol is still discussed. Sclareol is generally thought to be produced by glandular trichomes like other essential oil components, but a study focused on sclareol localization at the surface of the calyx suggests that it may be secreted by other epidermal cells [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Before accumulating at the surface of the epidermis, sclareol must be transported through the plasma membrane, the cell wall and the cuticle, but the molecular mechanism underlying sclareol secretion remains unknown. Passive transport, active transport together with cuticular components or active transport involving ABC transporters can be hypothesized [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF].

Terpene production in roots

In addition to terpenes produced in aerial parts, clary sage plants produce diterpenes in roots. The abietane-type diterpenes aethiopinone, 1-oxoaethiopinone, salvipisone and ferruginol are generally the most abundant ones (Kuźma et al., 2009a;[START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF][START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF][START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF] (Figure 16). These diterpenes display a range of interesting pharmacological activities, notably antibacterial, anti-proliferative, anti-inflammatory and antifungal properties [START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF]. For example, salvipisone and aethiopinone display bactericidal activity against Staphylococcus aureus (Kuźma et al., 2009a), and aethiopinone is active against different solid tumor cell lines, particularly human melanoma cells [START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF]. Given their potential medical use, several studies have focused on enhancing their biosynthesis in clary sage hairy root cultures (Kuźma et al., 2009a;[START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF][START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF][START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF]. However, the biological function of clary sage root diterpenes remains unknown.

Aim of the project

Multiple strategies aiming at increasing terpenoid production in plants through targeted genetic improvement have been developed in diverse plant species like Nicotiana benthamiana or Artemisia annua [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF][START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF][START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF].

The success of these targeted approaches depends on diverse parameters which strongly differ in each case, such as the process of accumulation of the terpenoid of interest, the activity of related metabolic pathways and the nature of regulation mechanisms. A better understanding of sclareol production in clary sage would be highly valuable to provide guidance for future breeding projects dedicated at improving clary sage performances.

When the metabolite of interest is secreted by glandular trichomes, manipulating plant development to increase glandular trichome density can significantly increase the production of this metabolite [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF]. This strategy can be successful only if the production of the metabolite of interest is localized in glandular trichomes. The presence of sclareol has been reported in glandular trichomes of clary sage calyces [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF], but other epidermal cells may also secrete sclareol [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. The first chapter of this manuscript provides new data about the respective contributions of glandular trichomes and other epidermal cells to sclareol accumulation at the surface of the calyx.

The biosynthesis of all terpenoids depends on prenyl substrate supply by MVA and/or MEP pathways. The pool of substrate available for the biosynthesis of a terpenoid of interest can be enlarged by increasing the activity of MVA and/or MEP pathways, or by blocking competing pathways using the same substrate [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF][START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF]. To design such modifications of metabolic fluxes, sufficient information about metabolic pathways connected to the pathway of interest must be gathered. Notably, it is important to know whether the terpenoid of interest derives from the MVA or from the MEP pathway, and to identify potential competing pathways. When transiently expressed in tobacco leaves, enzymes involved in sclareol biosynthesis are targeted to plastids [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF], the cellular compartment where the MEP pathway is localized. However, respective contributions of MVA and MEP pathways to sclareol biosynthesis have not been reported to date. The second chapter of this manuscript focuses on the metabolic origin of sclareol and another abundant terpene, linalyl acetate, in clary sage calyces.

Terpenoid biosynthesis is activated in response to developmental or environmental stimuli through the action of specific signaling pathways. Enhancing the activity of these signaling pathways represents another possibility to increase the production of a terpenoid of interest [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF]. This strategy requires the prior identification of signaling pathways stimulating the biosynthesis of the terpenoid of interest. Signaling mechanisms involved in sclareol biosynthesis regulation have not been explored so far. Since the production of many terpenoids is enhanced by jasmonates [START_REF] Colinas | Combinatorial Transcriptional Control of Plant Specialized Metabolism[END_REF][START_REF] Wasternack | Jasmonates are signals in the biosynthesis of secondary metabolites -Pathways, transcription factors and applied aspects -A brief review[END_REF], the third chapter of this manuscript investigates the impact of jasmonates on sclareol production in clary sage calyces.

Environmental conditions and genetic polymorphisms are responsible for variations in terpenoid content between different populations of the same plant species. The selective pressure exerted by growth conditions can lead to the selection of genetic modifications modulating terpene levels and making the plant more adapted to its environment [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF][START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Wild clary sage naturally grows in diverse environments in countries of the Mediterranean basin and Western Asia [START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF]. Cultivated clary sage populations probably represent only a subset of clary sage natural diversity in terms of genetic variation and sclareol content. The fourth chapter of this manuscript reports the study of genetic and phenotypic diversity among Croatian wild populations of clary sage. The exploration of this natural diversity may ultimately lead to the identification of new ways to increase clary sage productivity.

Chapter 1

Mass spectrometry imaging to localize sclareol biosynthesis

Introduction

Information about the precise localization of sclareol in clary sage has already led to improvements in clary sage cultivation practices. Since more than 90% of sclareol is contained in floral scapes and less than 10% in the rosette, the cutting height used for clary sage harvest was increased from 20 cm to 70 cm. This modification enhanced sclareol yield from 1.67% to 2.25% (source: Philippe Gallois, 7èmes Rencontres du Végétal, 2013). In the perspective of clary sage breeding, knowing the fact that sclareol is mainly present in floral parts suggests that increasing the reproductive/vegetative biomass ratio may be an efficient way to improve clary sage productivity. Therefore, obtaining more knowledge concerning the process of sclareol accumulation in clary sage is key to improve the yield of sclareol production and to design new clary sage breeding strategies. Today, the identity of cells involved in sclareol production and the mechanism of sclareol secretion remain elusive [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. The purpose of this chapter is to improve our understanding of sclareol production by clary sage with new information about its localization in calyces.

Calyces have been previously shown to be the organs of clary sage with the highest sclareol content [START_REF] Lawrence | Production of Clary Sage Oil and Sclareol in North America[END_REF][START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. They are also characterized by a very high density of glandular trichomes [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF], which are known to produce high amounts of terpenes in diverse plant species [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. Hence, sclareol is generally thought to be produced in these specialized epidermal structures. This hypothesis is reinforced by single gland GC-MS analyses of glandular trichome content performed by [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. Although gland content seemed to be highly variable from gland to gland, including between glands of the same type, subcuticular storage cavities of capitate and peltate glandular trichomes generally contained substantial amounts of sclareol. In average, the content of calyx capitate and peltate glands consisted of 61.1% and 48.6% sclareol, respectively, suggesting that sclareol is synthesized by both glandular trichome types [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF].

A few years later, a study analyzed sclareol localization at the surface of the calyx using a set of microscopy and analytical chemistry techniques [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. This study highlighted the presence of abundant epicuticular crystals at the surface of the calyx (Figure 17) and identified these crystals as being composed of almost pure sclareol. The authors hypothesized that sclareol may be produced by all epidermal cells, like other cuticular secretions, and suggested that single gland analyses performed by Schmiderer et al. may have been biased by contaminations with sclareol originating from epidermal crystals [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. To shed some light on the process of sclareol accumulation at the surface of the calyx, we analyzed sclareol localization with different analytical techniques than those used by Schmiderer et al. and Caissard et al. The presence of sclareol at the surface of the calyx was assessed using mass spectrometry imaging and its distribution was compared with that of linalyl acetate, a compound considered to be produced in glandular trichomes. Indeed, glandular trichomes are known to be specialized in the production of essential oil terpenes [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF][START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF] and linalyl acetate is generally abundant in clary sage essential oil [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Sharopov | The essential oil of salvia sclarea L. from Tajikistan[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF]. This analysis was complemented by the characterization of a glabrous mutant line of clary sage.

Materials and methods

Plant material

Clary sage seeds from the "Vatican White" population were purchased from Jelitto (Germany). The "glabrous" clary sage mutant was generated after treatment of "Vatican White" seeds with ethyl methanesulfonate (EMS) to induce the formation of random point mutations in the genome (Drevensek et al., unpublished data). The first generation of mutants was grown in the field at the French National Conservatory of Perfume, Medicinal, Aromatic and Industrial Plants (CNPMAI) in Milly-la-Forêt, France. Seeds from this first generation were harvested in 2016 and sown in 2017 to obtain the second generation of mutants. Wild-type plants and mutants of the second generation were grown in pots for 1 month in growth chamber (16h of day; 27 °C during the day, 21 °C at night; 60% hygrometry) and then 6 months in the greenhouse (24 °C from 6 a.m. to 10 p.m., 20 °C from 10 p.m. to 6 a.m.; 60% hygrometry). Pots were then moved outdoor in December 2017 for natural vernalization and left outdoor until flowering in June-August 2018. Wild-type plants were moved back to the greenhouse in October 2018 before scanning electron microscopy and LDI-FT-ICR experiments.

Scanning electron microscopy

Mature calyces were collected from a wild-type "Vatican White" clary sage plant cultivated in the greenhouse (24 °C from 6 a.m. to 10 p.m., 20 °C from 10 p.m. to 6 a.m.; 60% hygrometry). A freshly cut calyx was introduced in the observation chamber of the scanning electron microscope (Helios NanoLab 660 instrument, FEI) without any sample preparation.

Observations were performed with the voltage set to 0.35 kV.

Metabolite localization by LDI-FT-ICR

Calyx samples were collected from a wild-type "Vatican White" clary sage plant cultivated in the greenhouse (24 °C from 6 a.m. to 10 p.m., 20 °C from 10 p.m. to 6 a.m.; 60% hygrometry). The zone located at the junction of the upper and the lower lip of the calyx, measuring approximately 2 mm x 4 mm, was dissected with a scalpel and fixed onto a conductive microscope slide with conductive double-sided tape. Samples were left on the bench for one night in a microscope slide box. Optical images showing top views of calyx samples were taken the day after sample preparation using a zoom stereomicroscope (AxioZoom V16, Zeiss). Samples were then analyzed the same day by mass spectrometry imaging. Laser-desorption ionization was performed using the MALDI source of the SolariX XR 9.4 Tesla Fourier-Transform Ion Cyclotronic Resonance (FT-ICR) mass spectrometer (Bruker). Sampling was performed using the Smart Beam II laser at 25% of its power to avoid sample damage. The distance between 2 sampling points was set to 25 µm. Data analysis was performed with the SCiLS Lab 2019b Pro software (Bruker). Data were normalized with the total ion current (TIC). To improve data visualization, for each ion of interest, the 0.3% spots with the highest signal were all displayed with the maximum brightness level using the "hotspot removal" function of the software.

Sclareol and linalyl acetate quantification by GC-MS

Clary sage calyces were immersed in hexane without grinding to extract metabolites present at the surface. 1.5 mL of solvent was used to extract metabolites from 1 mature calyx. 10undecen-1-ol was employed as internal standard and was added in the solvent before extraction at a concentration of 0.75 mM. After 2 h of agitation, 1 mL of extract was centrifuged at 13,000 rpm for 10 min to eliminate potential dust or debris. Samples were directly analyzed by gas chromatography coupled to mass spectrometry (GC-MS) using a 7890B/5977A GC-MS system from Agilent and according to a protocol adapted from Laville et al. [START_REF] Laville | Amphilectane diterpenes from Salvia sclarea: Biosynthetic considerations[END_REF]. The system was equipped with a 10 m guard column and an Rxi-5Sil MS column (length, 30 m; inner diameter, 0.25 mm; film thickness, 0.25 μm) (Restek, Bellefonte, PA, USA). 1 µl of sample was injected with a split ratio of 30:1. Oven temperature was set to 110 °C for 2 min, then increased to 270 °C at a rate of 10 °C/min, and finally set to 270 °C for 2 min. Other temperatures were set as follows: injector, 270 °C; transfer line, 270 °C; source, 230 °C; quadrupole, 150 °C. The carrier gas was helium at a constant flow of 1 mL/min. The quadrupole mass spectrometer was switched on after a solvent delay of 5 min and was programmed to scan from 35 u to 350 u. Data analysis was carried out using the MassHunter Quantitative Analysis software from Agilent. Sclareol and linalyl acetate were identified by comparison with analytical standards purchased from Sigma-Aldrich. Absolute quantification of sclareol and linalyl acetate was performed using a calibration curve prepared with analytical standards.

Results

Different accumulation patterns for sclareol and linalyl acetate

The chemical composition of clary sage calyx surface was analyzed by mass spectrometry imaging. Ions were generated from calyx surface using the laser-desorption ionization (LDI) method. In most cases, this method requires the application of a matrix on the sample to facilitate compound ionization (matrix-assisted laser-desorption ionization, MALDI). In our case, compounds of interest were detectable without matrix and calyx samples were analyzed in a state as close as possible to their native state. Ion detection and quantification was performed by Fourier-transform ion cyclotronic resonance (FT-ICR), a high-resolution mass spectrometry technique allowing reliable identification of compounds in a complex mixture of chemicals such as plant epidermis surface. The most abundant ion on all calyx samples was an ion of exact mass equal to 347.23556 Da (Figure 18). Analysis of sclareol pure standard confirmed the identity of this ion as being an adduct between a molecule of sclareol and a potassium ion ([sclareol-K] + ). In addition to [sclareol-K] + , we focused our analysis on two other ions of exact masses equal to 235.10917 Da and 655.50810 Da, respectively. Analysis of linalyl acetate pure standard indicates that the ion of 235.10917 Da is an adduct between a molecule of linalyl acetate and a potassium ion ([linalyl acetate-K] + ). According to its exact mass, the ion of 655.50810 Da is an adduct between two molecules of sclareol and a potassium ion K + ([(sclareol) 2 -K] + ).

[Linalyl acetate-K] + ions are mainly present in the form of spots which often coincide with a capitate glandular trichome on the optical image (Figure 19a-b and 20a-b, full white arrows). This observation suggests that linalyl acetate is produced by capitate glandular trichomes. This result is in accordance with the literature, which indicates that essential oil components are generally produced in glandular trichomes [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF][START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF].

Surprisingly, [sclareol-K] + ion distribution differs from [(sclareol) 2 -K] + (Figure 19c-d and20cd). [Sclareol-K] + ions are found over most of the calyx surface, particularly on the veins bordering the analyzed zone. By contrast, [(sclareol) 2 -K] + ions are detected as spots which are often associated with a capitate glandular trichome, in a similar way than [linalyl acetate-K] + ions. This discrepancy between [sclareol-K] + and [(sclareol) 2 -K] + distributions could be explained by a higher sclareol/K + ratio at the surface of capitate glandular trichomes compared to the rest of the epidermis. Indeed, [(sclareol) 2 -K] + ions may form upon laser impact only if many sclareol molecules are present around a single K + ion. The sclareol/K + ratio could be higher at the surface of capitate glandular trichomes if K + ions are less abundant than on the rest of the epidermis, for reasons which remain to be elucidated.

Conversely, the sclareol/K + ratio could also be higher if sclareol is more abundant at the surface of capitate glandular trichomes than on the rest of the epidermis. This would be the case if capitate glandular trichomes produce more sclareol than other epidermal cells. Thus, the presence of [(sclareol) 2 -K] + spots associated with capitate glandular trichomes supports the hypothesis that sclareol is mainly produced by glandular trichomes. However, since [sclareol-K] + ions are detected all over calyx surface, the hypothesis that other epidermal cells contribute to sclareol production cannot be excluded.

Distinct roles of different trichome types in sclareol production

Scanning electron microscopy observation of a freshly cut mature calyx suggests that capitate glandular trichomes spontaneously secrete their content. Indeed, spills of electrondense material were visible on many capitate glandular trichome stalks (Figure 21b). The use of a pin to puncture a glandular head confirmed that this electron-dense material comes from capitate glandular trichome heads (Figure 21c-g). If we hypothesize that sclareol is produced only by glandular trichomes, the spilling of capitate glandular trichome content could explain why sclareol is present all over calyx epidermis while not being produced by pavement cells. The absence of [linalyl acetate-K] + signal on calyx surface except on capitate glandular trichomes could be due to the higher volatility of linalyl acetate, which probably evaporates quickly after its secretion by capitate glandular trichomes. Metabolic activity may vary from gland to gland due to differential expression of biosynthetic enzymes. Consequently, each gland may contain specialized metabolites in different proportions, as suggested by single gland analyses performed by Schmiderer et al. [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF].

A large number of capitate glandular trichomes appear intact on scanning electron microscopy images, with no visible secretion along their stalks (Figure 21a). This observation may explain why some capitate glandular trichomes visible on optical images are not associated with any [sclareol-K] + , [linalyl acetate-K] + or [(sclareol) 2 -K] + signal on mass spectrometry images (Figure 19 and20). The impact of the laser used for compound ionization may not be sufficient to break the cuticle and access glandular head content.

Similarly, no [sclareol-K] + , [linalyl acetate-K] + or [(sclareol)2-K] + signal is visible at the position of most peltate glandular trichomes (Figure 19 and 20, yellow arrows), although these trichomes were previously shown to contain high amounts of sclareol and linalyl acetate [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. The thick cuticle covering the storage cavity of peltate glandular trichomes [START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF] may prevent their content from being ionized upon laser impact. Chapter 1 -Mass spectrometry imaging to localize sclareol biosynthesis 55 Intriguingly, some [sclareol-K] + signal seems to be associated with non-glandular trichomes implanted on calyx veins and at the edge of the calyx (Figure 22a-b). Moreover, drops of electron-dense material can sometimes be seen dripping down non-glandular trichomes on scanning electron microscopy images (Figure 22c-d). These observations suggest that "nonglandular" trichomes may produce some sclareol and may even have a secretory activity, although this affirmation needs to be confirmed by further observations.

A clary sage glabrous mutant produces less sclareol and linalyl acetate

A clary sage "glabrous" line was identified in a collection of clary sage mutant lines obtained by EMS-generated random mutagenesis (Drevensek et al., unpublished data) (Figure 23a).

According to careful numbering of different trichome types (Drevensek et al., unpublished data), the "glabrous" aspect of calyces from these plants is essentially due to a drastic reduction of the number of large capitate glandular trichomes, while the number of peltate and small capitate glandular trichomes remains unchanged (Figure 23b). Metabolite quantification by GC-MS indicates that sclareol and linalyl acetate content is strongly reduced in glabrous calyces compared to wild-type calyces (Figure 23c-d). This lower metabolite content is likely due to the nearly absence of large capitate glandular trichomes, an idea reinforcing the hypothesis that capitate glandular trichomes are primarily responsible for calyx sclareol production. However, distinct EMS-induced point mutations could be the cause of the decrease in metabolite content and the reduced density of capitate glandular trichomes, respectively. The analysis of the co-segregation of these two

Discussion

Sclareol is mainly produced by glandular trichomes

Glandular trichomes of the Lamiaceae are known to be specialized in the production of essential oil terpenes, mainly mono-and sesquiterpenes [START_REF] Werker | Function of essential oil-secreting glandular hairs in aromatic plans of Lamiacea-a review[END_REF][START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. In Nicotiana species, glandular trichomes produce diterpenes, for example sclareol in Nicotiana glutinosa [START_REF] Guo | Biosynthesis of labdenediol and sclareol in cell-free extracts from trichomes of Nicotiana glutinosa[END_REF]. These elements led the scientific community to assume that sclareol accumulated in clary sage calyces is produced by glandular trichomes.

We provide here several lines of evidence reinforcing this hypothesis. (Figure 24). Sclareol present on the major part of calyx epidermis, detected in the form of [sclareol-K] + ions, could initially be produced in glandular heads and spread over pavement cells along with the rest of glandular head content. This process of accumulation was shown to occur in the case of the diterpene duvatrienediol in Nicotiana tabacum [START_REF] Chang | Structural Organization of Tobacco Leaf Polar Cuticular Lipids[END_REF][START_REF] Keene | Direct demonstration of duvatrienediol biosynthesis in glandular heads of tobacco trichomes[END_REF]. Tobacco capitate glandular trichomes are known to regularly exude their content [START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF] and our scanning electron microscopy images highlight the presence of glandular head content dripping down clary sage trichome stalks (Figure 21). Gene expression was quantified by reverse transcription-quantitative PCR (RT-qPCR) on RNAs purified from intact buds, intact calyces and purified glandular trichomes. TPS1: sclareol-synthase gene [START_REF] Günnewich | A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate[END_REF]. SS: sclareol-synthase gene [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. TPS1132: sclareolsynthase gene [START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF]. LPPS: LPP-synthase gene [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. Bars represent the average of measurements performed on 2 replicates and error bars correspond to standard deviation.

Our scanning electron microscopy observations (Figure 21) do not confirm the presence of sclareol crystals described by Caissard et al. at the surface of calyx epidermis [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF] (Figure 17). We employed conventional scanning electron microscopy, while Caissard et al. performed environmental scanning electron microscopy observations. However, we did not apply any fixation or preparation protocol to calyces before observations. Samples were submitted to higher vacuum compared to environmental scanning electron microscopy, but it is unlikely that the totality of sclareol crystals were ripped off calyx surface during vacuum pumping. It is important to note that samples used in the study conducted by [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Consistently, we could see sclareol crystals rapidly forming when calyces were heated by a lamp during observations with a binocular. The absence of crystals on our samples could be due to milder culture conditions. Indeed, culture conditions were controlled in the greenhouse (24 °C from 6 a.m. to 10 p.m., 20 °C from 10 p.m. to 6 a.m.; 60% hygrometry); it is thus likely that hygrometry was higher and temperature was lower compared to the field. Sclareol crystals observed by Caissard et al. were not evenly distributed at the surface of the epidermis and were clustered in patches [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Similarly, splashes of gland content that we observed by scanning electron microscopy were scattered over calyx epidermis (Figure 21).

In the field, sclareol crystals may form progressively from splashes of gland content upon dehydration and evaporation of more volatile compounds like linalyl acetate. This hypothesis was already suggested by Caissard et al. [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF].

Some sclareol could also be produced by other epidermal cells

As previously underlined, splashes of gland content observed by scanning electron microscopy did not cover all epidermis (Figure 21). By contrast, [sclareol-K] + signal is detected almost everywhere on calyx surface. Splashes of glandular head content may not account for all the sclareol present on the epidermis. In addition to capitate glandular trichomes, other epidermal cells may contribute to sclareol production, albeit at a lower extent compared to capitate glandular trichomes. Mass spectrometry imaging is a powerful technique, but false-negatives are possible if chemicals cannot be correctly ionized by the laser. In particular, the cuticle present at the surface of epidermal cells could prevent laser access to metabolites. Indeed, it is surprising that no sclareol and linalyl acetate signals could be associated with peltate glandular trichomes (Figure 19 and 20), given the fact that these trichomes are known to produce volatile monoterpenes [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF] and were previously shown to contain large amounts of sclareol [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. The analysis of the surface of glabrous calyces by LDI-FT-ICR could provide valuable information about the respective contributions of peltate trichomes, large capitate trichomes and pavement cells to sclareol production. Since large capitate trichomes are almost absent on glabrous calyces, signal potentially associated with peltate trichomes or pavement cells would be easier to distinguish.

Our results suggest that "non-glandular" trichomes may have a secretory activity. Indeed, [sclareol-K] + signal seems to be associated with "non-glandular" trichomes, and electrondense material similar to the glandular head content of capitate trichomes can sometimes be seen dripping down the sides of these trichomes (Figure 22). Similarly, recent publications suggest that T-shaped trichomes of Artemisia annua, generally considered nonglandular, may produce specialized metabolites [START_REF] Soetaert | Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua[END_REF][START_REF] Judd | Artemisinin biosynthesis in non-glandular trichome cells of Artemisia annua[END_REF]. Tshaped trichomes were shown to express genes involved in artemisinin biosynthesis and mass spectrometry imaging confirmed the presence of artemisinin in these cells [START_REF] Judd | Artemisinin biosynthesis in non-glandular trichome cells of Artemisia annua[END_REF]. Filamentous trichomes like T-shaped trichomes of Artemisia annua or "nonglandular" trichomes of clary sage are generally considered to be involved in plant mechanical defense against insects via hindering their locomotion [START_REF] Soetaert | Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua[END_REF].

Even if their structure is not fully adapted to metabolite secretion, they could secrete metabolites, albeit in smaller amounts compared to glandular trichomes dedicated to this function. In clary sage, metabolites like sclareol could be secreted along with cuticular lipids via the same secretion pathway, as previously suggested [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Further investigation is needed to assess "non-glandular" trichome secretory activity and, if confirmed, to understand the process of metabolite secretion by these trichomes. Notably, the expression of genes involved in specialized metabolite biosynthesis could be compared in "non-glandular" and glandular trichomes sorted by laser microdissection, as performed previously in Artemisia annua [START_REF] Soetaert | Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua[END_REF].

Engineering higher glandular trichome density to increase sclareol yield

Since sclareol is mainly produced in glandular trichomes, increasing glandular trichome density appears to be a promising approach to enhance sclareol production in clary sage.

Higher glandular trichome density can be engineered through the manipulation of transcription factors regulating glandular trichome initiation. This strategy is currently being developed to enhance the production of the anti-malarial sesquiterpene artemisinin in Artemisia annua [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF][START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. Genes involved in artemisinin biosynthesis were previously shown to be preferentially expressed in glandular trichomes, highlighting the major role played by glandular trichomes in artemisinin production [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF]. Transcription factors positively regulating glandular trichome initiation were later identified: the MIXTA-like transcription factor AaMIXTA1 [START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF] and the homeodomain leucine zipper (HD-ZIP) transcription factors AaHD1 and AaHD8 [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. The overexpression of each of these positive regulators significantly enhanced glandular trichome density and also artemisinin yield, without any adverse effect on plant growth and fitness [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. The highest increase was observed after the overexpression of AaMIXTA1, which doubled artemisinin content [START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF].

Some regulators of glandular trichome initiation and density appear to be conserved in different plant lineages. For example, the tomato homolog of AaHD8, SlCD2, is also a positive regulator of glandular trichome density in tomato [START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF].

Homologs of conserved regulators could be looked for in the available transcriptome of clary sage calyces [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF]. If further gene expression analysis shows that identified transcription factors are differentially expressed in glandular trichomes, these transcription factors would be good candidates for an involvement in glandular trichome initiation in clary sage. Enhancing or repressing the expression of positive or negative regulators of glandular trichome initiation, respectively, could increase glandular trichome density and thus sclareol yield in clary sage.

Chapter 2 A retro-biosynthetic approach to decipher sclareol metabolic origin 1. Introduction

Metabolic crosstalk between MVA and MEP pathways

In plants, MVA and MEP pathways are localized in two different subcellular compartments:

the cytosol and the plast, respectively. Consequently, their activity supplies two physically separated pools of IPP and DMAPP. In accordance with the subcellular localization of biosynthetic enzymes, biosynthesis of downstream products generally depends on only one of these two pools [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF]. Most sesquiterpenoids originate from the MVA pathway, while the majority of mono-and diterpenoids are of MEP pathway origin [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF].

However, a growing number of studies describe the production of terpenoids of mixed origin, i.e. made of five-carbon units originating from both pathways [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF]. Indeed, prenyl intermediates can be exchanged between the two compartments, opening up the possibility of cooperation between MVA and MEP pathways [START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF]. Metabolite transfer from plasts to cytosol, and conversely, has been previously highlighted in BY-2 tobacco cells [START_REF] Hemmerlin | Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells[END_REF], but can also occur in whole plants, like for example in young cotton seedlings [START_REF] Opitz | Both methylerythritol phosphate and mevalonate pathways contribute to biosynthesis of each of the major isoprenoid classes in young cotton seedlings[END_REF]. In tobacco cells, the percentage of precursors exchanged from plasts to cytosol can reach 100% and the percentage of precursors exchanged from cytosol to plasts can attain 80% [START_REF] Hemmerlin | Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells[END_REF][START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF]. A unidirectional proton symport system has been suggested to transport prenyl diphosphates from plastids to cytosol [START_REF] Bick | Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: Unidirectional transport of intermediates across the chloroplast envelope membrane[END_REF]. However, the identity of exchanged prenyl intermediates and the exact molecular mechanism responsible for their transport remain unknown [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF].

Crosstalk between MVA and MEP pathways happens when prenyl intermediates start to accumulate in a given compartment [START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF]. In that case, the excess of intermediates produced by one of the two pathways can be transferred to another compartment to supply the other pathway. This situation is encountered upon treatment with an MVA-or MEP pathway-specific inhibitor, but also under particular physiological conditions in specific tissues or at given developmental stages. Notably, transport of prenyl intermediates from plastids to cytosol frequently occurs in photosynthetic organs [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF], whereas transport from cytosol to plastids can be substantial in the absence of light:

in young cotton seedlings, the MVA pathway provides as much as 50% of the substrate used for phytol side chain and carotenoid biosynthesis [START_REF] Opitz | Both methylerythritol phosphate and mevalonate pathways contribute to biosynthesis of each of the major isoprenoid classes in young cotton seedlings[END_REF]. It has been previously

suggested that in developing seedlings, prenyl diphosphate precursors produced in the cytosol by the MVA pathway are transported to plastids to sustain carotenoid and gibberellin production [START_REF] Rodríguez-Concepción | Distinct Light-Mediated Pathways Regulate the Biosynthesis and Exchange of Isoprenoid Precursors during Arabidopsis Seedling Development[END_REF].

Towards clary sage metabolic engineering

Metabolic engineering approaches aim at increasing the production of a compound of interest in an organism by genetically optimizing metabolic fluxes and regulatory processes.

The compartmentalization of plant terpenoid biosynthesis pathways and the possibility of crosstalk between MVA and MEP pathways have already been taken in account to engineer terpenoid production in plant systems [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF][START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF][START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF][START_REF] Mafu | Plant diterpenoid metabolism for manufacturing the biopharmaceuticals of tomorrow: prospects and challenges[END_REF][START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF]. A good understanding of the metabolic network including the pathway of interest is required to design a successful metabolic engineering strategy. In particular, it is important to know which metabolic pathways provide substrate to the pathway of interest, and to identify potential competing pathways [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF].

Respective contributions of MVA and MEP pathways to sclareol production in clary sage have not been assessed so far. Terpene synthases responsible for sclareol biosynthesis have been fused to GFP and expressed in tobacco plants (Nicotiana benthamiana) in order to analyze their subcellular localization. Fusion proteins were specifically localized in plasts, suggesting that sclareol biosynthesis occurs in plasts and that sclareol precursors may be derived from the plastidial MEP pathway [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. However, the MVA pathway may also sustain sclareol biosynthesis by producing prenyl intermediates which would then be transferred to plasts. The possibility of prenyl intermediate transport from cytosol to plasts has already been evidenced in spike lavender, another plant belonging to the Lamiaceae family [START_REF] Mendoza-Poudereux | Metabolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender[END_REF].

The MVA contribution to the biosynthesis of mainly MEP-derived terpenoids can be high in non-photosynthetic organs like roots [START_REF] Opitz | Both methylerythritol phosphate and mevalonate pathways contribute to biosynthesis of each of the major isoprenoid classes in young cotton seedlings[END_REF]. Clary sage calyces are photosynthetic, but it is unclear whether glandular trichomes, the multicellular epidermal structures producing the majority of calyx sclareol (see Chapter 1), are photosynthetic or not. Contrasted situations are encountered in other plant species. Tomato type VI trichomes are photosynthetic but still rely on sucrose imported from other leaf cells to achieve their high metabolic productivity [START_REF] Balcke | Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites[END_REF]. Artemisia annua glandular trichomes have both photosynthetic and non-photosynthetic cells [START_REF] Duke | Development and Fine Structure of the Glandular Trichomes of Artemisia annua L[END_REF], while peltate glandular trichomes of Mentha x piperita, a Lamiaceae species closely related to clary sage, are not photosynthetic [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. If the photosynthetic activity of clary sage glandular trichomes is low, MVA-derived prenyl diphosphate precursors may be transported from the cytosol to the plast in glandular trichomes secretory cells, but this remains to be demonstrated.

To elucidate the metabolic origin of sclareol produced by clary sage calyces, we analyzed the impact of MVA and MEP pathway-specific inhibitors and carried out isotope labeling experiments based on the use of 1-13 C-glucose and 13 C-NMR spectroscopy. Eventually, knowing which metabolic pathways provide substrate for sclareol biosynthesis or compete with sclareol biosynthesis will help to design ways to redirect carbon fluxes towards sclareol biosynthesis.

Materials and methods

Plant material

Clary sage seeds from the "Vatican White" population were purchased from Jelitto (Germany). Clary sage plants were grown in pots for 1 month in growth chamber (16h of day; 27 °C during the day, 21 °C at night; 60% hygrometry) and then 3 months in the greenhouse (24 °C from 6 a.m. to 10 p.m., 20 °C from 10 p.m. to 6 a.m.; 60% hygrometry).

Pots were then moved outdoor in December 2017 for natural vernalization and left outdoor until flowering in June-August 2018.

Treatment with MVA or MEP pathway-specific inhibitors

Mevinolin and fosmidomycin were purchased from Sigma-Aldrich. Stock solutions (50 mM)

were prepared in ethanol and water, respectively. Nutritive solution containing 0, 1, 10 or 100 µM of inhibitor was prepared in 5 mL vials (Murashige and Skoog basal salt mixture (Sigma-Aldrich); glucose 5% m/v (Euromedex); PPM TM 0.1% v/v (Plant Cell Technology); ethanol 0.2% v/v). Clary sage inflorescence axillary stems were cut between the 3 rd and the 4 th node (about 10 cm from the apex). Each stem was immediately placed in a vial in a way that its extremity was touching the bottom of the vial and dipped in nutritive solution (Figure 25). Treatment was performed by leaving inflorescence stems on the bench for 6 days. One calyx was sampled from each inflorescence stem, immediately frozen in liquid nitrogen and stored at -80 °C before sclareol and linalyl acetate quantification by GC-MS.

Sampled calyces were at stage 2 at the beginning of the treatment and had reached stage 4 or 5 after 6 days.

Sclareol and linalyl acetate quantification by GC-MS

Clary sage calyces were immersed in hexane or ethanol without grinding to extract metabolites present at the surface. 1.5 mL of solvent was used to extract metabolites from 1 mature calyx. 10-undecen-1-ol was employed as internal standard and was added in the solvent before extraction at a concentration of 0.75 mM. After 2 h of agitation, 1 mL of extract was centrifuged at 13,000 rpm for 10 min to eliminate potential dust or debris.

Samples were directly analyzed by gas chromatography coupled to mass spectrometry (GC-MS) using a 7890B/5977A GC-MS system from Agilent and according to a protocol adapted from Laville et al. [START_REF] Laville | Amphilectane diterpenes from Salvia sclarea: Biosynthetic considerations[END_REF]. The system was equipped with a 10 m guard column

Isotope labeling and analysis by 13 C-NMR or GC-MS

Isotope labeling and sample preparation

Nutritive solution containing 1-13 C-glucose was prepared in 5 mL vials (Murashige and Skoog basal salt mixture (Sigma-Aldrich); 1-13 C-glucose 5% m/v (Sigma-Aldrich); PPM TM 0.1% v/v (Plant Cell Technology); ethanol 0.2% v/v). Clary sage inflorescence axillary stems were cut between the 3 rd and the 4 th node (about 10 cm from the apex). Each stem was immediately placed in a vial in a way that its extremity was touching the bottom of the vial and dipped in nutritive solution. Feeding was performed by leaving inflorescence stems on the bench for 5 days. Young calyces (stage 4 or younger) were sampled from each inflorescence stem, immediately frozen in liquid nitrogen and stored at -80 °C before further analysis.

Calyces collected on one inflorescence stem were immersed in 8 mL of hexane without grinding to extract metabolites present at the surface. After 2 h of agitation, the extract was centrifuged at 13,000 rpm for 10 min to eliminate potential dust or debris. 300 µL of the extract were saved for GC-MS analysis of isotope cluster distortion and the rest of the extract was prepared for 13 C-NMR analysis. Hexane was evaporated by leaving the samples under the fume hood for 2-3 days. Evaporation needed to be slow in order to avoid linalyl acetate volatilization. Dry extract was resuspended in 600 µL of CDCl 3 for 13 C-NMR analysis. adjusting the phase and applying a baseline correction. The attribution of each peak of the spectrum to a carbon of sclareol or linalyl acetate (Table 1) was performed using 1 H, COSY, HSQC and HMBC experiments and inspired by previously published data [START_REF] Fowler | Plant terpene biosynthesis. The biosynthesis of linalyl acetate in Mentha citrata[END_REF][START_REF] Chain | FT-IR, FT-Raman, UV-Visible, and NMR spectroscopy and vibrational properties of the labdane-type diterpene 13-epi-sclareol[END_REF]. Calibration curves with known concentrations of sclareol and linalyl acetate analytical standards (purchased from Sigma-Aldrich) confirmed that the acquisition was quantitative, i.e. that peak area was proportional to the amount of 13 C present in the sample (R 2 > 0.98 for sclareol and R 2 > 0.96 for linalyl acetate). Potential incorporation of 13 C at a given position was evaluated using the labeling ratio. This ratio was calculated by dividing peak area measured for labeled samples by peak area measured for unlabeled samples. In blue: carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis; in grey, carbons predicted to be unlabeled whatever the pathway involved in IPP biosynthesis.

Analysis of isotope cluster distortion by GC-MS

Extracts saved for GC-MS analysis were processed using the protocol described above (see page 67). During GC-MS analysis, compounds contained in the sample are fragmented and each fragment produces a signal in the form of a group of peaks known as isotope cluster.

The mass spectrum of a given compound displays several isotope clusters, which correspond to the different fragments generated in the mass spectrometer. The different peaks of an isotope cluster represent ions with the same elemental composition, but different isotopic compositions: heavier peaks correspond to ions which contain one or more heavy isotopes like 18 O or 13 C. The isotope cluster is present even without artificial isotope labeling because 18 O and 13 C are present in nature, although in low abundance compared to light isotopes 16 O and 12 C (0.2 % and 1.1 % versus 99.8 % and 98.9 %). The incorporation of additional 13 C atoms leads to a distortion of the isotope cluster, with ions containing heavy isotopes becoming more abundant. For each compound, the distortion of several isotope clusters of the mass spectrum was evaluated by calculating their distortion ratio, which corresponds to the sum of heavy ions peak areas divided by light ion peak area. For β-caryophyllene, 134+135+136/133 and 148+149/147 ratios were calculated, and for linalyl acetate, 81+82+83/80, 94+95+96/93 and 122+123/121 ratios were calculated. A higher distortion ratio reveals the incorporation of 13 C atoms in the compound.

Results

Sclareol and linalyl acetate are highly abundant terpenes of clary sage calyx surface

The solid-liquid extraction of sclareol from plant material is usually performed with an organic solvent like hexane [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF][START_REF] Chain | FT-IR, FT-Raman, UV-Visible, and NMR spectroscopy and vibrational properties of the labdane-type diterpene 13-epi-sclareol[END_REF][START_REF] Dufour | Purification of sclareol by supercritical CO2 fractionation process[END_REF].

Sclareol is then quantified by GC-MS. This protocol is suitable for terpenes with a small hydrocarbon backbone harboring few decorations, displaying relatively low polarity and relatively high volatility [START_REF] Jiang | Extraction and Analysis of Terpenes/Terpenoids[END_REF]. Therefore, it was used to quantify sclareol and at the same time identify other metabolites present at the surface of clary sage calyces.

According to GC-MS analyses, two metabolites are particularly abundant in clary sage calyx hexane extracts: sclareol and linalyl acetate (Figure 26a). 13 C-NMR analysis confirmed that calyx hexane extracts are essentially composed of sclareol and linalyl acetate (Figure 26c).

Hexane is a strongly nonpolar solvent, which preferentially extracts molecules displaying low polarity. To enable the extraction of more polar metabolites, ethanol was used instead of hexane. Again, sclareol and linalyl acetate were the main metabolites visible on the GC-MS chromatogram, respectively accounting for 54% and 43% of total peak area (Figure 26b).

Linalool could not be detected in hexane or ethanol extracts either by GC-MS or 13 C-NMR, although it is often reported as a major monoterpene of clary sage essential oil [START_REF] Lattoo | Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea[END_REF][START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF][START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF][START_REF] Sharopov | The essential oil of salvia sclarea L. from Tajikistan[END_REF][START_REF] Nasermoadeli | Comparison of Salvia sclarea L . Essential Oil Components in Wild and Field Population[END_REF]. However, in a study comparing dichloromethane extracts and essential oil composition, sclareol, linalyl acetate and linalool respectively accounted for 74%, 22% and 1.5% of peak area in dichloromethane extracts, versus 6%, 63% and 18% in essential oil [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. The absence or very low abundance of linalool in hexane and ethanol extracts is consistent with these previously published data.

Given the abundance of linalyl acetate in calyx extracts, we considered the hypothesis that linalyl acetate biosynthesis may significantly compete with sclareol biosynthesis for prenyl diphosphate substrates. For this reason, we focused our study on both sclareol and linalyl acetate biosynthesis and tried to decipher the metabolic origin of both metabolites. To investigate the contribution of MVA and MEP pathways to the production of IPP and DMAPP necessary for sclareol and linalyl acetate biosynthesis, we used specific inhibitors of each pathway, respectively mevinolin and fosmidomycin. Mevinolin is a competitive inhibitor of HMGR, a rate-limiting enzyme of the MVA pathway, while fosmidomycin competitively inhibits DXR, a key enzyme of the MEP pathway [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF].

Cut inflorescence stems were fed with different concentrations of inhibitor and sclareol and linalyl acetate content was quantified by GC-MS after 6 days. Mevinolin treatment does not lead to any significant modification of sclareol and linalyl acetate content (Figure 27a). By contrast, fosmidomycin treatment inhibits sclareol and linalyl acetate production in a dosedependent way (Figure 27b). A significant drop in sclareol content can already be observed with a concentration of 1 µM of fosmidomycin in the feeding solution and the strongest inhibition is observed with 100 µM of fosmidomycin, with a decrease of 66% in sclareol content. A significant drop in linalyl acetate content (by 93%) can be observed with 100 µM of fosmidomycin. These results indicate that sclareol and linalyl acetate biosynthesis mainly depends on the MEP pathway.

However, 34% of sclareol content remains after 6 days of treatment with 100 µM of fosmidomycin (Figure 27b). The inhibition of the MEP pathway may be incomplete, or this amount of sclareol may have been produced before the start of the treatment. Alternatively, the MVA pathway may be activated and produce a part of the IPP and DMAPP needed for sclareol biosynthesis when the MEP pathway is inhibited by fosmidomycin. To test this hypothesis, we employed another approach to study the contribution of MVA and MEP pathways: a retro-biosynthetic approach based on the use of 1-13 C-glucose. sclareol [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF][START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF][START_REF] Günnewich | A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate[END_REF] and linalyl acetate [START_REF] Fowler | Plant terpene biosynthesis. The biosynthesis of linalyl acetate in Mentha citrata[END_REF][START_REF] Zaks | Biosynthesis of linalyl acetate and other terpenes in lemon mint (Mentha aquatica var. citrata, Lamiaceae) glandular trichomes[END_REF].

Cut inflorescence stems were fed with 1-13 C-glucose and 13 C-labeling patterns obtained on sclareol and linalyl acetate were analyzed by 13 C-NMR. According to theoretical sclareol 13 Clabeling patterns, carbons 2, 6, 11 and 15 should be labeled only if the MEP pathway was involved in the production of IPP and DMAPP, while carbons 1, 3, 5, 7, 9, 12, 14 and 18 should be labeled only if the MVA pathway was involved (Figure 28b). After 1-13 C-glucose feeding, carbons 2, 6, 11 and 15 are strongly labeled while carbons 1, 3, 5, 7, 9, 12, 14 and 18 are weakly labeled (Figure 29). Interestingly, carbons 4, 8, 10 and 13, which should not be labeled whatever the pathway involved in IPP and DMAPP biosynthesis, show a weak labeling comparable to carbons specific of the MVA pathway. Similar results were obtained for linalyl acetate: carbons specific of the MEP pathway are strongly labeled whereas carbons specific of the MVA pathway are weakly labeled, and carbons which should not be labeled whatever the pathway involved also show a weak labeling (Figure 30). These results

suggest that both sclareol biosynthesis and linalyl acetate biosynthesis mainly depend on the MEP pathway. In grey, carbons predicted to be unlabeled; in blue, carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis. In grey, carbons predicted to be unlabeled; in blue, carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis.

A B C

Feeding with 1-13 C-glucose together with MVA and MEP pathways inhibitors confirms sclareol and linalyl acetate metabolic origin

To further investigate the contribution of each pathway, mevinolin or fosmidomycin were added to the feeding solution together with 1-13 C-glucose. In presence of fosmidomycin, all carbons of sclareol show a comparable very weak labeling with a labeling ratio ranging between 1.0 and 1.9 (Figure 31a). This result indicates that the MEP pathway is mainly responsible for the labeling observed at all carbon positions of sclareol, whether weak or strong. By contrast, labeling ratios observed in presence of mevinolin are comparable to the control (Figure 31a). Interestingly, the weak labeling of carbons specific of the MVA pathway is retained in presence of mevinolin, suggesting that this labeling is not due to MVA pathway activity. Similar results are obtained for linalyl acetate (Figure 31b). Taken together, these results exclude a possible minor contribution of the MVA pathway and show that sclareol and linalyl acetate biosynthesis fully depends on IPP and DMAPP produced by the MEP pathway. Given the fact that glucose is a central intermediate in primary metabolism, the very weak labeling observed for all sclareol carbons in presence of fosmidomycin is probably due to an aspecific labeling of all cell metabolites. Consistently, in the context of first clary sage feeding tests using 1-13 C-glucose, an incorporation of 13 C was detected in fructose, serine and palmitic acid (Drevensek et al., unpublished data).

Comparison of results obtained for sclareol and linalyl acetate

Higher labeling ratios (ranging between 16.7 and 20.2) are observed for labeled carbons of linalyl acetate compared to labeled carbons of sclareol (labeling ratios ranging between 7.3 and 13.0) (Figure 31). This observation suggests that during the feeding, more IPP and DMAPP units were targeted towards linalyl acetate biosynthesis compared to sclareol biosynthesis. Consistently, in presence of fosmidomycin, linalyl acetate carbons specific of the MEP pathway are more labeled than sclareol carbons specific of the MEP pathway (labeling ratios ranging between 2.0 and 3.0, versus labeling ratios ranging between 1.0 and 1.9). This result indicates that the MEP pathway is not completely inhibited by fosmidomycin, and that remaining MEP pathway activity produces IPP and DMAPP that is preferentially used for linalyl acetate biosynthesis compared to sclareol biosynthesis.

Although linalyl acetate biosynthesis seems to be preferred in presence of fosmidomycin according to 13 C-labeling data, the drop in linalyl acetate content obtained in presence of 100 µM of fosmidomycin is more important than the drop in sclareol content (93% versus 66%) (Figure 27b). This apparent discrepancy between the two experiments could be explained by a higher turn-over of the linalyl acetate pool. Indeed, without inhibitor treatment, linalyl acetate content of calyx organic extracts is comparable to sclareol content.

However, linalyl acetate is more volatile than sclareol and probably evaporates more rapidly at the surface of the calyx. A higher carbon flux through linalyl acetate biosynthesis is probably necessary to maintain a linalyl acetate content equivalent to sclareol content in spite of higher linalyl acetate evaporation. In presence of fosmidomycin, it is likely that linalyl acetate produced before the start of the treatment evaporates whereas sclareol remains at the surface of the calyx. This would explain why only 7% of linalyl acetate content remains after 6 days of treatment, versus 34% in the case of sclareol.

The case of the acetate group of linalyl acetate

Carbons 11 and 12 of linalyl acetate correspond to the acetate moiety. In lemon mint (Mentha aquatica var. citrata, Lamiaceae), a linalool acetyltransferase activity has been detected and uses acetyl-CoA as the acetyl group donor to produce linalyl acetate from linalool [START_REF] Zaks | Biosynthesis of linalyl acetate and other terpenes in lemon mint (Mentha aquatica var. citrata, Lamiaceae) glandular trichomes[END_REF]. It is likely that a similar activity exists in clary sage and that acetyl-CoA is used as the acetyl group donor for the production of linalyl acetate. After 1-13 Cglucose processing through glycolysis and pyruvate oxidation, 13 C-labeling is expected to be found on carbon 2 of acetyl-CoA, which corresponds to carbon 12 of linalyl acetate [START_REF] Carvalho | Noninvasive measurement of murine hepatic acetyl-CoA 13 C-enrichment following overnight feeding with 13 C-enriched fructose and glucose[END_REF][START_REF] Jones | Identifying Sources of Hepatic Lipogenic Acetyl-CoA Using Stable Isotope Tracers and NMR[END_REF]. Peak 25 of the calyx extract 13 C-NMR spectrum integrates for 2 carbons of linalyl acetate: carbon 12 and carbon 5 (Table 1). Carbon 5 is expected to be labeled only if the MEP pathway contributes to the biosynthesis of IPP and DMAPP required for linalyl acetate biosynthesis. On the spectrum obtained for samples fed with 1-13 C-glucose without inhibitor, the area measured under peak 25 is about twice the area measured under peaks corresponding to single labeled carbons (carbons 1, 8 and 10), indicating that both carbon 12 and carbon 5 are labeled (Figure 30). This result is in agreement with acetyl-CoA being the acetyl group donor and with the involvement of the MEP pathway in linalyl acetate biosynthesis. In grey, carbons predicted to be unlabeled; in blue, carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis.

The mixed origin of the sesquiterpene β-caryophyllene

Sclareol and linalyl acetate are major compounds produced by clary sage calyces (Figure 26), but calyx organic extracts also contain minor compounds such as the sesquiterpenes germacrene D and β-caryophyllene [START_REF] Schmiderer | Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae)[END_REF]. The abundance of these minor compounds is too low for them to generate a signal in 13 C-NMR, but β-caryophyllene 13 Clabeling was successfully analyzed by GC-MS. 13 C-labeling was estimated through the analysis of isotope cluster distortion induced by the incorporation of 13 C atoms. The shapes of two isotope clusters of β-caryophyllene mass spectrum and three isotope clusters of linalyl acetate mass spectrum were evaluated by calculating the ratio of heavy ions peak areas over light ion peak area. As expected, feeding inflorescence stems with 1-13 C-glucose leads to a distortion of β-caryophyllene and linalyl acetate isotope clusters in favor of heavy ions: the distortion ratio is multiplied by a factor 2 to 5 depending on considered cluster (Figure 32). This result indicates that 1-13 C-glucose has been metabolized by plant cells and progressively incorporated into β-caryophyllene and linalyl acetate, although this observation alone does not provide information about which metabolic pathways have been involved in the production of each metabolite. Interestingly, terpene biosynthesis inhibitors have contrasted effects on the distortion of β-caryophyllene and linalyl acetate isotope clusters. Unlike mevinolin, fosmidomycin almost completely abolishes the distortion of linalyl acetate isotope clusters induced by 1-13 C-glucose feeding (Figure 32a). This result is in agreement with 13 C-NMR data and demonstrates that linalyl acetate biosynthesis essentially depends on IPP and DMAPP production by the MEP pathway. By contrast, fosmidomycin and mevinolin both reduce the distortion of β-caryophyllene isotope clusters, suggesting that both MVA and MEP pathways contribute to the production of IPP and DMAPP required for β-caryophyllene biosynthesis (Figure 32b).

Figure 32: Estimation of linalyl acetate and β-caryophyllene 13 C-labeling after feeding with 1-13 Cglucose and terpenoid biosynthesis inhibitors. Clary sage cut inflorescence stems were fed with a solution containing 1-13 C-glucose (Labeled) or standard glucose (Unlabeled) with 100µM of mevinolin (MEV), 100µM of fosmidomycin (FOS) or no inhibitor (Control) for 5 days. Linalyl acetate and β-caryophyllene 13 C-labeling was estimated by GC-MS analysis of isotope cluster distortion induced by the incorporation of 13 C atoms. Distortion ratio was calculated by dividing the sum of heavy ions peak areas by light ion peak area. Bars correspond to the average of distortion ratio measurements performed on 3 biological replicates and error bars correspond to standard deviation.

Biosynthetic origin of mono-, sesqui-and diterpenes in clary sage and other plants

Isotope labeling strategies are powerful tools to study metabolic fluxes and have been successfully employed to characterize the metabolism of many plant species [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF]. Notably, the specific labeling patterns obtained on terpenoids after labeling with 1-13 C-glucose are useful to analyze the contribution of MVA and MEP pathways to the production of terpenoids of interest (Figure 28). Here, we employed this strategy to assess the biosynthetic origin of 3 terpenoids produced by clary sage calyces: the diterpene sclareol, the sesquiterpene β-caryophyllene and the monoterpene linalyl acetate.

The strong impact of a treatment with the MEP pathway inhibitor fosmidomycin already suggested a strong involvement of the MEP pathway in sclareol and linalyl acetate biosynthesis (Figure 27). However, one could argue that the drop in sclareol and linalyl acetate content is due to a decrease in photosynthetic activity caused by the inhibition of carotenoid synthesis by fosmidomycin [START_REF] Lipko | Isoprenoid generating systems in plants -A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process[END_REF]. The feeding of clary sage inflorescence stems with 1-13 C-glucose together with fosmidomycin rules out this hypothesis and clearly demonstrates that sclareol and linalyl acetate both originate from the plastidial MEP pathway (Figure 31), like the majority of plant monoterpenes and diterpenes [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF]. These results are consistent with the plastidial localization of clary sage terpene synthases involved in sclareol biosynthesis [START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. Isotope labeling experiments had previously shown that linalyl acetate is MEP-derived in lemon mint (Mentha citrata) [START_REF] Fowler | Plant terpene biosynthesis. The biosynthesis of linalyl acetate in Mentha citrata[END_REF]. Comparable results were obtained for other monoterpenes and diterpenes in other species of the Lamiaceae family. In Salvia divinorum, the same labeling strategy based on the use of 1-13 C-glucose demonstrated that the diterpene salvinorin A is MEP-derived [START_REF] Kutrzeba | Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway[END_REF]. In spike lavender (Lavandula latifolia), the main monoterpenes contained in the essential oil, 1,8-cineole and camphor, both originate from the MEP pathway, as illustrated by pulse-chase experiments based on 13 CO 2 labeling [START_REF] Mendoza-Poudereux | Dynamics of Monoterpene Formation in Spike Lavender Plants[END_REF].

Sesquiterpenes are usually MVA-derived. However, the GC-MS analysis of isotope cluster distortion after labeling with 1-13 C-glucose suggests that in clary sage calyces, the sesquiterpene β-caryophyllene is of mixed origin (Figure 32). A similar GC-MS analysis was previously employed to investigate the contribution of MVA and MEP pathways to the biosynthesis of mono-and sesquiterpenes in snapdragon flowers [START_REF] Dudareva | The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers[END_REF].

Other plants produce sesquiterpenes with IPP stemming from both MVA and MEP pathways, like for example the artemisinin-producing plant Artemisia annua [START_REF] Towler | Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways[END_REF]. The contribution of the plastidial MEP pathway to the biosynthesis of a sesquiterpene in the cytosol suggests that the MEP pathway is highly active in glandular trichomes of clary sage calyces, although these epidermal structures have been reported to be nonphotosynthetic in other Lamiaceae species like mint [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. The analysis of clary sage glandular trichome ultrastructure by transmission electron microscopy would provide useful information about the structure-function adaptation of secretory cells. In particular, it would help to determine whether glandular trichome plasts are functional chloroplasts like in tomato type VI capitate trichomes [START_REF] Balcke | Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites[END_REF], or nonphotosynthetic leucoplasts similar to those of mint peltate trichomes [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. If clary sage glandular trichomes are not photosynthetic, carbon assimilates must be transported from neighboring cells to glandular trichomes to supply MEP pathway activity. A recent study demonstrated that although being photosynthetic, tomato type VI glandular trichomes rely on leaf sucrose to maintain their high metabolic activity [START_REF] Balcke | Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites[END_REF].

This conclusion emerged from the comparison of 13 C incorporation in primary metabolites of leaves and isolated type VI glandular trichomes during 13 CO 2 labeling. The same approach could be employed on clary sage to analyze the contribution of non-glandular cells sucrose to glandular trichome metabolism.

Metabolic engineering of terpene production in plants

Different strategies can be conceived in order to increase the production of a compound of interest in plant cells. A first category of approaches attempts to increase the amount of substrate available for the biosynthesis of the molecule of interest by removing bottlenecks in upstream pathways [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF]. Suppressing competition by other metabolic pathways using the same substrate is another way to reach the same goal (Lu et al., 2016;[START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF]. Such strategies have been developed in heterologous terpene production platforms like Nicotiana benthamiana [START_REF] Reed | Engineering terpenoid production through transient expression in Nicotiana benthamiana[END_REF], but significant results have also been obtained directly in species naturally producing the compound of interest, like Artemisia annua for the anti-malarial compound artemisinin [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF].

Increasing the production of prenyl intermediates

When the metabolite of interest is a diterpene, a promising metabolic engineering approach is to attempt to enlarge the pool of GGPP, the universal precursor of diterpene biosynthesis.

Strategies aiming at increasing GGPP biosynthesis have been tested in different plant systems with contrasted results. In some cases, the overexpression of a GGPP-synthase alone was enough to enhance diterpene biosynthesis, for example tanshinone biosynthesis in Salvia miltiorrhiza hairy roots [START_REF] Kai | Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures[END_REF]. By contrast, in Nicotiana benthamiana, the overexpression of a GGPP-synthase in addition to a cembatrien-ol synthase did not increase cembatrien-ol content compared to plants overexpressing the cembatrien-ol synthase alone, suggesting that GGPP-synthase activity is not limiting [START_REF] Brückner | High-level diterpene production by transient expression in Nicotiana benthamiana[END_REF]. In such cases, enlarging the IPP and DMAPP pool may be necessary.

The majority of plant diterpenoids being mainly MEP-derived, efforts in enlarging the IPP and DMAPP pool available for diterpenoid production have historically focused on increasing the activity of the MEP pathway rather than the MVA pathway. Enzymes catalyzing the first steps of the MEP pathway, DXS and DXR, are often reported as rate-limiting [START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF].

For this reason, enhancing their activity appears to be a good strategy to increase IPP and DMAPP production by the MEP pathway. In Salvia miltiorrhiza hairy roots, overexpression of DXS or DXR could enhance tanshinone content by up to 5-fold [START_REF] Kai | Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures[END_REF][START_REF] Shi | Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase[END_REF]. In Nicotiana benthamiana, the overexpression of DXS2 in addition to a cembatrien-ol synthase did not increase cembatrien-ol content compared to plants overexpressing the cembatrien-ol synthase alone. However, the overexpression of DXS2 and a GGPP-synthase together with the cembatrien-ol synthase led to a 3.5-fold increase of cembatrien-ol content [START_REF] Brückner | High-level diterpene production by transient expression in Nicotiana benthamiana[END_REF]. In Vitis vinifera, a specific DXS allele with better catalytic activity is responsible for the distinct aroma of Muscat cultivars, due to the higher monoterpene content of berries [START_REF] Battilana | Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation[END_REF]. The overexpression of the muscat allele of DXS1 increased monoterpene production in microvine (Dalla [START_REF] Costa | Induction of Terpene Biosynthesis in Berries of Microvine Transformed with VvDXS1 Alleles[END_REF].

Enhanced MVA pathway activity can also positively impact MEP-derived terpene biosynthesis. In Lavandula latifolia, the overexpression of HMGR increased the biosynthesis of 1,8-cineole and camphor, the major monoterpenes contained in the essential oil:

camphor and 1,8-cineole contents were enhanced by 30% and 18%, respectively [START_REF] Mendoza-Poudereux | Dynamics of Monoterpene Formation in Spike Lavender Plants[END_REF]. Surprisingly, both metabolites remained produced with IPP stemming only from the MEP pathway, suggesting that the MVA pathway affects monoterpene production otherwise than by exporting prenyl intermediates to plastids [START_REF] Mendoza-Poudereux | Dynamics of Monoterpene Formation in Spike Lavender Plants[END_REF]. This crosstalk may be due to protein prenylation with MVA-derived prenyl groups, which could positively regulate the expression of monoterpenoid biosynthesis genes as previously evidenced in Catharanthus roseus cell cultures [START_REF] Courdavault | CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathwayin Catharanthus roseus cells[END_REF].

Suppressing competing pathways

Another way to increase the amount of substrate available for the biosynthesis of the compound of interest is to eliminate competition by pathways using the same substrate. In Artemisia annua, several metabolic pathways compete with the biosynthesis of artemisinin.

For example, the biosynthesis of three other sesquiterpenes, β-caryophyllene, β-farnesene and germacrene A use the same MVA-derived substrate FPP. Downregulation of a βcaryophyllene synthase, a β-farnesene synthase or a germacrene A synthase by RNA interference increased artemisinin content by 77%, 77% and 103%, respectively [START_REF] Lv | Branch Pathway Blocking in Artemisia annua is a Useful Method for Obtaining High Yield Artemisinin[END_REF]. Reducing the flux through a competing pathway has also proved to be an efficient strategy to enhance the production of MEP-derived terpenoids. For example, in sweet potato, lutein biosynthesis competes with the biosynthesis of β-carotene for the use of the same MEP-derived substrate, lycopene. To increase β-carotene production, the enzyme catalyzing the first step of lutein biosynthesis from lycopene, the lycopene ε-cyclase, was downregulated through RNA interference. Lutein content was reduced to undetectable levels in sweet potato transgenic calli, while β-carotene content was approximately 21-fold higher [START_REF] Kim | Downregulation of the lycopene ϵ-cyclase gene increases carotenoid synthesis via the β-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli[END_REF]. These results highlight the potential of blocking competing pathways to enhance the yield of a compound of interest.

Perspectives for clary sage metabolic engineering

Increasing MEP pathway activity

Since sclareol is clearly MEP-derived in clary sage calyces (Figure 31), enhancing MEP pathway activity may be a straightforward approach to increase sclareol content. This strategy has already been employed successfully to enhance the production of abietane diterpenes by clary sage roots [START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF]. The ectopic expression of AtDXS and AtDXR genes in hairy roots led to a 2.2-fold and 3.3-fold increase in abietane diterpene content, respectively. Some hairy root lines overexpressing AtDXS also grew significantly slower. However, this negative effect on growth is not due to increased abietane diterpene accumulation, because AtDXR overexpression led to even higher abietane diterpene levels without any adverse effect on root growth. Some lines overexpressing AtDXR even grew faster than the control, thereby combining higher abietane diterpene content and improved growth [START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF].

Increasing MVA pathway activity

At first sight, blocking the MVA pathway appears to be a better strategy than enhancing it, because it would block sesquiterpene biosynthesis, which theoretically competes with sclareol biosynthesis for carbon assimilates. However, this strategy would probably be inefficient for two reasons. First, in clary sage calyces, sesquiterpenes are produced in very low amounts compared to sclareol (Figure 26), and thus divert very few carbon precursors away from the MEP pathway and sclareol biosynthesis. Secondly, MVA and MEP pathways rather cooperate than compete, due to the existence of metabolic crosstalks [START_REF] Hemmerlin | A raison d'être for two distinct pathways in the early steps of plant isoprenoid biosynthesis?[END_REF][START_REF] Liao | The potential of the mevalonate pathway for enhanced isoprenoid production[END_REF]. Therefore, boosting MVA pathway activity may also elevate sclareol content. In Salvia miltiorrhiza hairy roots, HMGR overexpression increased the biosynthesis of the presumably MEP-derived diterpene tanshinone by up to 3-fold [START_REF] Kai | Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures[END_REF][START_REF] Shi | Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase[END_REF]. Moreover, an even higher elevation of tanshinone content (by up to 5-fold) was obtained by co-overexpressing HMGR together with DXR [START_REF] Shi | Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase[END_REF],

indicating that strategies based on MEP or MVA pathway enhancement can be successfully combined.

Blocking monoterpene biosynthesis

In addition to being both MEP-derived (Figure 31), the monoterpene linalyl acetate and the diterpene sclareol are mainly produced in the same cells of clary sage calyces: glandular trichome secretory cells (see Chapter 1). Hence, their respective biosynthesis pathways are in competition for the use of the same IPP and DMAPP pool produced by the MEP pathway in glandular trichomes plasts. Reducing or blocking the biosynthesis of linalyl acetate may therefore increase sclareol production, because IPP and DMAPP not converted to linalyl acetate would be available for sclareol biosynthesis.

In lemon mint (Mentha aquatica var. citrata, Lamiaceae), linalyl acetate is obtained after acetylation of the monoterpene linalool, which is produced in one step from GPP through the action of a linalool-synthase [START_REF] Zaks | Biosynthesis of linalyl acetate and other terpenes in lemon mint (Mentha aquatica var. citrata, Lamiaceae) glandular trichomes[END_REF]. To our knowledge, no linalool acetyltransferase gene was identified yet, whereas several linalool-synthase genes were characterized in Lamiaceae species including lavender [START_REF] Landmann | Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia)[END_REF] and sweet basil [START_REF] Iijima | The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil[END_REF]. Clary sage genes homologous to these linalool-synthase genes probably encode enzymes responsible for linalool biosynthesis in calyces. The suppression of linalool-synthase activity would lead to GPP accumulation. The accumulation of its product may in turn inhibit GPP-synthase activity, thereby increasing the amount of IPP and DMAPP available for GGPP biosynthesis. However, multiple linalool-synthase isoforms might be functional in calyx glandular trichomes. In that case, the reduction of linalool-synthase activity would require the inactivation of several, if not all, linalool-synthase isoforms, otherwise remaining isoforms would probably complement the loss of function. Moreover, in absence of linalool-synthase activity, other terpene synthases may convert GPP into other monoterpenes, again diverting prenyl intermediates away from sclareol biosynthesis.

A way to block the biosynthesis of all monoterpenes would be to suppress GPP-synthase activity. In Mentha piperita and Antirrhinum majus, two species relatively closely related to Salvia sclarea, GPP-synthases are heteromeric, comprising a large catalytic subunit and a small regulatory subunit [START_REF] Burke | Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer[END_REF][START_REF] Tholl | Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases[END_REF]. In absence of the regulatory subunit, the catalytic subunit produces GGPP instead of GPP [START_REF] Burke | Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate[END_REF]. If clary sage GPP-synthases are also heteromeric, inactivating regulatory subunits would at the same time repress GPP biosynthesis and increase GGPP-synthase activity, because catalytic subunits would display GGPP-synthase activity and work together with authentic GGPP-synthases. Alternatively, inactivating catalytic subunits would also suppress GPP biosynthesis, thereby increasing the amount of IPP and DMAPP available for GGPP production by GGPP-synthases. As mentioned previously in the case of linalool-synthases, these strategies may be difficult to implement if multiple GPP-synthase isoforms are functional in calyx glandular trichomes, because the inactivation of several, if not all, GPPsynthase isoforms would be necessary. In any case, this approach requires the characterization of genes encoding clary sage GPP-synthase catalytic subunit(s) and putative regulatory subunit(s), and the identification of GPP-synthase isoform(s) active in calyx glandular trichomes. Transcriptomic data obtained from clary sage calyces are a precious resource for such analyses. Interestingly, only one GPP-synthase isoform was identified in the available clary sage calyx transcriptome, suggesting that only one GPP-synthase isoform is expressed in the calyx [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF]. If further gene expression analysis on isolated glandular trichomes confirms that only one GPP-synthase isoform is expressed in calyx glandular trichomes, the inactivation of this isoform would be a promising and straightforward strategy to redirect carbon fluxes towards sclareol biosynthesis.

Engineering increased sclareol secretion

If sclareol production is strongly induced, existing transport mechanisms responsible for sclareol secretion may not be sufficient to export all sclareol out of glandular trichome cells.

Sclareol may accumulate inside cells and impact glandular trichome productivity negatively.

Indeed, sclareol has been shown to display cytotoxic activity to animal an fungal cells, but also to plant cells, as evidenced in Arabidopsis thaliana [START_REF] Van Den Brûle | The ABC transporter SpTUR2 confers resistance to the antifungal diterpene sclareol[END_REF]. An efficient induction of sclareol biosynthesis may require the engineering of adapted transport systems. Today, the mechanism of sclareol secretion in clary sage remains elusive and may involve passive diffusion and/or active transport systems [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. A transporter belonging to the Pleiotropic Drug Resistance-like (PDR-like) subfamily of ATP-Binding Cassette (ABC) transporters was shown to export exogenous sclareol out of Arabidopsis thaliana cells [START_REF] Van Den Brûle | The ABC transporter SpTUR2 confers resistance to the antifungal diterpene sclareol[END_REF]. In Nicotiana plumbaginifolia, an ABC transporter of the same PDR-like subfamily is involved in plant defense and is more expressed in leaf glandular trichomes and flower petals [START_REF] Stukkens | NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense[END_REF]. If sclareol transport proves to be limiting, increasing the expression of PDR-like ABC transporters in clary sage may further enhance sclareol secretion by clary sage.

Chapter 3

Is sclareol biosynthesis regulated by jasmonates?

1. Introduction

Regulation of plant specialized metabolism by jasmonates

Jasmonates are phytohormones involved in the regulation of plant stress responses and other various developmental processes. For example, they induce the production of specialized metabolites upon herbivore feeding or attack by a necrotrophic pathogen [START_REF] Goossens | Jasmonates: signal transduction components and their roles in environmental stress responses[END_REF], but also play a role in primary root growth, flower development and leaf senescence [START_REF] Huang | Jasmonate action in plant growth and development[END_REF]. The jasmonate family of phytohormones includes jasmonic acid and its inactive and active forms, among which a volatile derivative, methyljasmonate (MeJA).

Endogenous jasmonic acid production by plant cells is induced by diverse biotic and abiotic stresses and developmental signals [START_REF] Wasternack | Jasmonates are signals in the biosynthesis of secondary metabolites -Pathways, transcription factors and applied aspects -A brief review[END_REF]. Jasmonic acid can undergo various chemical modifications including conjugation with amino acids and methylation. Jasmonic acid conjugation with isoleucine leads to the formation of the active form JA-Ile. JA-Ile is transported to the nucleus and specifically interacts with the SCF COI1 -JAZ co-receptor complex [START_REF] Goossens | Jasmonates: signal transduction components and their roles in environmental stress responses[END_REF]. JASMONATE ZIM DOMAIN (JAZ) proteins interact with positively-acting transcription factors to repress their action. SCF COI1 is an E3ubiquitin ligase complex which is part of the ubiquitin-proteasome degradation machinery and specifically targets JAZ proteins. Upon binding of JA-Ile, SCF COI1 mediates the degradation of JAZ proteins by the proteasome, thus removing the repression exerted by JAZ proteins. Positively-acting transcription factors are released and bind to JA responsive elements located in JA response gene promoters, thus triggering the expression of these genes [START_REF] Goossens | Jasmonates: signal transduction components and their roles in environmental stress responses[END_REF].

JA response genes include transcription factors which control the JA-induced accumulation of specialized metabolites. These regulators often belong to the AP2/ERF, bHLH, MYB and WRKY transcription factor families [START_REF] Zhou | Jasmonate-responsive transcription factors regulating plant secondary metabolism[END_REF]. For example, in Catharanthus roseus, monoterpene indole alkaloid production is controlled by AP2/ERF transcription factors of the ORCA family and by the bHLH transcription factors BIS1 and BIS2, which are all JA-inducible [START_REF] Wasternack | Jasmonates are signals in the biosynthesis of secondary metabolites -Pathways, transcription factors and applied aspects -A brief review[END_REF]. In Artemisia annua, the biosynthesis of the sesquiterpene artemisinin is activated in response to JA through the action of the bHLH transcription factor MYC2 and also the WRKY transcription factor GSW1.

GSW1 is expressed specifically in glandular trichomes, which are the site of artemisinin production in Artemisia annua [START_REF] Shen | The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua[END_REF]Chen et al., 2017b).

JA-triggered accumulation of specialized metabolites is primarily achieved through the induction of biosynthetic gene expression. In the case of JA-inducible terpenoids, the expression of downstream pathway genes like terpene synthase genes is enhanced, as well as MVA or MEP pathway genes. For example, in Artemisia annua, JA increases the expression of a DXS gene, an FPP-synthase gene and a cytochrome P450 gene, which respectively encode the first enzyme of the MEP pathway, the enzyme producing FPP (the linear precursor of artemisinin) and an enzyme involved in the conversion of FPP to artemisinin [START_REF] Maes | Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants[END_REF]. When specialized metabolites are produced in glandular trichomes, JA can also impact the yield of a compound of interest through the induction of glandular trichome development, as demonstrated in Artemisia annua [START_REF] Maes | Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants[END_REF] and tomato [START_REF] Chen | Type VI glandular trichome density and their derived volatiles are differently induced by jasmonic acid in developing and fully developed tomato leaves: Implications for thrips resistance[END_REF].

How to use jasmonate signaling to enhance the production of compounds of interest

Many terpenoids exploited in industry are specialized metabolites produced by plants in response to biotic and abiotic stresses. Strategies based on the induction of plant stress responses have therefore been designed to increase the biosynthesis of terpenoids of interest in plant systems [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF]. Genes involved in the same biosynthetic pathway are often co-regulated. Hence, manipulating biosynthesis pathway regulation offers the possibility to upregulate all biosynthetic genes together in appropriate proportions, a goal which is more difficult to reach when targeting biosynthetic enzymes one by one (Wang et al., 2016).

Methyljasmonate (MeJA) is a volatile derivative of jasmonate which activates JA signaling after conversion to JA-Ile in plant cells. Commercial MeJA is often used as an elicitor to enhance the biotechnological production of terpenes of interest in plant systems [START_REF] Lu | Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids[END_REF]. For example, MeJA has been successfully employed at industrial scale to increase the production of the anti-cancer molecule paclitaxel (Taxol®) in Taxus cuspidata cell cultures.

The combination of MeJA elicitation and cell culture optimization methods led to high paclitaxel titers of up to 900 mg/L, in spite of the negative impact of MeJA on cell growth [START_REF] Patil | Methyl jasmonate represses growth and affects cell cycle progression in cultured Taxus cells[END_REF].

Other strategies aiming at increasing terpenoid production employ genetic modifications to enhance JA signaling. In tomato, constitutive activation of JA signaling was achieved through the overexpression of the precursor protein of systemin, a peptide hormone known to activate JA signaling in tomato, and led to the accumulation of the phenylpropanoidpolyamine conjugate caffeoyl putrescine [START_REF] Chen | Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato[END_REF]. Different components of JA signaling have also been targeted to enhance the production of JA-inducible terpenoids. In Salvia miltiorrhiza hairy roots, the production of the diterpene tanshinone was increased by up to 2.5-fold through downregulation of JAZ repressors [START_REF] Shi | Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins[END_REF]. In Catharanthus roseus cells, overexpression of the JA-inducible transcription factors ORCA4, BIS1 or BIS2 led to an increase in monoterpene indole alkaloid content [START_REF] Wasternack | Jasmonates are signals in the biosynthesis of secondary metabolites -Pathways, transcription factors and applied aspects -A brief review[END_REF].

Artemisinin content of Artemisia annua plants was enhanced by up to 50% through the overexpression of either of the AP2/ERF transcription factors AaERF1, AaERF2 or AaORA1 [START_REF] Yu | The Jasmonate-Responsive AP2/ERF Transcription Factors AaERF1 and AaERF2 Positively Regulate Artemisinin Biosynthesis in Artemisia annua L[END_REF][START_REF] Lu | AaORA , a trichome-specific AP2/ERF transcription factor of Artemisia annua , is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea[END_REF], or through the overexpression the bHLH transcription factor AaMYC2 [START_REF] Shen | The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua[END_REF]. Moreover, the overexpression of the WRKY transcription factor AaWRKY1 could increase artemisinin content by 80% [START_REF] Han | Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants[END_REF].

Regulation of clary sage specialized metabolism by jasmonates: state of the art

Several studies demonstrate a positive impact of JA signaling on specialized metabolite content in different clary sage aerial and underground organs. Increased levels of metabolites belonging to the phenylpropanoid family (caffeic acid, rosmarinic acid, luteolin and apigenin) were detected in clary sage leaves 3 days after spraying with MeJA at a concentration of 7.5 mM [START_REF] Hao | Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea[END_REF]. A limited, but significant increase in rosmarinic acid content was also visible 6 days after spraying with MeJA at a concentration of 0.75 mM.

Besides, clary sage hairy roots treated with MeJA produced higher levels of abietane diterpenes (aethiopinone, 1-oxoaethiopinone, salvipisone and ferruginol) (Kuźma et al., 2009a;[START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF]. The strongest induction was observed for aethiopinone content, which could be enhanced by up to 25-fold [START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF]. The increase in abietane diterpene content was strongly correlated to enhanced transcription of biosynthetic genes, including MEP pathway genes (especially DXS2 and DXR) and GGPPsynthase genes [START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF]. Interestingly, MeJA treatment could elevate total abietane diterpene content by more than 20-fold, i.e. to much higher levels than AtDXS and AtDXR ectopic expression in hairy roots, which only led to a 2 to 3-fold increase in abietane diterpene content [START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF][START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF].

Abietane diterpene content in clary sage hairy roots was recently increased by ectopic expression of the JA-responsive transcription factors AtMYC2, AtWRKY18 or AtWRKY40 [START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF]. Ectopic expression of each of these transcription factors enhanced the transcription of MEP pathway genes, suggesting that JA stimulates abietane diterpene production partially through increasing substrate supply [START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF]. Best results

were obtained through the ectopic expression of AtMYC2, which increased total abietane diterpene content by more than 5-fold. This lower increase compared to results obtained by direct MeJA treatment could be explained by the fact that MeJA enhances abietane diterpene production through the upregulation of more than one transcription factor [START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF].

Taken together, results obtained on clary sage hairy roots highlight the potential of JA signaling manipulation to engineer higher terpenoid production in clary sage. However, the impact of JA signaling on terpenoid production in clary sage aerial organs, including calyces, has not been assessed so far. Here, we treated clary sage inflorescences with MeJA in order to determine whether sclareol biosynthesis in calyces is regulated by JA.

Materials and methods

Calyx development stages

Early developmental stages were defined according to the size of the bud: stage 1 corresponds to a small bud (less than 3 mm long) and stage 2 corresponds to a large bud (more than 4 mm long). Later developmental stages were defined according to the appearance of the corolla and the calyx. At stage 3, calyx has opened but the corolla is still closed; at stage 4, calyx and corolla have both opened; at stage 5, corolla is wilted or has fallen; and at stage 6, the calyx has completely dried (Figure 33a).

MeJA treatment by feeding

Methyljasmonate (MeJA) was purchased from Sigma-Aldrich and stock solutions were prepared in ethanol. Nutritive solution containing 0 µM, 20 µM, 100 µM, 500 µM, 1 mM, 3 mM, 5 mM or 7.5 mM of MeJA was prepared in 5 mL vials (Murashige and Skoog basal salt mixture (Sigma-Aldrich); sucrose 2% m/v (Euromedex); PPM TM 0.1% v/v (Plant Cell Technology); ethanol 0.4% v/v). Clary sage inflorescence axillary stems were cut between the 3 rd and the 4 th node (about 10 cm from the apex). Each stem was immediately placed in a vial in a way that its extremity was touching the bottom of the vial and dipped in nutritive solution. Treatment was performed by leaving inflorescence stems on the bench for 2 or 6 days. At the end of the treatment, one mature calyx (stage 4 or 5) was sampled from each inflorescence stem (at the 2 nd node, starting from the base), immediately frozen in liquid nitrogen and stored at -80 °C before sclareol and linalyl acetate quantification by GC-MS.

MeJA treatment by spraying

Spraying solution containing 3 mM of MeJA was prepared in water. Ethanol was added to the control solution to reach the same concentration as in the solution containing MeJA (0.4% v/v). All aerial parts of clary sage plants were sprayed with control or MeJA solution, including rosette leaves, calyces and inflorescence stems. 75 mL of spraying solution were sprayed on one plant in order to saturate the surface of all aerial organs with solution. Plants were placed in the shade during the evaporation of the spraying solution. After 2 days, mature calyces (stage 4 or 5) were sampled from each plant, immediately frozen in liquid nitrogen and stored at -80 °C before sclareol and linalyl acetate quantification by GC-MS.

Sclareol and linalyl acetate quantification by GC-MS

Clary sage calyces were immersed in hexane without grinding to extract metabolites present at the surface. 1.5 mL of solvent was used to extract metabolites from 5-6 calyces at stage 1, 2 calyces at stage 2 or 1 calyx at stage 3, 4, 5 or 6. The extraction was performed without removing the developing corolla from the bud at stages 1 and 2 and without removing achenes at stages 5 and 6. 10-undecen-1-ol was employed as internal standard and was added in the solvent before extraction at a concentration of 0.75 mM. After 2 h of agitation, 1 mL of extract was centrifuged at 13,000 rpm for 10 min to eliminate potential dust or debris. Samples were directly analyzed by gas chromatography coupled to mass spectrometry (GC-MS) using a 7890B/5977A GC-MS system from Agilent and according to a protocol adapted from Laville et al. [START_REF] Laville | Amphilectane diterpenes from Salvia sclarea: Biosynthetic considerations[END_REF]. The system was equipped with a 10 m guard column and an Rxi-5Sil MS column (length, 30 m; inner diameter, 0.25 mm; film thickness, 0.25 μm) (Restek, Bellefonte, PA, USA). 1 µl of sample was injected with a split ratio of 30:1. Oven temperature was set to 110 °C for 2 min, then increased to 270 °C at a rate of 10 °C/min, and finally set to 270 °C for 2 min. Other temperatures were set as follows: injector, 270 °C; transfer line, 270 °C; source, 230 °C; quadrupole, 150 °C. The carrier gas was helium at a constant flow of 1 mL/min. The quadrupole mass spectrometer was switched on after a solvent delay of 5 min and was programmed to scan from 35 u to 350 u.

Data analysis was carried out using the MassHunter Quantitative Analysis software from Agilent. Sclareol and linalyl acetate were identified by comparison with analytical standards purchased from Sigma-Aldrich. Absolute quantification of sclareol and linalyl acetate was performed using a calibration curve prepared with analytical standards. Calyx dry weight was measured after lyophilization of dry material remaining after extraction. For dry weight measurements, achenes were removed from stage 6 calyces, but not from stage 5 calyces (because of their very small size at this stage, their mass was considered negligible).

Results

Regulation of sclareol biosynthesis during calyx development

Calyces have been shown to be the organs of clary sage where sclareol accumulates at highest levels [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF], but the dynamic of sclareol production during calyx development has not been investigated yet. To explore the temporal dimension of sclareol accumulation in the calyx, sclareol content was quantified by GC-MS at different stages of calyx development. Linalyl acetate, another major metabolite produced by the calyx, was quantified at the same time (Figure 33).

Sclareol and linalyl acetate production starts early during calyx development as these metabolites are already detectable at stage 1 (Figure 33c), where they already represent respectively 7.5% and 4.5% of calyx dry mass (Figure 33d). Metabolite content then gradually increases until reaching its maximum at stage 5, where sclareol and linalyl acetate are present in comparable amount (Figure 33c), each of them accounting for about 6% of calyx dry mass (Figure 33d). A decrease in metabolite content is observed at stage 6 (Figure 33c) whereas calyx dry weight at stage 6 is higher than at stage 5 (Figure 33b). The decrease in linalyl acetate content can be explained by an evaporation of this volatile molecule. Given its high boiling point (398 °C), sclareol is unlikely to have evaporated, but sclareol crystals formed during calyx drying may have fallen because of the wind. Consequently, stage 6 was not considered to be the best stage to measure metabolite content and stage 5 was used instead. Hexane extraction was performed without removing the developing corolla from the bud at stages 1 and 2 and without removing achenes at stages 5 and 6. To measure dry weight, achenes were removed from stage 6 calyces, but not from stage 5 calyces (because of their very small size at this stage, their mass was considered negligible). MeJA [START_REF] Tamogami | Methyl jasmonate is transported to distal leaves via vascular process metabolizing itself into JA-Ile and triggering VOCs emission as defensive metabolites[END_REF]. Thus, we could reasonably expect that in our experiment, MeJA provided in the feeding solution would be transported to clary sage calyces through the inflorescence stem vasculature, and that it may impact calyx metabolism. MeJA concentration in the feeding solution was 7.5 mM, the same concentration as that used for leaf spraying by [START_REF] Hao | Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea[END_REF]. Inflorescence stems were fed with MeJA during 6 days in order to let enough time for the youngest calyces to fully develop from bud stage to mature stage.

After 6 days, cut inflorescence stems fed with 7.5 mM of MeJA displayed a brown color and were wilted, whereas control stems were still green (Figure 34). During other feeding experiments with solutions containing glucose or terpene inhibitors, cut inflorescence stem length visibly increased, indicating that internode elongation is to some extent maintained during the feeding. By contrast, after 6 days of feeding, stems fed with MeJA were clearly shorter than control stems, suggesting that the presence of MeJA in the feeding solution led

to an interruption of stem growth during the feeding (Figure 34). The browning of organs (stem, bracts and calyces) located at the apex supports our initial assumption that MeJA is able to reach calyces and to induce a response in calyx cells after transport through the vasculature (Figure 34, white arrows). However, some calyces that were already mature at the beginning of the feeding remained green, and some calyces that were still at bud stage at the beginning of the feeding did not get brown (Figure 34, black arrows), underlying the possibility that all calyces may not receive the same amount of MeJA. In general, all calyces located at the second node (starting from the base) browned, except very young ones.

Consequently, in further feeding experiments, sampling was performed among mature calyces located at the second node. Protocol optimization tests were carried out in order to find milder treatment conditions that could potentially impact calyx metabolism without impairing inflorescence stem growth and fitness. Browning and wilting of inflorescence stems is still observed after 6 days of feeding with 3 or 5 mM of MeJA, whereas inflorescence stems fed with only 1 mM of MeJA for 6 days remain green and turgescent like control stems. Therefore, 2 independent 6-dayfeeding experiments with MeJA concentrations lower than 1 mM were conducted. Five different MeJA concentrations were tested in order to detect a potential dose-dependent effect of MeJA. Sclareol and linalyl acetate content was quantified by GC-MS and the 2 experiments yielded the same results (results of the second experiment are shown in Figure 35a): no impact of MeJA was detected on sclareol and linalyl acetate content.

Concentrations tested may be too low to trigger any modification of calyx metabolism.

Further optimization tests were conducted, with higher MeJA concentrations while reducing the length of the treatment. Inflorescence stems fed with 5 mM of MeJA were completely brown and wilted in less than 48h, whereas browning and wilting started only after 3 days with 3 mM of MeJA. Therefore, a 2-day-feeding experiment with 3 mM of MeJA was performed. Again, no impact on sclareol and linalyl acetate content was detected by GC-MS analysis (Figure 35b).

6-day-feeding

A B C

MeJA treatment on flowers by spraying

A treatment protocol closer to that used by Hao et al. on leaves (Hao et al., 2015) was tested on the inflorescence. Inflorescence stems were sprayed with a solution containing MeJA without separating the stems from the rest of the plant. In order to avoid any impact on calyx growth and fitness, a concentration of 3 mM was used (instead of 7.5 mM in the work of Hao et al.) and calyces were sampled 48h after spraying (instead of 3 days in the work of Hao et al.) for GC-MS analysis. As expected, no browning and wilting was observed on floral organs. Again, no modification of sclareol and linalyl acetate content was detected with this protocol (Figure 35c).

To interpret the results of this last experiment and of the 2-day-feeding, it is important to note that all GC-MS analyses were performed on calyces at mature stage (stage 4 or 5). At the beginning of 2-day experiments (spraying or feeding), calyces intended to be sampled were already almost at mature stage (late stage 3 or stage 4). At this stage, in normal conditions, sclareol content has almost reached its maximum (Figure 33c). Since sclareol accumulates at the surface of the calyx, a modification of sclareol production rate between late stage 3 and stage 5 may have a limited impact on final sclareol content.

Discussion

MeJA apparently does not impact sclareol production, but further protocol optimization is needed

The phytohormone MeJA has already been shown to induce abietane diterpene biosynthesis in clary sage hairy roots (Kuźma et al., 2009a;[START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF][START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF] and phenylpropanoid production in clary sage leaves [START_REF] Hao | Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea[END_REF]. For these reasons, we hypothesized that MeJA may increase terpenoid production in clary sage calyces, including sclareol biosynthesis. The treatment protocols that we employed to test this hypothesis do not highlight any clear impact of MeJA on sclareol and linalyl acetate content in clary sage calyces (Figure 35). Sclareol and linalyl acetate biosynthesis may be insensitive to jasmonate signaling. Alternatively, sclareol and linalyl acetate biosynthesis might be controlled by jasmonates, but the experimental design we used may not be appropriate to demonstrate the role of these phytohormones. Further optimization of treatment and sampling protocols may be necessary to highlight an impact of MeJA on sclareol and linalyl acetate production.

We tested two types of treatment protocol: cut inflorescence stem feeding with nutritive solution containing MeJA, and whole plant spraying with MeJA dissolved in water. Whole plant spraying is closer to physiological conditions compared to the stress caused by stem cutting. However, it is difficult to spray a reproducible amount of MeJA solution on each part of the plant, while inflorescence stem feeding requires less plant material and is easily reproduced. Unfortunately, feeding with MeJA had a negative impact on cut inflorescence stem growth and fitness (Figure 34). This result is not surprising, given the well reported negative effect of jasmonate signaling on plant growth and its ability to induce leaf senescence [START_REF] Huang | Jasmonate action in plant growth and development[END_REF]. Notably, MeJA inhibits plant cell division by inducing cell cycle arrest at G2 phase in Arabidopsis thaliana cell cultures [START_REF] Pauwels | Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells[END_REF]. The impact of MeJA on cut inflorescence stem growth and fitness was more or less marked depending on the concentration of MeJA in the nutritive solution and the duration of the treatment. This observation is consistent with studies conducted on clary sage hairy roots:

treatment with MeJA for 7 days had no visible impact on hairy root fitness, while a longer treatment of 28 days severely impaired growth [START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF].

Due to the strong effect of MeJA feeding on clary sage cut inflorescence stem growth and fitness, a milder treatment protocol based on whole plant spraying may be preferable to test the impact of MeJA on calyx specialized metabolism. This type of protocol was successfully employed on tomato to demonstrate the positive effect of jasmonate signaling on type VI glandular trichome density on leaves [START_REF] Boughton | Methyl Jasmonate Application Induces Increased Densities of Glandular Trichomes on Tomato, Lycopersicon esculentum[END_REF][START_REF] Chen | Type VI glandular trichome density and their derived volatiles are differently induced by jasmonic acid in developing and fully developed tomato leaves: Implications for thrips resistance[END_REF]. Glandular trichome density was significantly increased 7 days after spraying with a solution of MeJA at a concentration of 7.5 mM. Here, we treated clary sage plants with a solution containing 3 mM of MeJA and collected calyces two days after spraying. The concentration we used may be too low and two days between the start of the treatment and sampling may be too short for glandular trichome development to be induced. Repeated applications of MeJA at higher concentration and a longer period between the first treatment and sampling may be necessary to impact calyx specialized metabolism and glandular trichome initiation.

A recent study on tomato highlighted a differential impact of JA on defense chemical production in leaves, depending on the developmental stage of the leaf: the increase in defense chemical content was higher in developing leaves compared to fully developed leaves [START_REF] Chen | Type VI glandular trichome density and their derived volatiles are differently induced by jasmonic acid in developing and fully developed tomato leaves: Implications for thrips resistance[END_REF]. A more detailed analysis showed that in developing leaves, the increase in defense chemical content was mainly explained by a higher type VI glandular trichome density, whereas it was essentially due to a higher metabolic activity of type VI glandular trichomes in fully developed leaves. Taken together, these results indicate that the impact of MeJA may be different depending on the developmental stage of the considered organ. In our study, we analyzed the impact of MeJA treatment on mature calyces which were already almost at mature stage at the beginning of the treatment. In further experiments, it may be interesting to focus the analysis on young calyces (stage 2 to 3) instead, because the possible effect of MeJA may be more marked than in mature calyces.

At the start of the MeJA treatment, a certain amount of sclareol is already present at the surface of younger calyces as well as of mature ones (Figure 33). Variations in this initial quantity of sclareol may mask a minor, but significant impact of MeJA on the amount of sclareol produced after the start of the treatment. To distinguish newly produced sclareol from sclareol initially present on the calyx, MeJA treatment may be combined with stable isotope labeling. For example, clary sage plants could be sprayed with MeJA and then exposed to 13 CO 2 in a similar way as the protocol previously used on lavender plants [START_REF] Mendoza-Poudereux | Dynamics of Monoterpene Formation in Spike Lavender Plants[END_REF]. MeJA could also be applied in gaseous form, as previously performed on Arabidopsis thaliana [START_REF] Thomma | Disease development of several fungi on Arabidopsis can be reduced by treatment with methyl jasmonate[END_REF].

Other potential ways to manipulate sclareol biosynthesis regulation

Besides jasmonates, other phytohormones may play a role in sclareol biosynthesis regulation. The phytohormone abscisic acid (ABA), which plays diverse roles in plant development and adaptation to environment, has been shown to induce terpenoid production in several plants. For example, it stimulates lycopene and β-carotene accumulation in tomato fruits and increases artemisinin production in Artemisia annua (Lu et al., 2016). In the latter, ABA signaling has already been successfully engineered in order to enhance artemisinin production. The expression of the transcription factor AabZIP1 was shown to be induced upon ABA treatment, and its overexpression led to a 2.5-fold increase in artemisinin content (Zhang et al., 2015b). The impact of phytohormones like ABA on sclareol production by clary sage calyces could be tested by feeding or spraying. If sclareol content is increased by ABA, homologs of AabZIP1 identified in clary sage available transcriptomes [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF][START_REF] Hao | Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea[END_REF] would be good candidates for an involvement in sclareol biosynthesis regulation.

Many regulators of plant specialized metabolism have been identified among transcription factors involved in plant stress response, but plant development regulators also participate in the spatiotemporal control of specialized metabolites biosynthesis [START_REF] Colinas | Combinatorial Transcriptional Control of Plant Specialized Metabolism[END_REF]. In Arabidopsis thaliana and rice, transcription factors of the YABBY family play diverse roles in plant development, but do not appear to regulate specialized metabolism (Wang et al., 2016). However, in spearmint (Mentha spicata), downregulation or upregulation of YABBY5 respectively increased or decreased the production of essential oil monoterpenes, indicating that YABBY5 is a negative regulator of terpenoid biosynthesis in this plant.

Interestingly, the ectopic expression of MsYABBY5 in another Lamiaceae (Ocimum basilicum), and in a Solanaceae (Nicotiana sylvestris) also decreased terpenoid biosynthesis, suggesting that the function of YABBY5 may be conserved in Lamiaceae and Solanaceae [START_REF] Wang | Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata)[END_REF]. Initially, the authors focused their study on YABBY5 because it is preferentially expressed in spearmint peltate glandular trichomes, which are responsible for the production of essential oil monoterpenes. Data available on genes expressed in glandular trichomes appear to be a precious resource for the identification of novel regulators of the biosynthesis of specialized metabolites produced by glandular trichomes. If a clary sage homolog of YABBY5 is more expressed in isolated glandular trichomes than in other calyx cells, it would be promising to decrease its expression and analyze the effect of this genetic modification on sclareol content.

Chapter 4

Clary sage natural diversity 1. Introduction

The use of natural genetic diversity for breeding

During the past century, agricultural practices caused a dramatic erosion of biodiversity in major crop plants like wheat or rice [START_REF] Kilian | NGS technologies for analyzing germplasm diversity in genebanks[END_REF]. The concomitant need for genetic improvement to enhance crop performance prompted the search for new genetic resources. Natural populations of wild species closely related to domesticated crop plants carry distinct alleles which can be introgressed into elite varieties. Huge seed collections of wild relatives of crop plants were established and rapid progress in molecular biology provided effective tools to tap into these genetic resources [START_REF] Kilian | NGS technologies for analyzing germplasm diversity in genebanks[END_REF].

Notably, it enabled the development of marker-assisted selection, whereby molecular markers are used for the indirect selection of traits of interest in crops [START_REF] He | Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding[END_REF].

Once markers have been associated with a trait of interest, breeding can be performed through selecting plants possessing these markers, instead of phenotyping the plants and selecting the ones presenting the desired phenotype. In recent years, next-generation sequencing technologies further increased the potential of genetic resources to enhance plant breeding programs [START_REF] He | Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding[END_REF]. For instance, Genome Wide Association Studies (GWAS) allow the identification of genetic loci involved in the quantitative variation of a trait of interest among a collection of populations. This approach is based on the use of a large set of genetic markers easily generated by high-throughput sequencing [START_REF] He | Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding[END_REF].

Deeper analysis of identified genetic loci can eventually lead to the characterization of genes regulating the variation of the trait of interest, thereby improving our understanding of plant development and physiology.

The raising global demand for sclareol currently encourages breeding initiatives aiming at enhancing clary sage productivity. In addition to sclareol content, flowering time is another important trait to take into account. Indeed, since sclareol is mainly produced in floral organs [START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF], the productivity of clary sage plants flowering late is potentially more impacted by summer drought. Early flowering is therefore a desirable trait.

The same idea has previously been suggested for the breeding of chamomile, a medicinal plant which also produces valuable terpenoids in flowers [START_REF] Otto | Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping[END_REF]. Wild Salvia sclarea populations found around the Mediterranean Sea represent a pool of potentially new alleles which could be introgressed into cultivated varieties to improve sclareol content and flowering time. However, the characterization of such alleles requires the identification of genetic loci involved in the quantitative variation of these traits of interest. If natural populations displaying distinct sclareol levels were identified, genetic loci impacting sclareol yield could be characterized using state-of-the-art technologies and approaches like GWAS.

Detailed study of these loci would be highly valuable to unravel regulatory mechanisms controlling sclareol production. As a first step towards this goal, we analyze here the sclareol content and flowering time of several wild populations of Salvia sclarea sampled in the region of Makarska in South Croatia (Figure 36).

Impact of environmental conditions on terpene production and genetic diversity

Specialized metabolite levels observed in plants result from the combination of physiological variations, environmental conditions and genetic factors [START_REF] Figueiredo | Factors affecting secondary metabolite production in plants: volatile components and essential oils[END_REF][START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Environmental conditions impacting metabolite content include abiotic factors, such as temperature and soil composition, and biotic factors, like the presence of specific pathogens. Altitude potentially influences plant specialized metabolism as a combination of abiotic and biotic factors. Indeed, temperature, hygrometry, wind speed and water availability change with altitude, promoting the development of distinct microbial and insect communities [START_REF] Mahzooni-Kachapi | The effects of altitude on chemical compositions and function of essential oils in stachys lavandulifolia vahl. (Iran)[END_REF][START_REF] Foutami | Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean[END_REF]. Higher elevation (3200 m or 2400 m instead of 1600 m above sea level) was shown to impact negatively the yield and quality of essential oil extracted from Stachys lavandulifolia, a species belonging to the Lamiaceae family [START_REF] Mahzooni-Kachapi | The effects of altitude on chemical compositions and function of essential oils in stachys lavandulifolia vahl. (Iran)[END_REF].

Plants harboring the same genome often have distinct chemical profiles if they are submitted to different growth conditions, due to up-or down-regulation of biosynthesis pathways in response to these different environments [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Over evolutionary time scales, the selective pressure exerted by environmental factors may lead to the selection of new genotypes determining a better chemical adaptation of the plant to its environment [START_REF] Foutami | Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean[END_REF]. Genetic mutations selected through this evolution process can determine the presence or absence of a metabolite, but in the large majority of cases, they are responsible for quantitative variations in metabolite content [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF].

Here, we study clary sage populations sampled in three different types of macroenvironment encountered around the Croatian city of Makarska: the coast, the mountain range alongside the coast and the plateau behind the mountain range (Figure 36). In situ, populations growing in these three macro-environments are submitted to different environmental conditions specific to coastal, mountain and plateau environments. Sampling sites are notably characterized by their distinct elevation (40-80 m for coastal and plateau sites and 360-550 m for mountain sites). The selective pressure associated with these environmental conditions may have led to the selection of genetic polymorphisms which could potentially have an impact on sclareol production. To assess the presence of genetic variation, genes involved in sclareol production were sequenced in individuals belonging to the different Croatian populations. Detected single nucleotide polymorphisms were used to estimate the genetic diversity found among Croatian populations and to compare it to genetic diversity found in reference populations. Finally, individuals from Croatian and reference populations were grown in the same environment in order to investigate whether genetic variation between the populations has an impact on sclareol content and flowering time.

Materials and methods

Plant material

Genetic diversity analysis

For each population, 3-4 individuals were grown in growth chamber (16h of day; 27 °C during the day, 21 °C at night; 60% hygrometry) for 6 weeks. DNA was extracted from leaves using the DNeasy 96 Plant Kit from Qiagen. Genomic fragments from DXS2, CMK and ITS loci were amplified by PCR using primers listed in Table 2. Sequences of PCR products were obtained through Sanger sequencing performed by Eurofins Genomics. For each individual, sequences from DXS2, CMK and ITS loci were concatenated in order to integrate data from the 3 loci in a single phylogenetic tree. Single nucleotide polymorphisms (SNPs) were located by aligning the sequences using the ClustalW algorithm in the MEGA7 software.

Heterozygous sites were visualized in Sanger sequencing chromatograms provided by Eurofins Genomics and manually annotated in MEGA7 using the IUPAC code. Encoding heterozygous sites using the IUPAC code was necessary to reconstruct haploid sequences using the PHASE algorithm in the DnaSP software. Haploid sequences obtained (2 per individual) were used to build a phylogenetic tree in MEGA7 using the Neighbor Joining method with 500 bootstrap repetitions.

Target Primer Sequence

DXS2

DXS2_F 5'-GCAGTTTCTTGCCATTGCTCC-3' DXS2_R 5'-TAATACGTACCTGGTGACCC-3'

CMK CMK_F 5'-CGAGAGGTACAGGTGGAGGA-3' CMK_R 5'-CAAGAGTGGTCGGGTGAGAT-3' ITS ITS_F 5'-GCATCGATGAAGAACGTAGC-3' ITS_R 5'-TCCTCCGCTTATTGATATGC-3'
Table 2: Primers used to amplify genomic fragments from DXS2, CMK and ITS loci.

Field trial

Seeds from Makarska, Ravca, Banja, Milly and Vatican White populations were sown on August 3 rd , 2017. Plantlets were grown in growth chamber (16h of day; 27 °C during the day, 21 °C at night; 60% hygrometry) until being transplanted in the field in Chemillé (France) on November 7 th , 2017. Plants were placed every 0.4 m x 0.7 m according to a randomized layout. Flowering time was defined as the date of opening of the first flowers and was recorded between May 31 st and June 29 th , 2018. For calyx sampling, inflorescences were cut on July 19 th , 2018 at the beginning of the afternoon. Mature calyces (harboring green achenes) were cut from all over the inflorescence, immediately placed in dry ice and stored at -80 °C at the end of the day until further analysis.

Sclareol and linalyl acetate quantification by GC-MS

Clary sage calyces were immersed in hexane without grinding to extract metabolites present at the surface. For each individual, 12 mL of solvent were used to extract metabolites from 8 mature calyces. 10-undecen-1-ol was employed as internal standard and was added in the solvent before extraction at a concentration of 0.75 mM. After 2 h of agitation, 1 mL of extract was centrifuged at 13,000 rpm for 10 min to eliminate potential dust or debris.

Samples were directly analyzed by gas chromatography coupled to mass spectrometry (GC-MS) using a 7890B/5977A GC-MS system from Agilent and according to a protocol adapted from Laville et al. [START_REF] Laville | Amphilectane diterpenes from Salvia sclarea: Biosynthetic considerations[END_REF]. The system was equipped with a 10 m guard column and an Rxi-5Sil MS column (length, 30 m; inner diameter, 0.25 mm; film thickness, 0.25 μm) (Restek, Bellefonte, PA, USA). 1 µl of sample was injected with a split ratio of 30:1. Oven temperature was set to 110 °C for 2 min, then increased to 270 °C at a rate of 10 °C/min, and finally set to 270 °C for 2 min. Other temperatures were set as follows: injector, 270 °C; transfer line, 270 °C; source, 230 °C; quadrupole, 150 °C. The carrier gas was helium at a constant flow of 1 mL/min. The quadrupole mass spectrometer was switched on after a solvent delay of 5 min and was programmed to scan from 35 u to 350 u. Data analysis was carried out using the MassHunter Quantitative Analysis software from Agilent. Sclareol and linalyl acetate were identified by comparison with analytical standards purchased from Sigma-Aldrich. Absolute quantification of sclareol and linalyl acetate was performed using a calibration curve prepared with analytical standards.

Results

Croatian populations show genetic polymorphism in MEP pathway genes

As mentioned above, we hypothesized that distinct environmental conditions associated with coastal, mountain and plateau environments may have led to the selection of genetic polymorphisms potentially impacting sclareol production. To support this hypothesis, we checked whether genetic polymorphisms can be found among Croatian populations in genes involved in sclareol biosynthesis. We looked for genetic polymorphisms in the sequences of 2 genes of the MEP pathway: DXS2 and CMK. These two genes were selected because substantial parts of their genomic DNA could be readily amplified by PCR and sequenced. In addition to sequences from the 6 Croatian populations, sequences from 3 reference populations were analyzed: Vatican White and Milly, which were both used as reference populations in the context of the PISTILL project, and Toscalia, which is a population of cultivated clary sage. Single nucleotide polymorphisms (SNPs) were detected in partial sequences obtained from DXS2 and CMK (respectively 4 and 9 SNPs) (Figure 37a). Five of these SNPs are found in individuals from Croatian populations but not in reference populations, indicating that Croatian populations represent a source of new alleles (Figure 37b). Most of these SNPs probably have no impact on enzyme activity, for example if they are located in introns or if they are silent due to genetic code redundancy. However, analyzed sequences are small and other SNPs could probably be found in non-sequenced parts of DXS2 and CMK genes and also in other genes submitted to an equivalent selection pressure. Since the existence of specific genetic polymorphisms in genes involved in sclareol biosynthesis has been assessed among Croatian populations, their sclareol content may be different compared to reference populations.

Population Individual

1 R Y W A C Y A R C R A R T 2 G C A A C C A A C A A A T 3 G C A A C Y A R C R A R T 4 R Y W A C Y A R C R A R T 1 G Y W W C T A G C G A G T 2 G T T T C Y A G S R R R T 3 G T T T C T A G C G A G T 4 G T T T C Y A G S R R R T 1 G Y W W C Y A R C R A R T 2 G C A A C C A A C A A A T 3 G C A A C Y A R C R A R T 1 A T T A C T A G C G A G T 2 A T T A C T A G C G A G T 3 A T T A C T A G C G A G T 4 R Y W A C Y A R C R A R T 1 G Y W W C Y A R C R A R T 2 G Y W W C Y A R C R A R T 3 G Y W W C Y A G S R R R T 4 R T T W C Y A G S R R R T 1 G Y W W C C A R S A R A T 2 G Y W W C C A G G A G A T 3 G C A A C C A R S A R A T 1 G T T T C C A A C A A A T 2 G T T T C C A A C A A A T 3 G T T T C C A A C A A A T 4 G T T T C C A R C A R A T 1 A T T A C Y A R C A R A W 2 A T T A S C W R C A A A T 3 R T T W C Y A G C A A A W 4 A T T A S C W R C A A A T 1 G T T T C C A R C A R A T 2 G T T T C C A R C A R A T 3 G T T T C C A R C A R A T 4 G T T T C C A R C A R

Choice of the populations to be analyzed in the field

To test whether genetic differences between Croatian and reference populations have an impact on sclareol production, a field trial was carried out in order to compare their sclareol content. The first step was the choice of populations which are the most relevant for the study. Indeed, Croatian populations being geographically close from each other (Figure 36), some of these populations may be almost genetically identical. Since the field trial is aimed at investigating the impact of genetic variation on plant phenotype, populations selected for the field trial should be genetically distinct. Moreover, the study of the link between genotype and phenotype is easier if each population used in the study is as genetically homogenous as possible.

Genetic diversity among Croatian and reference populations and genetic homogeneity of each population were evaluated using SNPs found in DXS2 and CMK sequences, along with 3 additional SNPs detected in Internal Transcribed Spacer (ITS) sequences. To integrate polymorphism data detected at these 3 loci, the 3 sequences obtained for each individual were concatenated. Raw concatenated sequences could not be used directly to build a phylogenetic tree because heterozygous sites are not properly taken in account by tree building algorithms. The phasing algorithm PHASE was used to reconstruct the 2 haploid sequences carried by each individual. In total, 6 or 8 haploid sequences per population were obtained, depending on the number of individuals sequenced (3 or 4, respectively). All haploid sequences were considered to be independent and used to build a phylogenetic tree (Figure 38). For example, the 4 individuals belonging to the Vatican White population are heterozygous and carry the same 2 haploid sequences (Figure 37b). As a consequence, the 8 haploid sequences of the Vatican White population form 2 groups of 4 identical sequences which cluster together in the tree (Figure 38). 38). For this reason, the 5 latter populations were considered more homogenous and selected for the field trial.

Comparison of flowering date and metabolite content of the different populations

In autumn 2017, 93 plants (between 15 and 23 per population) were transplanted in the field using a random layout. In total, 73 plants (between 10 and 22 per population) flowered between May 31 st and June 29 th , 2018, that is, between 301 and 330 days after sowing.

These flowering dates are consistent with data available in the literature; indeed, clary sage is usually reported to flower in June-July [START_REF] Wagner | Changes in the Qualitative and Quantitative Composition of Essential Oils of Clary Sage and Roman Chamomile During Steam Distillation in Pilot Plant Scale[END_REF]. The date of flowering does not significantly vary between the three Croatian populations (Figure 39). Interestingly, Vatican White plants flowered significantly earlier than Milly and Makarska plants (10 days earlier in average) (Figure 39).

Complex relationships between genetic diversity, chemical diversity and environmental conditions

The production of specialized metabolites by plants is known to be impacted by environmental conditions. In Salvia desoleana, a sage species closely related to Salvia sclarea, sclareol content was shown to be influenced by soil chemical composition: plants grown on soil rich in silt produced less sclareol compared to plants grown on soil rich in sand [START_REF] Rapposelli | Relationship Between Soil and Essential Oil Profiles in Salvia desoleana Populations: Preliminary Results[END_REF]. Other abiotic factors such as temperature or hygrometry may influence sclareol production by clary sage. Here, we study Croatian populations of clary sage naturally growing in three different types of environment: coast, mountain and plateau.

These three types of environment are probably submitted to distinct climatic conditions, notably because they are located at different altitudes above sea level (40-80 m for coast and plateau, 360-550 m for mountain) (Figure 36).

The impact of altitude on clary sage terpene production was recently investigated using herbarium samples. Sampling sites were located in several European and Middle Eastern countries at different altitudes, ranging from 34 m to 1300 m [START_REF] Foutami | Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean[END_REF].

According to this study, altitude had no significant effect on clary sage sample chemical composition. Another study focused on the impact of environmental conditions on terpene production in a related species, Salvia officinalis [START_REF] Russo | Chemical composition and anticancer activity of essential oils of Mediterranean sage (Salvia officinalis L.) grown in different environmental conditions[END_REF]. Plants were cultivated at 18 sites located around Campobasso in Italy and grouped in three categories according to global environmental conditions: lowland, low hill and high hill. Terpene production was evaluated through the analysis of essential oil chemical composition. The main essential oil components were always α-thujone, camphor, borneol, γ-muurolene and sclareol, but their relative abundances in essential oil varied according to cultivation site. Notably, the percentage of sclareol in essential oil ranged between 5.9 and 23.1%, although these variations could not be attributed to a particular environmental factor such as altitude or water availability [START_REF] Russo | Chemical composition and anticancer activity of essential oils of Mediterranean sage (Salvia officinalis L.) grown in different environmental conditions[END_REF].

In addition to the influence of environmental conditions, the production of specialized metabolites by plants also depends on genetic factors. Relationships between genetic diversity, chemical diversity and environmental conditions were investigated in 50 Sardinian populations of the terpene-producing plant Helichrysum italicum [START_REF] Melito | Genetic and Metabolite Diversity of Sardinian Populations of Helichrysum italicum[END_REF].

Genetic diversity was estimated using the AFLP (Amplified Fragment Length Polymorphism)

DNA fingerprinting method, and chemical diversity was evaluated through the characterization of leaf essential oil composition. AFLP-based clades were in general congruent with the geographical localization of Helichrysum italicum populations, indicating limited gene exchanges between populations. The genetic structure was best explained by environmental conditions at sampling site, suggesting an involvement of adaptive selection in shaping genetic diversity. The amount of oxygenated monoterpenes present in the essential oil was significantly correlated with AFLP-based clades. Therefore, genetic factors may be partly responsible for differences in metabolite content. However, the strong association of the genetic structure with environmental conditions at sampling site prevented the analysis of the respective contributions of genetic factors and environmental conditions to chemical diversity [START_REF] Melito | Genetic and Metabolite Diversity of Sardinian Populations of Helichrysum italicum[END_REF].

In our study, we cultivated three Croatian populations in the same environment (field trial)

to focus on differences in metabolite content attributable to genetic variation.

Perspectives for the study of clary sage natural diversity

Different types of genetic markers can be developed and employed to study the genetic diversity of plant populations. For example, SSR (Simple Sequence Repeats, also known as microsatellites) and ISSR (Inter Simple Sequence Repeats) markers have been developed for the study of patchouli populations [START_REF] Sandes | Molecular characterization of patchouli (Pogostemon spp) germplasm[END_REF], and the AFLP method (Amplified Fragment Length Polymorphism) was used to analyze 50 Sardinian populations of Helichrysum italicum [START_REF] Melito | Genetic and Metabolite Diversity of Sardinian Populations of Helichrysum italicum[END_REF]. The AFLP method is cheap and does not require any genomic data about the species of interest, but provides limited information due to the relatively low number of markers generated by this approach. For instance, only 125 AFLP markers were generated to analyze the genetic structure of the 50 Sardinian populations of Helichrysum italicum [START_REF] Melito | Genetic and Metabolite Diversity of Sardinian Populations of Helichrysum italicum[END_REF].

Generation of SNP markers through genotyping-by-sequencing provides much higher marker density compared to other methods [START_REF] He | Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding[END_REF]. This approach was recently employed to evaluate the genetic diversity and reveal the genetic structure of cultivated chamomile populations [START_REF] Otto | Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping[END_REF]. 16,972 SNPs were simultaneously identified and genotyped by sequencing the genome of 2-4 individuals from each of the 33 populations used in the study (91 individuals in total). In absence of a reference genome, the chamomile genome was assembled de novo with sequences obtained from the 91 individuals. Unlike other methods like AFLP or SSR marker identification, genotyping-by-sequencing requires significant genome-wide sequencing efforts, but technological advances continuously reduce the cost of high-throughput sequencing [START_REF] He | Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding[END_REF]. Due to their abundance in genomes and their increasingly affordable genotyping, SNPs tend to gradually replace other markers [START_REF] Kilian | NGS technologies for analyzing germplasm diversity in genebanks[END_REF].

The recent study of chamomile diversity mentioned above illustrates how genotyping-bysequencing and GWAS approaches can contribute to increase knowledge on non-model plants and facilitate their breeding [START_REF] Otto | Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping[END_REF]. The flowers of chamomile contain numerous medicinally active specialized metabolites, notably the sesquiterpene α-bisabolol.

Therefore, flowering time and α-bisabolol content are important traits for chamomile breeding. SNP markers generated through genotyping-by-sequencing were used for GWAS, leading to the identification of genetic loci controlling flowering time and α-bisabolol content [START_REF] Otto | Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping[END_REF]. A similar approach could be employed to identify genetic loci controlling flowering time and sclareol content in clary sage. However, to perform successful GWAS, the collection of populations must be large enough and display sufficient genetic diversity [START_REF] Kilian | NGS technologies for analyzing germplasm diversity in genebanks[END_REF]. To meet these criteria, clary sage seed sampling should be extended to a larger geographic area than the region of Makarska in Croatia. Indeed, more natural genetic diversity is probably present in the native area of clary sage and would complement new alleles already provided by Croatian populations used in this study (Figures 37 and38). It could also be relevant to sample cultivated populations of clary sage grown around the world.

Vatican White plants flower earlier and produce less specialized metabolites

Phenotyping of flowering date and metabolite content during the field trial highlighted Vatican White plants may also be due to a lower glandular trichome density. In Artemisia annua, the amount of artemisinin found in leaves is tightly correlated to glandular trichome density [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF]. Careful measurements of glandular trichome density are needed to test this hypothesis.

Conclusion 1. Context and objectives of the project

Clary sage produces sclareol, a terpenoid widely used in perfume industry for the hemisynthesis of ambroxide, a valued perfume component with remarkable fixative

Perspectives for research on sclareol production in clary sage

In the first chapter of this manuscript, we provide several lines of evidence supporting the hypothesis that sclareol is mainly secreted by calyx glandular trichomes. Mass spectrometry imaging suggests that sclareol is most abundant on large capitate glandular trichomes, and clary sage plants lacking these trichomes produce less sclareol. First, it is important to assess whether the absence of large capitate glandular trichomes and the lower sclareol content are caused by the same mutation, and if so, to identify this mutation by positional cloning.

The characterization of this mutation would improve our understanding of biological processes underlying glandular trichome development and sclareol production in clary sage.

Approaches developed in the second chapter of this manuscript underline the respective contributions of MVA and MEP pathways to the biosynthesis of three terpenoids in clary sage calyces: sclareol, linalyl acetate and β-caryophyllene. Sclareol and linalyl acetate both originate from the plastidial MEP pathway, while the sesquiterpene β-caryophyllene seems to be of mixed origin. However, the origin of carbon assimilates needed for MVA and MEP pathways activity is still unknown. They may be produced by photosynthesis in clary sage glandular trichomes, or be imported from neighboring cells. The ultrastructural and metabolic specificities of glandular trichomes and their relationships with other calyx cells could be investigated through transmission electron microscopy and dynamic isotope labeling experiments.

Experiments described in the third chapter of this manuscript could not demonstrate any role of jasmonates in the regulation of sclareol production. Two possibilities remain plausible: either jasmonates are not involved in sclareol biosynthesis regulation, or the experimental protocol needs further optimization. In the latter case, employing a mild treatment protocol and focusing the study on young developing calyces may help to highlight an impact of jasmonates on sclareol content. The role of other phytohormones like ABA, known to control terpenoid biosynthesis in other plants, could also be investigated.

The fourth chapter of this manuscript explores genetic and phenotypic diversity of Croatian wild populations originating from three different types of environment: coast, mountain and plateau. Although genetic polymorphisms were detectable in sclareol biosynthesis genes, Croatian populations were not different in terms of sclareol content and flowering time when grown in the same environment. Croatian climatic conditions apparently did not result in genetic differentiation affecting sclareol production. However, the difference in sclareol content observed between "Vatican White" and "Milly" reference populations deserves further investigation. It could be the consequence of a global downregulation of specialized metabolism or of a decrease in glandular trichome density in "Vatican White" plants. To test these hypotheses, glandular trichome density and expression levels of biosynthetic genes should be compared between the two reference populations.

Perspectives for clary sage genetic improvement

Several strategies aiming at enhancing sclareol production in clary sage emerge from results described in this manuscript. Sclareol being MEP-derived and mainly produced in glandular trichomes, increasing glandular trichome density or boosting MEP pathway activity are promising ways to elevate sclareol content. Moreover, since linalyl acetate is also MEPderived and produced in glandular trichomes, blocking linalyl acetate biosynthesis may increase sclareol production through reducing competition for substrate. The MEP pathway and linalyl acetate biosynthesis could be manipulated through genetic modifications targeting enzymes involved in these pathways. Similarly, glandular trichome density could be increased through the manipulation of glandular trichome initiation regulators. Candidate genes putatively encoding DXS, DXR and GPP-synthase enzymes have been identified in the transcriptome previously obtained from clary sage calyces [START_REF] Legrand | One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L[END_REF], but transcription factors involved in glandular trichome initiation remain to be characterized.

Candidate genes could be identified in the same transcriptome by looking for genes displaying high sequence similarity with conserved regulators of glandular trichome initiation.

Clary sage breeding programs could be enhanced through an efficient exploitation of clary sage natural diversity. Croatian populations studied in the fourth chapter of this manuscript represent a valuable genetic resource because they have been shown to carry new alleles compared to reference populations. Extending clary sage seed sampling to the whole Mediterranean basin, where clary sage is naturally growing, would increase the amount of genetic resources available for clary sage breeding and for the study of sclareol biosynthesis regulation.

The feasibility of approaches described above will depend on the development of tools dedicated to clary sage genetic improvement. The sequencing of clary sage genome and robust genetic markers would enable the identification of genetic loci controlling traits of interest and the rapid introgression of new alleles into elite varieties. The development of a clary sage transformation protocol would also facilitate the functional characterization of candidate genes involved in the quantitative variation of traits of interest. Clary sage transgenic hairy roots can be obtained (Kuźma et al., 2009a;[START_REF] Vaccaro | Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots[END_REF][START_REF] Vaccaro | Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming[END_REF][START_REF] Alfieri | Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors[END_REF], but to our knowledge, no protocol allowing stable transformation of whole clary sage plants has been described so far. Genomic data and biotechnological tools specific to clary sage would be highly valuable for basic and applied research projects aiming at understanding and improving this perfume plant.

Annex

Review: Genetic control of glandular trichome development Scents, pigments, medicines… Life would be bleaker and more difficult for humans if plants

were not producing such a wide variety of compounds. These chemicals are critical for the capacity of plants to adapt to their environment and to overcome the various challenges they are facing every day, like pollinator attraction or defense against pathogen attacks. Due to their frequent self-toxicity, many of these compounds are produced by specialized secretory structures, for example glandular trichomes. Glandular trichomes are quite common as they can be found in approximately 30% of all vascular plants [START_REF] Fahn | Structure and Function of Secretory Cells[END_REF][START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]. The development of these multicellular structures originating from the epidermis has been suggested as a model to study plant cell differentiation [START_REF] Yang | Trichomes as models for studying plant cell differentiation[END_REF]. Glandular trichomes are also remarkable for their very high metabolic productivity [START_REF] Balcke | Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites[END_REF][START_REF] Tissier | Plant secretory structures: more than just reaction bags[END_REF].

Compounds produced by glandular trichomes are harnessed by human industries that benefit from their various properties. Organic synthesis often made it possible to dispense with the initial plant source. However, total synthesis can be complex and expensive, and many compounds of interest are still directly extracted from plants. For example, the perfume ingredient ambrox is synthesized from sclareol extracted from Salvia sclarea [START_REF] Yang | One-pot synthesis of (-)-Ambrox[END_REF], and world supply in the anti-malarial drug artemisinin still relies on artemisinin extraction from Artemisia annua [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF][START_REF] Tissier | Plant secretory structures: more than just reaction bags[END_REF]. The amount of specialized metabolites produced by a plant is often tightly correlated to the density of glandular trichomes present at the surface of the epidermis [START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF][START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF]. Increasing glandular trichome density has recently emerged as a new plant breeding strategy to enhance the yield in compounds of interest for the pharmaceutical sector [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF].

This strategy could also be used to breed crops with improved resistance to herbivores [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]. In some cases, decreased trichome density is also desirable in order to reduce the amount of compounds toxic to humans, like gossypol in cotton [START_REF] Ma | Genetic basis for glandular trichome formation in cotton[END_REF].

Engineering glandular trichome density requires reliable data about the genetic network controlling glandular trichome initiation. Several lines of evidence indicate that glandular (and in general multicellular) trichome formation is probably controlled by a different network than the one controlling non-glandular trichome formation in Arabidopsis thaliana [START_REF] Yang | Trichomes as models for studying plant cell differentiation[END_REF]. Compared to non-glandular trichome formation, our knowledge about genes involved in glandular trichome formation is still limited. Nevertheless, recent studies have led to significant advances, which are summarized in this review.

Which species could serve as model(s)?

As no glandular trichomes are found in Arabidopsis thaliana, research on glandular trichome development has been carried out on various other plant species. From the careful analysis of recent literature, three species emerge as main working materials: tomato (Solanum lycopersicum, Solanaceae), sweet wormwood (Artemisia annua, Asteraceae) and cucumber (Cucumis sativus, Cucurbitaceae). In tomato, glandular trichomes are essential for plant defense against herbivores [START_REF] Kang | Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry[END_REF][START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]; in Artemisia annua, they produce the anti-malarial drug artemisinin [START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF]; and in cucumber, their size and number are important fruit quality traits [START_REF] Liu | Trichome-Related Mutants Provide a New Perspective on Multicellular Trichome Initiation and Development in Cucumber (Cucumis sativus L)[END_REF][START_REF] Yang | CsMYB6-CsTRY module regulates fruit trichome initiation in cucumber[END_REF]. Tomato and Artemisia annua have both already been suggested as good potential models for the study of glandular trichome development [START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF]. Tomato has long been established as a model plant in other fields of plant research; sequenced genome, reliable genetic tools and extensive genetic resources are available for Solanum lycopersicum and other related species [START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF]. Research efforts focusing on Artemisia annua are more recent, but genetic transformation protocols are available [START_REF] Shen | The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua[END_REF] and a draft assembly of the genome has been recently published [START_REF] Shen | The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis[END_REF].

Eight trichome types have been described on tomato leaves, among which four are glandular: type I, type IV, type VI and type VII. Type VI glandular trichomes are the most abundant and secrete mainly terpenoids, whereas type I and type IV are involved in acyl sugar biosynthesis [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]. The current classification of tomato glandular trichomes is still discussed; for instance, type I and type IV were suggested to be the same type according to the molecules they secrete [START_REF] Mcdowell | Comparative functional genomic analysis of Solanum glandular trichome types[END_REF], but are generally considered to be different according to their morphology and patterning [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF][START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF]. Artemisia annua leaves display two types of trichomes: T-shaped non-glandular ones and glandular ones, which are able to accumulate artemisinin along with various other compounds [START_REF] Duke | Development and Fine Structure of the Glandular Trichomes of Artemisia annua L[END_REF][START_REF] Xiao | Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin[END_REF][START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF]. Eight trichome types have been recently characterized on cucumber fruits including two glandular types: type I and type VI, type I being the most frequent of the two [START_REF] Xue | Classification of fruit trichomes in cucumber and effects of plant hormones on type II fruit trichome development[END_REF]. Type I glandular trichomes are also called bloom trichomes and are believed to be involved in fruit cuticle formation and in the secretion of mineral substances [START_REF] Xue | Classification of fruit trichomes in cucumber and effects of plant hormones on type II fruit trichome development[END_REF]. Given the fact that the genetic control of multicellular trichome development in cucumber fruits has been recently reviewed [START_REF] Liu | Trichome-Related Mutants Provide a New Perspective on Multicellular Trichome Initiation and Development in Cucumber (Cucumis sativus L)[END_REF], this review focuses on recent discoveries concerning genes involved in glandular trichome development in tomato and Artemisia annua.

Description of glandular trichome morphology and development

Glandular trichome morphology has been described in a large number of plant species and this abundant literature highlights their tremendous diversity of shape and size [START_REF] Tissier | Glandular trichomes: What comes after expressed sequence tags?[END_REF]. Nevertheless, a common organization scheme shared by most glandular trichomes arises from the description of their structure. Glandular trichomes are usually multicellular and composed of 3 parts: a base, a stalk and a gland. The gland is responsible for the secretion of specialized metabolites. Gland cells are highly differentiated, with an ultrastructure adapted to their very high metabolic activity. Ultrastructural adaptations depend on the nature of secreted compounds and have been previously reviewed by Lange and Turner [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF]. The stalk is the structure bearing the gland and the base connects the stalk to surrounding epidermal cells. Each of these 3 parts can be unicellular or multicellular and cells can be more or less elongated. This variability in cell number and shape accounts for a large part of the high morphological diversity found among glandular trichomes and is also responsible for their wide variety of sizes. For example, 10celled glandular trichomes of Artemisia annua have a biseriate structure of only 40-50 µm long [START_REF] Duke | Development and Fine Structure of the Glandular Trichomes of Artemisia annua L[END_REF], whereas tomato type I glandular trichomes are 2-3 mm long with a long stalk [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF].

Secreted metabolites often accumulate in a storage cavity. This storage cavity can be subcuticular: in that case, molecules secreted at the top of gland cells accumulate under the cuticle which is gradually pushed away from the cell wall, as seen in Artemisia annua glandular trichomes [START_REF] Duke | Development and Fine Structure of the Glandular Trichomes of Artemisia annua L[END_REF]. The storage cavity can also be intercellular, as seen in type VI glandular trichomes of tomato [START_REF] Bergau | The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites[END_REF]. The size of the storage cavity has an impact on glandular trichome shape. For example, in cultivated tomato, type VI glandular trichome glands have a four-leaf clover shape due to the small size of the storage cavity, whereas in the wild tomato species Solanum habrochaites, a larger storage cavity is responsible for their spherical shape [START_REF] Bergau | The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites[END_REF]. An abscission zone between the stalk and the gland, allowing quick separation of the gland from the rest of the trichome, has been described in tomato type VI glandular trichomes [START_REF] Bergau | The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites[END_REF].

In addition to the wide inter-specific diversity, intra-specific morphological diversity of glandular trichomes can also be very high, as illustrated by the distinct morphologies of the four glandular trichome types described in tomato [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]. The proportion of each trichome type can be different in closely related species of tomato [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF] and is highly dependent on leaf developmental stage [START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF]. For example, in cultivated tomato, type IV trichomes are present on juvenile leaves and absent in adults leaves [START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF]. These developmental variations being rarely well described, the description of a modification of the proportion of each trichome type is difficult. For example, woolly mutant tomato lines have been known for decades as "hairy" mutants of tomato, but the precise phenotype of certain lines in terms of glandular trichome type proportions is controversial [START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF].

Given their common organization scheme, glandular trichomes must share common key developmental events. A number of studies have attempted to describe the different steps of glandular trichome development in different plant species [START_REF] Duke | Development and Fine Structure of the Glandular Trichomes of Artemisia annua L[END_REF][START_REF] Bergau | The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites[END_REF][START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF]. Trichome initiation occurs when an epidermal cell acquires a trichome identity according to signals received from surrounding cells. This cell then undergoes tightly controlled cell divisions; the number and the orientation of these divisions and the extent of cell elongation contribute to shape various trichome morphologies. These first developmental steps are common to glandular and non-glandular trichomes. Additionally, in glandular trichomes one or more cells differentiate into gland cells. The acquisition of the secretory activity implies a profound remodeling of cell ultrastructure [START_REF] Lange | Terpenoid biosynthesis in trichomes-current status and future opportunities[END_REF] and the activation of specialized metabolism pathways, for example terpenoid biosynthesis in Artemisia annua glandular trichomes [START_REF] Soetaert | Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua[END_REF]. All these developmental events are critical for glandular trichome patterning, morphogenesis and differentiation, but their genetic control remains poorly understood [START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF]. However, a certain number of genes involved in glandular trichome initiation have been recently characterized in Artemisia annua and tomato, along with a few genes involved in glandular trichome morphogenesis. These genes and their respective functions are detailed below.

Genes controlling glandular trichome initiation

Several transcription factors involved in glandular trichome initiation have been identified

both in tomato and Artemisia annua. They belong to various families of transcription factors, but the majority of them belong to two subfamilies: the R2R3-MYB subfamily and the HD-ZIP IV subfamily.

Transcription factors

R2R3-MYB transcription factors

Transcription factors of the R2R3-MYB subfamily have two DNA-binding MYELOBLASTOSIS-RELATED (MYB) domain repeats. Different members of this subfamily are known to be involved in the regulation of secondary metabolism [START_REF] Chezem | Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs[END_REF] or in the regulation of epidermal cell fate, for example MIXTA and MIXTA-like genes [START_REF] Brockington | Evolutionary Analysis of the MIXTA Gene Family Highlights Potential Targets for the Study of Cellular Differentiation[END_REF]. The first MIXTA gene was characterized in snapdragon (Antirrhinum majus) and controls the differentiation of conical epidermal cells from flat epidermal cells [START_REF] Brockington | Evolutionary Analysis of the MIXTA Gene Family Highlights Potential Targets for the Study of Cellular Differentiation[END_REF].

In Artemisia annua and tomato, three members of the R2R3-MYB subfamily have been characterized as positive regulators of glandular trichome initiation: AaMYB1 and AaMIXTA1

in Artemisia annua [START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF] and SlMX1 in tomato [START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Ewas | RNA-seq reveals mechanisms of SlMX1 for enhanced carotenoids and terpenoids accumulation along with stress resistance in tomato[END_REF] (Figure 41). Indeed, Artemisia annua plants overexpressing

AaMYB1 show an increase in glandular trichome density [START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF].

Moreover, down-or upregulation of AaMIXTA1 in Artemisia annua [START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF] and SlMX1 in tomato [START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Ewas | RNA-seq reveals mechanisms of SlMX1 for enhanced carotenoids and terpenoids accumulation along with stress resistance in tomato[END_REF] respectively decreases and increases glandular trichome density. AaMIXTA1 and SlMX1 are both MIXTA-like genes [START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF] whereas AaMYB1 belongs to another clade of the R2R3-MYB subfamily [START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF].

3.1.2. HD-ZIP IV transcription factors HD-ZIP transcription factors are characterized by a homeodomain (HD) DNA-binding domain and a leucine-zipper (ZIP) motif responsible for protein dimerization [START_REF] Gao | Comprehensive analysis and expression profile of the homeodomain leucine zipper IV transcription factor family in tomato[END_REF]Chen et al., 2017a). Members of the HD-ZIP IV subfamily of transcription factors also have a START domain and are known to be involved in epidermal cell differentiation in plants, including cuticle biosynthesis and patterning of trichomes and stomata [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF][START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF].

In Artemisia annua, two HD-ZIP IV transcription factors, namely AaHD1 and AaHD8, have recently been shown to positively regulate glandular trichome initiation (Figure 41).

Overexpression of AaHD1 [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF] or AaHD8 [START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF] increases glandular trichome density, whereas downregulation of any of the two genes has the opposite effect.

AaHD8 acts upstream of AaHD1 by directly promoting its expression [START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF].

The closest homolog of AaHD8 in tomato is CUTIN DEFICIENT 2 (SlCD2) [START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. A loss-of-function mutation in SlCD2 is responsible for the phenotype of the sticky peel mutant of tomato, which displays a lower number of glandular trichomes (especially type VI) [START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF]. Therefore, the function of AaHD8/SlCD2 in positive regulation of glandular trichome initiation seems to be conserved between tomato and Artemisia annua.

Another HD-ZIP IV transcription factor, WOOLLY (Wo), appears to be an important regulator of glandular trichome initiation in tomato (Figure 41). Dominant point mutations in the Cterminus part of Wo are responsible for the phenotype of woolly mutants, which show dramatically increased trichome density [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF][START_REF] Yang | Transcriptome profile analysis of cell proliferation molecular processes during multicellular trichome formation induced by tomato Wo v gene in tobacco[END_REF]. According to the first characterization of Wo, type I glandular trichome density is increased in plants carrying dominant woolly mutations and reduced in Wo-RNAi plants, suggesting that Wo enhances type I glandular trichome initiation [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF]. However, a recent re-analysis of the phenotype of woolly mutants indicates instead that woolly mutants show a higher density of type III and type V non-glandular trichomes and a lower density of type IV glandular trichomes in adult leaves [START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF]. According to this study, the effect of the dominant woolly point mutation is different depending on leaf developmental stage: indeed, a higher density of type IV glandular trichomes was observed in juvenile leaves of woolly mutants, whereas it was lower in adult leaves compared to the wild-type [START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF].

Cyclins

The induction of cell divisions in early steps of glandular trichome development requires not only transcription factors, but also cell cycle regulators like cyclins. The tomato gene SlCycB2 encodes a B-type cyclin, which is a type of cyclin promoting the G2/M transition [START_REF] Gao | The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense[END_REF]. Type I glandular trichome density is reduced in SlCycB2-RNAi plants, highlighting the role of SlCycB2 in glandular trichome initiation [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF][START_REF] Gao | The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense[END_REF] (Figure 41). SlCycB2 may promote a shift from endoreduplication to mitosis in epidermal cells, thereby inducing the first cell divisions of type I glandular trichome development [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF]. However, SlCycB2 overexpression does not seem to be an efficient tool to increase glandular trichome density because it inhibits the initiation of type I and type VI glandular trichomes [START_REF] Gao | The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense[END_REF]. SlCycB2 expression is upregulated in Wo-overexpressing plants,

Regulatory complexes

Non-glandular trichome initiation in Arabidopsis thaliana is known to be controlled by a MYB-bHLH-WD40 complex [START_REF] Pattanaik | An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis[END_REF]. An important output of recent efforts in dissecting the genetic network underlying glandular trichome development was the identification of 2 complexes controlling glandular trichome initiation in tomato and Artemisia annua. In Artemisia annua, the expression of the positive regulator of glandular trichome initiation AaHD1 was recently shown to be enhanced by a complex formed by two transcription factors: the HD-ZIP IV protein AaHD8 and the R2R3-MYB MIXTA-like protein AaMIXTA1 [START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF] (Figure 41). In tomato, the closest homolog of AaHD8 is SlCD2 [START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. SlCD2 may interact with a MIXTA-like transcription factor, maybe SlMX1, to form a complex similar to AaHD8-AaMIXTA1, but this remains to be demonstrated. A direct interaction between the C2H2 zinc-finger protein SlH and the HD-ZIP IV transcription factor Wo was recently detected, suggesting that SlH and Wo act as a heterodimer to induce type I glandular trichome formation [START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF] (Figure 41). Moreover, Wo and the B-type cyclin SlCycB2 physically interact, supporting the hypothesis that these two proteins also act together to induce type I glandular trichome formation [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF] (Figure 41). An H-Wo-CycB2 complex may be involved in the positive regulation of type I glandular trichome initiation in tomato, but has not been detected yet [START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF].

Genes involved in hormonal signaling

Glandular trichome initiation is known to be regulated by plant hormones in various plant species [START_REF] Maes | Hormone-mediated promotion of trichome initiation in plants is conserved but utilizes species-and trichome-specific regulatory mechanisms[END_REF]. In particular, jasmonates (JA) are able to induce glandular trichome initiation in tomato [START_REF] Maes | Hormone-mediated promotion of trichome initiation in plants is conserved but utilizes species-and trichome-specific regulatory mechanisms[END_REF] and Artemisia annua [START_REF] Maes | Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants[END_REF]. Consistently, proteins involved in JA signaling have recently been shown

to impact glandular trichome initiation in both species. In Artemisia annua, the transcriptional activity of the positive regulator of glandular trichome initiation AaHD1 is repressed by a direct interaction with AaJAZ8, which belongs to the JAZ family of JA signaling repressors [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF] (Figure 41). In tomato, the overexpression of the JA signaling repressor SlJAZ2 decreases glandular trichome density, indicating that SlJAZ2 is a negative regulator of glandular trichome initiation [START_REF] Yu | The Jasmonate ZIM-domain protein gene SlJAZ2 regulates plant morphology and accelerates flower initiation in Solanum lycopersicum plants[END_REF]. The expression of Wo and SlCycB2 is strongly repressed in plants overexpressing SlJAZ2, suggesting that SlJAZ2 inhibits glandular trichome development by downregulating the positive regulators Wo and SlCycB2 [START_REF] Yu | The Jasmonate ZIM-domain protein gene SlJAZ2 regulates plant morphology and accelerates flower initiation in Solanum lycopersicum plants[END_REF] (Figure 41). The induction of glandular trichome initiation by JA in Artemisia annua and tomato probably proceeds through the JA-triggered degradation of the repressors AaJAZ8 and SlJAZ2 by the proteasome, respectively. Consistently, SlJAZ2 was shown to directly interact with the F-box protein SlCOI1 in a yeast two-hybrid screen [START_REF] Thines | JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling[END_REF] and SlCOI is itself a positive regulator of glandular trichome development [START_REF] Li | The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development[END_REF]. In addition to jasmonate signaling, other hormonal signaling pathways control glandular trichome development. For instance, two genes involved in auxin signaling have been shown to impact glandular trichome development: SlARF3 (Zhang et al., 2015b) and SlIAA15 [START_REF] Deng | The tomato SlIAA15 is involved in trichome formation and axillary shoot development[END_REF].

Genes controlling later steps of glandular trichome development

Cytoskeleton regulators

Following the initiation step, glandular trichome morphogenesis starts. Various cellular components are at play to define glandular trichome shape, which is essential for its correct functioning. Indeed, the alteration of type VI glandular trichome morphology caused by the hairless mutation in tomato leads to impaired synthesis of defense metabolites and decreased resistance to herbivores [START_REF] Kang | Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue[END_REF]. The mutation responsible for the observed bending and swelling of type VI glandular trichomes has been located in the gene encoding the SRA1 subunit of the WAVE regulatory complex [START_REF] Kang | Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue[END_REF]. This complex is highly conserved among eukaryotes and controls actin filaments nucleation and polymerization [START_REF] Kang | Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue[END_REF]. Therefore, actin cytoskeleton remodeling seems to play a critical role in glandular trichome morphogenesis. This hypothesis is reinforced by the recent analysis of the tomato inquieta mutant [START_REF] Jeong | Genetic analysis of the tomato inquieta mutant links the ARP2/3 complex to trichome development[END_REF]. Glandular trichomes of this mutant display similar morphological defects as glandular trichomes of the hairless mutant.

This phenotype has been associated with a mutation in the homolog of the ARPC2A gene of Arabidopsis thaliana, which is another important actor of actin cytoskeleton polymerization [START_REF] Jeong | Genetic analysis of the tomato inquieta mutant links the ARP2/3 complex to trichome development[END_REF].

Cuticle deposition regulators

Several studies have highlighted a tight link between cuticle deposition and non-glandular trichome development in Arabidopsis thaliana, with many genes involved in both processes [START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF]. Similarly, the correct accumulation of cuticle may be crucial for glandular trichome development in tomato and Artemisia annua. In Artemisia annua, downregulation of the AP2/ERF transcription factor gene AaTAR1 by RNAi leads to an altered cuticular wax deposition and an increase in cuticle permeability [START_REF] Tan | TRICHOME and ARTEMISININ REGULATOR 1 is required for trichome development and artemisinin biosynthesis in Artemisia annua[END_REF]. Interestingly, glandular trichomes of AaTAR1-RNAi plants have an abnormal morphology: the top of the gland is swollen and gland cell number is reduced [START_REF] Tan | TRICHOME and ARTEMISININ REGULATOR 1 is required for trichome development and artemisinin biosynthesis in Artemisia annua[END_REF]. In tomato, downregulation of the R2R3-MYB transcription factor gene SlMX1 by RNAi decreases cuticle deposition along with trichome density, whereas the opposite is observed in lines overexpressing SlMX1 [START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Ewas | RNA-seq reveals mechanisms of SlMX1 for enhanced carotenoids and terpenoids accumulation along with stress resistance in tomato[END_REF]. Likewise, the sticky peel mutant, which carries a mutation in the HD-ZIP IV transcription factor gene SlCD2, is impaired in cutin accumulation and displays a lower glandular trichome density at the same time [START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF]. These examples highlight a link between cuticle formation and glandular trichome initiation in tomato and Artemisia annua, but it is unclear whether cuticle deposition is necessary for proper glandular trichome morphogenesis, or whether these two processes are simply coregulated. The analysis of glandular trichome morphology in plants harboring mutation in cuticle biosynthesis genes could help to answer this question.

Evolution of glandular trichome development regulators

Evidence supporting the conservation of glandular trichome development regulators in the Solanaceae

Several lines of evidence suggest that multicellular trichome formation, including glandular ones, may be controlled by the same regulatory network in all Solanaceae. Indeed, ectopic expression of Wo v , a strong allele of Wo, is able to induce multicellular trichome formation in tobacco and potato, and tobacco homologs of Wo and SlCycB2 were upregulated in Wo voverexpressing tobacco plants [START_REF] Yang | Transcriptome profile analysis of cell proliferation molecular processes during multicellular trichome formation induced by tomato Wo v gene in tobacco[END_REF]. The ectopic expression of SlH in tobacco also triggers trichome formation [START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF]. Conversely, ectopic expression of pepper or tobacco orthologues of SlH in tomato plants induces trichome formation [START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF]. Moreover, the overexpression of the tobacco gene NtCycB2 in tomato and tobacco led to a phenotype comparable to the overexpression of SlCycB2 in both species [START_REF] Gao | The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense[END_REF]. Taken together, these results support the idea that the function of at least H and CycB2, and probably Wo, is conserved among Solanaceae species.

Some regulators appear to be conserved in distant plant families, while the others are likely to have evolved independently

In Arabidopsis thaliana, non-glandular trichome development also involves transcription factors belonging to R2R3-MYB and HD-ZIP IV subfamilies. The R2R3-MYB transcription factor GLABRA 1 (AtGL1) interacts with bHLH and WD40 proteins to form a MYB-bHLH-WD40 complex [START_REF] Pattanaik | An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis[END_REF]. This complex is able to induce the expression of the HD-ZIP IV transcription factor GLABRA 2 (AtGL2), which positively regulates non-glandular trichome initiation. Besides, single repeat R3-MYB transcription factors repress non-glandular trichome initiation [START_REF] Pattanaik | An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis[END_REF].

The positive regulator of glandular trichome development AaMYB1 increases AtGL1 and

AtGL2 expression and induces non-glandular trichome initiation when ectopically expressed in Arabidopsis thaliana [START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF]. The orthologue of AaMYB1 in Arabidopsis thaliana is AtMYB61. Interestingly, non-glandular trichome density is reduced in myb61 mutants, indicating that AtMYB61 positively regulates non-glandular trichome initiation in Arabidopsis thaliana [START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF]. Another study shows that a functional orthologue of the R3-MYB gene AtTRY may be present in tomato: indeed, SlTRY is able to inhibit trichome initiation when expressed in Arabidopsis thaliana [START_REF] Tominaga-Wada | Control of Plant Trichome and Root-Hair Development by a Tomato (Solanum lycopersicum) R3 MYB Transcription Factor[END_REF]. Taken together, these results suggest that several genes may have a conserved function in the regulation of trichome initiation in Arabidopsis thaliana, Artemisia annua and Solanum lycopersicum.

However, other studies suggest that the network controlling non-glandular trichome initiation in Arabidopsis thaliana is different from the one controlling glandular trichome initiation in the Solanaceae. AtGL1 does not have any impact on glandular trichome development when expressed in tobacco, and the expression of AmMIXTA, a gene of Antirrhinum majus closely related to AaMIXTA1 and SlMX1, could not rescue the phenotype of the gl1 mutant of Arabidopsis thaliana [START_REF] Yang | Trichomes as models for studying plant cell differentiation[END_REF]. Moreover, although AtGL2

and Wo are both HD-ZIP IV transcription factors, the closest homolog of Wo in Arabidopsis thaliana is not AtGL2 but PROTODERMAL FACTOR 2 (AtPDF2), a gene involved in shoot epidermal cell differentiation but not in trichome initiation [START_REF] Yang | Trichomes as models for studying plant cell differentiation[END_REF][START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF]. Some regulators involved in early steps of trichome development may be shared among a wide range of dicots, whereas other regulators are likely to have evolved several times independently among dicots and monocots. This statement raises the following question: which regulatory networks could have served as starting material for the evolution of trichome development regulators?

Different scenarios for the evolution of glandular trichome development regulators

MIXTA-like and HD-ZIP IV transcription factors are known to be key regulators of epidermal cell fate in different plant species [START_REF] Brockington | Evolutionary Analysis of the MIXTA Gene Family Highlights Potential Targets for the Study of Cellular Differentiation[END_REF][START_REF] Takada | Specification of epidermal cell fate in plant shoots[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. For example, several HD-ZIP IV transcription factors are known to be involved in cuticle biosynthesis and stomata patterning [START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF]. Glandular trichomes being specialized epidermal structures, some genes initially involved in the differentiation of other epidermal structures may have acquired a function in glandular trichome development during the course of evolution. In agreement with this hypothesis, several regulators of glandular trichome development characterized in Artemisia annua and tomato also have additional functions related to epidermal cell differentiation. AaMYB1 and AtMYB61 have been shown to impact not only trichome density, but also stomatal aperture in Arabidopsis thaliana [START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF]. Similarly, SlCD2 and SlMX1 in tomato and the AaHD8-AaMIXTA1 complex in Artemisia annua seem to be involved in cuticle development in addition to glandular trichome initiation [START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF][START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Ewas | RNA-seq reveals mechanisms of SlMX1 for enhanced carotenoids and terpenoids accumulation along with stress resistance in tomato[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF].

The main function of glandular trichomes being the production of specialized metabolites, some genes initially involved in the regulation of specialized metabolism could also have acquired a function in glandular trichome development. Some members of the R2R3-MYB subfamily are specialized metabolism regulators [START_REF] Chezem | Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs[END_REF] and several bHLH transcription factors have been reported to activate terpene biosynthesis in various plant species [START_REF] Xu | SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells[END_REF]. In agreement with this hypothesis, the bHLH transcription factor SlMYC1 differentially regulates mono-and sesquiterpene biosynthesis in leaves and stems of cultivated tomato in addition to its role in glandular trichome initiation [START_REF] Xu | SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells[END_REF].

Lastly, since the production of specialized metabolites by glandular trichomes is a part of plant response to biotic and abiotic stresses, genes initially involved in stress response may have acquired a function in glandular trichome development. Consistently, JAZ repressors have been shown to repress glandular trichome initiation, linking this developmental process to JA signaling, a well-known stress signaling pathway [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF][START_REF] Yu | The Jasmonate ZIM-domain protein gene SlJAZ2 regulates plant morphology and accelerates flower initiation in Solanum lycopersicum plants[END_REF].

Perspectives

Almost all genes recently shown to be involved in glandular trichome development in tomato and Artemisia annua also impact the development of non-glandular trichomes [START_REF] Deng | The tomato SlIAA15 is involved in trichome formation and axillary shoot development[END_REF][START_REF] Tan | TRICHOME and ARTEMISININ REGULATOR 1 is required for trichome development and artemisinin biosynthesis in Artemisia annua[END_REF][START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Kang | Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue[END_REF][START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF][START_REF] Matías-Hernández | AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[END_REF][START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF][START_REF] Gao | The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF][START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF]. SlMYC1 is the only gene identified so far which seems to affect only glandular trichome development [START_REF] Xu | SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells[END_REF]. It would be of high interest to identify other genes specifically controlling glandular trichome development, in particular genes involved in the acquisition of the secretory activity. In tomato, such genes may be found among the targets of SlMYC1, which remain to be identified. Alternatively, a comparative study of type IV and type V trichomes could lead to insights in the genetic regulation of glandular cell differentiation, because these two trichome types are morphologically very similar, except for the apical cell which is glandular in type IV and non-glandular in type V trichomes [START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]. In Artemisia annua, given the fact that HD-ZIP transcription factors act only as dimers, AaHD1 may interact with distinct HD-ZIP transcription factors to induce glandular or non-glandular trichome initiation. Therefore, the identification of AaHD1 interactors may lead to the identification of regulators specific to glandular trichome initiation [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF].

In tomato, the elucidation of the regulatory network underlying the initiation and differential morphogenesis of all trichome types may be challenging, but this diversity of trichome types could be highly valuable for the study of the fine regulation of trichome differentiation. Laser microdissection could be a powerful tool for comparative analyses of glandular trichome types. It is not easy to isolate protruding organs with this technique.

However, it has already been done successfully on Artemisia annua to compare glandular and non-glandular trichomes [START_REF] Soetaert | Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua[END_REF]. Besides, the recent identification of quantitative trait loci (QTLs) controlling the shape of type VI glandular trichomes in tomato represents a precious information for the future characterization of more genetic regulators of glandular trichome morphogenesis [START_REF] Bennewitz | QTL Mapping of the Shape of Type VI Glandular Trichomes in Tomato[END_REF].

A better understanding of glandular trichome development will open exciting avenues for the targeted improvement of agronomical traits. For example, tomato lines with more type IV trichomes or bigger type VI trichome secretory cavities could produce more acylsugars and terpenes, respectively, and thus show better resistance to herbivores [START_REF] Vendemiatti | Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF][START_REF] Bennewitz | QTL Mapping of the Shape of Type VI Glandular Trichomes in Tomato[END_REF]. The yield of high value-added compounds produced in plant glandular trichomes could also be increased, with benefits for perfume and pharmaceutical industries. Results obtained on Artemisia annua, which produces the antimalarial drug artemisinin, are encouraging. The overexpression of AaMIXTA1, AaHD1 or AaHD8 significantly enhanced artemisinin production, without any adverse effect on plant growth and fitness [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua[END_REF][START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF]. The highest increase was observed with the overexpression of AaMIXTA1, which doubled artemisinin content [START_REF] Shi | The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF].

Figure 1 :

 1 Figure 1: Chemical formulas of ambrein, ambroxide and sclareol.

France

  , the Interprofessional Technical Institute of Perfume, Medicinal, Aromatic and Industrial Plants (ITEIPMAI) is currently conducting a clary sage breeding program and has already obtained several improved varieties (see page 20). The development of new clary sage varieties with enhanced sclareol yield is the purpose of the PISTILL project driven by the Flower and Carpel Development Group at the Institute of Plant Sciences Paris-Saclay. The work presented here is intended to improve our understanding of sclareol biosynthesis and of mechanisms regulating sclareol production in clary sage. This knowledge will ultimately guide plant breeding approaches developed in the context of the PISTILL project.

Figure 2 :

 2 Figure 2: Wild clary sage growing in the region of Makarska, Croatia (July, 2016).

Figure 3 :

 3 Figure 3: Sclareol is mainly produced in clary sage calyces. A: Clary sage calyx. B: Apex of a clary sage inflorescence[START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. C: Sclareol quantification in different flower organs of clary sage[START_REF] Caissard | Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)[END_REF]. Legends: Br, bract; FB, flower bud; Co, corolla; Ca, calyx; Le, leaf.

Figure 4 :

 4 Figure 4: Exploitation of clary sage in perfume industry.

  growing demand for sclareol encouraged the development of clary sage breeding programs in countries where clary sage is cultivated, including Hungary, Bulgaria, Moldavia, Romania and France (source: Community Plant Variety Office). In France, the Interprofessional Technical Institute of Perfume, Medicinal, Aromatic and Industrial Plants (ITEIPMAI) has obtained several improved clary sage varieties through recurrent selection targeting sclareol yield and flowering time. Recurrent selection is a plant breeding approach involving successive cycles of three steps: intermating between best-performing populations, evaluation of traits of interest, and selection of best-performing individuals.

  Figure 6: Examples illustrating the diverse biological functions of terpenes in plants.

Figure 7 :

 7 Figure 7: Pharmacologically active diterpenoids and their respective source plants[START_REF] Mafu | Plant diterpenoid metabolism for manufacturing the biopharmaceuticals of tomorrow: prospects and challenges[END_REF].

3. 4

 4 .1. Production of five-carbon building blocks by MEP and MVA pathways IPP and DMAPP are the universal five-carbon building blocks from which all terpenes are derived. The co-existence of two metabolic pathways for the production of IPP and DMAPP is an atypical feature of plants: most bacteria use only the MEP pathway, and animals, fungi

  photosynthetic and floral tissues and are thought to be mostly involved in primary metabolism, whereas DXS2 enzymes have more restricted spatiotemporal expression patterns and are generally involved in specialized metabolism[START_REF] Tholl | Biosynthesis and Biological Functions of Terpenoids in Plants[END_REF]. The second step of the MEP pathway corresponds to the conversion of DXP into 2-methyl-D-erythritol-4phosphate (MEP) through the action of the DXP-reductoisomerase (DXR). The MEPcytidylyltransferase (MCT) then couples MEP to CDP to form 4-diphosphocytidyl-2-methyl-Derythritol (CDP-ME), which is subsequently phosphorylated by the CDP-ME kinase (CMK).The obtained 4-diphosphocytidyl-2-methyl-D-erythritol-2-phosphate (CDP-ME2P) is cyclized by the MEcPP synthase (MDS) to form 2-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP).

Figure 8 :

 8 Figure 8: Production of five-carbon building blocks by MEP and MVA pathways (adapted from Tholl, 2015). AACT, acetoacetyl-CoA thiolase; CDP-ME, 4-diphosphocytidyl-2-methyl-D-erythritol; CDP-ME2P, 4diphosphocytidyl-2-methyl-D-erythritol-2-phosphate; CMK, CDP-ME kinase; CoA, coenzyme A; DMAPP, dimethylallyl pyrophosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXR, DXPreductoisomerase; DXS, DXP-synthase; GAP, glyceraldehyde-3-phosphate; HDR, HMBPP-reductase; HDS, HMBPP-synthase; HMBPP, 4-hydroxy-3-methylbut-2-enyl-diphosphate; HMG-CoA, hydroxymethylglutaryl-CoA; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IDI, IPP isomerase; IPP, isopentenyl pyrophosphate; MCT, MEP-cytidylyltransferase; MDS, MEcPP synthase; MEcPP, 2-methyl-D-erythritol-2,4-cyclodiphosphate; MEP, 2-methyl-D-erythritol-4-phosphate; MK, mevalonate kinase; MPDC, mevalonate-diphosphate decarboxylase; MVA, mevalonate; PMK, phospho-mevalonate kinase.

Figure 9 :

 9 Figure 9: The role of prenyltransferases in terpene biosynthesis. DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl pyrophosphate.

Figure 11 :

 11 Figure 11: Seasonal variations of terpene production in Crithmum maritimum[START_REF] Barroso | Seasonal Variation in the Composition of the Essential Oil of Crithmum maritimum L. Flavour Fragr[END_REF].

Figure 12 :

 12 Figure 12: Structure of a peltate glandular trichome[START_REF] Lange | The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity[END_REF]

Figure 13 :

 13 Figure 13: Glandular trichomes of clary sage calyces observed by scanning electron microscopy. A: Developing calyx (stage 1). B: Mature calyx (stage 5). Calyx development stages: see page 97. Full white arrows indicate large capitate glandular trichomes; empty white arrows indicate small capitate glandular trichomes; full yellow arrows indicate peltate trichomes; empty yellow arrows indicate non-glandular trichomes. Scale bars: 100 µm.

Figure 14 :

 14 Figure 14: Diurnal variations in clary sage essential oil yield[START_REF] Lawrence | Production of Clary Sage Oil and Sclareol in North America[END_REF].

Figure 15 :

 15 Figure 15: Molecular mechanism of sclareol biosynthesis (adapted from Schalk et al., 2012). GGPP, geranylgeranyl pyrophosphate; LPP, labda-13-en-8-ol diphosphate; LPPS, LPP-synthase; SS, sclareol-synthase.

Figure 16 :

 16 Figure 16: Chemical formulas of the main abietane diterpenes found in clary sage roots.

Figure 17 :

 17 Figure 17: Epidermal crystals observed by Caissard et al. at the surface of clary sage calyces (Caissard et al., 2012). Clary sage calyces were observed by environmental scanning electron microscopy. Arrows indicate sclareol crystals. Scale bars: 100µm.

Figure 18 :

 18 Figure 18: Average mass spectrum of the surface of a clary sage calyx. The surface of a mature clary sage calyx was analyzed by mass spectrometry imaging (LDI-FT-ICR). This mass spectrum corresponds to the average of mass spectra obtained at each position of the calyx sample.

Figure 19 :

 19 Figure 19: Localization of sclareol and linalyl acetate at the surface of a clary sage calyx. The chemical composition of the epidermal surface of a clary sage calyx was analyzed through laser desorption-ionization followed by FT-ICR mass spectrometry (LDI-FT-ICR). A: Top views of three clary sage calyx samples observed with a zoom stereomicroscope. B-D: Spatial distribution of [linalyl acetate-K] + (green), [sclareol-K] + (blue) and [(sclareol) 2 -K] + (red) ions at the surface of calyx samples shown in A. White arrows (full or empty) indicate capitate glandular trichomes. Yellow arrows indicate peltate glandular trichomes. Scale bars: 500 µm.

Figure 20 :

 20 Figure 20: Localization of sclareol and linalyl acetate at the surface of a clary sage calyx (detail). The chemical composition of the epidermal surface of a clary sage calyx was analyzed through laser desorption-ionization followed by FT-ICR mass spectrometry (LDI-FT-ICR). A: Top view of a clary sage calyx sample observed with a zoom stereomicroscope. B-D: Spatial distribution of [linalyl acetate-K] + (green), [sclareol-K] + (blue) and [(sclareol) 2 -K] + (red) ions at the surface of the calyx sample shown in A. White arrows indicate capitate glandular trichomes and yellow arrows indicate a peltate glandular trichome. Scale bars: 250 µm.

  Although [linalyl acetate-K] + and [(sclareol) 2 -K] + distribution indicates that they are involved in linalyl acetate and sclareol production, capitate glandular trichomes are not systematically associated with both [linalyl acetate-K] + and [(sclareol) 2 -K] + signals. Some capitate glandular trichomes visible on optical images are associated only with a [linalyl acetate-K] + spot without any [(sclareol) 2 -K] + signal, and conversely (Figure 19 and 20, empty white arrows).

Figure 21 :

 21 Figure 21: Epidermal surface of a clary sage calyx observed by scanning electron microscopy. Scale bars: A, 500 µm; B-G, 100 µm. White arrows indicate glandular head content spilling out of the trichome.

Figure 22 :

 22 Figure 22: Potential secretory activity of calyx "non-glandular" trichomes. A: Top view of a clary sage calyx sample observed with a zoom stereomicroscope. Scale bar: 1 mm. B: Spatial distribution of [sclareol-K] + ions at the surface of the calyx sample shown in A, detected through laser desorption-ionization followed by FT-ICR mass spectrometry (LDI-FT-ICR). Scale bar: 1 mm. Yellow dotted lines indicate the position of "non-glandular" trichomes. C-D: "Non-glandular" trichomes of a clary sage calyx observed by scanning electron microscopy. Yellow arrows indicate spills of electron-dense material similar to the glandular head content of capitate trichomes. Scale bars: C, 200 µm; D, 100 µm.

  phenotypes and the cloning of their causal mutations should shed light on this question and lead to insights in the understanding of glandular trichome development and sclareol production in clary sage.

Figure 23 :

 23 Figure 23: A glabrous mutant of clary sage displays reduced sclareol and linalyl acetate content. A: Observation of stem and calyces from wild-type and glabrous plants with a binocular. B: Density of different glandular trichome types at the surface of calyces from wild-type and glabrous plants. P: peltate glandular trichomes; LC: long capitate glandular trichomes; SC: small capitate glandular trichomes. Glandular trichomes were classified according to Schmiderer et al., 2008. Bars represent the average of 9 (WT) or 10 (glabrous mutant) measurements and error bars correspond to standard deviation. Stars indicate the results of Student's t-test (***: p<0.001). (Drevensek et al., unpublished data) C and D: Sclareol (C) and linalyl acetate (D) were quantified by GC-MS after hexane extraction. Bars represent the average of 3 replicates and error bars correspond to standard deviation.

  First, spots of [linalyl acetate-K] + and [(sclareol) 2 -K] + ions were detected on calyx samples by mass spectrometry imaging and coincide with capitate glandular trichomes (Figure19 and 20). [Sclareol-K] + ions were also detected on the rest of the epidermis (Figure19and 20). However, [(sclareol) 2 -K] + ions probably form only where sclareol is most abundant and may thus indicate the main sites of sclareol production. Secondly, a clary sage line with fewer capitate glandular trichomes produces less linalyl acetate and sclareol (Figure23). Due to the presence of thousands of EMS-induced point mutations in this line, the two phenotypes may be caused by distinct mutations, but we can suppose that they have the same causal mutation. Since peltate and small capitate trichomes are unchanged compared to the wild-type (Figure23b), this mutation may be located in a gene encoding a transcription factor specifically inducing large capitate trichome development. Taken together, these results support the idea that glandular trichomes are the main site of sclareol production. This theory is further underpinned by gene expression analyses performed on isolated glandular trichomes, showing that sclareol-synthase and LPP-synthase genes are more expressed in isolated glandular heads compared to the rest of the calyx(Drevensek et al., unpublished data)

Figure 24 :

 24 Figure24: Expression level of sclareol biosynthesis genes in isolated glandular trichomes.(Drevensek et al., unpublished data) Glandular trichomes (GT) were isolated from flower buds or calyces by abrasion with liquid nitrogen. Gene expression was quantified by reverse transcription-quantitative PCR (RT-qPCR) on RNAs purified from intact buds, intact calyces and purified glandular trichomes. TPS1: sclareol-synthase gene[START_REF] Günnewich | A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate[END_REF]. SS: sclareol-synthase gene[START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. TPS1132: sclareolsynthase gene[START_REF] Schalk | Toward a Biosynthetic Route to Sclareol and Amber Odorants[END_REF]. LPPS: LPP-synthase gene[START_REF] Caniard | Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture[END_REF]. Bars represent the average of measurements performed on 2 replicates and error bars correspond to standard deviation.

Figure 25 :

 25 Figure 25: Feeding of a clary sage inflorescence stem.

  2.4.2. Analysis of 13 C-labeling patterns by 13 C-NMR 13 C Nuclear Magnetic Resonance ( 13 C-NMR) experiments were performed using an Ascend 400 MHz NanoBay instrument equipped with an AVANCE III HD console and a Prodigy BBO cryoprobe ( 31 P-15 N) (Bruker). Analyses were performed at 277 K without tube spinning, using a proton-decoupled (decoupling sequence waltz16) carbon pulse program (zgig) with 90° pulses of 9.75 µs at 41 W for 13 C. The relaxation delay (D1) was set to 15 s in order to enable complete acquisition of the signal corresponding to 13 C nuclei relaxation. To get good signalto-noise ratio, 5096 scans were done, representing 23 h 34 min of analysis per sample. Data were analyzed using the TopSpin software (Bruker). Free induction decay signal was processed by performing an exponential line broadening, calculating the Fourier transform,

Figure 26 :

 26 Figure 26: Analysis of the composition of clary sage calyx organic extracts by GC-MS and 13 C-NMR. A, B: GC-MS chromatograms of clary sage calyx extracts obtained after extraction in hexane (A) or ethanol (B). Values indicated in B correspond to average peak area expressed as a percentage of total peak area ± standard deviation (8 biological replicates). ISTD: internal standard (10-undecen-1-ol). C: 13 C-NMR spectrum of sclareol and linalyl acetate analytical standards and of a clary sage calyx hexane extract.

Figure 27 :

 27 Figure 27: Impact of mevinolin or fosmidomycin treatment on sclareol and linalyl acetate content. Clary sage cut inflorescence stems were fed with a solution containing mevinolin (A) or fosmidomycin (B) for 6 days. Sclareol and linalyl acetate content was quantified by GC-MS. Bars correspond to the average of measurements performed on 4 biological replicates; error bars correspond to the standard deviation and stars indicate the results of Student's t-test (*: p<0.05, **: p<0.01).

Figure 28 :

 28 Figure 28: Theoretical 13 C-labeling patterns of sclareol and linalyl acetate after 1-13 C-glucose processing through MVA and MEP pathways.In grey, carbons predicted to be unlabeled; in blue, carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis.

  Figure 29: Sclareol 13 C-labeling after feeding with 1-13 C-glucose.

Figure 30 :

 30 Figure30: Linalyl acetate 13 C-labeling after feeding with 1-13 C-glucose. Clary sage cut inflorescence stems were fed with a solution containing 1-13 C-glucose (Labeled) or standard glucose (Unlabeled) for 5 days. Linalyl acetate 13 C-labeling was analyzed by quantitative 13 C-NMR after hexane extraction. Bars correspond to the average of peak area measurements performed on 3 biological replicates and error bars correspond to the standard deviation. In grey, carbons predicted to be unlabeled; in blue, carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis.

  Figure 31: Sclareol and linalyl acetate 13 C-labeling after feeding with 1-13 Cglucose and terpene biosynthesis inhibitors. Clary sage cut inflorescence stems were fed with a solution containing 1-13 C-glucose or standard glucose with 100µM of mevinolin (MEV), 100µM of fosmidomycin (FOS) or no inhibitor (Control) for 5 days. Sclareol (A) and linalyl acetate (B) 13 C-labeling was analyzed by quantitative 13 C-NMR after hexane extraction. The labeling ratio is calculated by dividing peak area measured for labeled samples by peak area measured for unlabeled samples. Bars correspond to the average of labeling ratio measurements performed on 3 biological replicates and error bars correspond to the standard deviation.In grey, carbons predicted to be unlabeled; in blue, carbons predicted to be labeled only if the MVA pathway is involved in IPP biosynthesis; in green, carbons predicted to be labeled only if the MEP pathway is involved in IPP biosynthesis; in orange, carbons predicted to be labeled whatever the pathway involved in IPP biosynthesis.

Figure 33 :

 33 Figure 33: Sclareol and linalyl acetate accumulation during calyx development.Sclareol and linalyl acetate content at each developmental stage of the calyx (A) were quantified by GC-MS after hexane extraction and are expressed in mg/calyx (C) or as a percentage of calyx dry weight (D). Dry weight was measured after hexane extraction (B). Hexane extraction was performed without removing the developing corolla from the bud at stages 1 and 2 and without removing achenes at stages 5 and 6. To measure dry weight, achenes were removed from stage 6 calyces, but not from stage 5 calyces (because of their very small size at this stage, their mass was considered negligible).

  treatment on flowers by feedingIn order to analyze the impact of MeJA on sclareol and linalyl acetate production by calyces, clary sage cut inflorescence stems were treated with MeJA by feeding. A study performed on Achyranthes bidentata has shown that MeJA can be transported to distal leaves through stem vasculature. Moreover, an increased emission of volatile organic compounds was detected in distal leaves of Achyranthes bidentata cut stems fed with a solution containing

Figure 34 :

 34 Figure 34: Impact of MeJA treatment on cut inflorescence stem growth and fitness. Clary sage cut inflorescence stems were fed with a solution containing 7.5mM of MeJA for 6 days. White arrows indicate brown calyces and black arrows indicate green calyces.

Figure 35 :

 35 Figure 35: Impact of MeJA treatment on sclareol and linalyl acetate content. A: Clary sage cut inflorescence stems were fed with a solution containing MeJA for 6 days. Bars represent the average of measurements performed on 4 biological replicates and error bars correspond to standard deviation. B: Clary sage cut inflorescence stems were fed with a solution containing 3 mM of MeJA for 2 days. Bars represent the average of measurements performed on 10 calyces (5 stems, 2 calyces per stem) and error bars correspond to standard deviation. C: Clary sage whole plants were sprayed once with a solution containing 3 mM of MeJA and calyces were sampled 2 days after treatment. Bars represent the average of measurements performed on 4 calyces (MeJA) or 8 calyces (Mock) and error bars correspond to standard deviation. Sclareol and linalyl acetate were quantified by GC-MS after hexane extraction.

Seeds from 6

 6 wild populations of clary sage were sampled in summer 2016 around the city of Makarska in Croatia. Localization of sampling sites is indicated in Figure 36, along with their elevation above sea level. For each population, seeds from 10 individuals were collected and stored at 4 °C and 10% humidity until sowing. Vatican White seeds were purchased from Jelitto (Germany). Toscalia and Milly seeds were kindly donated by the French National Conservatory of Perfume, Medicinal, Aromatic and Industrial Plants (CNPMAI).

Figure 37 :

 37 Figure 37: Single nucleotide polymorphisms (SNPs) found in DXS2 and CMK sequences from Croatian and reference clary sage populations. A: SNPs found in DXS2 and CMK sequences are highlighted in yellow and encoded using the IUPAC code. B: Genotype of individuals used to estimate genetic diversity. Heterozygous genotypes are encoded using the IUPAC code (R = A or G; S = C or G; W = A or T; Y = C or T).

Figure 38 :

 38 Figure 38: Estimation of genetic diversity found in Croatian and reference clary sage populations.The tree was built with the Neighbor Joining method using concatenated haploid sequences from ITS, DXS2 and CMK loci. The 2 haploid sequences carried by each individual were considered to be independent. The percentage of replicate trees in which haploid sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches.

  Calyces from each individual were sampled in the field in summer 2018 and sclareol and linalyl acetate were quantified by GC-MS (Figure40). No significant difference in sclareol and linalyl acetate content of calyces could be detected between the three Croatian populations, which produce the same amount of sclareol and linalyl acetate as Milly plants. Interestingly, Chapter 4 -Clary sage natural diversity 120 a significant difference was detected between the 2 reference populations: Vatican White plants produce 20% less sclareol and 30% less linalyl acetate than Milly plants (Figure40).

Figure 39 :Figure 40 :

 3940 Figure 39: Flowering of Croatian and reference clary sage populations. A, B: Flowers of representative Vatican White (A) and Milly (B) clary sage plants. C: Date of flowering, expressed in days after sowing. The day of opening of the first flowers was considered as the flowering date. Letters indicate the result of a Kruskal-Wallis rank test: values harboring the same letter are not significantly different.

  Beforehand, alignment of ITS, DXS2 and CMK sequences underlined the presence of SNPs in the genome of individual plants belonging to Croatian populations (Figure 37). Haploid sequences (harboring various combinations of SNPs) were used to estimate genetic diversity among Croatian populations (Figure 38). Each haploid sequence was present in at least two populations, highlighting the existence of gene exchanges between Croatian populations, or pointing to an incomplete genetic differentiation from the same initial pool of alleles.Frequencies of the different haploid sequences varied among Croatian populations, suggesting that Croatian populations were not genetically equivalent. However, flowering time, sclareol content and linalyl acetate content were not significantly different between the three populations analyzed in the field trial (Figures39 and 40). Climatic conditions encountered in coastal, mountain and plateau environments may not have led to significant genetic differentiation of clary sage populations. If the impact of selective pressure is limited, it may be masked by potential gene flows due to pollen transport between populations. The extent of gene exchanges may have been underestimated due to the limited number of genetic markers used to evaluate genetic diversity among Croatian populations.

  significant differences between the two clary sage populations used as reference populations, Vatican White and Milly. Vatican White plants flowered 10 days earlier and produced 20% less sclareol and 30% less linalyl acetate than Milly plants (Figures39 and 40).For this reason, if a GWAS approach is planned in the context of clary sage breeding, it would be judicious to include Vatican White and Milly populations in the collection of clary sage populations.The Milly population is a French population of clary sage maintained by the French National Conservatory of Perfume, Medicinal, Aromatic and Industrial Plants (CNPMAI), whereas the Vatican White population is propagated and commercialized for ornamental purposes, notably owing to the distinct color of its flowers. Indeed, Vatican White flowers are generally white or light purple, whereas flowers of other populations, including Milly and Croatian populations, display darker shades of pink and purple (Figure39). In other plant species, the pink or purple color of flowers is often due to the presence of specialized metabolites belonging to the flavonoid family[START_REF] Tanaka | Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids[END_REF]. The lighter color of Vatican White flowers may be the consequence of reduced flavonoid biosynthesis.A simultaneous decrease in flavonoid biosynthesis and terpenoid biosynthesis may be explained by a global downregulation of specialized metabolism in Vatican White flowers. If this downregulation is operated at transcriptome level, it would be relevant to conduct a comparative transcriptomic study of Milly and Vatican White flower organs. A differential expression of biosynthetic genes would validate the initial hypothesis, and differentially expressed transcription factors would be good candidates for an involvement in specialized metabolism regulation or flowering time control. Since sclareol and linalyl acetate are mainly produced by glandular trichomes (Chapter 1), the lower metabolite content observed in

  properties. In response to the development of ambroxide use in functional perfumery, the global demand for sclareol currently raises, prompting efforts aiming at increasing sclareol supply. The PISTILL project, driven by the Flower and Carpel Development Group at the Institute of Plant Sciences Paris-Saclay, aims at increasing the yield of sclareol production from clary sage through targeted genetic improvement. The purpose of the work presented in this manuscript was to enhance knowledge on sclareol biosynthesis and its regulation in clary sage, with a dual objective: orientating the PISTILL project towards promising strategies to boost sclareol yield, and improving our general understanding of terpenoid production in plants. We investigated the cellular and metabolic origin of sclareol in clary sage calyces, tried to identify regulation mechanisms controlling sclareol accumulation, and started to explore how clary sage natural diversity could be exploited. This study opens up perspectives for basic research aiming at understanding sclareol production in clary sage, and at the same time highlights interesting avenues for targeted genetic enhancement of clary sage performances.

3. 1

 1 .3. Other transcription factorsTwo other transcription factors involved in glandular trichome initiation and belonging neither to the R2R3-MYB subfamily, nor to the HD-ZIP IV subfamily, have also been recently characterized in tomato: the C2H2 zinc-finger protein HAIR (SlH)[START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF] and the bHLH protein MYELOCYTOMATOSIS-RELATED 1 (SlMYC1)[START_REF] Xu | SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells[END_REF] (Figure41). Downregulation of SlMYC1 by RNAi or missense mutations in SlH reduce type VI or type I glandular trichome density, respectively. Moreover, type VI glandular trichomes are absent in myc1 knockout mutants and SlH knockout leads to a hair-absent phenotype[START_REF] Xu | SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells[END_REF][START_REF] Chang | Hair , encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF]. These results indicate that SlMYC1 positively regulates the initiation of type VI glandular trichomes and that SlH is a key positive regulator of all glandular trichome types. Interestingly, type VI glandular trichomes have smaller glands and shorter stalks in SlMYC1-RNAi plants, suggesting that SlMYC1 is also an important regulator of later steps of type VI glandular trichome morphogenesis in tomato[START_REF] Xu | SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells[END_REF].

Figure 41 :

 41 Figure 41: Simplified model of glandular trichome initiation in sweet wormwood (Artemisia annua) and tomato (Solanum lycopersicum).In Artemisia annua, glandular trichome initiation is positively regulated by the HD-ZIP IV transcription factor HD1. The expression of HD1 is enhanced by a complex formed by the R2R3-MYB transcription factor MIXTA1 and the HD-ZIP IV transcription factor HD8. The R2R3-MYB transcription factor MYB1 is another positive regulator of glandular trichome initiation. Conversely, the JA signaling repressor JAZ8 represses HD1 transcriptional activity, thereby inhibiting glandular trichome initiation. In Solanum lycopersicum, glandular trichome initiation is positively regulated by the HD-ZIP IV transcription factor WOOLLY. WOOLLY has been shown to interact with the B-type cyclin CycB2 and with the C2H2 zinc-finger protein HAIR, which is itself a positive regulator of glandular trichome initiation. A HAIR-WOOLLY-CycB2 complex may exist, but has not been detected yet. The bHLH transcription factor MYC1, the R2R3-MYB transcription factor MX1 and the HD-ZIP IV transcription factor CD2 are 3 other positive regulators of glandular trichome initiation. CD2 is the closest tomato homolog of the transcription factor HD8 of Artemisia annua. Like HD8, CD2 may interact with a MIXTA-like protein, maybe MX1. MX1 and WOOLLY both induce the expression of CycB2. Conversely, the JA signaling repressor JAZ2 inhibits WOOLLY and CycB2 expression, thereby inhibiting glandular trichome initiation.

  Knowing to what extent gene networks controlling glandular trichome development are conserved among the plant kingdom will be critical to develop plant breeding strategies based on glandular trichome phenotype in other plant species. Targeted mutagenesis approaches like CRISPR-Cas9 or TILLING could be used to investigate whether the functions of already identified genes are conserved or not, and to characterize new regulators and actors of glandular trichome development.Université Paris-SaclayEspace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Biosynthèse du sclaréol et sa régulation chez la sauge sclarée Mots-clés : biologie des plantes, terpène, ingénierie métabolique, sclaréol, sauge sclarée, métabolisme spécialisé des plantes Résumé : Le sclaréol est un diterpène produit par les organes floraux de la sauge sclarée (Salvia sclarea, Lamiaceae). Il est utilisé en parfumerie pour l'hémisynthèse de l'ambroxide, une substance caractérisée par une odeur ambrée et une grande capacité de fixation des parfums. L'augmentation de la demande mondiale en sclaréol stimule actuellement les tentatives d'accroître le rendement de la production de sclaréol à partir de la sauge sclarée. L'objectif du travail présenté dans ce manuscrit était d'améliorer notre compréhension de la biosynthèse du sclaréol et de sa régulation chez la sauge sclarée, afin de mettre en évidence des stratégies d'augmentation du contenu en sclaréol de la sauge sclarée. L'analyse de la surface des calices de sauge sclarée par imagerie par spectrométrie de masse suggère que le sclaréol est principalement sécrété par des structures épidermiques spécialisées appelées trichomes glandulaires. De plus, nous avons mis en évidence les contributions respectives des deux voies de biosynthèse des terpènes présentes chez les plantes, les voies MVA et MEP, à la biosynthèse de trois terpènes de la sauge sclarée. Des expériences de marquage au 13 C indiquent que le sclaréol et l'acétate de linalyle sont tous deux issus de la voie MEP, alors que le βcaryophyllène semble être d'origine mixte. Nous avons également étudié le rôle potentiel d'une phytohormone, le méthyljasmonate, dans la régulation de la production de sclaréol chez la sauge sclarée. Enfin, nous avons exploré la diversité génétique et phénotypique de populations croates de sauge sclarée sauvage, et montrons que ces populations représentent une ressource génétique distincte par rapport aux populations de référence. L'ensemble de ces résultats met en évidence des pistes prometteuses pour l'amélioration génétique ciblée des performances de la sauge sclarée.Title: Sclareol biosynthesis in clary sage and its regulationKeywords: plant biology, terpenoid, metabolic engineering, sclareol, clary sage, plant specialized metabolism Abstract: Sclareol is a diterpene produced by floral organs of clary sage (Salvia sclarea, Lamiaceae). It is used in perfume industry for the hemisynthesis of ambroxide, a high-valued perfume component characterized by an amber scent and a high perfume fixation capacity. The global demand for sclareol currently rises, prompting attempts at increasing the yield of sclareol production from clary sage. The purpose of the work presented in this manuscript was to improve knowledge on sclareol biosynthesis and its regulation in clary sage, in order to highlight strategies aiming at enhancing clary sage sclareol content. The analysis of the surface of clary sage calyces by mass spectrometry imaging suggests that sclareol is mainly secreted by specialized epidermal structures called glandular trichomes. Moreover, we have highlighted the respective contributions of the two terpenoid biosynthesis pathways present in plants, MVA and MEP pathways, to the biosynthesis of three terpenoids of clary sage. 13 C-labeling experiments indicate that sclareol and linalyl acetate both originate from the MEP pathway, whereas β-caryophyllene seems to be of mixed origin. We have also investigated the potential role of a phytohormone, methyljasmonate, in the regulation of sclareol production in clary sage. Finally, we have explored the genetic and phenotypic diversity of Croatian wild clary sage populations and show that these populations represent a distinct genetic resource compared to reference populations. Taken together, these results highlight promising avenues for targeted genetic enhancement of clary sage performances.

Table 1 : Interpretation of the 13 C-NMR spectrum of clary sage calyx extracts: attribution of peaks to carbon positions in sclareol and linalyl acetate.

 1

		Sclareol	Linalyl acetate δ [ppm]
	Peak		Carbon 11	170,89
	Peak	Carbon 14		146,50
	Peak		Carbon 6	142,51
	Peak		Carbon 7	132,66
	Peak		Carbon 2	124,44
	Peak		Carbon 1	113,86
	Peak	Carbon 15		112,11
	Peak		Carbon 3	83,58
	Peak	Carbon 8		75,62
	Peak	Carbon 13		74,60
	Peak	Carbon 9		62,20
	Peak	Carbon 5		56,69
	Peak	Carbon 7		45,51
	Peak	Carbon 12		44,83
	Peak	Carbon 3		42,63
	Peak		Carbon 4	40,55
	Peak	Carbon 1		40,24
	Peak	Carbon 10		39,86
	Peak	Carbon 18		34,20
	Peak	Carbon 4		33,83
	Peak	Carbon 16		27,90
	Peak		Carbon 9	26,51
	Peak	Carbon 17		25,00
	Peak		Carbon 10	24,16
	Peak		Carbons 5+12	23,01
	Peak	Carbon 19		22,21
	Peak	Carbon 11		21,19
	Peak	Carbon 6		19,75
	Peak	Carbon 2		19,11
	Peak		Carbon 8	18,24
	Peak	Carbon 20		16,12

δ: 13 C-NMR chemical shift.

  Sclareol and linalyl acetate were quantified by GC-MS after hexane extraction.

		7%				6%
	Sclareol (% calyx DW)	1% 2% 3% 4% 5% 6%			Linalyl acetate (% calyx DW)	1% 2% 3% 4% 5%
		0%				0%
			Mock MeJA	MeJA	MeJA	MeJA		Mock MeJA	MeJA	MeJA	MeJA
			20µM	100µM	500µM	1mM		20µM	100µM	500µM	1mM
			2-day-feeding			spraying
	Metabolite content (% calyx DW)	0% 1% 2% 3% 4% 5% 6% 7%		Mock MeJA 3mM	DW) calyx (% Metabolite content	0% 1% 2% 3% 7% 6% 5% 4%	Mock MeJA 3mM
			Sclareol Linalyl				Sclareol Linalyl
			acetate					acetate

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