Tina Ilc

Danièle Werck-Reichhart

Nicolas Navrot

Gautier Arista

Raquel Tavares

Frédéric Choulet

Etienne Paux

Marc Fischer

Philippe Hugueney

Cam Ille Rustenholz

ife r a CYPo m e

Abs tract

Wine arom a strongly influences wine quality, yet its com position and evolution during the winem aking process are poorly understood. Volatile com pounds that constitute wine arom a are traditionally divided into three classes according to their origin: grape, ferm entation and m aturation arom a. We challenge this view with m eta-analysis and review of grape and wine volatiles and their precursors from 82 profiling experim ents. We com piled a list of com m on grape and wine volatiles and quantitatively com pared 43 of them . Our work offers insight into com plex relationships between biosynthesis of arom a in grapes and the changes during the winem aking process. With a particular focus on m onoterpenes we show that the diversity of these com pounds in wines is m ainly due to the oxidative m etabolism of linalool.

Furtherm ore, we dem onstrate that m ost of the linalool produced in grapes is converted to these oxidized derivatives.

Ackn o w le d ge m e n ts

I would like to thank Daniele Werck for initiating this project and giving m e the great opportunity to do m y PhD thesis under her supervision. Thank you for your trust, for the opportunity to bring m y own ideas to the project and for your continuous availability for discussion. I also wish to thank Nicolas Navrot for his day-to-day supervision, support and encouragem ent, as well as all of his help when I cam e to Strasbourg 3 years ago.

I am grateful to Prof Sylvie Baudino, Prof Matthias Wüst and Dr Hubert Schaller for accepting to participate in m y jury. This work would not be possible without strong collaborations. I am grateful to all the collaborators from INRA Colm ar, for teaching m e a lot about grape and arom a in particular, for the m any useful discussions, as well as the practical help: Philippe Hugueney, Cam ille Rustenholz, Eric Duchene, Gautier Arista, Gisele Butterlin, Andrea Ilg and Marc Fischer. I want to thank Laurence Miesch and her team for synthesis of standards, as well as for m any interesting discussions. I am also grateful to Raquel Tavares from University of Lyon for the collaboration on phylogeny.

I am grateful to all the m em bers of Marie Curie ITN "P4fifty". It was a great pleasure to take part in this network and to get to know so m any excellent scientists. In particular, I would like to thank everyone who welcom ed m e for one of m y secondm ents: Fanny Lam bert (Mane S.A.); Sabine Flitsch, Slavom ira Husarova (University of Manchester), Bernhard Hauer, Stefan Ham m er and Silke Bastian (University of Stuttgart). I also wish to thank m y m entor Birger Lindberg Møller for m any interesting discussions and useful guidelines. Special thanks also to Margaret Cafferky for adm inistrating the network and being always available for help. I also thank the European Union for funding the "P4fifty" project. I want to thank the current and form er colleagues from the "Cytochrom e P450 " team :

Francois Bernier, Benoit Boachon, Carole Gavira, Thierry Heitz, Lucie Kriegshauser, Agn ès Lesot, Rozenn Menard, Fabienne Philippon, Em m anuelle Pineau, Franck Pinot, Hugues Renault, Pascaline Ullm an, Gaetan Verdier, and Em ilie Widem ann. Thank you all for your help, all the discussions, useful tips and challenging questions. My very special thanks go to Annette Alber, Claire Parage, J uliana Iglesias and Zhenhua Liu for always taking tim e for m y scientific as well as "real-life" problem s. I also wish to thank everyone from the Metabolom ics platform (Dim itri Heintz, J ulien Delecolle, Arnaud Agin, Maxim ilien Nuel, J ulie Zum steg and Raphael Lugan) and Gene expression platform (Malek Alioua) for your help and training. I want to thank m y fam ily for teaching m e the im portance of education in m y m ind from an early age. Thanks to m y grandm a for bringing m e up to be so stubborn. I also want to thank v m y friends in Slovenia for not letting the distance com e between us. Finally, a m assive thank you to m y big "Strasbourg fam ily" who have supported m e throughout this PhD and m ade Strasbourg m y hom e.

vi

Pre face

This work was part of the Marie Curie Initial Training Network "P4fifty", the goal of which was the application of cytochrom e P450 enzym es for white biotechnological processes. My principal role in this network was the discovery of P450 enzym es with novel functions. Wine is a rich source of chem ically diverse arom a com pounds, yet the underlying enzym es are largely unknown. The aim of this work is therefore to identify cytochrom es P450 that synthesize arom a in grapes. These cytochrom es P450 could be used for industrial production of flavor and fragrance com pounds. In addition, grapevine is one of the m ost im portant crops in France and worldwide and arom a is of crucial im portance for the quality of the final product. The knowledge of arom a biosynthetic pathways could thus also help to im prove the quality of grapes and wines. This work was also supported by the funding of collaboration with the team of Dr Philippe Hugueney of the French National Institute for Agricultural Research (INRA) in Colm ar by ANR (the French National Agency of Research).

In the first part of m y thesis, I review the current knowledge on wine arom a and its biosynthesis in the form of a m eta-analysis of 19 published datasets of grape and wine arom a profiles. The objective of this work was to identify arom a com pounds that are possibly synthesized in grapes by cytochrom es P450 . This reviewing and com piling study, presented here as Ch a pte r 1: Meta-analysis of grape and wine arom a (to be subm itted to J ournal of Agricultural and Food Chem istry), revealed an elaborate oxidative m etabolism of the m onoterpenol linalool, and convinced us to focus our efforts on biotransform ations of this com pound in grape and wine.

After briefly introducing biosynthesis of terpenoids (Ch a pte r 2 ) and cytochrom es P450 (Ch apte r 3 ), I review in detail recent progress in oxidations of m onoterpenols by cytochrom es P450 in Ch ap te r 4 : Cytochrom e P450 -catalyzed oxidations of m onoterpenols in plants (invited publication in Frontiers in Plant Science).

In collaboration with the INRA group, we built an exhaustive and reliable annotation of cytochrom e P450 genes in the reference grapevine genom e. We then used this annotation to study the expression of these genes using publically available and INRA's newly-generated transcriptom ic data (Ch a p te r 5 : Annotation, classification, genom ic organization and expression of the Vitis vinifera CYPom e, to be subm itted to BMC Genom ics). This allowed us to identify several gene candidates for biosynthesis of arom a in grapes. In addition, it pointed to other cytochrom es P450 of interest, involved for instance in the plant interactions with pathogens, and will provide the argum ent for future projects.

The second part of the results and m ain experim ental section addresses the form ation of an im portant wine arom a com pound, the wine lactone (Ch apte r 6 : CYP76F14 catalyzes vii biosynthesis of wine lactone precursor from linalool, to be subm itted to New Phytologist). I first show that this potent arom a is form ed during wine ageing from a grapevine-derived precursor, (E)-8-carboxylinalool. I then dem onstrate that (E)-8 -carboxylinalool is synthesized from linalool by the grapevine cytochrom e P450 CYP76F14. The CYP76F14 gen e is highly expressed in grape berries at late ripening stages, indicating that it is likely the dedicated enzym e for production of (E)-8-carboxylinalool in grapes.

In Ch a pte r 7 (Additional results) I introduce the results that were obtained in other parts of the project and were not included in the preceding chapters. These include a study of grape genes expressed in the early stages of berry developm ent, a study of grapevine cytochrom es P450 from other fam ilies, and a sensory study showing im portance of wine lactone in white wine.

In the final chapter (Ch apte r 8 ) I discuss the conclusions and perspectives of m y work, including perspectives in the wine arom a research and flavor and fragrance biotechnology.

viii Ré s u m é e n fran çais L'arôm e est l'un des principaux critères de qualité du vin. Il est donc très im portant de com prendre l'origine et l'évolution des arôm es du vin dans le but de contrôler et de prédire leur qualité. Bien que de nom breux com posés arom atiques apparaissent lors de la ferm entation par les levures, les com posés arom atiques les plus puissants et les plus caractéristiques des cépages sont form és dans la baie de raisin (Vitis vinifera). Un grand nom bre de ces com posés appartiennent à la fam ille des m onoterpénoïdes, qui sont des m olécules isopréniques à 10 atom es de carbone. Les m onoterpénoïdes participent à l'arôm e et au parfum de nom breuses plantes.

Les m onoterpènes sont synthétisés par des enzym es appartenant à la fam ille des terpènes synthases. Un certain nom bre d'entre elles ont déjà été caractérisées chez la vigne. Une analyse bibliographique systém atique a par ailleurs m ontré qu'une grande partie des m onoterpènes trouvés chez la vigne et dans le vin sont des dérivés oxydés du linalool. Le dérivé de linalool dont l'arôm e est le plus puissant est la lactone du vin (wine lactone), un e m olécule qui présente une odeur sucrée de noix de coco. D'après la littérature, la wine lactone se form e par une réaction non-enzym atique à partir du précurseur (E)-8carboxylinalool au cours du vieillissem ent du vin. Chez de nom breuses plantes, com m e Arabidopsis thaliana ou Catharanthus roseus, les m onoterpènes sont oxydés par les cytochrom es P450 et en particulier les m em bres des fam illes CYP71 et CYP76. Les cytochrom es P450 , contrairem ent aux terpènes synthases, n'ont pas été décrits en détail chez la vigne. L'objectif de m a thèse est d'étudier le rôle des cytochrom es P450 dans la biosynthèse des arôm es du raisin, et plus particulièrem ent la form ation de la wine lactone et de son précurseur le (E)-8-carboxylinalool.

La partie expérim entale principale de m a thèse a consisté à étudier la form ation de la wine lactone et de son précurseur au cours de la m aturation des raisins et la vinification, au travers de l'identification et de la caractérisation des gènes im pliqués dans la biosynthèse de ces com posés. Les concentrations de la wine lactone dans les vins sont très faibles et, en conséquence, cette m olécule n'est pas détectée par les m éthodes habituellem ent utilisées pour l'analyse des arôm es du vin. J 'ai donc développé une m éthode d'extraction et de détection de la wine lactone par LC-MS/ MS qui m 'a perm is de quantifier ce com posé dans tous les échantillons de vins analysés. Par LC-MS/ MS j'ai égalem ent pu quantifier le (E)-8carboxylinalool et son ester de glucose. L'analyse de ces trois com posés dans différents échantillons représentatifs des vins d'Alsace dém ontre que les quantités de ces trois com posés sont fortem ent corrélées (Figure 1). Alors que les concentrations en ester de glucose de (E)-8-carboxylinalool sont élevées dans les vins jeunes (1 ou 2 ans), l'ester est com plètem ent hydrolysé en (E)-8-carboxylinalool dans les vins plus vieux. Les ix concentrations en wine lactone sont plus élevées dans les vins vieux, de m êm e que dans les vins où la concentration en (E)-8 -carboxylinalool est élevée. La conversion du (E)-8carboxylinalool en wine lactone suit une réaction du prem ier ordre avec un taux de conversion très faible de 0 ,0 0 0 12 an -1 . Cette valeur peut nous perm ettre d'estim er la concentration en wine lactone d'un vin à partir de la concentration en précurseur et de l'âge du vin. Cette annotation a révélé 279 gènes com plets, 20 gènes partiels, 11 pseudogènes putatifs et 227 pseudogènes de cytochrom es P450 chez le cultivar séquencé. Souvent ces gènes form ent des « clusters » qui regroupent jusqu'à 35 séquences (Figure 3). Ces clusters regroupent des gènes très sim ilaires qui généralem ent appartiennent à la m êm e fam ille de cytochrom es xi P450 (Figure 4). En conséquence, la plupart des gènes qui font partie d'un cluster ont leur hom ologue le plus proche au sein du m êm e cluster. Ces clusters ont été probablem ent form és par duplication en tandem et dans certains cas par duplication segm entale. La fam ille CYP82, qui est form ée de 25 gènes com plets, constitue la plus vaste fam ille de cytochrom es P450 chez la vigne. Des données de séquençage d'ARN ont ensuite été com parées à cette annotation. Elles ont perm is de m ettre en évidence les gènes candidats exprim és dans la baie en fin de m aturation, ainsi que dans les feuilles infectées par des agents pathogènes com m e le m ildiou et l'oïdium .

Cette analyse d'expression a m ontré qu'une grande partie des gènes de P450 s étaient activés par l'infection par des agents pathogènes et suggère que ce derniers pourraient être im pliqués dans la biosynthèse de com posés de défense.

De nom breux gènes de cytochrom es P450 sont surexprim és dans les baies m ûres, parm i eux les gènes de la fam ille CYP75, qui participent à la pigm entation (biosynthèse des flavonoïdes). Une attention toute particulière a été portée aux différentes fam illes susceptibles de contribuer aux propriétés arom atiques du raisin et vin, plus particulièrem ent les fam illes CYP76 et CYP71 qui ont récem m ent été décrites com m e participant au m étabolism e des m onoterpénols chez autres espèces végétales (Figure 2). Six gènes candidats fortem ent exprim és dans les baies m ûres ont été sélectionnés : CYP76F12, CYP76F14, CYP76T21, CYP76Y1, CYP76Y2 et CYP71AT7. Le niveau d'expression de ces gèn es a ensuite été évalué par qRT-PCR à cinq stades de développem ent de la baie. L'accum ulation de (E)-8 -carboxylinalool, libre ou conjugué a été quantifiée dans les m êm es échantillons. Les xii candidats pour une analyse fonctionnelle ont ensuite été sélectionnés en com parant profils d'expression et l'accum ulation du (E)-8-carboxylinalool. Le (E)-8 -carboxylinalool s'accum ule sous form e conjuguée dans la baie de raisin. La concentration en glucose ester de (E)-8-carboxylinalool dans les baies de Gewurztram iner augm ente durant la m aturation de la baie de raisin. L'analyse de l'expression des gènes dans la baie durant la m aturation m 'a perm is de m ettre en évidence 5 gènes de la fam ille CYP76 et un de la fam ille CYP71 dont l'expression augm ente égalem ent durant la m aturation. Plus xiii précisém ent, l'expression de ces gènes est activée lors des stades tardifs du développem ent, en particulier au stades m i-m ature et m ature. Le candidat le plus exprim é est CYP76F14.

Les candidats ont été clonés et exprim és dans la souche WAT11 de Saccharom y ces cerevisiae, qui exprim e une P450 réductase d'origine végétale (A. thaliana). Les cytochrom es P450 sont des enzym es ancrées sur les m em branes du réticulum endoplasm ique. J 'ai donc utilisé les m em branes m icrosom ales isolées de ces levures recom binantes pour effectuer les tests d'activité in vitro. Les produits d'oxydation du linalool ont été analysés par chrom atographie en phase gazeuse et liquide couplée à la spectrom étrie de m asse (GC-MS et LC-MS/ MS). Afin d'évaluer l'activité des candidats in planta, ces derniers ont aussi été exprim és transitoirem ent dans les feuilles de Nicotiana bentham iana.

Pour com penser la faible production de linalool par cette plante, un gène de linalool synthase de vigne a été co-exprim é pour form er le substrat. Cependant, les enzym es diffèrent en ce qui concerne le degré d'oxydation des produits form és : seule l'une des enzym es, CYP76F14, peut catalyser la triple oxydation du linalool en xv (E)-8-carboxylinalool, le précurseur de la wine lactone (Figure 6). La conversion de (E)-8hydroxylinalool (alcool) en (E)-8-oxolinalool (aldéhyde) sem ble être l'étape cruciale qui fait de CYP76F14 l'enzym e la plus efficace pour la form ation de (E)-8-carboxylinalool. Le voie de biosynthèse du (E)-8 -carboxylinalool a aussi été reconstituée in planta. J 'ai pu m ontrer que les feuilles de N. bentham iana exprim ant la linalool synthase et CYP76F14 produisaient du (E)-8-carboxylinalool sans accum ulation de produits interm édiaires. En revanche, les feuilles exprim ant la linalool synthase et CYP76T21 produisent à la fois du (E)-8carboxylinalool et du (E)-8-hydroxylinalool, tandis que l'expression de CYP76F12 ne perm et de détecter aucune trace de (E)-8 -carboxylinalool. Les (E)-8-hydroxy-et (E)-8-carboxylinalool ne sont que deux des dérivés oxygénés de linalool qui peuvent être détectés dans la baie de raisin (Figure 7). Cependant, aucune des En conclusion, les cytochrom es P450 jouent un rôle im portant dans la form ation des arôm es du vin. L'enzym e CYP76F14 catalyse l'oxydation du linalool en (E)-8 -carboxylinalool. Cette m olécule est ensuite lentem ent convertie en wine lactone, une m olécule odorante puissante, au cours du vieillissem ent du vin. Ce gène et ses paralogues pourront être utilisés com m e m arqueurs pour la sélection de nouvelles variétés de vigne présentant un profil arom atique souhaité. De plus, ces enzym es ou les levures recom binantes qui les exprim ent pourront être utilisées pour produire des arôm es par des procédés biotechnologiques. Cependant, la conversion chim ique du (E)-8-carboxylinalool en wine lactone dans des conditions am biantes est très lente. Deux solutions sont possibles pour accélérer sa form ation: soit réaliser la réaction dans des conditions plus drastiques de tem pérature et (ou) de pH, soit identifier des enzym es qui perm ettent cette cyclisation. Enfin, le (E)-8-carboxylinalool xvii pourrait être utilisé com m e un arôm e à libération lente dans les cosm étiques ou autres produits.

Cette étude a, d'autre part, dém ontré l'im portance d'une analyse ciblée des com posants de l'arôm e du vin. Une m éta-analyse de l'arôm e du vin et du raisin m 'a perm is de m ontrer que, m êm e si l'analyse non ciblée perm et la quantification d'un grand nom bre de com posés volatils, de nom breux com posés im portants, tels que la wine lactone, ne sont pas détectés en raison de leur faible concentration. J 'ai, par conséquent, développé une m éthode ciblée pour l'analyse de la wine lactone qui m 'a perm is de m ettre en évidence la lente transform ation du (E)-8-carboxylinalool en wine lactone, ainsi que les différences de concentration de ces com posés entre les cépages.

Enfin, l'annotation fine des gènes de cytochrom e P450 dans génom e de la vigne ouvre la voie à une exploration des autres rôles des cytochrom es P450 dans la résistance ou le développem ent de la vigne et la qualité du vin, par exem ple dans la résistance aux pathogènes, la taille ou la pigm entation de la baie. L'analyse de l'expression des gènes a d'ores et déjà m is en évidence plusieurs gènes de cytochrom es P450 qui sont activés dans les feuilles infectées par les pathogènes.

In tro d u ctio n

Arom a is a crucial determ inant of the wine quality, but we are still far from understanding its com position, let alone the influence of genetic factors and environm ent on its developm ent.

Yet we would ultim ately want to understand and control wine arom a to ensure consisten t production of high quality wines (1). Wine arom a is a com plex m ixture of volatile organic com pounds. These are sm all, nonpolar m olecules that readily enter the gas phase and reach our nasal cavity while we sm ell or drink a glass of wine. Different volatile organic com pounds in wine span a large range of concentrations. While it is tem pting to think that the m ore abundant com pounds im pact the arom a m ore than the trace com pounds, it is not necessarily the case. We can sm ell som e of the com pounds at very low concentrations, whereas others rem ain undetected even at high levels. The strength of arom a of a particular com pound is expressed as an odor detection threshold, which is the lowest concentration perceivable to the hum an sm ell. Com pounds with low sensory thresholds are often responsible for the characteristic sm ell of a particular food (2).

Wine arom a com pounds differ in their origin and evolution during the winem aking process.

Many authors have classified wine arom a com pounds into three categories based on their origin: grape (or varietal) arom a, ferm entation arom a and ageing arom a, also called the wine bouquet (3)(4)(5). However, these three classes are not so clear-cut: ultim ately m ost of the arom a precursors (even the sim ple ones) originate from grapes and are in som e way m odified by the ferm entation process or ageing.

The grape arom a is synthesized in grape berries by a variety of enzym es, including terpene synthases, O-m ethyl transferases, carotenoid cleavage dioxygenases, cytochrom es P450 and probably other not yet characterized enzym es. Genetic variation in arom a biosynthesis genes cause differences in arom a between grapevine varieties: an allelic variant of 1-deoxy-Dxylulose-5-phosphate synthase, a terpenoid biosynthetic gene, causes accum ulation of terpenoids in Muscat and Gewurztram iner grapes. An abundance of terpenoids gives these wines a distinct floral arom a (6)(7)(8). In another exam ple, differential expression of an Om ethyl transferase gene results in higher production of m ethoxypyrazines, com pounds evoking the typical capsicum arom a in Sauvignon wines (9). The genetic factors underlying the arom a typicity of all other grapevine varieties-only in Europe over 20 0 0 (10 )-rem ain unexplored.

Grape berries store m ost of the volatiles they produce as glycosides (11). Possible functions of glycosylation are sequestration, detoxification and decrease of volatility and reactivity (12). Since glycosides are not volatile, they do not directly contribute to wine arom a. They do, however, affect the arom a indirectly: they form a precursor pool from which volatile aglycons can be released during yeast and m alolactic ferm entation, during vinification by addition of exogenous glycosidases, during wine ageing by the low pH (13) and, as dem onstrated recently, by enzym atic hydrolysis in the m outh, catalyzed by the enzym es in the saliva (14). Arom a glycosides in grapes have either one or two sugar m oieties attached to the aglycon e (m ono and diglycosides, respectively). The first sugar m oiety, directly attached to the aglycone, is in all cases glucose. The m ajority of glycosides (at least in the case of terpenoids) are present in the form of diglycosides, in which a second sugar-arabinose, apiose or rham nose-is attached to the glucose (13). The diglycosides cannot be hydrolyzed by a ǃglucosidase, and require other glycosidase enzym es to release the volatile aglycon (15).

The winem aking process profoundly influences the wine arom a. Hydrolysis of glycosides is not the only effect ferm entation has on the arom a com position. Yeast (Saccharom y ces cerevisiae) produces m any com ponents of wine arom a, particularly alcohols and esters, from prim ary m etabolites. Different yeast strains can produce rem arkably different arom a profiles (16). After alcoholic ferm entation wines are som etim es subjected to m alolactic ferm entation by Oenococcus oeni, the principal role of which is to reduce tartness or acidity of wine by converting m alic acid to lactic acid. These bacteria can also alter the com position of arom a by, for exam ple, prom oting deglycosylation (17).

The evolution of wine arom a continues after the ferm entation process. In addition to above m entioned acid hydrolysis of arom a glycosides during wine ageing, low pH can cause other im portant chem ical changes. William s et al. postulated that som e hydroxylated linalool derivatives undergo cyclisation or other rearrangem ents at low pH (18). In addition, during storage in oak barrels com pounds from the wood penetrate the wine and influence its taste and arom a.

Standardized sam ple preparation (19,20 ) and analysis procedure is routinely used for analysis of grape and wine volatiles and their precursors: grape juices or wines are extracted to a non-ionic solid phase, free volatiles are eluted by a non-polar solvent (pentane or a m ixture of pentane and dichlorom ethane), and glycosidically bound volatiles are eluted by a m ore polar solvent (ethyl acetate or m ethanol). Free fraction can then be directly analyzed by gas chrom atography, whereas the bound fraction is enzym atically hydrolyzed prior to the analysis. Gas chrom atography allows for sim ultaneous analysis of hundreds of volatiles with good resolution of structurally sim ilar m olecules. Furtherm ore, coupling to a m ass spectrom eter enables reliable identification of com pounds by searching m ass spectra databases even when analytical standards are not available.

Many research laboratories used this m ethod to investigate how arom a is influenced by grape variety, grape ripening, environm ent and different winegrowing and winem aking techniques. While these studies have without doubt answered som e of these im portant questions, a global picture of wine arom a using an ensem ble of these valuable data has not been painted yet. We collected, curated and analyzed these data to answer the following questions: what are the com ponents of grape and wine arom a, at which concentration are they present and to what extent are they glycosylated. We com pared these param eters between grapes and wines. We then com pared these data to the existing knowledge on the origin and evolution of these arom a com ponents with a particular focus on their biosynthesis. This m eta-analysis will hopefully add to our understanding of wine arom a com position and developm ent. Analysis of 19 publications describing grape or wine arom a revealed 385 different volatile organic com pounds. More than half of them were only identified in one or two studies, and were elim inated from further analysis to account for the possibility of incorrect peak annotation. Som e of the elim inated com pounds m ight be im portant varietal com pounds, but this study focuses on the sim ilarities, not the differences between the varieties. The rem aining 141 validated volatile com pounds (Table S1) were assigned to one of the 12 classes based on their chem ical structure and biosynthetic origin. Authors of m ost reviews m ake a clear distinction between grape and ferm entation derived wine arom a (4,5,21,22) and therefore use a different classification of wine arom a com ponents. Classes defined in this study are based on classifications from other authors, while trying to contain all the m olecules and to m inim ize the overlap between classes. The m ost represented classes (with respect to the num ber of validated com pounds) were aliphatic alcohols, aliphatic esters, m onoterpenes and volatile phenols (Figure 1). Not all volatiles contribute to the arom a equally. Their im pact depends on their concentration, as well as arom a intensity. The latter is usually expressed as the odor detection threshold, which is the lowest concentration of a particular com pound that can be detected by the hum an sm ell. In a recent m eta-analysis, Dunkel et al. (2) com piled a list of food volatiles occurring at concentrations exceeding their odor detection threshold.

Co m p o s itio n o f w in e aro m a

Som ehow surprisingly, a total of only 220 key food odorants are responsible for the arom a of m ost of the foods and beverages we consum e, and am ong those fewer than 40 contribute to arom a of an individual food item . Another m eta-analysis identified 57 supra-threshold volatiles in wines (all but 2 of these volatiles are also included am ong the 220 key food odorants). Only 60 % (N=35) of those key wine odorants overlap with our set of com m on wine volatiles, com piled from profiling experim ents (Figure 2). The other 40 % (N= 22) of key wine odorants are overlooked in volatile profiling experim ents because of their low concentrations. In particular, sulfur-containing volatiles and fatty acid lactones appear system atically overlooked (Table S2). Monoterpenes are one of the m ost represented classes of m olecules in our study, but two odor-active m onoterpenes, cis-rose oxide and win e lactone, are still system atically overlooked in profiling experim ents. Conversely, the highim pact aliphatic alcohols, acids and esters, as well as phenols and benzenoids, are apparently present in sufficient concentrations to be easily detectable in profiling experim ents. A group of 15 com m on wine volatiles features on the list of key food odorants, but not the key wine odorants. We labeled those com pounds "potential" key odorants (Table S2). The progress in analytical chem istry will likely perm it discovery of m ore key wine odorants in future.

Rotundone, the odorant conferring the peppery character to Shiraz wines, was only discovered recently (23) and was not included am ong the key food odorants nor the key wine odorants.

Although odor detection threshold is com m only used to describe an influence of a particular com pound on arom a, this value needs to be used with caution for describing com plex m ixtures, including wines. Both synergistic and antagonistic effects can occur in odor perception: som etim es a m ixture of com pounds can be perceived even if all com ponents are at sub-threshold concentrations; conversely, som e com pounds can m ask the perception of other com pounds, so they rem ain undetected at supra-threshold concentrations (24,25). In addition, odor detection threshold is averaged across the population. Because of the variability in odor receptor genes in hum an population, each individual has a highly personalized odor perception (26). Finally, threshold values are not known for all the wine volatiles. For these reasons we decided to include all the volatiles, not just the key wine odorants, in our m eta-analysis. (27). While the overlap between the three studies is sizeable, m any key odorants are overlooked in volatile profiling experim ents. Each tile represents one volatile m olecule and its color corresponds to the m olecular class.

Origin a n d e vo lu tio n o f w in e aro m a

During the winem aking process the arom a undergoes m ajor changes, in particular deglycosylation of arom a com pounds synthesized in grapes and biosynthesis of new com pounds. To analyze these changes quantitavely we selected 43 com pounds with sufficient num ber of available data points (>30 ) across the 8 2 volatile profiling datasets. For each com pound we com puted the total concentration (the sum of free and bound concentration) and percentage that is glycosylated (bound/ total concentration), and tested for differences between these values in grapes and wines (Figure 3). These differences in concentration an d degree of glycosylation are largely characteristic of each class of m olecule.

Aliph atic a lco h o ls

Aliphatic alcohols are a diverse group of com pounds that can originate both from grapes and yeast ferm entation. C6-alcohols, a com m on group of plant volatiles with six carbon atom s, sm ell characteristically "green", rem iniscent of leaves and fresh cut grass. They are derived from corresponding C6-aldehydes, also im portant arom a com pounds, by alcohol dehydrogenase enzym es. C6-aldehydes are products of hydroxyperoxide lyase (CYP74) enzym es (28), which were recently characterized in grapevine (29). C6 alcohols can be consum ed by the yeast during ferm entation (30 ), which m ay explain why concentration of (E)-2-hexen-1-ol is lower in wines com pared to grapes. Short chain alcohols, also known as fusel alcohols, can add a negative im pact to the wine arom a at high concentrations (31). They are form ed by the yeast during ferm entation from am ino acid catabolism (30 ). A typical exam ple is isoam yl alcohol, the concentration of which is m uch higher in wines com pared to grapes and is also the m ost abundant com pound in this study (Figure 3a). Aliphatic alcohols also contribute to the arom a as precursors of esters, which are discussed below.

Aliph atic a cid s

The yeast produce aliphatic acids during ferm entation (5) from prim ary m etabolites: fatty acids (C6 or longer), am ino acids (short and branched) or sugar (acetic acid). Arom a of volatile fatty acids is generally unpleasant, ranging from sweaty and cheesy to goaty and rancid. Although all aliphatic acids included in this study were detected in both grapes an d wines, their concentration was significantly higher in wines (Figure 3a), confirm ing they are predom inantly a ferm entation product.

Longer aliphatic acids (C8 and C10 ) in grapes are glycosylated at least to som e extent, which is unexpected because they lack a hydroxyl group to which the sugar m oiety is usually attached. Instead, they are probably stored as glucose esters, a less com m on type of glycoconjugate, where sugar and aglycone are connected via an ester bond. Fatty acid glucose esters have been described in other plants (32), but their role as wine arom a precursors has not yet been extensively studied.

Aliphatic acids can be transform ed to m ore pleasant sm elling com pounds, such as esters (described below) and lactones. Five fatty acid derived lactones (gam m a-nonalactone, gam m a-decalactone, gam m a-dodecalactone, gam m a-(Z)-6-dodecenolactone and (Z)-oak lactone, also known as whisky lactone) are am ong the key wine odorants, but are system atically overlooked in the volatile profiling experim ents, presum ably because of their low concentration in wines (27).

Alip h atic e s te rs

Esters are a group of volatiles that contribute to fruity notes of wine and other ferm ented beverages. They are produced during ferm entation from alcohol and acyl-CoA by yeast alcohol acyltransferase enzym es. The two m ajor groups of esters in wine are ethyl esters and acetate esters. Their concentrations in grapes are negligible.

The concentration of ethyl esters of m edium -chain fatty acids depends on the concentration of the fatty acid precursor (33). Our data confirm s this observation: concentrations of ethyl hexanoate and ethyl octanoate are strongly positively correlated to concentrations of their precursors, hexanoic and octanoic acids, respectively (Figure 4). Sim ilar relationship was recently found for C9 and, to a lesser extent, C12 ethyl esters (34). While hexanoic acid is predom inantly a ferm entation product, non-negligible am ounts are present in grapes as well.

The biosynthesis of hexanoic acid in grapes could thus influence the concentration of a typical ferm entation product, ethyl hexanoate, in wine. Conversely, precursor concentration does not determ ine the concentration of acetate esters.

The lim iting factor in the production of acetate esters by S. cerevisiae is expression of the alcohol acetyltransferase gene in yeast (35). Indeed, isoam yl acetate and hexyl acetate do not correlate to the concentrations of their precursors (Figure 5).

Mo n o te rp e n e s

Monoterpenes are a large class of plant specialized m etabolites. They are built from two isoprenoid units, giving them a backbone of 10 carbon atom s. These com pounds give m any fruits, flowers, herbs and spice their characteristic arom a. Most of the wine m onoterpenes contribute towards floral and citrusy notes. For exam ple, m onoterpenols and their derivatives give the characteristic arom a to Muscat (36) and Gewurztram iner (37) wines.

High m onoterpenol concentration in these varieties results from a m utation in an early terpenoid biosynthesis gene deoxy-D-xylulose synthase (6)(7)(8). Our data show that m onoterpenols span a large concentration range in grapes and wines (Figure 3a) which points to their role as varietal arom a com pounds in som e grapevine varieties. Unlike other classes of volatiles described here their concentrations in grapes and wines are sim ilar, as expected for grape-derived com pounds. Conversely, the glycosylated percentage of m any terpene com pounds is lower in wines com pared to grapes, suggesting ferm entation nonetheless affects the m onoterpene content in wine by releasing volatile m onoterpen es from their glycosylated precursors. Monoterpenes are products of terpene synthase enzym es. The terpene synthase gene fam ily has expanded in grapevine, which underlines the im portance of terpenoids in this species (38). More than half of terpene synthase genes were functionally characterized (38) and were found to produce a large num ber of different m ono-and sesquiterpene backbones in vitro.

Interestingly, this variability is not reflected in the volatile profiles of grapes and wines in our selected studies. Sesquiterpenes do not appear in our dataset of 141 validated volatiles.

However, a labeling study revealed a production of 14 sesquiterpenes in grape skins of two different varieties, suggesting the sesquiterpene m etabolism in grapes is nonetheless active (39). The concentrations of sesquiterpenes in grapes and wines are probably too low to be detected in non-targeted profiling experim ents, but they do contribute to wine arom a of at least som e varieties: rotundone, an oxygenated sesquiterpene, is responsible for the peppery arom a of Shiraz wines (23). Monoterpenes, on the other hand, are one of the largest m olecular classes in our study, with 22 different m olecules identified in grapes or wines (Figure 1). Interestingly, this large chem ical variability am ong wine m onoterpenes does not result from the variability of different backbones synthesized by terpene synthases. Half of the validated m onoterpenes in this study are derivatives of the sam e m onoterpene: linalool.

Seem ingly the variability of m onoterpenes in grapes arises from the enzym es that oxygenate linalool at different positions.

Elaborate oxidative linalool m etabolism in grapevine was previously dem onstrated by feeding experim ents (40 ), (Figure 7). All of the described linalool derivatives, with the exception of nerol oxide, 6,7-epoxylinalool and 6,7-dihydroxylinalool are also present on our list of validated volatiles. Com m on m etabolic origin of m onoterpenes, in particular linalool derivatives, is also apparent from the correlation m atrix of grape and wine volatiles (Figure 6). Linalool is positively correlated to all the m onoterpenes included in the study, which supports its role as a central m onoterpene m etabolite in grapes. Enzym es catalyzing linalool oxidation in grapes have not yet been identified, but in other plants enzym es from cytochrom e P450 superfam ily were shown to oxidize m onoterpenes (41,42), including linalool (43,44). Hydroxylated linalool derivatives were discovered in grapes in early 1980 s (11,45). These com pounds have a very weak odor so they are unlikely to contribute to the wine arom a directly, but they m ay have an indirect contribution. They were found to spontaneously transform to com pounds with strong arom a in conditions m im icking win e m aturation: in acidic conditions they undergo spontaneous elim ination of water and rearrangem ent to either linear (hotrienol) or cyclic com pounds (linalool oxides) (18).

Although low concentrations of hotrienol were detected in grapes, its concentration in wines is significantly higher (Figure 3a), which supports the hypothesis of acid-catalyzed form ation from 7-hydroxylinalool during winem aking and wine m aturation. In addition, concentrations of 7-hydroxylinalool and hotrienol are strongly correlated (Figure 6). We do not find, however, the sam e am ount of evidence for non-enzym atic form ation of linalool oxides from 6,7-dihydroxylinalool or 6,7-epoxylinalool. Concentrations of linalool oxides in wines and grapes are com parable. In addition they are glycosylated to a very high degree in grapes, which suggests they are form ed in m etabolically active grape berries, as dem onstrated previously in the feeding study (40 ). Form ation through acid-catalyzed cyclisation during wine m aturation is therefore probably of lesser significance. From a single profiling experim ent it is difficult to estim ate what proportion of the total linalool pool is transform ed to oxygenated derivatives, m ainly because not all derivatives are quantified in all the experim ents. Strong correlation between concentrations of m onoterpenes in the studies included in this m eta-analysis (Figure 6) allowed us to describe and predict relationships between concentrations of linalool and oxygenated linalool derivatives with a set of linear m odels (Table S3). In the investigated concentration range (0 .0 0 1-10 NjM) m ost of linalool is oxygenated (Figure 8). At low concentrations virtually all linalool (97%) is oxygenated and the m ain linalool derivative is (E)-8 -hydroxylinalool. At high concentrations oxygenated derivatives represent 52% of the com plete linalool pool, and the m ost abundant derivative is 7-hydroxylinalool. 6-hydroxylinalool and linalool oxides represent a m inor part of linalool derivatives. The total fraction of oxygenated linalool derivatives in the linalool pool is underestim ated since not all linalool derivatives were included in the calculation.

Monoterpenes in grapes are predom inantly glycosylated, although variation between the sam ples is high (Figure 3). Since only free com pounds can im pact the wine arom a, this variability could be im portant for varietal characteristics. Several m onoterpenes are glycosylated to a lesser extent in wines as com pared to grapes. This can be attributed to hydrolysis of glycosides by the yeast during ferm entation, as well as acid hydrolysis during wine m aturation.

In spite of the large num ber of m onoterpenes included in this study, the list presented here is not exhaustive. The lowest lim it of quantification in the analyzed set of profiling experim ents is around 10 -7 Njg L -1 (Figure 3) and concentrations of som e m onoterpenes, including rose oxide or wine lactone, in wines are below this lim it. Nonetheless, because of their low sensory threshold, they still contribute to wine arom a as one of the key wine odorants even at these low concentrations (27). Furtherm ore, another linalool derivative, (E)-8carboxylinalool, has been detected in grapes and wines, but is not detectable by gas chrom atography, hence the absence from the data collected here. This com pound also indirectly influences wine arom a as a precursor to wine lactone. S3 ).

N o ris o pre n o id s

Norisoprenoids are a group of carotenoid-derived m etabolites. Sim ilarly to m onoterpenes, their arom a is m ostly described as floral or fruity, although som e, for exam ple 1,1,6trim ethyl-1,2-dihydronaphthalene (TDN) which is described as petrol or kerosene-like, can have a negative im pact on arom a (46). Norisoprenoids are synthesized by carotenoid cleavage dioxygenases, enzym es that cleave carotenoid substrates at different in-chain positions, yielding products of different sizes.

Most of the norisoprenoids in grapes are derived from ǃ-carotene or neoxanthin (Figure 9) (47) and have 13 carbon atom s (C13-norisoprenoids), with the exception of 4-oxoisosporone (C9). So far only one grapevine carotenoid cleavage dioxygenase has been characterized (48).

It was shown to cleave zeaxanthin, a m inor grape carotenoid, whereas neoxanthin and ǃcarotene were not tested as substrates. A hypothetical reductase in the ǃ-dam ascenone pathway (Figure 9a) has to our knowledge not yet been identified.

The chem ical diversity of norisoprenoids in grapes appears to originate from different nonenzym atic reactions, including photooxygenation, therm al degradation or acid hydrolysis (47). Although norisoprenoids are considered grape-derived m etabolites, our data show higher concentrations of two norisoprenoids (3-hydroxy-ǃ-dam ascone and 3-oxo-Į-ionol) in wines as com pared to the grapes (Figure 3). However, it is unlikely these com pounds would form during ferm entation or wine m aturation, since they are glycosylated to a large extent. It is unknown if non-enzym atic transform ations occur also on glycosylated derivatives of these com pounds.

Vo latile p h e n o ls

Volatile phenols are a diverse group of wine volatiles with respect to both their origin an d im pact on wine arom a. Many of them are com m on plant volatiles, derived from ferulic acid or related m etabolites, and contribute to pleasant spicy arom a notes. Although enzym es catalyzing their biosynthesis have not yet been characterized in grapes, they have been studied in other plants. Exam ples include clove arom a eugenol, which is synthesized by an enzym e reducing coniferyl acetate in basil or petunia flowers (49), or vanillin, synthesized from ferulic acid by a hydratase/ lyase enzym e in vanilla pods (50 ). Most of volatile phenols are stored in grapes as glycosides, and can be hydrolyzed during winem aking. It is noteworthy that vanillin in wine can also originate from ageing in oak barrels (51). Not all volatile phenols are associated with pleasant arom a: som e of them , for exam ple guaiacol, are described as sm okey, ashy or m edicinal and are considered as off flavors in wine. These com pounds can originate from ferm entation, contam ination with spoilage yeast Brettanom y ces (52) or sm oke exposure of grapes, for exam ple from nearby forest fires (53). Concentration of guaiacol is m uch higher in wines com pared to grapes (Figure 3), which confirm s ferm entation origin of this com pound. Interestingly, som e authors suggested that glycosylated precursors from grapes represent only a m inor source of p-vinyl guaiacol in wine (54), but our data suggest this contribution can be substantial.

Be n ze n o id s

Benzenoid com pounds in wines are cinnam ic acid derivatives with varying side chain lengths and oxidation states. Phenylethanol and phenylacetaldehyde have a weak floral arom a. The high odor detection threshold of phenylethanol is com pensated by its high concentration.

Although these volatiles can be produced by both plants (55) and yeast (56), their concentration in wines is generally m uch higher than in grapes, which suggests that contribution from yeast is larger (Figure 3). An exception is phenylacetaldehyde, which is reduced by the yeast to yield 2-phenylethanol.

Co n clu s io n s

Analysis of volatile profiles of grapes and wines and their precursors from 20 publications resulted in a list of 141 com m on grape and wine volatiles (Table S1). A com parison to the previously published list of key wine odorants (27) revealed an overlap of 35 com pounds, whereas 22 key wine odorants are overlooked in the volatile profiling experim ents we reviewed. In particular, sulfur-containing volatiles and fatty acid-derived lactones are consistently overlooked. This result points to the need of targeted analysis of low-abundance com pounds.

To explore the com plex origin and evolution of wine arom a, 43 volatiles with sufficient num ber of data points were analyzed in greater detail. Som e com pounds occur in sim ilar concentration range in both grapes and wines. These com pounds are m ost likely derived from grapes and their concentration rem ains constant during ferm entation and wine m aturation. Concentrations of other com pounds differ dram atically between grapes and wines, because they are either produced or consum ed by the yeast during ferm entation . Som e glycosylated com pounds are also hydrolyzed during ferm entation, which is reflected as a lower glycosylated fraction in wines com pared to grapes.

The boundaries between the roles of grape m etabolism , ferm entation and ageing in the form ation of arom a are blurred because m any com pounds are affected by all three of these processes. Monoterpenoids, typically considered grape-derived volatiles, accum ulate in grapes as glycosides, which can be hydrolysed during the ferm entation and can undergo chem ical rearrangem ents during wine m aturation. Hotrienol is a m onoterpene thought to be form ed from 7-hydroxylinalool by a non-enzym atic acid-catalyzed reaction. Although it is present in grapes, its concentration is significantly higher in wines, which is in agreem ent with its wine m aturation origin. Yet its concentration also depends on the concentration of its precursor, 7-hydroxylinalool, which is synthesized in the grapes. What is m ore, 7hydroxylinalool is partially de-glycosylated during ferm entation, which m ay also affect hotrienol concentration. To understand and be able to predict wine arom a, all these different factors will need to be taken into consideration.

Monoterpenes are one of the largest groups of volatiles in wines, but their chem ical diversity in wines is not due to the different backbones synthesized by a large fam ily of grapevine terpene synthases. Half of the m onoterpenes in this study are oxidized derivatives of the sam e m onoterpene-linalool-with m ain derivatives being 7-and (E)-8-hydroxylinalool. We showed that most of linalool produced in grapes is oxygenated. Linalool oxygenases thus not only influence wine arom a by form ation of new arom a com pounds, but also by depletion of their substrate, one of the key wine odorants.

Although considerable progress in biosynthesis of wine arom a has been m ade since the sequencing of Vitis vinifera genom e in 20 0 7 (57), m any enzym atic steps leading to arom a com pounds rem ain to be identified. This knowledge is im portant for the breeding of new grape varieties, and could as well be used to produce ingredients for flavor and fragrance biotechnology. We collected published datasets on free and glycosylated grape and wine volatiles. We used search term »grape arom a profiling free glycosylated« on Google Scholar and reviewed citations of included publications. We reviewed 10 4 publications, 45 of them were excluded because they only contained inform ation on free volatiles, and 8 because they only contained inform ation on glycosylated volatiles. To ensure the com pounds were annotated correctly, only publications that contained inform ation of Kovats retention index were included.

Mate ria l an d m e th o d s D ata co lle ctio n

Further 31 publications were thus excluded, which resulted in 19 selected publications.

Publications or sam ples that described Vitis species other than Vitis vinifera subsp. vinifera

were excluded from all quantitative analysis, but used for validation of retention indices.

D ata cu ratio n

Molecule nam es and their Kovats retention indices were aggregated in one file and m anually checked for duplicates (synonym s), which resulted in 385 unique m olecules. They were assigned to one of the 16 classes of volatiles (aliphatic alcohol, ketone, aldehyde, ester or acid, m onoterpene, norisoprenoid, sulfur containing, phenol, arom atic alcohol, ketone, aldehyde, ester or acid, nitrogen containing or other). 244 of unique m olecules appeared in less than three publications and were excluded from further analysis because of insufficient inform ation. Kovats retention indices of the rem aining 141 com pounds were com pared and in the cases where standard deviation of the retention indices exceeded 50 , outliers were excluded (annotation of a com pound was considered incorrect in the particular publication and was excluded from the dataset).

We paid particular attention to geom etric isom ers of som e com pounds, which elute close to each other and have sim ilar or identical m ass spectra, so they are often m isannotated. Such pairs of com pounds are: pyranic linalool oxides, furanic linalool oxides and 8hydroxylinalools. In case of pyranic linalool oxides, the two isom ers were incorrectly annotated in the first paper reporting their structure (78). This error was identified and corrected later (79), but som e authors continued to use the incorrect annotation. All the data were curated so that the first eluted furanoid linalool oxide was annotated as trans isom er, and the second one as cis. Sim ilarly, first eluted pyranoid linalool oxide that elutes first was annotated as trans and the second one as cis (80 ). Retention indices of (Z) and (E)-8hydroxylinalool were first reported in (81) and the author claim ed the NMR spectra were in agreem ent with previously published data. However in a subsequent publication (82), which is also cited in the widely used database of volatiles Pherobase (http:/ / www.pherobase.com / ), the retention indices of the two com pounds are inverted even if the author received standards from the author of (81). Recent publication (44) confirm s the correct elution order is (Z)-, followed by (E)-8-hydroxylinalool, as reported in the original publication (81).

Statis tical an alys is

Statistical analysis was perform ed using the R software version 3.0 .2 (8 3). For quantitative analysis 43 m olecules with m ore than 30 data points were selected from the pool of 141 validated com pounds. Concentrations of these com pounds in free and glycosidically bound form s were then extracted from the 20 publications. If a com pound was only detected in the free fraction, it was assigned a concentration of 0 in the glycosylated fraction, and vice versa.

Labels »not detectable«, »not quantifiable« and »trace« were all converted to zero concentration. Concentrations of free and bound form of each m olecule were then added to calculate the »total concentration«. »Fraction glycosylated« was calculated by dividing the concentration of glycosylated com pound with the total concentration. Concentration units were not the sam e in all studies. All concentrations in wines were given as Njg L -1 , som e concentrations in grapes were given as Njg L -1 and som e as Njg kg -1 . In addition, grape juice and wine have different, so the concentrations are not directly com parable, but since we com pared them on a logarithm ic scale we considered these differences negligible and did not adjust for them .

Log-transform ed concentrations were com pared using a two-sided t-test. Only m olecules with 5 or m ore data points in each category (grape and wine) were tested stastistically. Included key wine odorants are com pounds that were detected in at least three profiling experim ents included in this m eta-analysis, as well as identified as one of the key wine odorants in the Francis and Newton m eta-analysis (27). Potential key wine odorants are com pounds that were detected in at least three profiling experim ents included in this m eta-analysis as well as identified as one of the key food odorants in the Dunkel et al. m eta-analysis (2). Overlooked key wine odorants are com pounds that were not detected in at least three profiling experiments included in this m eta-analysis, but were identified as one of the key wine odorants in the Francis and Newton m eta-analysis (27).

Su p p le m e n tary in fo rm atio n

Class

Bio s yn th e s is o f te rp e n o id s in p lan ts

Terpenes (also isoprenoids or terpenoids) are the largest group of plant natural products with an incredible structural and functional diversity (1). Regular terpenes have a backbone com posed of a variable num ber of 5-carbon (5C) isoprene units. Terpenes are grouped by the num ber of isoprene units: m onoterpenes have 2 isoprene units, sesquiterpenes 3, diterpenes 4, until tetraterpenes which have 8 isoprene units (Figure 1). The nam e hem iterpene is som etim es used for isoprene, a 5-carbon com pound which som e plants em it in large quantities as protection from abiotic stress (2). Monoterpenes and sesquiterpenes (10 C and 15C isoprenoids, respectively), are a large and im portant class of volatile organic com pounds: they are em itted by flowers to attract pollinators, deter herbivores and prevent fungal infections. They are also produced in fruits to attract seed dispersers (3). Diterpenes (20 C isoprenoids) are not volatile, but nonetheless have im portant ecological roles: they serve in plant defense as phytoalexins (antim icrobial com pounds) and antiherbivore com pounds (4).

Sesterterpenes (C25) are present in som e plants ( 5) but their function is not very well docum ented. Triterpenes (30 C isoprenoids) are im portant for m em brane structure, as well as in plant defense (e.g. saponins, which probably act through m em brane disruption) (6).

The m ost im portant function of tetraterpenes (40 C, also known as carotenoids) is light harvesting in thylakoid m em branes. Natural rubber, an econom ically im portant high m olecular weight polym er, is produced by polym erization of 5C building blocks (7). In addition to the above m entioned functions, m any isoprenoids act as plant horm ones, e.g. gibberellins (C20 ), brassinosteroids (C30 ), abscisic acid and strigolactones (both C40derived).

Com m on precursors of all terpenoids are two 5-carbon isom ers dim ethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These precursors can be synthesized via two independent and spatially separated pathways: the cytosolic m evalonate (MVA) pathway an d the chloroplastic 2-m ethyl-D-erythrol-4-phosphate (MEP) pathway (8, 9) (Figure 1). The next step in terpene biosynthesis pathway is condensation of the C5 units, a reaction catalyzed by short-chain prenyltransferases. Farnesylpyrophosphate synthase (FPPS), which synthesizes the 15C precursor to sesquiterpenes and triterpenes, is located in the cytosol, whereas geranylpyrophosphate synthase (GPPS) and geranylgeranylpyrophosphate synthase (GGPPS), which synthesize C10 and C20 precursors to m onoterpenes and diterpenes, respectively, are located in the chloroplast (3). Squalene, the C30 precursor, and phytoene, the C40 precursor, are synthesized by head-to-head condensation of FPP and GGPP, respectively. Because of com partm entalization of short-chain prenyltransferases, the m evalonate pathway m ostly supplies precursors for sesqui-and triterpenes biosynthesis, whereas m ono-, di-and tetraterpenes source their precursors from the MEP pathway. Until recently precursors with all-trans configuration were considered the only com m on terpen e precursors. However, cis-short-chain-prenyltransferases were found to synthesize neryldiphosphate (NPP) and Z,Z-farnesyldiphosphate in tom ato species (10 , 11). The final step in terpene biosynthesis (C10 -C20 ) is catalyzed by terpene synthases (TPS).

These enzym es use isoprenyl diphosphate substrates and are able to transform them into a rem arkable variety of cyclic and acyclic structures. The reaction proceeds via a carbocation interm ediate, which can undergo cyclisations or rearrangem ent reactions in the TPS active site, until the reaction is stopped by proton loss or nucleophile (water) addition (12). In the latter case the resulting product contains a hydroxyl functional group. Biosynthesis of som e diterpenes proceeds in two steps: first a class II diTPS synthesizes a bicyclic diphosphate interm ediate, which is then used as a substrate by the class I diTPS (4). Recently, TPSindependent biosynthesis of geraniol has been discovered in rose petals: a so-called Nudix hydrolase (NUDX1) hydrolyses geraniol diphosphate to geraniol phosphate, which is then further hydrolyzed by a phosphatase to yield geraniol (13). Many terpenes in plants undergo further m odifications: about 97% of the known terpen es are oxygenated ( 14), a large part of which are likely products of cytochrom es P450 . These enzym es are known to oxidize m ono-to tetraterpenes. P450 s act on different terpenoids across plant species and their genes often cluster on genom es with those of terpen e synthases from the sam e pathways (15). They usually introduce a hydroxyl functional group, but also carbonyl or carboxyl products are known. Som e P450 s also catalyze oxidative cleavage of terpenoids (16). Another very com m on m odification of terpenes is glycosylation, catalyzed by fam ily 1 glycosyltransferases, also known as UDP-glycosyltransferases or UGTs.

Glycosylation increases water solubility of terpenes and can regulate their hom eostasis, storage and transport (17). Other m odifications are rarer and often restricted to particular classes of terpenes, but are nonetheless im portant. For exam ple, oxidative cleavage of carotenoids by carotenoid cleavage dioxygenases produces apocarotenoids, which include horm ones strigolactones and norisoprenoids, an im portant class of arom a com pounds (18).

Chapter 3

Cyto ch ro m e s P4 5 0

Cytochrom es P450 are a large enzym e fam ily of m onooxygenases present in all kingdom s of life. They catalyze the insertion of an oxygen atom to relatively inert positions in organic m olecules. Their substrates are structurally very diverse, which also reflect the diversity of their functional roles: detoxification of xenobiotics, including drugs in hum ans, to biosynthesis of basic and specialized m etabolites.

Cytochrom es P450 are nam ed after their unusual spectral properties. In the 1950 s and 1960 s a carbon m onoxide-binding proteinaceous pigm ent was discovered in rat and rabbit liver m icrosom es (1-3). Like other hem e-containing proteins (or cytochrom es) this newlydiscovered pigm ent had an intense absorption peak around 40 0 , also called Soret peak or band. Unlike other cytochrom es, this peak shifted to 450 nm upon binding of carbon m onoxide to the reduced form of the protein, hence the nam e "P-450 ". Based on this property, a sim ple spectrophotom etric m ethod, also called CO-difference spectroscopy, is still widely used for characterization of cytochrom es P450 : the difference in m olar extinction coefficient (İ P0 -1 cm -1 ) between 450 and 490 nm in the difference spectrum allows for the estim ation of cytochrom e P450 concentration (4). Enzym atic activity of cytochrom es P450 was first dem onstrated in 1965 (5).

At the heart of the rem arkable P450 catalytic activity is their ability to bind and activate m olecular oxygen. Their active site contains a hem e prosthetic group; in addition to the four porphyrine nitrogen atom s, the iron ion is coordinated with a cysteine residue of the enzym e via a thiolate bond. In the P450 resting state the sixth ligand of the Fe(III) ion is usually a water m olecule Figure 1-1). This m olecule can be displaced by the substrate, which influences the redox potential of the iron atom and facilitates its reduction to ferrous (III) state (Figure 123). This reduced interm ediate binds m olecular oxygen, which is first reduced again and then protonated to form the ferric hydroperoxo interm ediate (Figure 1 transfer the second electron (9). Finally, CPRs are not specific for their P450 substrate, as higher plants have no m ore than three reductase genes (usually two) and m ore than 40 0 P450 genes (10 ). Despite very low sequence identity between distantly related P450 s, num erous 3D structures solved to date show that their structural fold is highly conserved: a two-dom ain triangular structure with centrally-bound hem e prosthetic group and adjacent substrate cavity (Figure 2). The largest dom ain, also nam ed Į-dom ain, m ostly consists of Į-helices, whereas the sm aller ǃ-dom ain is rich in ǃ-sheets. Į-helices in P450 s have been assigned labels from A to L. I-helix (Figure 2) participates in binding of the oxygen m olecule, whereas F and G helices m odulate the access to the active site. The loop between F and G helices is highly variable and participates in substrate recognition. The m ost com m on m onoterpenols in plants are linalool, nerol and geraniol, the latter two being cis and trans isom ers, respectively (Figure 2). Linalool is an optically active com pound, present in the flower scent of both m onocotyledonous and dicotyledonous plants (3). Both enantiom ers, 3S-and 3R-linalool, occur naturally at enantiom eric excess ranging from 10 0 % (3S) to 97.5% (3R) in different plant species (4). Irregular m onoterpenols, i.e. built from nonhead-to-tail isoprenyl condensation, such as chrysanthem ol, lavandulol or artem isinol are outside the scope of this review, in which we focus on linalool, geraniol and their oxygenated derivatives.

Monoterpenols differ from other aliphatic m onoterpenes in their chem ical properties. The presence of an alcohol functional group m akes them not only m ore polar and, therefore, soluble in water ( 5), but also m ore chem ically reactive. Reports show m onoterpenols isom erize to one another in acidic aqueous solvents (6,7), although these harsh conditions are probably not achieved in biological system s. The alcohol functional group also m akes m onoterpenols m ore am enable to secondary transform ations, such as oxidation, glycosylation (8-10 ), esterification (11) or m ethylation (12). Am ong these transform ations, glycosylation is the m ost ubiquitous: m any plants store m onoterpenols as water-soluble glycosides. Plants presum ably glycosylate m onoterpenols to decrease their toxicity, facilitate their storage and transport between plant organs (13). In this review we focus on the recent advances in understanding m onoterpenol oxidative m etabolism , in particular m ediated by cytochrom e P450 oxygenases. These enzym es can greatly diversify the structures of m onoterpenols and change their chem ical properties. This diversity is reflected in different roles these com pounds have in plants, as well as their different econom ical uses.

Eco lo gical an d e co n o m ic im p o rtan ce o f m o n o te rp e n o l d e rivative s Irid o id s are ge ran io l d e rivative s w ith im p o rtan t m e d icin al p ro pe rtie s

Only few oxygenated linear geraniol derivatives have been reported in plants. Those include 8-hydroxygeraniol, foliam enthoic acid and carboxygeranic acid, conjugated to hexose, pentose, m alonyl or acetyl groups. These com pounds were detected upon expression of cytochrom es P450 CYP76C4 and CYP76B6 in N. bentham iana (14), but their biological relevance and occurrence in vivo has not yet been dem onstrated.

The best docum ented oxidized derivatives of geraniol are iridoids. Those are cyclic derivatives, with a fused cyclopentane and pyran ring system bearing either a hydroxyl or a glucosyloxy groups at position C-1 (e.g. 7-deoxy-loganetic and loganic acids, respectively, in Figure 5). In plants, iridoids are m ostly found as conjugates (iridoid glycosides or IGs). They were first isolated in the m id-180 0 s from the root of Rubia tinctorum (15), and were nam ed according to their structural sim ilarity and biosynthetic origin to iridodial and iridom yrm ecin found in the ants of the genus Iridom y rm ex (16,17). Iridoids are found in m ore than 57 dicot fam ilies ( 16) that belong to Asteridae, such as Apocynaceae, Rubiaceae, Lam iaceae, Loganiaceae, Verbenaceae, Valerianaceae, Gentianaceae or Scrophulariacea. Because of their wide distribution and diversity they can be used as chem otaxonom ic m arkers. Iridoid producing plants have been used in folk m edicine for the treatm ent of m any diseases such as parasitoses, inflam m ation, diabetes and others (18,19).

The num ber of plant iridoids is estim ated to reach 30 0 0 m olecules according to a thorough literature-based inventory perform ed by Dinda and coworkers (20 -23), and, due to their proven or potential pharm acological properties, the num ber of studies on these m olecules is rising quickly. Many iridoids and iridoid-derived com pounds, such as m onoterpene indole alkaloids (MIAs) are used in m odern m edicine for their antibacterial, anti-inflam m atory, anti-tum oral, chem opreventive and various others activities (24,25). Am ong this variety of m olecules, vindoline and vincristine, extracted from Madagascar periwinkle (Catharanthus roseus) are used directly or as precursor for the synthesis of drugs for treatm ent of lung cancer and lym phom a. Other extensively used MIAs include com pounds as diverse as the antitum oral cam ptothecin (26), the antihypertensive ajm alicin or the classical antim alarial drug quinine (27) (Figure 3). This review focuses on the role and origin of m onoterpenol iridoids in plants. Description of the m edicinal properties of plant iridoids and their derivatives will not be detailed in this work, and can be found in a num ber of dedicated reviews (24,25). Iridoids accum ulate in plants as a protection against various herbivores and pathogens. A toxic effect of isolated com pounds against vertebrate and invertebrate predators has been reported (16, 28 , 29). They have also antibacterial (30 -32) and antifungal activities (30 , 33-35). The toxic and antim icrobial effect of the IGs is due to activity of their aglycone m oeity, which is released through enzym atic or non-enzym atic acidic hydrolysis (36). It has been

shown that the antim icrobial activity strictly requires enzym atic hydrolysis to liberate the

DJO\FRQH ,Q WKLV UHJDUG D VLQJOH UHSRUW GHVFULEHV D ǃ-glucosidase from leaves of

Ligustrum obtusifolium able to convert the secoiridoid glucoside m oiety of oleuropein into a glutaraldehyde-like structure that shows strong protein denaturing, protein-crosslinking, and lysine-alkylating activities (38).

It was recently suggested that IGs could also have a role during oxidative stress, such as drought stress. Drought stress increases the accum ulation of the IGs catalpol, aucubine, harpagide and harpagoside in roots of the m edicinal plant Scrophularia ningpoensis (39).

Based on the protective activities of catalpol and aucubin observed in anim al studies (40 ), it was suggested that this increase in IGs content m ay help the plant cell to deal with oxidative stress (41). An increase in indole alkaloids concentrations and particularly of the antioxidant alkaloid ajm alicine in Catharanthus roseus plants subm itted to drought stress has also been reported (42,43).

The toxic effect of iridoids towards insects is not general and has to be exam ined from a coevolutionary point of view. They can have a clear deterrent effect for non-adapted insects, but can be phagostim ulants for adapted ones (44). Feeding generalist insects with iridoid glycosides induces a reduced growth rate, an increase in larval stages duration and a dim inution of survival rates (28,29,45). As for the deterrent effect, it is still not clear if it is due to the glycosides or to the release of the algycones via acid-hydrolysis or insect-GHULYHG ǃglycosidases in the insect m idgut (34). Contrary to generalists, adapted insects can feed on iridoids producing plants, with even beneficial effects. Iridoids can act as oviposition stim ulants (46,47) and feeding stim ulants (48) for both adults and larvae. The iridoid resistance of specialist insects can be conferred by the ability to absorb and substract them from the gut before hydrolysis can occur (36). The uptaken iridoids can either be degraded, as observed in noctuid and geom etrid larvae (49), or sequestrated as observed in m any adapted Coleoptera, Hom optera, Hym enoptera or Lepidoptera, thereby conferring them obvious advantages against herbivores, parasites or pathogens (50 -52). Only a few iridoids, such as aucubin and catalpol, are sequestrated, suggesting either highly specific transport m echanism s from the insects gut or a differential degradation process between com pounds (36). Other insects have been shown to exploit the protective iridoid properties through de novo biosynthesis, as observed in som e species of Chry som eline (53). De novo synthetized iridoids can constitute a part of the defensive chem ical arsenal of insects, and they can also participate to a tightly regulated sex pherom one signal, as observed in som e species of aphids (54,55).

Mo n o te rp e n o l d e rivative s in fo o d an d be ve rage aro m a

Developm ent of gas chrom atography in the 1950 s (56) triggered interest for the analysis of volatile constituents of food, beverages and arom atic plants. Many of them included putative oxygenated linalool derivatives, but they were always found in com plex m ixtures so unam biguous identification was im possible. In 1963 a m ethod for synthesis of 6,7epoxylinalool was reported (57). This epoxide could be further transform ed in acid to form furanic or pyranic linalool oxides (structures with 5-or 6-m em bered ring, respectively, Figure 4) in cis or trans diastereom ers. In addition, each of these linalool oxides exists in two enantiom ers, giving a total of 8 stereoisom ers. Interestingly, pairs of enantiom ers have different sensory properties, depending on whether they form ed from (S)-or (R)-linalool: linalool oxides derived from (S)-linalool are described as sweet, floral and cream y, and those derived from (R)-linalool as earthy or leafy (59). Availability of synthetic reference standards enabled identification of linalool oxides in green and black tea (58). After this pioneering work on tea arom a, linalool oxides were discovered in m any other foodstuffs (see Table 1).

Other oxygenated linalool derivatives were later discovered in other plants, such as 6-and 7hydroxylinalool in cam phor tree (Cinnam om um cam phora) (60 ). Analysis of glycosylated volatiles allowed for identification of additional m onoterpenol derivatives, such as 8-and 9hydroxylinalool in birch (Betula alba) and J apanese quince (Chaenom eles japonica) (61). shown to cyclize in acidic m edium to form a potent sweet and coconut-like odorant wine lactone (63) (Figure 4). This reaction is thought to occur during wine ageing. Rose oxide is another m onoterpenol derivative with a strong rose-like arom a, which was first discovered in rose flowers (64) and later as a constituent of fruits and other foodstuffs (Table 1). Rose oxide differs from other com pounds described above in that it is not derived from linalool, but citronellol, a reduced geraniol/ nerol derivative.

Lin alo o l a n d its d e rivative s in plan t-in s e ct in te ra ctio n s

Linalool has been extensively investigated for its role in plant-insect interactions, including pollinator attraction (3,92,93), defense (94)(95)(96), and involvem ent in m ulti-trophic interaction by attraction of herbivory predators and parasites (96)(97)(98). However, the role of a large num ber of com pounds deriving from linalool oxidative m etabolism rem ains elusive. A few studies showed that pyranoid and furanoid linalool oxides from Clarkia brew eri

Firs t in s igh ts in to ge ran io l an d lin alo o l o xid ative m e tabo lis m

Battersby and coworkers first dem onstrated in labelling experim ents that geraniol was cyclized and then transform ed to loganin on the pathways leading to MIAs (112). Since then, efforts have been devoted to understanding the steps leading to the com m on MIA precursor secologanin, as well as other species-specific downstream alkaloids. The Dam toft and Inouye groups paved the way in the 80 s (113-117), with extensive precursor feeding experim ents in which the patterns of labelled carbon scram bling were studied for m any plants and pathways, thus asserting the different iridoid pathways leading from the m onoterpenol geraniol (and citronellol in a few cases) to iridodial and alkaloids (reviewed in details in (118)). In particular, they dem onstrated that geraniol is the precursor of m ost of the iridoids, and established that deoxyloganic and epi-deoxyloganic acids are crucial interm ediates in the synthesis of MIAs, each present in specific pathways. The putative pathway leading to these com pounds was predicted to involve a succession of oxido-reductions, but until recently the exact sequence and m ost of the enzym es involved rem ained unknown.

Active linalool oxidative m etabolism in plants was initially observed when feeding linalool to tobacco cell cultures led to the production 8 -hydroxylinalool (120 ). In Clarkia, both linalool and linalool oxides are em itted from flowers, but linalool oxides are likely form ed in other specific tissues from an enzym atic linalool oxidation pathway (3,121). The existence of this oxidative m etabolism in plants was then further supported by the detection of an increased production of both volatile and soluble oxygenated linalool derivatives upon heterologous expression of linalool synthases in different plant species including tom ato, carnation and Arabidopsis (122-124).

The biosynthetic route and fate of linalool in the plant was m ore thoroughly investigated using labeled precursors. The first investigations, carried out with lilac flowers using both deuterated and 18 O labeled precursors indicated that lilac com pounds derived from linalool via a plastidial (MEP) pathway and proceeded with low enantioselectivity, but with high conservation of the (R) or (S) configuration of the linalool precursor (125, 126). The pathway appeared to sequentially involve 8-hydroxylinalool, 8 -oxolinalool, and lilac aldehydes converted into lilac alcohols. Interestingly, labeled lilac com pounds were found associated with the plastids only when feeding was perform ed on intact plant tissues but not isolated plastids. Linalool feeding experim ents in kiwi (Actinidia arguta) flowers revealed form ation of lilac alcohol epoxides (128). (S)-linalool in kiwi flower petals is stereospecifically converted to lilac com pounds (129).

Luan and coworkers investigated the diversity of m etabolism of linalool in grape berries by feeding labeled linalool analogues (127). Linalool was shown to be the precursor of linalool oxides, 8-hydroxylinalool, 7-hydroxylinalool, 6-hydroxylinalool, hotrienol and nerol oxide, as well as their glycoconjugates. Hydroxylation at the position 7 is stereoselective, which strongly suggests that it results from an enzym atic and not a photooxydation reaction. They also proved that linalool oxides are preferentially form ed via a 6,7-epoxylinalool interm ediate, and also to a m inor extent through 6,7-dihydroxylinalool, thus m ost likely via an enzym atic reaction. They showed in addition that the total oxygenation activity was stronger at the beginning of the ripening period and that stereospecific form ation of furanoid and pyranoid linalool oxides occurred at different stages of berry m aturation. Interestingly, the sam e group also investigated m etabolism of deuterated geraniol in grape, but hydroxylated geraniol derivatives could not be detected (119). Som e geraniol was, however, converted to rose oxide, cyclic ether derived from oxidative m etabolism of citronellol, a reduced derivative of geraniol. It thus appears that geraniol is m ostly oxidized at the term inal positions and subsequently turned into iridodial and epi-iridodial, and not m uch is known about other natural single-hydroxylated derivatives. On the contrary, oxidation of linalool occurs at different positions in the carbon chain form ing a wide diversity of hydroxylated com pounds.

Re ce n t ad van

Ge ran io l m e tabo lis m in Ca t h a r a n t h u s r o s e u s

Early This was confirm ed when deep-sequencing and proteom ic approaches led to the elucidation of the com plete secologanin pathway in C. (Figure 5). These studies led to the characterization of CYP76A26 from C. roseus as a nepetalactol (iridodial) oxygenase, catalyzing three consecutive oxygenation steps and to the form ation of 7-deoxyloganic acid (136, 138). Interestingly, this enzym e was also found capable to hydroxylate m onoterpenols (such as nerol, citronellol), although with low efficiency. CYP76A26, however, did not hydroxylate geraniol, possibly preventing com petition with CYP76B6. The sam e or sim ilar strategies led the characterization of the com plete sequence of enzym es form ing the secologanin pathway (Figure 5).

Lin alo o l m e tabo lis m in Ar a b id o p s is t h a lia n a flo w e rs

The first lead to linalool oxygenases em erged from in silico analysis of gene co-expression. Finally, the subcellular localization of CYP76C1, CYP76C3 and CYP71B31 was investigated in parallel with localization of TP10 and TPS14 (10 3, 151). In all cases the two linalool synthases were detected in vesicular structures associated with the plastids whereas the P450 s appeared associated with plastid-wrapping endoplasm ic reticulum sheets. This m ost likely explains the initial observations of Kreck and coworkers (125), who found that labeled lilac com pounds associated with the plastids only when feeding was perform ed on intact plant tissues, but not isolated plastids.

Pe rs p e ctive s an d o p e n qu e s tio n s

Recent work started to reveal the core oxidative m etabolism of m onoterpenols in higher

Backgro u n d

Grapevine (Vitis vinifera L.) is one of the oldest (1) and econom ically the m ost im portant (2) fruit crops in the world. The m ajority of grapes produced worldwide are used in winem aking.

Modern cultivated grapevine has been shaped by thousands of years of selection for traits such as berry size, sugar content or skin color (3), but today viticulture is facing new challenges. In addition to pathogen pressure, it has to deal with clim ate change (4-6), and shift of consum er preference towards higher quality wines with a lower environm ental im pact (7,8 ). Traditional breeding is extrem ely difficult to apply in grapevine because of its long lifecycle, reduced fitness of progeny and com plexity of quality traits (9). Sequencing of the grapevine genom e in 20 0 7 (10 ) and advances in the 'om ics' techniques (11) set the stage for m ore efficient breeding solutions. The next crucial step towards im proved grapevine varieties is the identification of genes underlying im portant traits, such as interactions with pathogens, fruit developm ent and quality.

Many developm ental as well as ecological functions in plants are controlled by cytochrom e P450 oxygenases (12,13). These enzym es catalyze regio-and stereospecific insertion of an oxygen atom into sm all, hydrophobic substrates that range from terpenoids and fatty acids to am ino acids and their derivatives, such as phenolic com pounds. In the m odel plant

Arabidopsis thaliana they control processes as diverse as plant growth and branching (14,15), flower (16) and fruit developm ent (17), form ation of lignin and surface biopolym ers (18 , 19), em ission of volatiles (20 ) or plant-pathogen and plant-insect interactions (21,22). This m akes cytochrom es P450 attractive targets for crop im provem ent.

Cytochrom es P450 in plants evolved into m any distinct fam ilies, which are usually defined as genes with 40 % or higher protein sequence identity. Within one P450 fam ily the biochem ical function is often conserved across the plant kingdom . For exam ple, enzym es from the CYP97 fam ily are involved in carotenoid hydroxylation, CYP79s in the N -hydroxylation of am ino acid to aldoxim es, CYP75s in the hydroxylation of flavonoids, and CYP70 4s in fatty acid hydroxylation to form the precursors to structural polym ers sporopollenin and cutin (23).

Mem bers of other fam ilies, however, have divergent functions: som e m em bers of CYP72 fam ily are involved in iridoid biosynthesis, whereas others oxidize triterpene substrates (23).

These differences stem from different evolutionary pressures on genes with different functions. Fam ilies with essential functions, such as horm one m etabolism or synthesis of biopolym ers, are usually m aintained at low copy num ber and high purifying selection, whereas fam ilies with adaptive functions expanded or "bloom ed" in certain taxa (24). A welldocum ented exam ple is a bloom of CYP76M subfam ily in rice (Ory za sativa), which consists of 11 genes and 2 pseudogenes. At least 4 m em bers of this subfam ily are involved in the biosynthesis of diterpenoid antifungal com pounds (25,26). They are clustered close together in the genom e, which is another com m on feature of recently duplicated P450 s and probably results from sequential tandem duplications (24). Interestingly, CYP76 m em bers from other plants, for exam ple Arabidopsis thaliana or Catharanthus roseus, have a different biochem ical function, nam ely oxidation of m onoterpenols or their iridoid derivatives (27,28). Recently expanded P450 fam ilies m ight therefore have interesting ecological functions, but those are m ore difficult to predict com pared to functions of conserved P450 fam ilies. In addition, function of m any P450 fam ilies is still unknown or poorly understood.

Previous annotation of P450 s has highlighted som e potentially interesting gene fam ilies in the highly heterozygous V. vinifera cv. Pinot Noir genom e (29)(30)(31). In this work we perform ed the first com plete m anual annotation of P450 s in the nearly hom ozygous V.

vinifera reference genom e PN40 0 24 (10 ). We discuss the structural organization of the genes with particular focus on gene clusters. We evaluate phylogenetic relationships between those genes to identify recently expanded gene fam ilies likely linked to adaptive traits or dom estication. Finally, we investigate spatio-tem poral gene expression patterns, with particular focus on berry developm ent and pathogen response to identify P450 s with potential roles in these im portant physiological processes. This work will support further functional characterization of cytochrom e P450 genes in grapevine.

Re s u lts

Ge n e an n o tatio n , cla s s ificatio n an d p h ylo ge n y

A sim ilarity search of the V. vinifera PN40 0 24 genom e with known P450 sequences revealed 579 putative P450 sequences. We m anually curated the sequences obtained with a gene prediction algorithm , and validated the annotation with grapevine ESTs and RNAseq reads (see Material and m ethods). We distributed them into four categories: genes, partial genes, putative pseudogenes and pseudogenes. This led to the identification of 279 full-length genes, which is fewer than 315 genes reported for the heterozygous Pinot Noir genom e on the The cytochrom e P450 fam ilies usually have conserved or sim ilar functions across species.

Grapevine P450 s can be assigned to 48 fam ilies based on sequence identities A phylogenetic analysis of either the full-length sequences or of a subset of conserved P450 sites confirm ed this classification for m ost of the fam ilies. One exception is the CYP90 B gene, which is clustered with CYP720 and CYP724 as previously observed (32). Other exceptions are the fam ilies CYP76 and CYP80 , which form a m onophyletic group (Figure 1, see Material and m ethods). We thus investigated the phylogeny of these two fam ilies in the broader context of selected angiosperm species (Figure S1). CYP80 clearly groups with CYP76 sequences, but the phylogenetical relations of the clades CYP8 0 , CYP76A/ G and the rest of CYP76 sequences (labeled core CYP76) rem ain uncertain. Within the CYP76A/ G clade, a eudicot duplication gave rise to the two subfam ilies CYP76A and CYP76G. Within the large "core CYP76" clade the uncertain position of both the m onocot and Am borella trichopoda CYP76s could be due to a problem of long-branch attraction. A specific core eudicot duplication gave rise to CYP76F/ B/ X on one side and CYP76T/ C/ E on other side. These tree topologies were obtained both with the full-length alignm ent and the partial alignm ent of conserved sites.

Although species-specific "bloom s" appeared in the whole CYP76/ 80 fam ily, they are particularly abundant in the "core CYP76" clade. Different subfam ilies "expanded" in different species.

Com parison of P450 fam ily sizes between species (Figure S2 The 579 cytochrom e P450 sequences are distributed on all the 19 chrom osom es. Som e chrom osom es, nam ely 18, 19 and 6, carry a high num ber of P450 s, whereas others, for exam ple chrom osom e 5, carry very few (Figure S3). 24 P450 sequences (7 genes, 6 partial genes, 11 pseudogenes) are located on the "Unknown" chrom osom e which is composed of scaffolds that could not be anchored on any of the 19 chrom osom es. Since the genom e is not com pletely hom ozygous (estim ated hom ozygosity is 93% (10 )), the "Unknown" chrom osom e m ay also contain eventual allelic variants of genes that are placed on the 19 chrom osom es.

We further investigated the distribution of cytochrom e P450 sequences in clusters or groups in close physical proxim ity (separated by less than 20 0 kb and 8 non-P450 genes (34,35)).

Our results show that P450 sequences are organized in clusters and not random ly distributed Most of the largest P450 fam ilies (CYP82, CYP71, CYP81, CYP76, CYP72 and others) are organized in clusters (Table S1).

Clustering by P450 fam ily already indicates that m ore sim ilar P450 sequences cluster in closer physical proxim ity. But m any P450 fam ilies are dispersed am ong several clusters. We thus wished to explore whether the closest paralogs belong to the sam e or different clusters (Figure 3). The m ajority of clustered P450 genes (86%) have their closest paralog (the best BLAST hit) in the sam e cluster. The second and third closest paralogs (second and third best BLAST hit) are in the sam e cluster for 58% and 49% of the clustered P450 genes. The sequence sim ilarities within the sam e cluster are thus higher than between clusters. For each circle, the grey bars correspond to the 19 grape chrom osom es and the "Unknown chrom osom e". The lines connect com plete P450 genes according to their sim ilarity. The lines outside the circles show the sim ilarity between genes of the sam e cluster, whereas the lines in the circle connect sim ilar genes of different clusters. Only P450 genes that from clusters com posed of at least two com plete genes are illustrated here. The seven largest clusters are labeled with num bers corresponding to Table 1. The lines are connecting the genes corresponding to the best BLAST hit (A), second best hit (B) or third best blast hit (C).

Large P4 5 0 clu s te rs in V. v in ife r a ge n o m e fo rm e d v ia d iffe re n t m e ch an is m s

To investigate the m echanism s underlying the form ation of large physical clusters of cytochrom e P450 genes, we further analyzed the sequence sim ilarity within clusters, taking into account not only the coding P450 sequences, but also the surrounding non-codingsequences. This allowed us to infer the m echanism of cluster form ation. We focused on the 7 largest physical clusters, which com prise of 11 to 35 P450 sequences (Table 1). Together, these 7 clusters contain 23% of all P450 genes, and a sim ilar fraction of total P450 sequences.

Most of the sequences in these clusters are part of "clan 71", which is a large clade of plant cytochrom es P450 often involved in the biosynthesis of specialized m etabolites (Figure 1).

Analysis of sim ilarity blocks within these clusters showed they differ rem arkably in their structures (Figure S5). One of the largest physical clusters, cluster 65, is characterized by low sim ilarities, both am ong the P450 sequences and surrounding non-coding regions. The sim ilarity blocks of two other large physical clusters, 71 and 171, are restricted to P450 sequences and do not extend to the intergenic regions. Single gene duplications were thus probably the m ain m echanism of form ation of these two clusters. The sim ilarity blocks of physical clusters 13 8 and 18 2 extend to the non-coding regions around the cytochrom es P450 annotations. This suggests the duplication events leading to form ation of these clusters happened relatively recently. High sim ilarity between the non-coding regions, which include the prom oter regions, should result in sim ilar expression profiles. Cluster 13 8 has the highest fraction (73%) of pseudogenes of all the seven large clusters. In physical clusters 9 2 and 19 0 , the sim ilarity blocks extend over even longer regions that include 3-4 cytochrom e P450 sequences and their intergenic regions (Figure 4). In addition, the type of annotation (gene or pseudogene) was also m aintained in the sam e order between duplicated blocks. This suggests these two clusters form ed through very recent segm ental duplications.

Table 2 . D e s crip tio n o f th e s e ve n large s t p h ys ical P4 5 0 clu s te rs in th e V. v in ife r a ge n o m e .

Labelsequential num ber of each cluster in the genome; Chr -chrom osom e num ber; Location -chrom osom e coordinates; Total seq. -num ber of P450 sequences in each cluster, including com plete and partial genes, putative pseudogenes and pseudogenes with their fam ily distribution; Com plete genes -num ber of com plete P450 genes in the cluster; Expressed sequences -num ber of expressed P450 sequences in the cluster, coexpression -expression pattern of the cluster ("-" signifies low co-expression within the cluster); Organizationdescription of structural organization and m echanism of form ation of each cluster.

Exp re s s io n pro file s o f gra pe vin e P4 5 0 s

To identify P450 genes with potential roles in pathogen resistance or biosynthesis of berry m etabolites we analyzed the expression of the 579 P450 sequences. Pseudogenes were included in the analysis of expression to account for sequences that m ight be functional in other varieties, as well as for recently pseudogenized sequences that m ay still be expressed to som e extent. We used 73 RNA-Seq datasets (Table S2) which describe gene expression in different tissues (flowers, berries, leaves) and different stages of berry developm ent, and pathogen infection. We grouped the 73 experim ents in 5 categories: flowers, green berries, ripe berries, leaves (control) and leaves under biotic stress. The latter category includes leaves infected with the powdery m ildew pathogen Ery siphe necator and the downy m ildew pathogen Plasm opara viticola.

To enable a m eaningful com parison of gene expression between different experim ents we calculated fragm ents per kilobase of transcript per m illion m apped reads (FPKM) for each P450 sequence in the 5 categories of experim ents. We grouped the expression levels into 4 classes (no expression, low, average or high expression). The m ajority of P450 sequences (494 or 85%) were expressed in at least one experim ent. Of the rem aining 85 non-expressed P450 sequences, only 4 were com plete genes. Expression of com plete P450 genes (m ean FPKM = 11, m edian FPKM = 0 .6) was higher com pared to pseudogenes and putative pseudogenes (m ean FPKM = 1.6, m edian FPKM = 0 ) or partial genes (m ean FPKM = 1.7, m edian = 0 .1). In each of the 5 categories, an average of 12% of the genes were not expressed, 54% had low, 20 % average and 14% high expression. Interestingly, in leaves exposed to biotic stress, the fraction of non-expressed genes drops from 28% to only 9%, whereas the fraction The analysis of gene expression highlighted som e of the P450 s with potential role in biotic stress response. From the 10 6 sequences specifically expressed in leaves (clusters N , O, W and X), 44 were upregulated and 7 were downregulated upon biotic stress, confirm ing an expression shift upon pathogen infection. The P450 fam ily with the highest num ber of upregulated genes was CYP82 (7 sequences, 4 com plete genes), followed by CYP81 and CYP71 with 6 sequences each (1 and 3 com plete genes, respectively). CYP87B25 is the m ost induced gene upon biotic stress and two other CYP87 genes were am ong the 7 m ost upregulated sequences (FPKM>20 ).

Another m ajor shift in the P450 expression pattern occurs during the grape berry ripening.

From 81 sequences specifically expressed in berries (clusters F, I, J , K and P), only 4 were upregulated in green berries (cluster I), com pared to 32 in ripe berries (cluster J ).

Interestingly, the m ost represented fam ilies cluster J were also CYP82 with 5 sequences (1 com plete gene) and CYP71 with 5 sequences (3 com plete genes). This cluster in addition featured the P450 gene with the highest expression in all experim ents: CYP78A41 with 1417 FPKM.

We m ore thoroughly investigated co-expression of cytochrom e P450 sequences within the 7 largest physical clusters (Table 1). In the previous section we identified 4 large clusters with high sim ilarity, not only am ong the coding, but also the non-coding regions. These noncoding regions presum ably include prom oter sequences, so the genes in these clusters are expected to have the sam e expression pattern. Indeed, the 20 kbp duplicated block within cluster 9 2 (Figure 4b) retained the sam e expression profile after the segm ental duplication.

The duplicated segm ent consists of three P450 sequences: two pseudogenes and one gene.

The first pseudogene in both blocks retained a very low level of expression in ripe berries, the second pseudogene in both blocks was not expressed at any experim ental conditions, whereas the two com plete genes (CYP82D17 and CYP82D20 v2) were co-expressed in leaves and slightly induced upon pathogen infection. 7 out of 24 sequences in this cluster shared this sam e expression pattern (expression clusters N and O), whereas 4 other P450 sequences in the sam e cluster were up-regulated in ripe berries (cluster J). Interestingly, cluster 13 8 is also com posed of CYP82 sequences, but these sequences were preferentially expressed in young berries. Another large physical cluster with coordinated expression pattern was cluster 71, which com prises 11 CYP89 genes and 5 pseudogenes. Four of these genes were specifically expressed in leaves (cluster O). Three CYP76 sequences in the physical cluster 19 0 , on the other hand, were co-expressed in flowers.

D is cu s s io n

We produced a reliable and validated m anual annotation of cytochrom es P450 in the genom e of the nearly hom ozygous grapevine (V. vinifera) accession PN40 0 24 (10 ). Cytochrom e P450 superfam ily in Vitis vinifera contains both very sim ilar and very divergent genes (sequence identity ranges from 10 % to alm ost 10 0 %), and often form clusters in very close physical proxim ity, which m akes it challenging for autom ated annotation algorithm s. Manual curation is therefore necessary to produce a reliable annotation, suitable for dem anding downstream applications such as phylogenetic or gene expression analysis. Grapevine P450 s have been previously m anually annotated (Cytochrom e P450 hom epage, http:/ / drnelson.uthsc.edu/ vitis.htm )

Several gene fam ilies involved in the biosynthesis of specialized m etabolites, such as terpene synthase genes (TPS) and stilbene synthase genes (STS), have expanded in grapevine genom e com pared to other species (10 , 36, 37). Although the total num ber of cytochrom e P450 genes in grapevine is com parable to other species, individual P450 fam ilies experienced sim ilar expansions. These expended fam ilies, sim ilarly to TPS and STS fam ilies, form large physical clusters of m ore than 10 hom ologous sequences. One of such fam ilies is CYP75, which together with CYP79 fam ily m em bers form s the largest physical cluster of 35 P450 sequences on chrom osom e 6. Expansion of CYP75 genes in grapevine was previously docum ented, but the presence of another P450 fam ily, CYP79, in the sam e cluster was not reported (33).

Clustered genes with low or no hom ology som etim es participate in the sam e biosynthetic pathway (38,39), but this is unlikely in the case of CYP75 and CYP79, since both fam ilies have well established roles in different biosynthetic pathways: CYP79 genes code for am ino acid N-hydroxylases (23), whereas CYP75A genes code for flavonoid 3',5'-hydroxylases (40 ), crucial enzym es in the biosynthesis of blue anthocyanins in the grape skin (33,41). We can, however, not exclude the recruitm ent of som e of these genes in other pathways. Interestingly, the sequencing of the genom e of the grapevine cultivar Tannat, characterized by its very deep color, revealed an even higher num ber of CYP75 genes com pared to the PN40 0 24 accession (42). Copy num ber of genes in a cluster can therefore vary between cultivars and influence in the highly heterozygous genom e of Pinot noir cultivar (29). Our annotation represents an im provem ent over the existing dataset for several reasons. The assem bly of PN40 0 24 genom e is of better quality com pared to the Pinot noir genom e: it contains fewer gaps and a higher fraction of anchored contigs. The hom ozygosity of the genom e not only enabled a better quality of the assem bly, but also assured that m ost of the annotated sequences are individual loci and not allelic variants. This can partially explain a lower num ber of cytochrom e P450 genes in our annotation-279com pared to the 315 genes reported on the Cytochrom e P450 hom epage. Additionally, the annotation on the Cytochrom e P450 hom epage classifies the sequences in only two categories, genes and pseudogenes, whereas we em ployed a m ore stringent classification into genes, partial genes, putative pseudogenes and pseudogenes. Lastly, we report the exact genom ic coordinates of the P450 sequences, which facilitate com parison to annotations of other genes, and provide insights into structural organization of the grapevine CYPom e. varietal characteristics. Other expanded P450 fam ilies in the grapevine genom e that form large clusters are CYP82, CYP76, CYP81 and CYP89.

Analysis of gene expression across several tissues and conditions provides a first hint to the putative P450 functions in grapevine. Pathogen infection causes a m ajor shift in the P450 expression, inducing m em bers from fam ilies CYP71, CYP81, CYP82 and CYP87. Their hom ologs in other species have been shown to participate in biosynthesis of highly specialized defense com pounds (Table S1). Interestingly, CYP736A25v1, which was shown to be upregulated upon infection with the Pierce disease pathogen Xy lella fastidiosa (43), is also upregulated upon infection with powdery m ildew and downy m ildew pathogens. Other genes from the CYP736 fam ily are not affected by biotic stress. Another large shift in expression occurs in developing grape berries. The m ost upregulated P450 fam ilies in the ripe-berry expression cluster are CYP82 and CYP71 (the two largest P450 fam ilies in grapevine). These P450 s are likely to participate in the biosynthesis of defense com pounds or com pounds im portant for the organoleptic properties of wine (arom a, colour, taste, m outhfeel). The m ost up-regulated P450 gene in ripe berries and the P450 gene with the overall highest expressions is CYP78A41. A m em ber of the sam e P450 fam ily in tom ato (S.

ly copersicum ) was selected during dom estication to increase fruit size (44). High expression of CYP78A41 in grape berries points to a sim ilar event in grapevine dom estication.

The phylogenetic and structural data suggest that som e P450 fam ilies underwent m ultiple tandem or segm ental duplications, which resulted in large physical clusters of hom ologous sequences. Most of these P450 fam ilies are involved in biosynthesis of highly specialized m etabolites in other plant species. These genes are often expressed in specific conditions and tissues, such as leaves upon pathogen infection. Our work thus lays the ground for discovery of interesting novel P450 functions in grapevine.

Mate ria l an d m e th o d s Ge n e an n o tatio n

We annotated the cytochrom es P450 using the 12x version of the assem bly of the Vitis vinifera cv PN40 0 24 genom e (10 , 45 The four m asked datasets were used to perform four independent TBLASTN analyses (47) against the PN40 0 24 12x sequence with an e-value cutoff of 1e-3. The TBLASTN outputs were parsed using a hom em ade script. The hits from the three grape datasets were kept if they were at least 50 am ino acids long with at least 70 % sequence identity. The hits from the Arabidopsis dataset were kept if they were at least 50 am ino acids long with an identity percentage of at least 50 %. The software Exonerate (version 2.2.0 , build October 20 0 8, 37)

was used to predict gene structures using the protein2genom e param eter and the sam e cutoff of sequence identity as above. A hom em ade script was used to reform at the output files from exonerate into files in the gff form at. These gff files were im ported to the Artem is genom e browser (49) to perform the m anual curation of the structures suggested by Exonerate. The parsed hits identified through TBLASTN were used to im prove or to com plete the Exonerate annotations. Every annotation starting with a start codon, ending with a stop codon and with correct exon-intron borders (GT-AG or som etim es GC-AG) was considered as a com plete "gene". Every annotation showing the previously described gene structure but with a single point m utation creating a fram eshift, a prem ature stop codon or a wrong exon-intron border was considered as a "putative pseudogene" also m arked "pseudogene?" because it m ay result from a m istake in the genom e assem bly. Every annotation interrupted by a gap in the genom ic sequence or including one was considered as a "partial" annotation. All the other annotations with wrong gene structure but showing a significant sim ilarity level with a cytochrom e P450 from one of the four datasets were annotated as "pseudogenes". unigenes were m apped on the PN40 0 24 12x sequence using GMAP version 20 13-11-27 (51) using the default param eters except for the form at param eter which was set to "gff3_ m atch_ cdna". The second transcript dataset was locally assem bled using six RNA-Seq experim ents (( 52 The com m and m askFastaFrom Bed v2.19.1 from the bedtools package ( 58) was used to m ask the regions of the PN40 0 24 12x sequence where we annotated cytochrom e P450 exons after having reform atted the gff file of the annotations into a bed file. We perform ed TBLASTN analyses of the four grape cytochrom e P450 datasets against the m asked PN40 0 24 12x sequence and parsing analyses using the sam e param eters and cutoffs than previously described. This step allowed to identify the region of the grape genom e for which a cytochrom e P450 sim ilarity was m issed during the m anual curation.

To validate the set of com plete genes of cytochrom es P450 that we annotated, a BLAST against non-redundant sequence database (NR) was perform ed and only the genes for which the best hit was a cytochrom e P450 were kept. For the pseudogenes, a BLASTX was perform ed against the set of com plete P450 genes that we annotated and we kept only the ones that aligned over at least 30 % of the query length with the percentage identity of 50 %.

The cytochrom e P450 annotations were transferred to the im proved version of the PN40 0 24 12x assem bly when it was released (PN40 0 24 12X.2; https:/ / urgi.versailles.inra.fr/ Species/ Vitis/ Data-Sequences/ Genom e-sequences) using a hom em ade script. The presence of physical clusters of cytochrom e P450 s in the grape genom e was tested based on the following definition of a cluster. Two consecutive P450

annotations are part of a cluster if they are separated by 20 0 kb and 8 non-P450 genes at the m ost (34,35). The two annotations also have to be located on the sam e scaffold which guaranties a precise estim ation of the intergenic distances. A bootstrap test was perform ed to check whether the cytochrom es P450 were m ore clustered than what is random ly expected. A hom em ade script was developed with R version 3.0 .2 (59). Ten thousand sam pling without replacem ent of 579 (num ber of P450 annotations) or 279 features (num ber of com plete P450 genes) were perform ed on the genom e annotation V1 stored in Grape Genom e Database hosted at CRIBI counting 29,971 features. The percentage of features organized in clusters was com puted using the sam e protocol as for cytochrom es P450 . The p-value was calculated by counting each tim e a percentage equal of greater than the percentage of P450 in clusters divided by 10 0 0 0 (num ber of iterations).

Sequence sim ilarity within and between clusters (Figure 3) was analyzed by perform ing a BLASTP search of translated com plete P450 genes against them selves. Only the genes that aligned over at least 70 % of the query length with the percentage identity of 40 % were kept.

The Circos software (60 ) was used to draw the figure. Clusters that contained less than two com plete genes were excluded from this analysis (i.e. clusters that contained partial genes, pseudogenes and putative pseudogenes with less than 2 com plete genes).

The dotter software version 4.23 (61) was used to draw the sequence sim ilarity graphs of the cluster 190 with its fasta sequence and annotations in a gff form at as an input.

Se qu e n ce clas s ificatio n

Cytochrom e P450 genes, partial genes and putative pseudogenes were aligned to the P450 sequences from the heterozygous Pinot Noir genom e, retrieved from the cytochrom e P450 hom epage (http:/ / drnelson.uthsc.edu/ Cytochrom eP450 .htm l). In the case of protein sequence identity above 95%, the original nam e was kept. New sequences were assigned a fam ily based on the best hit am ong already nam ed grapevine P450 s. 22 sequences were given a new CYP nam e, and genes previously annotated as m em bers of CYP81V subfam ily were reclassified to CYP81Q subfam ily.

Ph ylo ge n y

Sequences from non-Vitis species were retrieved from the cytochrom e P450 hom epage (http:/ / drnelson.uthsc.edu/ Cytochrom eP450 .htm l). Pseudogenes and incom plete genes were excluded from the analysis. 279 Vitis vinifera CYP (Figure 1) and 191 CYP76, 80 and 70 6

protein sequences from Aquilegia caerulea, N elum bo nucifera, M im ulus guttatus, Solanum ly copersicum , Am borella trichopoda, Ory za sativa, Brachy podium distachy on, Arabidopsis thaliana, M edicago trunculata, Populus trichocarpa and Vitis vinifera (Figure S1) were aligned with MUSCLE (62) im plem ented in Seaview (63,64). Conserved sites were selected in the alignm ent using Gblocks (65) using the less stringent option param eters. Maxim um likelihood phylogenies were obtained from the full-length alignm ents and from the subset of m ore conserved sites alignm ents (all Vitis CYP: 166 sites and 11 species CYP alignm ent: 278 sites) using RAxML (v 8. were visualized and colored using Figtree (http:/ / tree.bio.ed.ac.uk/ software/ figtree). The species cladogram in (Figure S1) was inferred from the APGIII system (69).

Ge n e e xpre s s io n

We retrieved raw grape RNA-Seq data from NCBI SRA public database Using all non-zero FPKM values, the 33th and 66th quantiles were calculated to assign the expression values to one of the four levels of expression chosen: no, low, average and high expression. The experim ents were grouped into five categories regarding the conditions under which the sam ples were obtained. These categories are: flowers, young berries, ripe berries, leaves (control) and leaves under biotic stress. An average expression per category was then calculated for each gene and assigned to one of the four levels of expression regarding its value: no expression if the average was zero, low expression between zero and the 33% quantile, average expression between 33% and 66% quantile and high expression for averages higher than the 66% quantile.

The average expression values for each P450 annotation were used to perform a clustering analysis using HCE version 3.5 (74) with a com plete linkage m ethod and a Pearson's correlation as distance m easure. The cut-off to define the clusters was set at a Pearson's correlation coefficient of 0 .795.

Abs tract

Monoterpenes constitute an im portant part of wine arom a, notably the floral notes of som e wine varieties. The m onoterpene in wine with the lowest sensory threshold is the win e lactone, a bicyclic lactone that can form via acid-catalyzed cyclization of (E)-8carboxylinalool (also known as m enthiafolic acid) during wine m aturation. In a sam ple of 23 white wines, we show that (E)-8 -carboxylinalool content correlates with the content in wine lactone and we estim ate the kinetic constant for the form ation of wine lactone from (E)-8carboxylinalool. The concentration of this precursor was higher in Gewurztram iner wines com pared to other two varieties studied. We explored the biosynthetic origin of (E)-8carboxylinalool in grapes and identified a set of candidate cytochrom e P450 s likely to contribute to the conversion of linalool into (E)-8-carboxylinalool based on their gene expression during grape berry m aturation. One of them , CYP76F14, which is highly expressed in the late stages of berry ripening, can efficiently oxidize linalool to (E)-8-carboxylinalool in vitro as well as in planta.

In tro d u ctio n

Grapevine (Vitis vinifera L.) is one of econom ically the m ost im portant crops worldwide with m ost of the 70 m illion tons of yearly grape harvest used for winem aking (1). The quality of wine to a large extent depends on its arom a, but its chem ical com plexity m akes it a difficult subject to study, let alone predict: it consists of several hundred different volatile com pounds at concentrations spanning several orders of m agnitude. The advances in analytical chem istry in the second half of the 20 th century allowed scientists to study this com plex chem ical m ixture in m ore detail. It gradually transpired that characteristic wine arom a does not depend as m uch on the m ost abundant com pounds, but rather on low-concentration com pounds with very strong odor. One is isobutyl m ethoxypyrazine, responsible for characteristic bell pepper scent of Cabernet Sauvignon and Sauvignon Blanc wines, where it reaches concentrations of about 30 pg/ L (about an order of m agnitude higher than its odor threshold) (2). Another exam ple is rotundone, a sesquiterpene which conveys the peppery arom a to Shiraz wines at concentrations as low as 20 ng/ L (3). Wine lactone, a bicyclic m onoterpene lactone, is another trace wine arom a com pound with a very low odor threshold (10 ng/ L) (4). Pure wine lactone has a sweet, woody and coconut-like arom a ( 5) and was shown to contribute to the arom a of Gewurztram iner wines (4,6). It m ight also contribute to the arom a profile of fresh orange (7) and grapefruit ( 8) juice.

As opposed to rotundone or isobutyl m ethoxypyrazine, wine lactone is not synthetized in grapes, but predom inantly during wine m aturation (9). It form s in a slow, non-enzym atic, acid-catalyzed cyclisation from an odorless precursor, (E)-8 -carboxylinalool or m enthiafolic acid (10 -12). It has therefore been suggested that wine lactone concentration is increasing with wine aging ( 13), but the data on both (E)-8-carboxylinalool and wine lactone concentrations in wine are scarce. Concentrations of wine lactone in wine are very low, which causes analytical difficulties (14), whereas (E)-8 -carboxylinalool is often overlooked because gas chrom atography is traditionally used for analysis of arom a com pounds, although it is not be appropriate for the analysis of m ore polar m olecules. The analysis is additionally im peded by the fact that (E)-8 -carboxylinalool is found both in grapes and wines in the form of a glucose ester, with the sugar m oiety attached to the carboxyl functional group (10 , 15, 16).

Monoterpene glycosides and glucose esters were suggested to act as a precursor pool for the form ation of arom a com pounds during wine ferm entation and m aturation, as they are hydrolyzed chem ically (at low pH) or enzym atically (17). Luan et al. (18) proposed that (E)-8-carboxylinalool in grapes is form ed from the m onoterpene linalool, another im portant arom a com pound, in a m ulti-step enzym atic reaction: linalool is first oxidized at C8 to yield (E)-8-hydroxylinalool, also a com m on grape m etabolite (19), which is then further oxidized at the sam e carbon atom to (E)-8carboxylinalool (Figure 1), followed by glycosylation to form the glucose ester.

Form ation of linalool by grapevine terpene synthases (TPS) has already been studied (20 ), but the enzym es catalyzing downstream oxidation to (E)-8-carboxylinalool rem ained unknown. In m any plants m onoterpenes are oxidized by cytochrom es P450 (21), hem e containing enzym es that use m olecular oxygen to oxidize sm all, non-polar substrates, usually with rem arkable regio-and stereospecificity. P450 s from fam ilies CYP76 and CYP71 often oxidize m onoterpene alcohols, such as linalool (22)(23)(24) or geraniol (25). CYP76C1, for exam ple, converts linalool into (E)-8-carboxylinalool in Arabidopis thaliana flowers (24).

Enzym es from CYP76 and CYP71 fam ilies thus em erged as likely candidates for (E)-8carboxylinalool synthesis in grapes.

The aim of this work was to elucidate the pathway of wine lactone form ation in wines, from the early enzym atic steps during grape berry developm ent to the non-enzym atic steps during wine m aturation and aging. Using liquid chrom atography coupled to m ass spectrom etry, we first establish the relationship between the concentrations of wine lactone and its precursors in a selection of wines of different ages. We show that the expression of a subset of P450 genes is induced in the later stages of berry ripening and dem onstrate that, am ong these, only CYP76F14, which is the m ost highly expressed CYP76 in ripe berry, efficiently catalyzes the conversion of linalool to (E)-8-carboxylinalool both in vitro and in planta. We therefore suggest that CYP76F14 is responsible for the synthesis of (E)-8-carboxylinalool, the wine lactone precursor in grapes. Low abundance of wine lactone m akes it a difficult analytical subject with only a few successful attem pts of wine lactone quantification in wines reported to date (6,14). We first attem pted analysis of wine lactone in concentrated wine extracts by gas chrom atography coupled to m ass spectrom etry (GC-MS), the conventional m ethod for analysis of volatile com pound. However, the lim it of detection was too high to allow for the analysis of wine lactone at the low concentrations expected in wines. We therefore developed a liquid chrom atography-tandem m ass spectrom etry (LC-MS/ MS) m ethod, which enabled the quantification of wine lactone in all 23 investigated wines (Figure S1). We also used LC-

Re s u lts

MS/ MS to analyze (E)-8-carboxylinalool by direct injection of wines without extraction

(Figure S2). With this m ethod we also detected a conjugated form of (E)-8-carboxylinalool, putatively identified as (E)-8-carboxylinalool glucose ester.

To determ ine the factors controlling wine lactone concentrations in wine, we analyzed wines of different ages and varieties for their content of wine lactone and its two precursors, (E)-8carboxylinalool and (E)-8-carboxylinalool glucose ester. This analysis showed that concentration of wine lactone was increasing with wine age (Figure 2a), as well as with concentration of (E)-8-carboxylinalool (Figure 2b). The concentration of (E)-8carboxylinalool was strongly dependent on wine variety, and m uch higher in Gewurztram iner than in Riesling.

Concentrations of wine lactone were generally about 10 0 0 tim es lower com pared to (E)-8carboxylinalool. Only a sm all proportion of (E)-8-carboxylinalool was thus converted to wine lactone, which was in good agreem ent with the relatively stable concentration of (E)-8carboxylinalool over tim e. Although the concentrations of wine lactone were low (in the nM range), they were all above the sensory threshold of wine lactone, which is 60 pM in m odel wine (4).

Principal com ponent analysis was perform ed on wine age and concentrations of (E)-8carboxylinalool, (E)-8-carboxylinalool glucose ester and wine lactone to further explore the relationships between these factors (Figure 2c-d). This analysis revealed a strong negative correlation between the concentrations of (E)-8-carboxylinalool glucose ester and (E)-8carboxylinalool, supporting a decrease in (E)-8-carboxylinalool glucose ester by acid hydrolysis yielding free (E)-8-carboxylinalool (Figure S3). In Gewurztram iner wines (E)-8carboxylinalool glucose ester was only detected in 1-year-old wines, but not in 6-year-and older wines. In the wines without (E)-8-carboxylinalool glucose ester, the concentration of (E)-8-carboxylinalool was independent of age. Wine lactone concentration correlated with wine age and concentration of (E)-8-carboxylinalool, supporting its slow form ation from (E)-

8-carboxylinalool.

We used the first order reaction rate law to m odel conversion of (E)-8-carboxylinalool to wine lactone. Using the approxim ation of a constant concentration of (E)-8-carboxylinalool, we determ ined a rate constant k = (0 .0 0 0 12 ± 0 .0 0 0 0 2) year -1 . Because the kinetic constan t was so low, we could use a linear approxim ation for the reaction rate, with [wine lactone] = k  [(E)-8-carboxylinalool]  wine age. As evident from the plot of observed vs. calculated values, the deviations from predicted values were higher in young wines (Figure S4). A possible explanation is form ation of wine lactone from (E)-8-carboxylinalool glucose ester, or in grape berries or by the yeast during ferm entation.

Metabolic profiling pointed to differences between varieties. Gewurztram iner wines contained m ore (E)-8-carboxylinalool than Riesling or Muscat, and, as a result, they were also enriched in wine lactone. To explore the underlying causes of this differential accum ulation we investigated the biosynthesis of (E)-8-carboxylinalool. The concentrations of m any grape volatiles, including oxygenated m onoterpene derivatives, are reported to be higher in the ripe com pared to green berries (26,27). To establish (E)-8carboxylinalool accum ulation profile we thus analyzed its content in developing

Exp

Gewurztram iner berries at five stages from green to ripe berry. Free (E)-8-carboxylinalool

was not detected at any developm ental stage. We could, however, detect it in a conjugated form , putatively identified as (E)-8 -carboxylinalool glucose ester. Its concentration increased during ripening of Gewurztram iner berries (Figure 3a).

We postulated that the expression of genes involved in the biosynthesis of (E)-8carboxylinalool glucose ester would parallel its accum ulation in ripening grape berries. In the first step, we com pared gene expression in Gewurztram iner berries at two developm ental stages (green and m id-ripe berry) by RNA sequencing, with m ain focus on m em bers of the CYP71 and CYP76 fam ilies recently shown to m etabolize linalool in A. thaliana (22)(23)(24).

Annotation of cytochrom es P450 in V. vinifera PN40 0 24 genom e revealed an expanded CYP76 fam ily with 20 full-length coding sequences (Ch ap te r 5 ). Grapevine CYP71 fam ily, on the other hand, is of com parable size or sm aller than CYP71 fam ilies in other plant genom es and com prises of 23 predicted genes (28). RNA sequencing of Gewurztram iner berries highlighted five ripening induced CYP76 (CYP76F12, CYP76F14, CYP76T21, CYP76Y1 and CYP76Y2) and one CYP71 (CYP71AT7) gene candidate (Figure S5). Analysis of expression of the CYP76 fam ily in the Corvina cultivar (29) confirm ed induced expression of gene candidates in ripe berries (Figure S6). Most of these genes are expressed to com parable levels in leaves, buds and roots. The expression level of the candidates was investigated in m ore detail at 5 developm ental stages (green, before veraison, after veraison, m id-ripe and ripe) of Gewurztram iner berries by qRT-PCR. Expression of all candidate genes increased during the ripening (Figure 3).

However, only expression of CYP71AT7, CYP76F12 and CYP76T21 kept increasing until the ripe stage. Expression of CYP76Y1 and CYP76F14 peaked in m id-ripe berries and rem ained constant in the ripe berries. Expression of CYP76Y2 peaked in m id-ripe berries as well, but dropped in ripe berries. CYP76F14 reached the highest expression level of all candidates in the late stages of Gewurztram iner berry ripening.

Expression of several CYP76 and one CYP71 genes therefore parallels the accum ulation of (E)-8-carboxylinalool throughout the grape ripening. Those were considered as strong candidates for (E)-8-carboxylinalool biosynthesis.

Se ve ral ca n d id ate e n zym e s m e tabo lize lin alo o l in v it r o

To evaluate the catalytic activity of the candidate P450 s, we cloned their cDNA from V. vinifera cv. Muscat Ottonel and expressed them in yeast together with the P450 reductase 1 from A. thaliana. CO-differential spectroscopy (31) confirm ed the expression of all of them , except CYP76Y1 (Figure S7). As previously observed for other m em bers of CYP76 fam ily (23), the yeast expression level of grape CYP76 enzym es was relatively low.

We evaluated the enzym e activities by incubating the recom binant yeast m icrosom al fractions with linalool, (E)-8-hydroxylinalool and (E)-8 -oxolinalool. Three out of five candidate enzym es (CYP76F14, CYP76F12 and CYP76T21) m etabolized linalool (Figure 4a)

and (E)-8 -hydroxylinalool (Figure 4b), CYP76F14 was the m ost active on both substrates.

Conversion of (E)-8-oxolinalool proved difficult to evaluate due to a com peting reduction of this substrate, probably catalyzed by a m icrosom al yeast enzym e (24).

Gas chrom atography of the reaction products (Figure S7) revealed that CYP76F12, CYP76F14 and CYP76T21 converted linalool to (E)-8-hydroxylinalool. Two additional products of the three enzym es (Figure S8) were identified as downstream products of (E)-8-oxolinalool ((E)-8-hydroxy-6,7-dihydrolinalool and (E)-8-oxo-6,7-dihydrolinalool), presum ably generated by an endogenous m em brane bound yeast enzym e (24). (E)-8-oxolinalool was som etim es detected as an interm ediate product, but was in m ost cases reduced to these two side products, which were also found in the negative control, reaction with m icrosom es from yeast transform ed with an em pty vector. CYP76F12 differed from the other two enzym es in that it produced both (E) and (Z) isom ers of 8-hydroxylinalool. No m ajor product could be detected for CYP71AT7, despite the consum ption of low am ounts of substrate. The reaction products were also analyzed by LC-MS/ MS. This analysis confirm ed the products already identified by GC and in addition allowed for the detection of the final product, (E)-8-carboxylinalool, which is not readily detectable by GC (13). It showed that only one of the candidate P450 s, CYP76F14, catalyzed the conversion of linalool to (E)-8carboxylinalool as the m ain product (Figure 5). Sim ilarly, when the next m olecule in the pathway, (E)-8-hydroxylinalool, was used as a substrate, only CYP76F14 could oxidize it to (E)-8-carboxylinalool. However, when (E)-8 -oxolinalool was used as a substrate, three enzym es, CYP76F14, CYP76F12 and CYP76T21, oxidized it to (E)-8-carboxylinalool (Figure S11). CYP76F14 is thus the only candidate that catalyzes the whole cascade of oxidations leading to the conversion of linalool into (E)-8-carboxylinalool, m ainly because of its ability to efficiently oxidize (E)-8-hydroxylinalool to (E)-8-oxolinalool. C. roseus and A. thaliana m em bers of the CYP76 fam ily were recently shown to m etabolize a broad set of m onoterpenols (23,32,33). We therefore also tested the candidate enzym es for their activity with geraniol, another m ajor m onoterpenol in grapes (Ch a p te r 1). All candidate enzym es converted at least sm all am ounts of geraniol. CYP76T21 was the m ost active on geraniol (Figure S12) and the m ain reaction product detected by GC analysis was (E)-8-hydroxygeraniol (Figure S13).

The functional analysis of the selected P450 candidates expressed in yeast thus indicates that, whereas several of them can potentially contribute to linalool or geraniol oxidation in m aturing grape berries, CYP76F14 is the m ost active on linalool and its oxidation products, and the only enzym e that can efficiently catalyze the whole cascade of oxidation reactions from linalool to (E)-8 -carboxylinalool.

Re co n s tru ctio n o f th e ( E) -8 -carbo xylin alo o l path w a y in N ico t ia n a b e n t h a m ia n a le ave s

We used Agrobacterium -m ediated transient transform ation of N. bentham iana leaves to investigate the activity of the selected P450 enzym es in planta. In order to provide the P450 enzym es with linalool substrate, a linalool synthase was first cloned from grapevine. cDNA sequences of putative LIS expressed in Gewurztram iner berries were reconstituted based on RNAseq data and on hom ology with LIS identified in the grapevine reference genom e (20 ). A full-length cDNA corresponding to a putative TPS56 was then am plified from RNAs prepared from m id-ripe Gewurztram iner berries. Transient expression this cDNA in leaves of N.

bentham iana resulted in the biosynthesis of significant am ounts of linalool (data not shown), dem onstrating that this cDNA encoded an active linalool synthase.

To test if the sim ultaneous expression of a grapevine linalool synthase and the candidate linalool oxidases were sufficient to generate significant am ounts of (E)-8-carboxylinalool in a plant cell environm ent, we transiently co-expressed LIS together with one of the three best candidate P450 genes in leaves of N. bentham iana. Linalool and its derivatives were analyzed in m ethanol leaf extracts by LC-MS/ MS (Figure 6). All the targeted m etabolites-linalool, (E)-8-hydroxylinalool and (E)-8 -carboxylinalool-were detected in both free and glycosylated form s. Neither free nor glycosylated (E)-8-oxolinalool could be detected.

Both CYP76F14 and CYP76T21 depleted all the linalool produced by LIS, but only CYP76F14 com pletely converted it to (E)-8 -carboxylinalool without accum ulation of interm ediary product, (E)-8-hydroxylinalool. The products of candidate enzym es in plant tissues therefore corroborate their in vitro activity, in particular the (E)-8-carboxylinalool synthase activity of CYP76F14. ). Free and bound form s of the sam e m etabolite were detected with the same channel so that peak areas are roughly com parable. One-way analysis of variance (ANOVA) revealed significant differences between groups (P < 0 .0 5 for linalool, P < 0 .0 1 for m alonylhexosyl linalool and P < 0 .0 0 1 for other four m etabolites). Letters on the right axes indicate significant differences between m eans calculated with Tukey HSD test (Į = 0 .0 5).

Ge n e p o lym o rph is m s be tw e e n grape vin e varie tie s

Due to the lim ited availability of plant m aterial we used two different V. vinifera varieties in our experim ents, Gewurztram iner (Gw) for the analysis of gene expression and (E)-8carboxylinalool content, and Muscat Ottonel (Mo) for functional gene characterization. To explore the nucleotide polym orphism s am ong the two varieties, we isolated and sequenced the candidate genes from both of them and com pared them to the PN40 0 24 reference genom e (Ap p e n d ix II). CYP76F14 was the gene with the m ost polym orphism s. Sequences in Gewurztram iner and in PN40 0 24 were identical, but differed from the Muscat Ottonel sequence (12 SNP, 6 of which are nonsynonym ous). The latter variant was previously nam ed CYP76F2 (34), but it is m ost likely an allelic variant of CYP76F14 and not a distinct locus.

To test the effect of this polym orphism on CYP76F14 enzym atic activity, the Gewurztram iner and Muscat Ottonel enzym e activities were com pared. Interestingly, we did not find any difference in product profiles, neither in vitro (Figure S15) nor in planta (Figure S16). The am ino acid differences observed between the two variants thus do not seem to alter the biochem ical activity of the two enzym es.

D is cu s s io n W in e la cto n e fo rm s in w in e fro m ( E) -8 -ca rbo xylin alo o l an d its glu co s e e s te r

To gain better insight into the form ation of wine lactone in wine, we m easured the content of (E)-8-carboxylinalool, (E)-8-carboxylinalool glucose ester and wine lactone in 23 white wine sam ples. Concentrations of (E)-8-carboxylinalool in wines were in m icrom olar range, com parable to the concentrations of other linalool derivatives (Ch a pte r 1). Concentrations of wine lactone, on the other hand, were m uch lower-in the nanom olar range-but higher than those reported previously (4, 9), which could be due to the fact that we profiled older wines. Wine lactone was shown to form from (E)-8-carboxylinalool via a slow non-enzym atic cyclisation (11). Wedler et al. (35) recently challenged this unusual m echanism and based of a quantum chem istry approach suggested an alternative process involving oxidation of cyclic terpenes such as lim onene or Į-terpineol. We dem onstrate here that the concentration of wine lactone correlates with both wine age and (E)-8-carboxylinalool concentration, which is consistent with the form ation from (E)-8-carboxylinalool. In addition, oxygenated lim onene or Į-terpineol derivatives have to our knowledge never been detected in wines. This does not, however, exclude the possibility that wine lactone m ay result from lim onene oxidation in plant tissues where lim onene is the m ain terpene constituent, such as Citrus fruits. The form ation of wine lactone from (E)-8-carboxylinalool in wine follows first-order reaction kinetics with an extrem ely low reaction rate constant k = 0 .0 0 0 12 year -1 . This slow reaction rate is in agreem ent with the high activation energy required for the unlikely hydride shift proposed in the reaction m echanism (11). The concentration of wine lactone can thus be estim ated from the wine age and the easily m easurable (E)-8 -carboxylinalool concentration, circum venting the tedious sam ple preparation for analysis of wine lactone.

All (E)-8 -carboxylinalool is present as a glucose ester in grape berries. In young wines it is present in both free and conjugated form s, suggesting som e of (E)-8-carboxylinalool m ight be released by the yeast during ferm entation (14). In older wines the content of (E)-8carboxylinalool glucose ester is lower whereas the content of free (E)-8-carboxylinalool is higher.

We found large differences in wine lactone contents between different wines, stem m ing from differences in wine age and (E)-8-carboxylinalool content. Of the three wine varieties analyzed, the highest concentrations were m easured in Gewurztram iner wines, which supports the role of wine lactone as Gewurztram iner varietal arom a com pound (4). Our data also support the hypothesis that this is due to the higher concentration of the wine lactone precursor, (E)-8-carboxylinalool, in Gewurztram iner wines. In addition, prelim inary data shows that the content of (E)-8 -carboxylinalool glucose ester is higher in berries of Gewurztram iner com pared to the wild grapevine Vitis vinifera subsp. sy lvestris (Figure S17).

This could suggest that wine lactone is an arom atic trait selected during grapevine dom estication.

Th e bio s yn th e s is o f ( E) -8 -carbo xylin alo o l in grape vin e

The wine lactone precursor, (E)-8-carboxylinalool, accum ulates in ripening grape berries. We chose a set of candidate genes potentially involved in the biosynthesis of (E)-8carboxylinalool in grape based on their preferential expression in ripening berries. Together, the activities of recom binant enzym es and the pathway reconstruction in N . bentham iana dem onstrate that a terpene synthase (LIS) and a cytochrom e P450 (CYP76F14) are sufficient to efficiently produce (E)-8-carboxylinalool from com m on plant precursors. We cannot, however, exclude the involvem ent of other redundant enzym es in the grape berries.

Grapevine genom e contains 69 terpene synthase genes, of which at least 7 have linalool synthase activity in vitro (20 ). Within the set of investigated P450 candidates expressed in ripening grape berries, two other enzym es, CYP76F12 and CYP76T21, could catalyze som e of the oxidation steps that lead to the conversion of linalool into (E)-8-carboxylinalool, and m ay thus also participate in the production of production of the wine lactone precursor (Figure 1).

Linalool and its first oxygenated product, (E)-8-hydroxylinalool, accum ulate in grapes as glycosylated derivatives (19), form ation of which requires the activity of UDP glycosyltransferases (UGTs). UGTs com pete with P450 enzym es for available linalool and (E)-8-hydroxylinalool product. They can therefore prevent further oxidation and the form ation of the final product. It is noteworthy that N. bentham iana leaves expressing CYP76F14 accum ulated only the final product, (E)-8-carboxylinalool, and no glycosylated linalool or (E)-8-hydroxylinalool. CYP76F14 is thus efficient enough to outcom pete UGTs and is efficiently coupled with LIS for the channeling of linalool. This was not the case for the other P450 candidates. N. bentam iana leaves co-expressing CYP76T21 or CYP76F12 accum ulated significant am ounts of glycosylated linalool or (E)-8-hydroxylinalool, and less (E)-8-carboxylinalool com pared to CYP76F14. The high efficiency of CYP76F14 in (E)-8carboxylinalool biosynthesis, together with its high expression level in ripening berries, thus support the role of CYP76F14 as the m ain (E)-8 -carboxylinalool synthase in grapes. It is also noteworthy that CYP76F14 and the other m onoterpenol oxidases identified in this work m ay influence wine arom a by depleting powerful odorants such as linalool and geraniol from the terpenoid pool. CYP76F14 and other berry-expressed P450 s could thus be used as m arkers for the selection of grapevine varieties producing desired am ounts of m onoterpenols and the wine lactone precursor.

Wine lactone has 3 chiral centers, resulting in 8 possible stereoisom ers. Only one of them , the (3S,3aS,7aR) form , is a powerful odorant and was until recently the only stereoisom er detected in wines (5). Interestingly, it was shown to be the cyclisation product (3R, 6E)-8carboxylinalool, whereas the m ain linalool enantiom er in wine is (3S)-linalool (18). Recent work confirm ed that the predom inant configuration of (E)-8-carboxylinalool is also (3S), although the ratio between enantiom ers varied between sam ples (14). Consistent with presence of both (E)-8 -carboxylinalool enantiom ers the sam e investigation also revealed the presence of two enantiom ers of wine lactone, the m ajor one being the less fragrant (3R,3aR,7aS) enantiom er. CYP76F14 is not stereoselective and equally m etabolizes both linalool enantiom ers to produce both (R) and (S)-carboxylinalool (Figure S18).

(E)-8-hydroxylinalool is an interm ediate in the biosynthesis of (E)-8-carboxylinalool and a product of three cytochrom es P450 investigated in this study. These enzym es, CYP76F12, CYP76F14 and CYP76T21 thus likely synthesize (E)-8-hydroxylinalool in grapes, where this com pound accum ulates in glycosylated form (36). Strauss et al. showed that (E)-8hydroxylinalool can act as a precursor to dill ether, another potent odorant, in acidic solutions (36). Despite non-negligible am ounts of its precursor, dill ether has (to our knowledge) never been detected neither in grapes nor in wines. We could confirm that (E)-8hydroxylinalool cyclizes to dill ether in acidic buffer, but we were also unable to detect dill ether in any of the wines we analyzed. This could be related to poor ionisation, low concentration, slow form ation, or the com bination of these factors. A m ore sensitive targeted analytical approach could give us m ore inform ation on concentrations of this com pound in wines and reveal new roles of CYP76 enzym es in wine arom a.

Other oxygenated linalool derivatives in grapes are 6-hydroxylinalool, 7-hydroxylinalool, (Z)-8-hydroxylinalool, furanic and pyranic linalool oxides. None of the CYP76 enzym es included in this study could synthesize these com pounds, with the exception of CYP76F12, which could produce (Z)-8-hydroxylinalool. While our findings confirm the role of the CYP76 fam ily in oxidation of m onoterpenols (22,23), they also hint at other P450 fam ilies potentially involved in oxidations at non-term inal positions.

We showed that (E)-8 -carboxylinalool accum ulates in grape berries in the form of glucose ester. (E)-8 -carboxylinalool was not included as a substrate in the recently reported characterization of grapevine glycosyltransferases (37,38), so the enzym e catalyzing this reaction rem ains to be discovered. Profiling of wines showed that hydrolysis of this glucose ester occurs both during ferm entation and during wine m aturation. The role of yeast in this process rem ains to be explored, but could be an im portant factor affecting the wine lactone form ation and m ore generally wine arom a.

Mate ria l an d m e th o d s Syn th e s is o f m o n o te rp e n o id s

(E)-8-hydroxylinalool, (E)-8-oxolinalool and (E)-8-carboxylinalool were synthesized and purified as described previously (22,24). Enantiom erically pure wine lactone was synthesized according to the procedure of Chavan et al. (39) with the following m odifications: hydroboration was accom plished following the procedure of Bode and Carreira (40 ) and separation of the diastereom ers was carried out after the dehydrohalogenation step, contrary to recom m endation in (39). Wine lactone displayed identical spectroscopic data to those reported previously.

W in e s a m ple s

Wines Glycosylated derivatives were putatively identified on the basis of their m ass spectra (Table S3). Principal com ponent analysis was perform ed in R with the package FactoMineR.

Se le ctio n o f can d id a te ge n e s

Cytochrom e P450 genes were annotated in the PN40 0 24 genom e as described in Ch apte r 5 .

RNAseq data from Riesling and Gewurztram iner grape berries from two tim e points (before and after veraison) were aligned to these gene m odels. For further investigations, we chose the genes with increased expression in the later tim e point (after veraison). 29Septem ber 20 14). Berries were flash frozen in liquid nitrogen and stored at -8 0 °C. They were crushed with a ham m er to allow for the rem oval of seeds. Then they were ground using Qiagen Teflon grinding jar set and TissueLyser for 30 s at 30 s -1 .

To tal RN

Total RNA was isolated from 1 g of berry powder following the protocol in (30 ) Prim ers for qPCR were designed using the Prim er3 plus website (Table S 2). Specificity of the prim ers was confirm ed in silico using the Prim er Blast website (NCBI) and by m easuring the m elting curve of the products. The reaction m ixture contained 1x SYBR® Green Master Mix, 250 nM of forward and reverse prim ers and 50 x diluted cDNA from reverse transcription reaction. The qPCR experim ent was perform ed on Roche LightCycler® 480 . The three reference genes, EF1-Į ;0B 8%4B/ ;0B DQG 0'+ P (XM_ 0 0 2278676.3), were selected from 6 constitutively expressed grapevine genes (30 ) using the GeNorm algorithm (41). Am plification efficiency were calculated for each set of prim ers using LinRegPCR (42). Relative gene expression was calculated as described in (43).

Each point was obtained from 3 technical and 3 biological replicates. CYP76F14 and CYP76F12 PCR products were cloned into the pYeDP60 yeast expression vector using restriction enzym es Bam HI and KpnI. The N-term inal segm ent of CYP76F14 was recoded taking into account yeast codon preference using a 150 nt forward prim er. The PCR product was first inserted into pGem ® -T Easy vector (Prom ega) before being transferred to pYeDP60 using the sam e restriction enzym es. CYP71AT7, CYP76T21 and CYP76Y2 were inserted into the yeast expression vector pYeDP60 u2 using the USER™ cloning m ethod (44). Several colonies obtained for each gene were verified by sequencing.

Am plificatio n o f co d in

Muscat Ottonel was used for sequence am plification and som e sequence divergence with the reference genom e (PN40 0 24) was expected. In the cases where sequence divergences were consistent in several colonies, they were considered as relevant and the corresponding am plicons were expressed in yeast. The list of prim ers used for sequence am plification is provided in Table S 2. The GwCYP76F14 coding sequnce was synthesized de novo by Genecust with codon optim ization for expression in yeast. CYP76F12, CYP76F14 and CYP76T21 were cloned into the pCAMBIA230 0 vector with the USER TM cloning m ethod.

Linalool synthase is a Gewurztram iner allele of Vitis vinifera TPS56 (HM80 7392 PN55M1) and was cloned into vector pMDC32.

H e te ro lo go u s e xp re s s io n in ye a s t

The Saccharom y ces cerevisiae strain WAT11 was transform ed with pYeDP60 :CYP or "em pty" pYeDP60 plasm id as described in (45). Yeast cultures were grown as described for the high density procedure in (46) with the following m odifications. A 20 0 m L culture volum e was inoculated with 15 m L of the overnight culture, and the gene expression was induced by the addition of 5 vol. % of 20 0 g L -1 galactose. The m icrosom al m em branes were isolated as described in (47). The expression of P450 was evaluated by CO differential spectroscopy of reduced m icrosom es (31) (Figure S7). The intensity of the peak at 450 n m was too low to allow for reliable quantification of enzym es.

P4 5 0 e n zym e as s ays

In vitro P450 enzym e assays were conducted in 20 m M Na + / K + phosphate buffer, pH 7.

Gas ch ro m ato grap h y an alys is

Ethyl acetate extracts were analyzed on a Varian 390 0 gas chrom atograph (Agilent Technologies) with flam e ionization detector, equipped with a DB5 colum n (Agilent Technologies). Injector tem perature was 250 °C and injection was splitless. Tem perature gradient was: 0 .5 m in at 50 °C, 10 °C/ m in to 320 °C, and 5 m in at 320 °C. Tem perature of the FID detector was 28 0 °C.

Su p p le m e n tary in fo rm atio n

Table S6 . Prim e rs u s e d fo r clo n in g. S3). S3). S3). bentham iana leaves represents an attractive alternative (2,3). When the enzym e substrate is not present in the leaves endogenously, it can be vacuum infiltrated into the leaf discs expressing the gene to be characterized. With this m ethod we tested three candidate P450 s (CYP76Y2, CYP76T21 and CYP76F14) with four grape m onoterpenols: linalool, geraniol, nerol and citronellol.

Gene

We expressed the candidate P450 s in N. bentham iana leaves as described in the Ch a p te r 6 , except that they were not co-expressed with a terpene synthase. Instead, we cut discs from the leaves expressing the P450 s, vacuum infiltrated them with a buffer solution containing the substrate (20 m M K + / Na + phosphate buffer pH 7.4, 40 0 NjM substrate). Ten discs of 1.4 cm diam eter were incubated in 10 m L substrate solution for 4 h. The buffer solution was then collected and extracted using solid phase extraction cartridges (Oasis HLB 3cc with 60 m g sorbent). Ethyl acetate eluent was concentrated under argon and analyzed using GC-FID (for quantification) and GC-MS (for product identification). GC-FID conditions were identical to those used in Ch a pte r 6 . GC-MS was perform ed on PerkinElm er Clarus 68 0 / 680 T system fitted with a HP5m s colum n (30 m , 0 .25 m m , 0 .25 µm ; Agilent technologies). Tem perature program was identical to the one used on GC-FID. Injector tem perature was 250 °C, ionization energy was 70 eV and m / z was collected in the 50 -30 0 range. Both CYP76T21 and CYP76F14 m etabolized linalool, and the m ain product was in both cases (E)-8-hydroxylinalool (Figure 1a, Figure 2a). A second product of CYP76F14 was identified as a downstream product, (E)-8-oxolinalool (Figure 1a, Figure 2a). Although the non-volatile fraction was not analyzed in this experim ent, it is inform ative to com pare this experim ent to the co-expression of linalool synthase and P450 s in N. bentham iana leaves (Ch apte r 6 ). In the latter case, CYP76F14 converted all the linalool to (E)-8-carboxylinalool without accum ulation of interm ediary products. But when substrate was fed to the leaf discs expressing CYP76F14, two interm ediary products, (E)-8-hydroxy-and (E)-8-oxolinalool, were accum ulating. A possible reason for this difference is a high substrate concentration used in the leaf disc experim ent. If the affinity for linalool is higher com pared to the downstream products, the enzym e is saturated with the first substrate and unable to perform the subsequent oxidation steps. The sam e two enzym es, CYP76F14 and CYP76T21, also m etabolized geraniol in the leaf disc experim ent. The m ain product was in both cases identified as (E)-8-hydroxygeraniol (Figure 1b, Figure 2b). CYP76T21 produced m ore of the (E)-8 -hydroxygeraniol, whereas CYP76F14 was the m ost efficient at linalool oxidation. These results are in agreem ent with in vitro experim ents, where CYP76F14 was the m ost efficient at linalool oxidation and CYP76T21 was the m ost efficient enzym e at geraniol oxidation.

Leaf discs expressing CYP76F14 and CYP76T21 also m etabolized the two other m onoterpenols assayed: nerol and citronellol. The products of both enzym es were the sam e and were putatively identified as (E)-8-hydroxynerol and (E)-8-hydroxycitronellol (Figure 3, Figure 4). Com parison of relative am ounts of products produced by different enzym es (Figure 5) revealed that while both enzym es, CYP76F14 and CYP76T21, produced com parable am ounts of (E)-8-hydroxynerol and (E)-8-hydroxycitronellol, they differed in their efficiency to hydroxylate the other two substrates: CYP76F14 produced m ore (E)-8-hydroxylinalool com pared to CYP76T21, whereas CYP76T21 produced m ore (E)-8-hydroxygeraniol.

En an tio s e le ctivity o f gra p e vin e lin alo o l s yn th as e

The linalool synthase used in the N. bentham iana coexpression experim ent had not been previously characterized in term s of its enantioselectivity. To determ ine whether it produces (R)-or (S)-linalool, we transiently expressed it in N. bentham iana leaves as described in the Ch a pte r 6 . We then collected headspace volatiles with 30 m g Porapak Type Q 50 -80 Mesh cartridges (Waters). After elution with hexane, the sam ples were run on the chiral colum n Agilent CycloSil B.

Com parison to authentic standards of (S)-and (R)-linalool showed that VvLIS produces exclusively (S)-linalool (Figure 6), which is the predom inant form of linalool found in grapes.

Fu n ctio n al ch aracte riza tio n o f grap e vin e CYP76 ge n e s e xp re s s e d in gre e n be rry

In grape berries, linalool undergoes m any different oxidation reactions, which produce a num ber of hydroxylated and cyclic linalool derivatives (6). Like other arom a com pounds in grapes, their concentration is generally reported to increase during grape berry ripening (7,8). Som e of the linalool oxides, however, are shown to peak in green berries and to decrease during the berry ripening (9). In addition, our previous work showed that although the concentration of (E)-8-carboxylinalool, the wine lactone precursor, is increasing during the grape ripening, it is already present in green berries. We therefore decided to explore cytochrom es P450 that m etabolize linalool in green grape berries.

Com parison of RNA sequencing on cDNA from green and m id-ripe Gewurztram iner berries highlighted two CYP76 gene candidates for linalool m etabolism in green berries: CYP76F10 and CYP76T12 (Ch apte r 6 , Figure S5). These two genes were cloned from Gewurztram iner green berry cDNA (Table 1). Sequences of several clones revealed that one of the genes, CYP76T12, was expressed as two splicing variants: CYP76T12v1 with two introns and CYP76T12v2 with one intron (Figure 8). We investigated the expression of the two genes in developing Gewurztram iner berries using qRT-PCR (Figure 9). Two sets of prim ers were designed for specific detection of the two alternative splicing variants of CYP76T12 (Table 1). Lim ited options for prim er design resulted in suboptim al prim er design with, as a consequence, a low signal also in the negative control (without cDNA). However, this signal was lower com pared to sam ples with cDNA.

All three investigated sequences showed the highest expression in green berries, which is consistent with the RNAseq experim ent. The two splicing variants of CYP76T12 had a nearly identical expression pattern. However, the expression level of these genes was m uch lower com pared to previously investigated genes expressed in ripe berries (Ch a p te r 6 , Figure 3). Expression of CYP76F14, the m ost highly expressed CYP76 gene in ripe berries, exceeded the expression of CYP76F10 and CYP76T12 even in the green berries. We expressed CYP76F10 and both variants CYP76T12 sequences in yeast (S. cerevisiae WAT11). CO-differential spectroscopy of reduced CYP76F10 m icrosom e preparation (Figure 10 ) revealed weak ƩA peak at 450 nm , sim ilar to that observed with the other two m em bers of this subfam ily previously characterized (CYP76F12 and CYP76F14). CYP76T12v2 had a m ore intense peak at 450 nm , but we could not detect a peak at 450 nm in the m icrosom al preparation of CYP76T12v1. Splicing of 93 nucleotides, which corresponds to 31 am ino acids, thus probably disrupts the protein structure and renders the enzym e non-functional. We tested the recom binant enzym es with linalool and (E)-8-hydroxylinalool. CYP76F10 and CYP76T12v2, m etabolized both of the substrates (Figure 11). GC-FID analysis revealed that both of these enzym es can hydroxylate linalool at the position (E)-8 and further m etabolize (E)-8-hydroxylinalool. CYP76T12v1 did not show any enzym atic activity, which confirm s the deleterious effect of the splicing of additional intron. LC-MS/ MS analysis of the reaction product showed that both enzym es produce (E)-8carboxylinalool from linalool (Figure 12a). In addition, CYP76T12v2 produced com pounds putatively identified as (Z)-8-hydroxylinalool 3 and (Z)-8-carboxylinalool 6 . Because analytical standards of these products were unavailable, they could not be unam biguously identified. Their putative identification is supported by the fact that they are not produced from (E)-8-hydroxylinalool (Figure 12b) or (E)-8-oxolinalool (Figure 12c). Upon incubation with these two substrates only (E)-8-carboxylinalool was produced in addition to the two side products, probably produced by an endogenous yeast reductase (4 and 5 ).

Oxid atio n o f o le fin ic m o n o te rp e n e s by CYP71AT7

Whereas several A. thaliana CYP76 enzym es have been described as m onoterpenol oxidases, only one of the functionally characterized CYP71 exhibited this activity. However, som e CYP71 enzym es from different plants catalyze hydroxylations of olefinic (hydrocarbon) m onoterpenes. For exam ple, CYP71D13 and CYP71D15 oxidize lim onene in pepperm int (10 ), and CYP71AR1 oxidizes Į-pinene in strawberry (11). This m otivated us to test the activity of CYP71AT7, the m ost highly expressed CYP71 in ripe berries, with olefinic m onoterpenes.

We tested five m onoterpenes: Į-pinene, p-cym ene, lim onene, ǃ-ocim ene and DŽ-terpinene Although the conversion was relatively low, oxygenated products could be detected for two of the substrates (Figure 15). Because of unavailability of analytical standards, the products were only tentatively identified on the basis of the best m atch in the NIST m ass spectral library search (Figure 16). CYP71AT7 catalyzed the epoxidation of Į-pinene at the double bond to yield 2,3-epoxypinane (7) and the hydroxylation of p-cym ene at the m ethyl group to yield 4-isopropylbenzyl alcohol or cum inol (9 ). The solution of p-cym ene already contained som e p-cym en-8-ol ( 8), but its concentration increased after incubation with CYP71AT7.

All of the tested m onoterpenes except p-cym ene were reported as in vitro products of grapevine terpene synthases (12), but to our knowledge neither of them has been detected in grapes or wines. Neither has any of the three products been detected in grapes or wines. In addition, the conversion was relatively low (Figure 15), so we concluded that the observed catalytic activity was unlikely relevant in vivo.

Ro le o f o th e r CYP fam ilie s in grap e a ro m a bio s yn th e s is

In Ch ap te r 6 and this chapter we showed that grapevine CYP76 enzym es oxidize linalool at positions (E)-8 and (Z)-8. However, none of these enzym es oxidized linalool at in-chain positions, despite num erous other oxygenated linalool derivatives com m only found in grapes. This led us to consider that other P450 fam ilies m ight be involved in linalool oxidations in grapes. RNA sequencing experim ent revealed other P450 candidates with increased expression in m id-ripe Gewurztram iner berries (Figure 17). Som e of the genes featuring in this list belong to P450 fam ilies with conserved functions and were not further evaluated. For exam ple, CYP90 are involved in m etabolism of brassinosteroids, CYP97 m etabolize carotenoids and CYP74 catalyze rearrangem ent of fatty acid hydroxyperoxides (13). Genes from the CYP82 fam ily appeared possible candidates for m onoterpenol oxidation.

Mem bers of this fam ily are involved in terpenol m etabolism in A. thaliana. They synthetize hom oterpenes (shortened terpenoids) by an unusual oxidative cleavage of nerolidol and geranylgeraniol (14). In addition, this fam ily is larger in grapevine com pared to other plant species, which could indicate expansion and subfunctionalisation. Two CYP82 genes are induced during the berry ripening: CYP82D13 and CYP82H22. We attem pted to clone both of them from Muscat Ottonel ripe berry skin cDNA, but only succeeded at am plifying CYP82D13. The expression of CYP82H22 m ight be too low (Figure 17).

We am plified the CYP82D13 sequence from different varieties: Muscat Ottonel, Gewurztram iner and Riesling, as well as wild grapevine V. sy lvestris subsp. sy lvestris.

Surprisingly, all of the sequences, except one of the putative alleles of V. sy lvestris subsp.

sy lvestris had a m utation that changed the m ost conserved residue in the P450 sequences, the hem e-binding cysteine, into a tyrosine residue (Figure 18). Until recently the cysteine residue was considered the only conserved am ino acid in all P450 sequences, but m ore recently P450 genes with variations at this position have been discovered, although it is still unclear whether they have an enzym atic activity or not (15). We expressed the Muscat Ottonel CYP82D13 gene in yeast. The reduced m icrosom es of yeast expressing CYP82D13 lacked the typical absorbance peak at 450 nm . A m inor peak at 420 nm was however observed (Figure 19), indicating that at least a m inor proportion of the protein bound the hem e in the active site. Nevertheless, no enzym e activity was observed when this enzym e was tested with m onoterpenols.

Other fam ilies considered for m onoterpenol oxidations were CYP8 0 , CYP81 and CYP89.

Mem bers of the CYP80 fam ily were shown to catalyze phenol coupling reactions in alkaloid biosynthesis in other plants, but we decided to review this fam ily because of its phylogenetic proxim ity with CYP76 (Ch a p te r 5 ). The fam ily CYP81 had the m ost representatives in the list of ripening-induced cytochrom e P450 s (along with CYP76), we therefore decided to test it for involvem ent in arom a form ation. Mem bers of CYP81 catalyze different reactions in specialized m etabolism of other species, such as isoflavonoid, glucosinolate or lignan biosynthesis (16). Finally, two CYP89 m em bers are induced in ripe berries. So far the biochem ical function of this fam ily is unknown. We chose to investigate the m ost highly expressed m em ber of each of these fam ilies: CYP80 E3, CYP81Q19 (previously nam ed CYP81V13) and CYP89A41. Their coding sequences were am plified and expressed in yeast as described in Ch a p te r 6 . The list of prim ers used for am plification is available in Table 2. All three P450 s were expressed in yeast at least to a m inor extend, as indicated by a peak at 450 nm in the CO-difference spectrum of recom binant yeast m icrosom es (Figure 19). However, none of the enzym es showed any m onoterpenol oxidation activity, which suggests these P450 s are involved in other ripening related processes.

GGTACC TTATTTCAACCTCGGAGATAAGTG GG

Effe ct o f w in e la cto n e o n s e n s o ry p ro p e rtie s o f w in e

Wine lactone was reported to have a very low odor detection threshold, which was determ ined at 60 pM in m odel wine (17). Model wine only contains water and ethanol, and is as such a very sim ple m ixture, as opposed to wines, which contain hundreds of different volatiles. We wished to investigate if wine lactone has an im pact on wine arom a at concentrations detected in old Gewurztram iner wines (10 nM).

We tested the null hypothesis that addition of 10 nM wine lactone does not affect the sensory properties of wine. We used the m ethodology of the triangle test (18), in which panelists were presented with three sam ples, of which two are identical and one different. The panelists were then asked to identify the different sam ples. In our experim ent all three sam ples were the wine that contained the lowest am ount of wine lactone (<1 year old Riesling from the producer Em ile Weingut, which contained 1.74 nM wine lactone), except that one of the sam ples was spiked with 10 nM wine lactone.

Our study included 48 volunteers from Institut de Biologie Moléculaire des Plantes ( 21fem ales and 27 m ales) between the age of 21 and 60 (m ean age 37, standard deviation 12).

They were presented with three coded glasses containing 10 m L wine, arranged in a triangular form , and ballot papers, which instructed them to circle the code that represented the different sam ple.

The m ajority of panelists (27) chose the correct answer; the two false answers got 9 and 12 answers. The exact binom ial test was calculated with the sensR package (19) in R software.

The results showed that the d-prim e param eter, which describes the sensory difference, is greater than 0 (p-value < 0 .0 0 1). The d-prim e param eter was estim ated to 1.8, which indicates a m edium to large sensory difference. We therefore could conclude that wine lactone im pacts the com plex wine arom a at concentrations above 10 nM.

Chapter 8

Co n clu s io n s an d p e rs p e ctive s

Ch e m ical co m p o s itio n o f w in e aro m a

Although econom ical im portance of wine m akes it one of the best researched beverages, we are still struggling to understand its chem ical com position. The quality of wine largely depends on its arom a, but its com plexity m akes it a difficult subject to study: wine arom a com prises a large num ber of chem ically diverse com pounds at very different concentrations.

We analyzed data from published grape and wine profiling experim ents and com piled a list of 141 com m on grape and wine volatiles. However, m any potent odorants in wine consistently slip under the radar of untargeted profiling because of their low concentrations. In term s of arom a, abundance of a com pound is rarely correlated to its im portance and for som e com pounds a hum an nose is a better detector than sophisticated analytical equipm ent.

An exam ple of low-abundance/ high-im pact arom a com pound is the wine lactone: a m onoterpene lactone with a sweet and coconut-like arom a, shown to be an im portant determ inant of arom a of som e wines. This com pound was not detected in any of the profiling experim ents in our m eta-analysis. In fact, in alm ost 20 years between its discovery in wine and this study, it has only been quantified in three wine sam ples (1, 2). Besides its low concentration (nM range), its relatively high polarity (theoretical logP ow = 2) decreases the efficiency of extraction to organic solvents, so the only successful attem pts of wine lactone quantification involved a laborious continuous extraction procedure. We developed an unconventional analytical m ethod, which exploits the unusually high sensitivity for wine lactone on LC-ESI-MS/ MS, sufficient to leverage for low extraction efficiency. Our workflow thus allowed for quantification of wine lactone in 23 sam ples. The differences in concentration of wine lactone between sam ples were over 10 -fold (from 2 to 32 nM) and always exceeded the odor detection threshold (60 pM in m odel wine) in all the analyzed wines. From the three analyzed wine varieties, Gewurztram iner had the highest wine lactone content.

D yn am ic ch an ge s in w in e aro m a

During the winem aking process wine arom a undergoes com plex chem ical and biochem ical changes. Although wine arom a is com m only divided into grape-derived, ferm entationderived and aging-derived, our m eta-analysis and review show that the sam e com pounds can be affected by all three of these processes. For exam ple, m any arom a com pounds are produced in the grapes as non-volatile glycosides, and released as volatile aglycone upon ferm entation, enzym atic hydrolysis, acid hydrolysis (during wine aging) or by enzym atic hydrolysis in hum an m outh. Som e volatile com pounds can undergo secondary transform ations, such as acid-catalyzed rearrangem ents, to yield com pounds with altered arom atic properties.

Developm ent of wine lactone follows both of these transform ations. Soon after discovery of wine lactone in wines, its putative precursor, (E)-8-carboxylinalool, was isolated from wines and shown to yield wine lactone in an acid-catalyzed chem ical rearrangem ent (3). This reaction was studied in the laboratory under som ewhat harsh conditions (sim ultaneous distillation-extraction). At realistic conditions, however, the reaction advanced extrem ely slowly, it was thus proposed that wine lactone form s from (E)-8-carboxylinalool during wine aging (4). Our LC-MS/ MS approach allowed for the quantification of both wine lactone and (E)-8-carboxylinalool in the sam e wine sam ples. Our results confirm ed the slow form ation of wine lactone from (E)-8-carboxylinalool during wine aging with an estim ated kinetic constant k = (0 .0 0 0 12 ± 0 .0 0 0 0 2) year -1 , m eaning that every year of wine aging only roughly one out of 10 0 0 0 (E)-8-carboxylinalool m olecules will transform into wine lactone. In spite of this extrem ely slow rate of form ation, wine lactone reaches concentrations above the odor detection threshold even in young wines, due to the high concentration of the precursor (E)-8-carboxylinalool.

Analysis of grape berry extracts showed that (E)-8-carboxylinalool is present in grapes exclusively in glycosylated form , previously characterized as a glucose ester (3,5). Wines, however, contain both free and conjugated (E)-8-carboxylinalool, pointing to partial hydrolysis of the glucose ester during the ferm entation. Additionally, the content of the glucose ester was higher in younger com pared to older wines, suggesting acid hydrolysis of the glucose ester takes place during wine aging. We could thus show that the form ation of wine lactone depends on the three processes: biosynthesis in grapes, ferm entation and aging.

Hydrolysis of (E)-8-carboxylinalool glucose ester during ferm entation and wine m aturation needs to be studied further to understand why it seem ingly occurs faster in som e wines (Gewurztram iner) com pared to the others (Riesling and Muscat).

Bio s yn th e s is o f w in e aro m a

The long history of grapevine dom estication and clonal selection resulted in cultivars with desired organoleptic properties, but at a cost of reduced fitness of the plants. This is reflected in a high pesticide use in viticulture. Genes that influence wine quality are therefore im portant for the creation of new pathogen resistant varieties with desired organoleptic properties. Inform ation about arom a biosynthetic genes can be applied in the process of m arker-assisted breeding, as well as in genetic engineering of new varieties. We identified and characterized the m ost likely candidate for the biosynthesis of (E)-8 -carboxylinalool, the wine lactone precursor in grapes. CYP76F14 is a grapevine cytochrom e P450 , which is highly expressed in ripe grape berries. We showed both in vitro and in N. bentham iana that CYP76F14 synthesizes (E)-8-carboxylinalool from linalool. Prelim inary genetic data, obtained at the INRA of Colm ar, indicate an overlap between the CYP76F14 locus and the quantitative trait locus (QTL) for (E)-8-carboxylinalool content (data not shown). This result consolidates the role of CYP76F14 as the m ain (E)-8-carboxylinalool synthase in grapevine.

As for m any arom a com pounds, chirality is detrim ental of the perceived arom a characteristic of wine lactone. Of all eight wine lactone diastereom ers, the one initially detected in wine (3S, 3aS, 7aR) has the lowest odor detection threshold (6). This isom er is derived from (3R, 6E)-8-carboxylinalool, whereas the predom inant configuration of linalool in grapes was found to be 3S (7). Only recently both enantiom ers of (E)-8-carboxylinalool-(3S) and (3R)-were found in wines, along with the both resulting enantiom ers of wine lactone-(3S, 3aS, 7aR) and (3S, 3aS, 7aR). We showed CYP76F14 can m etabolize both (3S)-and (3R)-linalool to corresponding carboxylic acid without clear stereoselectivity. The ratio of (3S, 6E)-to (3R, 6E)-8 -carboxylinalool, and therefore the am ount of the fragrant enantiom er of wine lactone, thus likely depend on the am ount of (3S)-and (3R)-linalool produced by the terpene synthases. In the functional analysis of the linalool synthase gene fam ily in grapevine, only one isoform was found to produce (3R)-linalool in vitro, as opposed to several 3S-linalool producing terpene synthases (8). The expression of the (3R)-linalool synthase m ight therefore be crucial for the production of the fragrant enantiom er of wine lactone.

The m anual annotation and expression analysis of the cytochrom e P450 genes present in the grapevine genom e was instrum ental to identify candidates for (E)-8-carboxylinalool biosynthesis. It also highlighted P450 genes that m ight be involved in biosynthesis of other organoleptic com pounds. Our m eta-analysis showed the presence of 6 other oxygenated linalool derivatives in grapes (6-hydroxylinalool, 7-hydroxylinalool and four linalool oxide isom ers), yet none of the enzym es we tested was able to produce any of these com pounds.

This indicates possible involvem ent of other P450 fam ilies, not previously im plicated in m onoterpenol m etabolism .

The genetic background of the differences between grapevine cultivars, including differences in arom a, is still largely unexplored. The reference grapevine genom e belongs to a highly inbred descendant of Pinot noir cultivar and therefore significantly differs, both genotypically and phenotypically, from cultivated varieties. In the reference genom e we identified 11

putative cytochrom e P450 pseudogenes, which only contain one nonsense or fram eshift m utation. Those m ight be functional genes in cultivated varieties and explain som e of the varietal differences. Another possible source of varietal differences is copy num ber variation.

Genom e and transcriptom e sequencing of the grapevine variety Tannat showed that m any of the genes contributing to its high polyphenol content, including the genes coding for flavonoid hydroxylases from the CYP75 fam ily, are not present in the PN40 0 24 genom e. We attem pted to am plify putative CYP76 varietal genes from Muscat Ottonel cDNA with degenerated prim ers designed on the m ost conserved CYP76 regions, but failed to am plify any novel genes (data not shown). This result does, however, not exclude the presence of other CYP76 genes in Muscat Ottonel or other genotypes. The dropping price of sequencin g will without doubt soon result in genom e and transcriptom e data of other grapevine varieties.

This will enable identification of varietal genes, involved in the biosynthesis of arom a and other com pounds.

Plant cytochrom es P450 are often involved in biosynthesis of defense com pounds. Our gene expression analysis highlighted grapevine P450 genes that are upregulated in response to pathogen infection. Studies in other species have shown that m any biosynthetic genes have "bloom ed" and produced large gene fam ilies involved in the biosynthesis of species-specific antim icrobial and other defense com pounds. The largest P450 fam ily in grapevine is CYP82, which underwent two separate bloom s in two different subfam ilies. One of them , CYP82D, is preferentially expressed in leaves upon pathogen infection, whereas the other CYP82H, is expressed in green berries. We attem pted to functionally characterize CYP82D13, which is expressed in ripe berries, but analysis of sequences from different grapevine varieties revealed that in m ost of them this gene bears a m utation in the codon coding for the m ost conserved am ino acid in cytochrom es P450 , the hem e-binding cysteine. This m ight either signify a gene inactivation or a dram atic change in catalytic activity.

Pe rs p e ctive s in flavo r an d fra gra n ce bio te ch n o lo gy

Over the last years the m arket dem and for natural flavors has been increasing, but the flavor m olecules concentrations in the source m aterials are often too low to allow for a sustainable production. This has boosted the research in flavor functional genom ics, since the identification of biosynthetic genes allows for the production of flavor com pounds in bioreactor. Our study adds five m ore enzym es to the repertory of known m onoterpenoloxidizing cytochrom es P450 , reviewed in the Ch ap te r 4 . CYP76F14 produces the precursor of the flavor com pound wine lactone, (E)-8-carboxylinalool. However, the chem ical conversion of (E)-8-carboxylinalool to wine lactone is likely too slow for industrial use even at harsher conditions. A synthetic biology approach could be used to com plem ent the grapevine biosynthetic pathway with a hypothetical (E)-8-carboxylinalool cyclizing enzym e and allow for the industrial production of wine lactone. We screened a library of bacterial squalene/ hopene cyclase m utants, which enable cyclization of non-phosphorylated terpenes, such as geraniol, with several linalool derivatives (9). None of these m utants was able to cyclize (E)-8-hydroxylinalool, (E)-8-oxolinalool or (E)-8-carboxylinalool (data not shown).

Surprisingly, before wine lactone was detected in wine, it had been discovered as a m etabolite in koala urine (10 ). Koalas feed on eucalyptus trees, which are rich in (E)-8-carboxylinalool conjugates (11). Koala liver m ight thus contain an enzym e capable of cyclizing (E)-8carboxylinalool to wine lactone.

Finally, volatile precursors m ay be used as a sm art m aterial for slow and controlled release of flavor or arom a. For exam ple, glycosylated arom a com pounds have been proposed for applications in cosm etics, where the skin m icrobiom e can catalyze the release of the volatile aglycone (12). Furtherm ore, the release of arom a from glycosylated precursors can be controlled by addition of glycosidase enzym es. Sim ilar applications could be developed for in situ conversion non-glycosylated arom a precursors, such as (E)-8-carboxylinalool. Although (E)-8-carboxylinalool conversion to wine lactone in wines is very slow, it can be controlled by the am ount of the precursor, pH, tem perature or a presence of catalyst. A patent describes a use of a different wine lactone precursor in a hydrogen tank coating for leak detection (13).

The precursor is covalently bound to a resin, and, upon contact with hydrogen, the precursor is released and converted to wine lactone. Wine lactone is suitable for these types of applications because of its low odor detection threshold. Wine has an im portant cultural, as well as econom ical value in Europe. Our appreciation of wine quality is profoundly shaped by its arom a, but arom a com position and form ation are still poorly understood. With a m eta-analysis of grape and wine arom a, we show that both are rich in m onoterpenes, in particular oxygenated linalool derivatives. We then review the recent advances in the oxidative m etabolism of the two m ajor plant m onoterpenols, linalool and geraniol. A thorough annotation of the P450 superfam ily in grapevine, revealed its genom ic organization, phylogeny and expression. Specifically, we identified genes showing an activated expression in the ripe grape berry, the stage during which the biosynthesis of m any arom a com pounds takes place. Am ong the known oxygenated m onoterpenols in grapevine, wine lactone has the lowest odor detection threshold and therefore the largest potential im pact on wine arom a. We dem onstrated that wine lactone is form ed during win e ageing via a slow non-enzym atic reaction from the precursor (E)-8 -carboxylinalool. We showed that the accum ulation of this precursor in grape berries parallels the expression of several cytochrom e P450 genes, am ong which CYP76F14 has the highest expression. We functionally characterized of the candidate enzym es in vitro and in the leaves of Nicotiana bentham iana. While three of them catalyzed som e of the oxidative steps from linalool to (E)-8-carboxylinalool, only CYP76F14 efficiently catalyzed the whole pathway. Taken together, CYP76F14 catalytic activity and expression pattern indicate that it is a prim e candidate for the form ation of the wine lactone precursor in grape berries. This work lays ground for the elucidation of novel roles of P450 s in the biosynthesis of arom a in grapevine, as well as other physiological processes, such as fruit developm ent or plant-pathogen interactions.

Figu re 1 .

 1 Figu re 1. Re latio n s e n tre l'âge d u vin e t le s co n ce n tratio n s e n w in e lacto n e , ( E) -8 -carbo xylin alo o l e t ( E) -8 -carbo xylin alo o l glu co s e e s te r. Les trois composés ont été quantifiés dans les m êmes vins par LC-MS/ MS (N=3). La concentration en wine lactone est élevée dans les vins âgés (a) ainsi que dans les vins présentant une concentration élevée de (E)-8-carboxylinalool. L'analyse en com posantes principales (ACP) des concentrations de m étabolites et de l'âge du vin confirm e cette observation : plan factoriel des variables (c) et des individus (d). Mus = Muscat, Rie = Riesling, Gew = Gewurztram iner. Le vin vieux de 29 ans est exclu de l'ACP. Il a été déjà suggéré que le précurseur de la wine lactone, le (E)-8 -carboxylinalool, est synthétisé par oxydation enzym atique du linalool. Plus récem m ent, les cytochrom es P450 , particulièrem ent les fam illes CYP76 et CYP71 ont été décrits com m e participant au m étabolism e des m onoterpénols chez Arabidopsis thaliana (Figure 2) et d'autres espèces végétales. Un préalable nécessaire à la caractérisation fonctionnelle d'une fam ille de gènes est une annotation correcte des gènes. Les annotations autom atiques peuvent contenir des erreurs, des artefacts ou encore des om issions. Cette partie de m on travail a eu donc pour objectif une annotation com plète et correcte des cytochrom es P450 à partir des données de séquençage d'un cultivar de référence de vigne Pinot Noir (V. vinifera PN40 0 24). Après

Figu re 2 .

 2 Figu re 2 . Le m é tabo lis m e d u lin alo o l p ar le s cyto ch ro m e s P4 5 0 ch e z Ar a b id o p s is t h a lia n a . Les enzymes recom binantes ont été produites dans la levure ou dans les feuilles de N . bentham iana. Les flèches en pointillés indiquent les étapes qui pourraient nécessiter d'autres enzym es non décrites.

Figu re 3 .

 3 Figu re 3 . Carte p h ys iqu e d e s s é qu e n ce s d e cyto ch ro m e s P4 5 0 s u r le s 19 ch ro m o s o m e s d e V. v in ife r a . Les cercles jaunes représentent les annotations isolées, les cercles bleus clairs représentent les clusters physiques com posés de mem bres d'une seule fam ille de P450 et les cercles violets les clusters physiques com posés de mem bres de 2-3 fam illes de P450 . La taille du cercle est proportionnelle au nom bre de séquences du cluster. Les num éros 1-19 notent les différents chrom osom es et « Un » est le « chrom osom e inconnu » qui contient des séquences dont l'em placem ent chrom osom ique est inconnu.

Figu re 4 .

 4 Figu re 4 . An alys e p h ylo gé n é tiqu e m o lé cu laire d e s cyto ch ro m e s P4 5 0 d e la vign e . L'alignem ent des séquences de protéines a été utilisé pour générer un arbre inféré au m axim um de vraisem blance. Le clade bleu foncé est le clan 71 qui contient souvent des gènes im pliqués dans le m étabolism e spécialisé. Les gènes colorés appartiennent aux sept plus grands clusters physiques.

Figu re 5 .

 5 Figu re 5 . Evo lu tio n d e la co n ce n tratio n e n e s te r d e glu co s e d e ( E) -8 -carbo xylin alo o l e t d e l'e xp re s s io n d e s gè n e s ca n d id ats p e n d an t la m atu ratio n d e baie s d e rais in . a) Concentration relative (± écart type) du glucose ester de (E)-8-carboxylinalool pendant le développem ent des baies de Gewurztram iner. L'analyse de variance m onofactorielle a montré des différences significatives (p=0 ,0 0 16) entre les stades de développem ent (« Green » -baies vertes, « Before veraison » -avant véraison, « After veraison » -après véraison, « Mid-ripe » -baies m i-m atures, « Ripe » -baies m ûres). Les lettres indiquent des différences significatives entre les m oyennes calculées par le test de Tukey HSD (Į = 0 ,0 5). b) Expression des gènes candidats dans les baies de Gewurztram iner, m esurée par RT-PCR quantitative. Les barres d'erreur représentent l'écart type de 3 échantillons. L'expression des gènes candidats est norm alisée par rapport à l'expression de 3 gènes constitutifs. J 'ai pu m ontrer que trois des enzym es candidates exprim ées dans la levure, CYP76F12, CYP76F14 et CYP76T21, m étabolisent le linalool in vitro. Toutes catalysent l'oxydation au niveau du carbone term inal en position (E)-8, et l'une d'entre elle en position (Z)-8.

Figu re 6 .

 6 Figu re 6 . Vo ie d e ( bio ) s yn th è s e p ro po s é e p o u r la w in e lacto n e . Les quatre prem ières étapes sont catalysées par les enzym es de la baie de raisin. Les deux dernières étapes ont lieu lors de la vinification et de la m aturation du vin.

Figu re 7 .

 7 Figu re 7. Mé tabo lis m e o xyd atif d u lin alo o l d a n s le s baie s d e V. v in ife r a cv. Mo rio Mu s cat. Le « Feeding » de baies de raisin avec un analogue deutéré de linalool a perm is d'identifier de nom breux produits de son m étabolism e oxydatif (Luan et al. Analy Chim Acta 20 0 6).

Figu re 8 .

 8 Figu re 8 . Clas s e s o f grap e an d w in e vo latile co m p o u n d s e valu ate d in th is s tu d y.

Figu re 9 .

 9 Figu re 9 . Eu le r d iagram o f w in e vo latile s in clu d e d in th is s tu d y an d ke y fo o d an d w in e o d o ran ts , id e n tifie d in tw o o th e r m e ta-an alys e s . Two other m eta-analyses identified volatile com pounds with the strongest im pact on arom a of food (2) and wine(27). While the overlap between the three studies is sizeable, m any key odorants are overlooked in volatile profiling experim ents. Each tile represents one volatile m olecule and its color corresponds to the m olecular class.

Figu re 10 .

 10 Figu re 10 . Co m p aris o n o f th e to tal co n ce n tratio n ( le ft) an d d e gre e o f glyco s ylatio n ( righ t) o f s e le cte d grap e an d w in e vo latile o rga n ic co m p o u n d s . Total concentration is the sum of free and bound concentration and percentage is glycosylated is bound divided by total concentration. Each point represents one grape or wine sam ple from one of the 19 publications included in the study. Non-detected com pounds (concentration zero) were assigned a concentration 0 .0 1 Njg L -1 to allow their representation on a logarithm ic scale. Grey dots on the right indicate significant difference between grapes and wines and their size is proportional to the p-value of the statistical test. Student's t-test was used on log-transform ed concentrations and Wilcoxon rank sum test was used on glycosylated fraction.

Figu re 11 .

 11 Figu re 11. Re latio n s h ip s be tw e e n th e co n ce n tratio n s o f e th yl e s te rs an d th e ir acid p re cu rs o rs in w in e s . a) Concentration of ethyl hexanoate is correlated to concentration of hexanoic acid (R=0 .780 , p-value<0 .0 0 1) and b) concentration of ethyl butanoate is correlated to concentration of octanoic acid (R=0 .830 , p-value<0 .0 0 1).

Figu re 12 .

 12 Figu re 12 . Re latio n s h ip s be tw e e n th e co n ce n tratio n s o f ace tate e s te rs an d th e ir p re cu rs o rs in w in e s . Concentration of the isoam yl acetate and hexyl acetate is not correlated to the concentration of neither the alcohol nor the acid precursor (Į=0 .0 1).

Figu re 13 .

 13 Figu re 13 . Co rre latio n m atrix o f s e le cte d vo latile co m p o u n d s in w in e s an d grap e s . Total concentrations were log-transform ed prior to calculation of correlation. Only the com pounds with m ore than 40 data points were included in the calculation. Color and dot size are proportional to the correlation coefficient. Only coefficients with p-value<0 .0 0 1 are displayed.

Figu re 14 .

 14 Figu re 14 . Oxid ative m e tabo lis m in Vit is v in ife r a cv. Mo rio Mu s cat grap e be rrie s . Feeding with deuterium -labelled linalool revealed com plex linalool m etabolism in grape berries. Adapted from (40 ).

Figu re 15 .

 15 Figu re 15 . Es tim ate d flu x o f lin alo o l to d iffe re n t o xyge n ate d d e rivative s . Relationship between log concentrations of each linalool derivative and linalool was described with a linear regression. These m odels were then used to estim ate the concentration of each derivative in the function of linalool concentration. (cis)-linalool oxide (F) and hotrienol were excluded from the figure because of low R 2 value (TableS3).

Figu re 16 .

 16 Figu re 16 . Fo rm atio n o f n o ris o p re n o id s fro m caro te n o id s . a) Form ation of dam ascenone-type derivatives from neoxanthin. b) Form ation of ǃ-ionone from ǃ-carotene via enzym atic or non-enzymatic pathway. CCD -carotenoid cleavage dioxygenase.

Figu re 17 .

 17 Figu re 17. Stru ctu ral d ive rs ity o f te rp e n o id s in p lan ts . Exam ples of different structural types of plant terpenoids.

Figu re 18 .

 18 Figu re 18 . Th e tw o bio s yn th e tic p ath w ays le ad in g to is o p re n o id s : th e cyto s o lic m e valo n ate p ath w ay ( MVA) an d th e ch lo ro p las tic 2 -m e th yl-D-e ryth ro l-4 -ph o s p h ate ( MEP) p ath w ay ( 2 ) . GA3P -glycerolaldehyde-3-phosphate, PEP -phosphoenolpyruvate, CoA -Coenzym e A, HMG-CoA -3-hydroxy-3m ethylglutaryl-coenzyme A, MVA-m evalonic acid, DMAPP -dim ethylallyl pyrophosphate, IPPisopentenylpyrophosphate, FPP -farnesyl pyrophosphate, DXP -1-deoxy-D-xylulose 5-phosphate, MEP -2m ethyl-D-erythritol 4-phosphate, GPP -geranyl pyrophosphate, GGPP -geranylgeranyl pyrophosphate, PQplastoquinone.

  -5 ). A second protonation causes the cleavage of the bond between the two atom s of oxygen and an exit of a water m olecule. The resulting radical ferryl oxo interm ediate, also nam ed com pound I (Figure1-6), abstracts a hydrogen atom from the substrate. The resulting iron-coordinated hydroxyl group rapidly recom bines with the substrate radical (Figure1-7). This unique reaction m echanism results in the insertion of an oxygen atom into relatively inert substrates, which are difficult to functionalize by other enzym es. P450 s usually catalyze C-hydroxylation reactions, but m any other types of P450 -catalyzed reactions have been described: epoxidation, heteroatom oxidation, heteroatom release, group m igration, C-C coupling and m any m ore(7, 8).

Figu re 19 . 1 .

 191 Figu re 19 . Th e cyto ch ro m e P4 5 0 catalytic cycle . A water m olecule is bound in the active site in the resting state 1. Substrate displaces the water m olecule 2 which triggers reduction of the Fe ion from (III) to (II) 3 . A m olecule of oxygen binds into the active site 4 and is protonated 5 after a second iron reduction. The bond between two atom s of oxygen is cleaved 5 and a water m olecule is released. The resulting ferryl oxo radical 6 abstracts a proton from the substrate 7. The reaction is term inated when the resulting hydroxide com bines with the substrate radical (6). The electrons required for oxygen cleavage are supplied from reduced cofactors nicotinam ide adenine dinucleotide (NADH) or nicotinam ide adenine dinucleotide phosphate (NADPH).Cytochrom es P450 require auxiliary enzym es, which transfer the electrons from the cofactor to P450 . In bacteria, the m ost com m on auxiliary enzym es are ferredoxin (Fe-S protein) or flavodoxin (a FMN-containing protein), which in turn receive electrons from ferredoxin or flavodoxin reductase, respectively. In eukaryotes the m ost com m on electron donor is an enzym e called P450 reductase (also CPR or POR), which contains both flavin m ononucleotide (FMN) and flavin adenine dinucleotide (FAD) prosthetic groups that transport the electrons from the NADPH to the P450 . Most eukaryotic P450 system s also differ from bacterial in the fact that both P450 and CPR are anchored to the endoplasm ic reticulum (ER) m em brane via a N-term inal transm em brane anchor, whereas all com ponents of bacterial P450 system s are soluble and localized in the cytosol. In som e cases, plant P450 s also require another C-term inally m em brane anchored electron donor, cytochrom e b 5 to

Figu re 2 0 .

 0 Figu re 2 0 . Th re e d im e n s io n al s tru ctu re o f th e m o d e l cyto ch ro m e P4 5 0 CYP10 1A1 ( P4 5 0 cam , 2 CPP) fro m Ps e u d o m o n a s p u t id a w ith bo u n d s u bs trate ( cam p h o r) . Hem e prosthetic group is displayed in red, cysteine residue participating in thiolate bond with iron in yellow and cam phor (substrate) in blue. The protein structure is displayed from three different angles: from the so-called (hem e) proxim al side (left), the distal side (centre) and a lateral view along the hem e axis.With the advances in sequencing techniques and increase in the num ber of sequenced genom es, the num ber of discovered P450 genes has skyrocketed in the last years. The NCBI RefSeq database currently contains alm ost 50 0 0 0 unique cytochrom e P450 genes. Although not present in all species (som e bacteria, for exam ple E. coli, do not have any P450 genes), they are spread in all kingdom s of life, including viruses, which suggests their com m on origin before accum ulation of oxygen in the Earth atm osphere. The m ost distant P450 s can now show less than 20 % sequence identity. More closely related P450 s are grouped into families which usually share at least 40 % am ino acid sequence identity; these fam ilies are further partitioned into subfam ilies with at least 55% am ino acid sequence identity. P450 s nom enclature is further refined based on phylogenetic analysis. The nam e of each P450 consists of a prefix CYP, num ber of the fam ily, letter of the subfam ily and num ber of the isoform .

Chapter 4 Plan t m o n o te rp e n o ls : ro le an d o xid ative m e tabo lis m

 4 Tina Ilc, Claire Parage, Benoît Boachon, Nicolas Navrot and Danièle Werck-Reichhart Institut de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, 670 0 0 Strasbourg, FranceAbs tractPlants use m onoterpenols as building blocks for production of functionally and structurally diverse m olecules, which they use for interactions with other organism s: pollinators, flower visitors, herbivores, fungal or m icrobial pathogens. For hum ans, m any of these m onoterpenol derivatives are econom ically im portant because of their pharm aceutical, nutraceutical, flavor or fragrance applications. The biosynthesis of these derivatives is to a large extent catalyzed by the enzym es from the cytochrom e P450 superfam ily. Here we review the knowledge on m onoterpenol m etabolism in plants with special focus on recent elucidations of oxidation steps leading to diverse linalool and geraniol derivatives. We evaluate the com m on points between oxidation pathways of these two m onoterpenols, such as involvem ent of the CYP76 fam ily, and highlight the differences. Finally, we discuss the m issing steps and other open questions in the biosynthesis of oxygenated m onoterpenol derivatives.In tro d u ctio nMonoterpenols and their derivatives are a group of plant secondary m etabolites with im portant ecological roles, such as flower or fruit scent. In addition they act as precursors to num erous allelochem icals such as iridoids or m onoterpenoid indole alkaloids (MIAs). They are thus an econom ically im portant raw m aterial in flavor or fragrance, nutraceutical and pharm aceutical industry. To accom plish such a variety of roles, diversification of chem ical structures is param ount. Monoterpenols belong to the large fam ily of m onoterpenes, which are com pounds com posed of two isoprenyl units (to form 10 -carbon structures).Monoterpenes are products of terpene synthase enzym es, which convert a m onoterpenyl diphosphate substrate (usually geranyl or neryl diphosphate, GPP or NPP respectively) into a variety of structures. The reaction starts with ionization of m onoterpenyl diphosphate, which yields a carbocation interm ediate, prone to rearrangem ents and cyclisation (Figure1). The carbocation then follows one of two fates, depending on the catalytic site of the terpene synthase: it either loses a proton and form s a cyclic or an aliphatic hydrocarbon, or is attacked by hydroxyl ion to form a m onoterpenol (1). Recently, a terpene synthaseindependent synthesis of geraniol was discovered in roses (Rosa x hy brida)(2). A cytosolic enzym e Nudix hydrolase (NUDX1), which hydrolyses geranyl diphosphate, is the m ain geraniol producing enzym e in rose flowers.

Figu re 2 1 .

 1 Figu re 2 1. Te rp e n e s yn th as e catalyze d bio s yn th e s is o f m o n o te rp e n o ls fro m ge ran yl d ip h o s p h ate ( GPP) ( ad ap te d fro m ( 1) ) . Terpene synthases cleave the diphosphate group from the substrate, which results in the form ation of a carbocation interm ediate that can further rearrange. The reaction can be term inated by SURWRQ DEVWUDFWLRQ ZKLFK \LHOGV K\GURFDUERQ PRQRWHUSHQHV KHUH LOOXVWUDWHG E\ P\UFHQH DQG ǃ-ocimene), or by addition of water, which yields m onoterpene alcohols, or m onoterpenols. The route leading to m onoterpenols is highlighted in green. GPP -geranyl diphosphate, LPP -linalyl diphosphate.

Figu re 2 2 . Plan t n o n -cyclic m o n o te rp e n e alco h o ls , w ith carbo n n u m be rin g u s e d in th is p ap e r s h o w n fo

 2 Figu re 2 2 . Plan t n o n -cyclic m o n o te rp e n e alco h o ls , w ith carbo n n u m be rin g u s e d in th is p ap e r s h o w n fo r ( R )-lin alo o l. 8-and 9-linalool oxidized derivatives are also referred to as (E)-8-and (Z)-8-linalool derivatives, respectively.

Figu re 3 .

 3 Figu re 3 . Exam p le s o f irid o id s ( au cu bin e an d catalp o l) an d irid o id -d e rive d alkalo id s .

Figu re 4 .

 4 Figu re 4 . Se le cte d m o n o te rp e n o l d e rivative s th at o ccu r in fo o d an d be ve rage s .Studies of grape (Vitis vinifera) volatiles revealed a rich linalool m etabolism . Som e of linalool diols in grape juices are unstable in acidic solutions and can spontaneously rearrange to m ore stable structures by cyclisation or elim ination of a water m olecule(62). These rearrangem ents can be accelerated by heating. The products of these rearrangem ents are m ore volatile and can have a stronger odor com pared to their precursors: for exam ple, hotrienol (Figure4), which form s from relatively odorless 7-hydroxylinalool upon acidification, has sweet tropical scent. Sim ilarly, a m onoterpene acid 8 -carboxylinalool was

  Many oxygenated m onoterpenol derivatives contribute to characteristic arom a of plants, am ong them m any agricultural crops. Other oxygenated m onoterpenols have a weaker odor but can rearrange in acidic m edium with help of elevated tem peratures. Given their ubiquitous presence, they m ight act as flavor precursor in foods and beverages during preparation or storage. Sim ilarly, m onoterpene glycosides can act as flavor precursors and release their volatile aglycons during food or beverage preparation, storage or ferm entation.

  (3) and Daphne m ezereum(92), as well as lilac com pounds in Silene latifolia (99-10 1) are im portant olfactory cues for pollinators. For exam ple, the m oth Hadena bicruris specifically recognizes lilac aldehydes em itted by Silene latifolia at night am ong the m any volatiles of the scent bouquet in a nursery pollination system . The m oth then lays its eggs in the flower and pollinates it at the sam e tim e (99-10 1). In another exam ple, a link was established between the em ission level of lilac aldehydes and attractiveness of the plants from the Asim itellaria lineage for different pollinators, nam ely short-and long-tongued fungus gnat (10 2).Olfactom eter trials dem onstrated that lilac aldehydes induced nectaring behavior of the longtongued fungus gnats but repelled short-tongued fungus gnats. The volatile com position of several Asim itellaria species is adapted to their specific pollinator species of fungus gnat, illustrating the scent-m ediated speciation of Asim itellaria to their pollinators. Moreover, pure lilac aldehydes and lilac alcohols were shown to repel thrips and hoverflies (10 3), suggesting linalool and its derivatives have a dual role of in attraction of beneficial and repellence of neutral or detrim ental insects. Arabidopsis thaliana flowers do not need pollinators to reproduce, although out-crossing events have been observed in natural populations since flowers are visited by insects such as solitary bees, hoverflies and thrips (10 4-10 6). As a consequence, A. thaliana chem ical profile m ay favor protective functions, such as defense against flower visitors and pollen thieves, over attractiveness.In addition to its role in scent production and plant-insect interactions, linalool oxidative m etabolism in plants m ight serve for linalool detoxification as was observed in insect guts(10 7, 10 8), fungus Botry tis cinerea(10 9) and soil fungi (110 ). Local m etabolism of linalool into its furanoid and pyranoid oxides in pistils of C. brew eri was proposed as a defense m echanism to protect pollen tube from linalool toxicity (3). Oxidation and subsequent glycosylation also increases the solubility of m onoterpenols and m ay favor both their sequestration and transport to other organs. Their presence in phloem m ight also serve as a line of defense against phloem feeding insect (111).

  ce s an d ro le o f cyto ch ro m e s P4 5 0 in ge ran io l an d lin alo o l m e tabo lis m

Figu re 5 .

 5 Figu re 5. Cyto ch ro m e s P4 50 in vo lve d in s e co lo ga n in p ath w ay in Ca t h a r a n t h u s r o s e u s . *OF ǃ-D)-glucopyranosyl m oiety.

  Two P450 enzym es CYP76C3 and CYB71B31 were tightly co-expressed with two terpen e synthases TPS10 and TPS14, ((R)-and (S)-linalool synthases, respectively) in A. thaliana flowers (151). Both P450 s were expressed exclusively in the upper segm ent of the anther filam ents and nectaries (and weakly in petals) upon anthesis. Both P450 s could m etabolize both linalool enantiom ers in yeast and after transient expression in Nicotiana bentham ina. CYP71B31 form ed 1,2-epoxylinalool and a m ixture of different diastereoisom ers of 4-and 5hydroxylinalool and CYP76C3 8-and 9-hydroxylinalool and a different m ixture of 4-and 5hydroxylinalool diastereoisom ers (Figure 6). However, altered expression of CYP76C3 and CYP71B31 in A. thaliana m utants had only a m inor im pact on overall floral linalool oxidative m etabolism , which prevented the identification of their end-chain endogenous products, as well as suggested the contribution of other enzym es in the floral linalool m etabolism . The other m em bers of the CYP76 fam ily in A. thaliana were m ore extensively investigated. At first their enzym e activity was tested in vitro in parallel with CYP76B6 from C. roseus and CYP76B1 from Helianthus tuberosus (152). All these enzym es m etabolized m onoterpenols, but am ong those showing a clear expression in yeast only CYP76C1, CYP76C2, CYP76C4 and CYP76B6 m etabolized linalool into 8-hydroxylinalool as m ajor product and 8hydroxylinalool as m inor product. In addition, CYP76C2 and CYP76C4 were shown to produce 1,2-epoxylinalool (Figure 6). This suggested a widespread m onoterpenol oxidation capacity in the CYP76 fam ily, and especially a linalool oxidase role of the CYP76C subfam ily in Brassicaceae (152).

Figu re 6 .

 6 Figu re 6 . Lin alo o l m e tabo lis m by A. t h a lia n a cyto ch ro m e s P4 5 0 . Recom binant enzym es were produced either in yeast or N . bentham iana leaves. Dotted arrows indicate reactions which m ay require other enzym es. CYP76C1 was by far the m ost efficient linalool-converting enzym e in vitro, appearing as a prim e linalool oxidase candidate in A. thaliana. Its enzym e activity and role was thus extensively investigated in vitro and in the plant (10 3). Like CYP71B31 and CYP76C3, CYP76C1 was co-expressed with TPS10 and TPS14 upon flower anthesis, but CYP76C1 prom oter :GUS transform ants revealed a m ore widespread expression in flower organs, including anthers, stigm a and petals. In vitro studies, transient expression in N.

Re fe re n ce s 1 .

 1 plants. Considering their widespread presence and their econom ic im portance, they are first rank targets in pathway discovery studies. The advances on linalool and geraniol oxidative pathways occurred in parallel, revealing som e sim ilarities but also differences, in part resulting from the intrinsic properties of the starting com pounds. Metabolism of both linalool and geraniol share com m on initial oxidation steps. The m ost com m on oxidation of both substrates occurs on the 8-carbon, and com m only involves several consecutive oxygenations via alcohol and aldehyde to acid as the final product. Both the alcohol and the acid are usually accum ulated in plants as glycosides. Conjugation and branching pathways indicate that each interm ediate is released from the enzym e. Polar derivatives, such as carboxylic acids and glycosides were long overlooked due to analytical procedures essentially restricted to GC. This fact points to the im portance of using com plem entary analytical approaches to get the full appraisal of a m etabolic pathway.In all the known m onoterpenol oxidative pathways the initial oxidation step is catalyzed by a cytochrom e P450 from CYP76 fam ily. These enzym es belong to different subfam ilies in different plant taxa and share quite low sequence identities (<55%). The branching point of the oxidation cascade is often, but not always, the aldehyde interm ediate. All the interm ediates of the oxidation cascade can be glycosylated, although the aldehyde interm ediate is usually not accum ulating in plants. The balance between the glycotransferase activity and the efficiency of the oxidation cascade m ay control the flux through the oxidation steps and consequently the distribution am ong the interm ediates. In Asteridae, the first elongation of the geraniol pathway led to em ergence of the large fam ilies of iridoids and seco-iridoids, followed by the second elongation to MIAs. This pathway is now elucidated up to the form ation of the seco-iridoid and strictosidine structures. In m any linalool producing plants, oxidation and cyclization leads to the production of cyclic com pounds, often stored in the plant as conjugates. It is currently unknown whether cyclic linalool derivatives are incorporated in m ore com plex structures, equivalent to MIAs.It is currently unclear whether cytochrom es P450 alone catalyze the form ation of cyclic linalool derivatives, such as linalool oxides or lilac aldehydes, or if additional enzym es are needed to cyclize activated precursors generated by P450 s. A hum an xenobiotic m etabolizing P450 CYP2D6 can efficiently convert linalool into cyclic linalool oxides (153). In plants, however, oxidation/ cyclisation activity of P450 s has not yet been confirm ed. A. thaliana CYP76C1 can form lilac aldehydes from 8-oxolinalool in vitro with low efficiency, but its overexpression in N. bentham iana was not sufficient to generate detectable am ounts of lilac com pounds. This could be either due to com peting N . bentham iana enzym es, but m ay as well signal requirem ent for additional cyclization enzym e(s), which rem ain to be identified. In addition, none of the cytochrom es P450 characterized so far has been able to produce linalool oxides, in spite of their widespread occurrence in plant kingdom .A com m on feature of CYP76 enzym es is their low regio-and stereoselectivity. The enzym es can usually oxidize a whole subset of m onoterpenols, the m ost striking exam ple being CYP76B6 from C. roseus that catalyzes the double oxidation of all linear m onoterpenols (geraniol, linalool, lavandulol, citronellol) with a very high efficiency(14). Other exam ples are CYP76C1 and CYP76C3, both which m etabolize (R)-and (S)-linalool with no significant enantioselectivity(10 3, 151). The low selectivity of CYP76C1 is also illustrated by its diverse product profile, including 8-carboxylinalool, lilac aldehydes or lilac alcohols. In addition, CYP76B1, CYP76C1, CYP76C2, and CYP76C4 were all found to m etabolize several phenylurea herbicides. Their herbicide-m etabolizing capacity was sufficient to confer an increased herbicide tolerance to plant transform ants(14, 154). This raises the question of prom iscuity of these enzym es with regard to other plant m etabolites and xenobiotics. The CYP76Ms of rice (Ory za sativa) evolved as m ultifunctional enzym es dedicated to the biosynthesis of labdane-related diterpenoid antifungal phytoalexins (155, 156). Structural studies are needed to understand different enzym e specificity and their apparent prom iscuity.Monoterpenol oxidative m etabolism usually controls the form ation of volatile or soluble oxygenated derivatives, in particular in flowers, fruits and young leaves, which require intensive protection against herbivores and other antagonists or predators. Together with glycosyl transferases, they also control the level of em ission of free m onoterpenols. There are now several exam ples where this activity sets the balance between attractiveness for pollinators and repellence of antagonists. In flowers (157) and fruits of Carica papay a(70 , 158, 159), as well as flowers of Clarkia brew eri(3) and Daphne m ezereum (92) linalool and linalool oxides appeared to play a role in insect attractions. The need for m aintaining a very high flexibility of this system (possibly in association with speciation or com plex interactions, including tri-trophic plant-insect interactions) could be responsible for the high versatility of this pathway. An alternative hypothesis is that m onoterpenol m etabolism evolved as a detoxification pathway since sim ilar pathways are reported in insects and m icroorganism s (3), which could provide another explanation for its high versatility. While the ecological role of linalool and its derivatives is already well established, the influence of iridoids and MIAs on plant adaptation and resistance to antagonists and m icroorganism s rem ains largely unexplored, in spite of the intense interest in pharm aceutical applications of these com pounds.The recent description of the core linalool and geraniol oxidative m etabolism in A. thaliana and C. roseus paves the way to the discovery and engineering of the m onoterpenol-derived pathways of plants of agronom ic and econom ic interest, not only for the production of m etabolites of interest but also for a direct plant protection effect. The reconstruction of these pathways in m icroorganism s for arom a, cosm etic and pharm aceutical industry is also in developm ent. Am ong the m ost appealing candidates are fruit, wine and tea arom a, as well as the large diversity of iridoids and alkaloids with docum ented therapeutic applications.Au th o rs ' co n tribu tio n sWriting -first draft: Introduction, Monoterpenol derivatives in food and beverages, First insights into geraniol and linalool oxidative m etabolism (TI); Geraniol derivatives and iridoids, First insights into geraniol and linalool oxidative m etabolism (CP); Role of linalool and its derivatives in plant-insect interactions, Linalool m etabolism (BB); Geraniol m etabolism (NN), Perspectives and open questions (DWR). Writing -review and editing (NN and DWR). Degenhardt J , Kollner TG, & Gershenzon J (20 0 9) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phy tochem istry 70 (15-16):1621-1637. 2. Magnard J L, et al. (20 15) Plant volatiles. Biosynthesis of m onoterpene scent compounds in roses. Science 349(6243):81-83. 3. Raguso RA & Pichersky E (1999) New Perspectives in Pollination Biology: Floral Fragrances. A day in the life of a linalool m olecule: Chem ical com m unication in a plantǦpollinator system . Part 1: Linalool biosynthesis in flowering plants. Plant Species Biol. 14(2):95-120 . 4. $SURWRVRDLH $& +ăQFLDQX 0 &RVWDFKH ,-I, & Miron A (20 14) Linalool: a review on a key odorant m olecule with valuable biological properties. Flavour and Fragrance Journal 29(4):193-219. 5. Weidenham er J D, Macias FA, Fischer NH, & William son GB (1993) J ust how insoluble are m onoterpenes? Journal of chem ical ecology 19(8):1799-180 7. 6. Baxter RL, Laurie WA, & Mchale D (1978) Transform ations of m onoterpenoids in aqueous acids: The reactions of linalool. geraniol, nerol and their acetates in aqueous citric acid. Tetrahedron 34(14):2195-other organism s. The m anual annotation of cytochrom e P450 genes in the genom e of Vitis vinifera PN40 0 24 revealed 579 P450 sequences, including 279 com plete genes. Most of the P450 sequences in grapevine genom e are organized in physical clusters, resulting from tandem or segm ental duplications. Although m ost of these clusters are sm all, som e of the P450 fam ilies, such as CYP76 and CYP82, underwent m ultiple duplications and form ed large clusters of hom ologous sequences. Analysis of gene expression revealed highly specific expression patterns, which are often shared within the genes in large physical clusters. Som e of these genes are induced upon biotic stress, which points to their role in plant defense, whereas others are specifically activated during grape berry ripening and m ight be responsible for the production of berry-specific m etabolites, such as arom a com pounds. Our com prehensive gene annotation and expression analysis provide groundwork for further functional characterization of this im portant gene fam ily in grapevine.

  Cytochrom e P450 hom epage (http:/ / drnelson.uthsc.edu/ Cytochrom eP450 .htm l), and suggests that som e sequences previously annotated as different genes are probably allelic variants. The num ber of cytochrom es P450 in grapevine is com parable to their num ber in other plants (e.g. 273 in Arabidopsis thaliana and Solanum ly copersicum , 30 9 in Ory za sativa). 20 sequences were annotated as partial genes, lacking a segm ent of the sequence due to gaps in the genom e assem bly. 11 putative pseudogenes only contain one nonsense m utation or fram e shift, which could originate from sequencing errors or be genuine and still be functional genes in som e varieties. Finally, the 269 pseudogenes are fragm ents, either containing m ultiple stop codons or fram eshift m utations, or sequences not aligning to the whole length of hom ologous P450 genes.

Figu re 2 3 .

 3 Figu re 2 3 . Mo le cu lar ph ylo ge n e tic an alys is o f grap e vin e cyto ch ro m e P4 50 . The alignm ent of full length cytochrom e P450 protein sequences was used to generate a m axim um likelihood tree. The dark blue clade is the clan 71, which often contains genes involved in specialized metabolism . The highlighted genes belong to the seven largest physical clusters.

  ) allowed us to identify fam ilies that potentially expanded in grapevine and m ight have a role in the production of speciesspecific specialized m etabolites: an expansion of the CYP75 fam ily, involved in anthocyanin biosynthesis, is already well docum ented(33), whereas the function of CYP82, the largest P450 fam ily in grapevine with 25 m em bers, is currently unknown in this species. Other fam ilies that are larger in grapevine than in m ost other species are: CYP76, CYP79, CYP80 , CYP81, CYP87, CYP89 and CYP716. Stru ctu ral o rgan izatio n o f th e P4 5 0 s in th e PN 4 0 0 2 4 ge n o m e

  Figure S3).

Figu re 2 4 .

 4 Figu re 2 4 . Ph ys ical m ap o f cyto ch ro m e P4 50 s e qu e n ce s o n th e 19 V. v in ife r a ch ro m o s o m e s . Yellow circles represent isolated annotations, light blue circles represent physical clusters com posed of m em bers of only one P450 fam ily and the purple circles represent physical clusters com posed of m em bers of 2-3 P450 fam ilies. The circle size is proportional to the num ber of sequences in the cluster. The num bers 1-19 are chrom osom e num bers and "Un" is "Unknown chrom osom e" which contains sequences with unknown chrom osom e location. Cytochrom e P450 fam ilies group genes with higher sequence sim ilarity ( SURWHLQ sequence identity) and often a sim ilar function. A m ajority of physical clusters are com posed of m em bers of only one P450 fam ily (63 clusters, 74%) and the rem aining clusters are com posed of up to 3 P450 fam ilies. The 4 largest clusters are com posed of several P450 fam ilies, whereas the clusters with single P450 fam ilies are sm aller (Figure 2, Figure S4).

Figu re 2 5 .

 5 Figu re 2 5 . Sim ilarity o f th e P4 5 0 ge n e s be tw e e n an d w ith in clu s te rs . For each circle, the grey bars correspond to the 19 grape chrom osom es and the "Unknown chrom osom e". The lines connect com plete P450 genes according to their sim ilarity. The lines outside the circles show the sim ilarity between genes of the sam e cluster, whereas the lines in the circle connect sim ilar genes of different clusters. Only P450 genes that from clusters com posed of at least two com plete genes are illustrated here. The seven largest clusters are labeled with num bers corresponding to Table1. The lines are connecting the genes corresponding to the best BLAST hit (A), second best hit (B) or third best blast hit (C).

Figu re 2 6 .

 6 Figu re 2 6 . D o t m atrix o f s e gm e n tal d u p licatio n s in th e p h ys ical clu s te r 9 2 . Physical cluster 92 is located on chrom osom e 18 and com prises 22 CYP82 sequences, one CYP74 sequence and one CYP70 4 sequence.The dots and the black lines represent the sequence sim ilarities in cluster 9 2 com pared to itself. The red rectangles on the sides of the graph represent cytochrom e P450 sequences. Com plete genes are labeled with their nam e and pseudogenes are labeled with "p" and the P450 fam ily. A) The sim ilarities for the whole cluster 9 2 . B) A zoom of the red squared region which contains two 20 -kbp-sequence blocks with very high sim ilarity. Analysis of gene expression showed that CYP82D17 and CYP82D20 v2 are co-expressed (expression cluster O, expression in leaves), and so are the first and the third pseudogene in the enlarged segm ent (expression cluster J, expression in ripe berries).

Figu re 2 7 .

 7 Figu re 2 7. H e at m ap a n d d e n d ro gram o f th e P4 50 s e qu e n ce s , clu s te re d acco rd in g to th e ir e xp re s s io n pro file . The expression levels were averaged over the experim ents classified in one of the five experim ental categories: flowers, young berries, ripe berries, control leaves and leaves under biotic stress. The dendrogram on the top of the figure shows 31 co-expression clusters, which include 494 expressed cytochrom e P450 sequences.We analyzed the expression patterns by clustering the expression profiles of the 494 expressed cytochrom e P450 sequences. A Pearsons' correlation coefficient cut-off of 0 .795 resulted in 31 expression clusters, shown in Figure5. Five expression clusters (I, J , N , X and Z) were up-regulated in one of the five experim ental categories. These five clusters contained 127 P450 s, showing that a quarter of the expressed P450 s were specifically up-regulated rather than constitutively expressed in the investigated conditions. The largest expression cluster was R, which consisted of 50 sequences with low basal level of expression in all investigated tissues and conditions. These m ay be sequences with constitutive expression, or with higher expression in conditions or organs not included in this study. Other large expression clusters were cluster N with 44 sequences specifically expressed in leaves undergoing biotic stress, cluster Z with 40 sequences specifically expressed in flowers and cluster E with 37 sequences expressed to a low level in reproductive organs (berries and flowers) as well as leaves under biotic stress.

  ), SRR519450 , SRR519456, SRR520 38 0 and SRR520 385;(53), all four sam ples;(54), SRR493740 -SRR493746;(42), SRR866544, SRR866570 , SRR866571 and SRR866576; (55), SRR522472, SRR522477 and SRR522478; and eight unpublished RNA-Seq datasets acquired by INRA. The software Tophat2 v2.0 .11(56) was used to m ap the RNA-Seq reads against the PN40 0 24 12x sequence using the following param eters: -p 5 -N 5 -read-edit-dist 5. The software Cufflinks v2.2.1 (57) was used to assem ble the transcripts from all the RNA-Seq experim ents. First the cufflinks com m and was used with the -p 5 param eter and then the cuffm erge com m and with the -p 15 param eter and using the fasta file of the PN40 0 24 12x sequence for the -s param eter. This assem bly led to 32,219 transcripts and to a gtf file showing their m apped location in the PN40 0 24 12x sequence. The two transcript datasets were form atted in gff form at com patible with the Artem is Browser so that the predicted gene structures of the cytochrom es P450 could be com pared with the transcripts and edited if needed.

2 . 4 )

 24 (66) via the CIPRES Science Gateway(67) and PhyML (im plem ented in Seaview v 4.5.4) (68). Bootstrap values are shown on the nodes of the Vitis all CYP phylogeny. Nodes with bootstrap values below 60 were m anually suppressed from the 11 species CYP phylogeny and are shown as trifurcations (unsolved topologies). The trees

( 4 (

 4 http:/ / www.ncbi.nlm .nih.gov/ sra). 73 sequence files generated in the fram ework of 7 different experim ents(42, 52, 53, 55, 70 , 71) and eight unpublished RNA-Seq datasets acquired by INRA were used. The data were form atted in the fastq form at using the fastqhttp:/ / www.ncbi.nlm .nih.gov/ books/ NBK158 90 0 ). Alignm ents of these reads against the PN40 0 24 12x sequence were then perform ed using GSNAP version20 13-11-27 (72) with the following param eters: -B 4, -N 1, -n 3, --nofails and the quality protocol according to the experim ent. These files were parsed to keep the best, unique and paired (if paired-end reads) alignm ents using a hom em ade script.The num ber of fragm ents aligned on each annotation from the genom e annotation V1 stored in Grape Genom e Database hosted at CRIBI and the cytochrom es P450 was counted using the com m and htseq-count from the HTSeq fram ework version 0 .6.0(73) with the following param eters: -m intersection-nonem pty and -s no. Using a hom em ade script, FPKMs (Fragm ents Per Kilo base of exon per Million fragm ents m apped) were calculated for every annotation.

Figu re S2 . Co m p aris o n o f th e n u m be r o f P4 5 0

 5 Figu re S2 . Co m p aris o n o f th e n u m be r o f P4 5 0 ge n e s p e r fam ily be tw e e n s p e cie s . Dot size is proportional to the relative fam ily size (num ber of genes per fam ily) in a given species com pared to Vitis vinifera (Vv = Vitis vinifera, Nn = N elum bo nucifera, Os = Oryza sativa, Bd = Brachy podium distachy on, Sl = Solanum ly copersicum , At = Arabidopsis thaliana, Pt = Populus trichocarpa, Gm = Gly cine m ax, Mt = Medicago truncatula). The num bers in the first colum n are the absolute fam ily sizes (num bers of genes per fam ily) in Vitis vinifera. The n um ber of genes per fam ily was retrieved from the cytochrom e P450 hom epage. Pseudogenes and fam ilies not present in V. vinifera (CYP83, CYP99, CYP70 2, CYP70 5, CYP70 8, CYP718 and CYP729) were excluded from the count.

Figu re 2 8 .

 8 Figu re 2 8 . Th e p ro p o s e d p ath w ay fo r w in e lacto n e fo rm atio n .

W in e la cto n e co n ce n tratio n in w in e is d e te rm in e d by w in e age an d p re cu rs o r co n ce n tratio nFigu re 2 9 .

 9 Figu re 2 9 . Re latio n s h ips be tw e e n w in e age an d co n ce n tratio n s o f w in e lacto n e , ( E) -8carbo xylin alo o l an d ( E) -8 -carbo xylin alo o l glu co s e e s te r. All three com pounds were quantified in the sam e wines by LC-MS/ MS (N=3). Concentration of wine lactone is higher in older wines (a) and wines with higher concentration of its precursor, (E)-8-carboxylinalool (b). Principal com ponent analysis of m etabolite concentrations and the age of wine confirm s this observation: (c) variables factor m ap and (d) individuals factor m ap (Mus = Muscat, Rie = Riesling, Gew = Gewurztram iner). The 29-year-old wine was excluded from PCA analysis.

re s s io n o f can d id ate P4 5 0

 5 ge n e s pa ra lle ls in cre as in g ( E) -8 -carbo xylin alo o l co n ce n tratio n d u rin g grape be rry ripe n in g

Figu re 3 0 .

 0 Figu re 3 0 . Co m p arative in cre as e in ( E) -8 -carbo xylin alo o l glu co s e e s te r co n te n t an d can d id ate P4 50 ge n e s e xp re s s io n d u rin g grap e be rry d e ve lo p m e n t. a) Relative concentration (±SD) of (E)-8carboxylinalool glucose ester in developing Gewurztram iner berries. One-way ANOVA revealed significant differences between developm ental stages (p=0 .0 0 16). Letters indicate significant differences am ong the m eans FDOFXODWHG ZLWK 7XNH\ +6' WHVW Į 5). b) Expression of candidate genes in Gewurztram iner grape berries as determ ined by qPCR. Error bars represent standard deviation of 3 technical and 3 biological replicates. Gene expression was norm alized to the expression of three constitutively expressed genes (30 ).

Figu re 3 1 .

 1 Figu re 3 1. Co n ve rs io n o f lin alo o l a n d ( E) -8 -h yd ro xylin alo o l by grap e vin e cyto ch ro m e s P4 5 0 e xp re s s e d in ye as t. Microsom al m embranes prepared from yeast expressing the candidate P450 s were incubated with substrates at 10 0 NjM concentration for 1h in the presence of NADPH. Reaction m ixtures were extracted and analyzed by GC-FID. Conversion (± SD, N=3) is calculated relative to the residual concentration of substrate in negative control (m icrosom al fraction from yeast transform ed with an em pty vector -EV) incubated under the same conditions. One-way analysis of variance (ANOVA) revealed significant differences (P < 0 .0 0 1) for both substrates. Letters on the right axes indicate significant differences between m eans calculated with Tukey HSD test (Į = 0 .0 5).

Figu re 3 2 .

 2 Figu re 3 2 . Ide n tificatio n o f th e p ro d u cts o f lin alo o l m e tabo lis m by re le van t can d id ate grap e P4 50 s e xp re s s e d in ye as t. LC-MS/ MS reaction profiles of three enzyme candidates in m ultiple-reaction-monitoring (MRM) m ode. Incubations are perform ed in the sam e conditions as for Figure 4. Each color represents an MRM channel for specific detection of target m olecules: blue for linalool (137>80 .7), green for (E)-8-hydroxylinalool (135>10 6.8 ), and red for (E)-8-carboxylinalool (167.2>92.8). Mass spectra of identified (Figure S9) and putative products (Figure S10 ) are available in supplem entary inform ation. Negative control is a reaction with m icrosom es from yeast transform ed with an em pty vector. 1-(E)-8-hydroxylinalool, 2 -(E)-8-carboxylinalool, 3 -(Z)-8hydroxylinalool, 4 -(E)-8-oxo-6,7-dihydrolinalool, 5 -(E)-8-hydroxy-6,7-dihydrolinalool. 4 and 5 are side products of yeast m etabolism of (E)-8-oxolinalool.

Figu re 3 3 .

 3 Figu re 3 3 . Qu an tificatio n o f o xid ize d lin alo o l m e tabo lite s in N . b e n t h a m ia n a le ave s e xp re s s in g grap e vin e lin alo o l s yn th as e an d can d id ate P4 50 ge n e s . Agrobacterium tum efaciens containing candidate P450 genes was infiltrated to N . bentham iana leaves Expression of green fluorescent protein (GFP) was used as a negative control. Am ounts of m etabolites are expressed as m eans of peak areas in the corresponding MRM channel (± standard deviation, N=3). Free and bound form s of the sam e m etabolite were detected with the same channel so that peak areas are roughly com parable. One-way analysis of variance (ANOVA) revealed significant differences between groups (P < 0 .0 5 for linalool, P < 0 .0 1 for m alonylhexosyl linalool and P < 0 .0 0 1 for other four m etabolites). Letters on the right axes indicate significant differences between m eans calculated with Tukey HSD test (Į = 0 .0 5).

  were purchased from Paul Ginglinger (Eguisheim , France) or produced by the INRA Colm ar. One sam ple (Riesling Weingut 20 14) was purchased at the superm arket. The INRA Gewurztram iner wines were m ade from grapes collected from the sam e vineyard localized in the "Grand Cru Osterberg" area in Ribeauvillé (France) in 1986, 1997, 1998, 20 0 1, 20 0 4, 20 0 5, 20 0 7 and 20 0 9. All wines were stored in the INRA cellar, which is m aintained at the constant tem perature. For the analysis of (E)-8-carboxylinalool and (E)-8-carboxylinalool glucose ester, wines were injected directly to LC-MS/ MS without being extracted or concentrated. For the analysis of wine lactone, 40 m L of wine was spiked with internal standard ((E)-8-oxolinalool), then extracted with 10 m L pentane: ethyl acetate (1:1 vol.). The extract was then evaporated to dryness and the residue resuspended in 20 0 NjL of m ethanol prior to LC-MS/ MS analysis. Liqu id ch ro m ato gra ph y co u ple d to ta n d e m m as s s p e ctro m e try Liquid chrom atography-m ass spectrom etry analyses were perform ed on a Waters Quattro Prem ier XE m ass spectrom eter equipped with an electrospray ionization source and coupled to an Acquity UPLC system (Waters USA). Chrom atographic separation was achieved using an AcqXLW\ 83/& EULGJHG HWK\O K\EULG & FROXPQ ā ā PP NjP :DWHUV DQG precolum n. The m obile phase consisted in (A) water and (B) m ethanol, both containing 0 .1% form ic acid. The solvent gradient was as follows: 95% A 0 -2 m in, linear gradient to 0 % A 2-12 m in, 0 % A 12-14 m in, linear gradient to 95% A 14-15 m in, 95% A 15-17 m in. The flow rate was 0 .350 m L m in -1 7KH FROXPQ ZDV KHDWHG WR & ,QMHFWLRQ YROXPH ZDV Nj/ IRU PXOWLSOH UHDFWLRQ PRQLWRULQJ 050 UXQV DQG Nj/ IRU QRQ-targeted (scan) runs. Nitrogen was usedas the drying and nebulizing gas. The nebulizer gas flow was set to 50 L/ h, and the desolvation gas flow to 90 0 L/ h. The interface tem perature was 40 0 °C and the source tem perature 135°C. The capillary voltage was set to 3.4 kV; for the scan runs the cone voltage was set to 25 V and the ionization was in positive or negative m ode. Low m ass and high m ass resolution was 15 for both m ass analyzers, ion energies 1 and 2 were 0 .5 V, entrance and exit potential were 50 V, and detector (m ultiplier) gain was 650 V. Parent and daughter ions used for MRM were: 137>8 0 .7 for linalool, 135>10 6.8 for (E)-8-hydroxylinalool, 151.2>92.8 for (E)-8-oxolinalool, 167.2>92.8 for (E)-8-carboxylinalool and 167.2>93 for wine lactone.

  A is o latio n fro m grap e be rrie s an d qPCR Grape berries were collected from the Gewurztram iner 643 clone from the am pelographic collection of INRA in Colm ar, France, at five different developm ental stages: green (4 J uly 20 14), before and after veraison (8 August 20 14), m id-ripe (8 Septem ber 20 14) and ripe (

  with the following m odifications: 7 m L of extraction buffer was used per g of berry powder and after LiCl precipitation the sam ples were treated as described above. The concentration of RNA, 260 / 28 0 and 260 / 230 ratios was m easured by Nano-Drop 20 0 0 spectrophotom eter (Therm o Scientific) and integrity of RNA was confirm ed by gel electrophoresis. cDNA was synthesized from 1 Njg total RNA using (dT)23 and Superscript® III Reverse Transcriptase (Life technologies) following m anufacturer's instructions. The resulting cDNA was diluted 5x and used as a tem plate in a quantitative PCR (qPCR) experim ent.

g s e qu e n ce s , co n s tru ctio n o f ve cto rs an d re co m bin an t e xpre s s io n

 n RNA was extracted from grape skins of V. vinifera cv. Muscat Ottonel following the protocol in(30 ), except that PVPP in extraction buffer was replaced with PVP. After LiCl precipitation and ethanol wash, the nucleic acids were digested using RQ1 RNase-Free DNase (Prom ega, &DW 0 7KH UHDFWLRQ PL[WXUH ZDV GLOXWHG WR Nj/ DQG H[WUDFWHG ZLWK HTXDO YROXPH of phenol: chloroform : isoam yl alcohol (25:24:1 vol.). The aqueous phase was extracted twice with an equal volum e of chloroform . RNA was precipitated with ethanol and resuspended in Nj/ RI 0LOOL4 ZDWHU cDNA was synthesized using Superscript® III Reverse Transcriptase (Life technologies) following m anufacturer's instructions, and was then used as a tem plate for PCR am plification of the target sequences.

  4, in the presence of 730 NjM reduced NADPH, 10 0 NjM of substrate and 10 % vol. of m icrosom al m em brane suspension. Reactions were conducted at 27°C for 1 hour under agitation and were stopped by the addition of solvent (ethyl acetate for sam ples to be analyzed by GC or m ethanol for sam ples to be analyzed by LC), vortexed for 30 seconds and centrifuged to pellet the m em branes and proteins. Ethyl acetate extracts were filtered over sodium sulfate to rem ove residual water. All assays were perform ed in triplicates.Tran s ie n t e xpre s s io n in N . b e n t h a m ia n aN. bentham iana plants were grown under 16 h day (24°C) / 8 h night (20 °C) conditions. The Agrobacterium tum efaciens strain LBA440 4 was transform ed with the pCam bia230 0 or pCam bia330 0 plasm ids by electroporation. Liquid cultures of transform ants (10 0 m L LB, 50 NjM rifam picin, 10 NjM gentam icin, 50 NjM kanam ycin) were grown overnight at 28°C (18 0 rpm agitation). The next day they were harvested by 10 m in centrifugation at 30 0 0 g and washed twice with tap water. Agrobacteria transform ed with the p19 gene of tom ato bushy stunt virus(48), linalool synthase and P450 genes were then m ixed with tap water in the sam e tube in 1:1:1 ratios, so that the OD 60 0 of each of the strains corresponded to 0 .4. The bacteria were then infiltrated into the abaxial epiderm is of 20 day-old N . bentham iana leaves with a 1 m L plastic syringe (without needle). For negative controls, strains for expression of either P450 or LIS were replaced with a GFP expression strain.Leaves tissues were collected 4 days post-infiltration, frozen in liquid nitrogen, and ground with a m etal bead (7 or 10 m m diam eter) in 12 m L plastic tubes. Leaves expressing GFP were also viewed under UV light to confirm successful agroinfiltration and gene expression. Leaf powder was stored at -80 °C and extracted with m ethanol (1:4 m / v) by vortexing. Extracts were then stored overnight at -20 °C and centrifuged just prior to analysis. The supernatant was transferred into a glass vials for LC-MS/ MS analysis.

  Figu re S6 . Targe te d LC-MS/ MS qu an tificatio n o f w in e lacto n e in w in e e xtracts . Specific MS/ MS transition (167.3 >92.8 m / z) was used for wine lactone detection.

Figu

  Figu re S8 . Re latio n s h ip be tw e e n ( E) -8 -carbo xylin alo o l glu co s e e s te r an d w in e age ( a) an d fre e ( E) -8 -carbo xylin alo o l ( b) in 2 3 w in e s am p le s . Both com pounds were quantified by LC-MS/ MS and are expressed as m eans (±SD, N=3). Due to unavailability of (E)-8-carboxylinalool glucose ester standard the concentration of this com pound is expressed in (E)-8-carboxylinalool equivalents.

Figu

  Figu re S11. Exp re s s io n o f CYP76 ge n e s in d iffe re n t o rgan s a n d d e ve lo p m e n tal s tage s o f Vit is v in ife r a cv. Co r v in a . Norm alized "grapevine expression atlas" data (29) was retrieved from Grape eFP Browser (49). Because of errors in gene annotation used for this dataset som e pairs of adjacent genes were annotated as one gene.

Figu

  Figu re S14 . Mas s s p e ctra ( ESI-MS) o f th e p ro d u cts id e n tifie d by co m p aris o n w ith au th e n tic s tan d ard s .. Peak num bers refer to those in Figure 5.

Figu

  Figu re S15 . Mas s s p e ctra ( ESI-MS) o f th e pro d u cts pu tative ly id e n tifie d bas e d o n MS fragm e n tatio n .Peak num bers refer to those in Figure5. 3 was identified on the basis of the sim ilarity of its m ass spectrum to that of 1. 4 and 5 were identified in(24).

Figu

  Figu re S16 . U PLC-MS an alys is o f th e p ro d u cts o f ( E) -8 -h yd ro xylin alo o l ( le ft) an d ( E) -8 -o xo lin alo o l ( righ t) m e ta bo lis m by th e ye as t-e xp re s s e d can d id ate P4 50 s catalyze d th e w h o le cas cad e o f lin alo o l o xid atio n s . LC-MS/ MS profiles of the products of three candidates enzym es in m ultiple-reaction-monitoring (MRM) m ode. Each color represents an MRM channel for specific detection of target m olecules: blue-linalool (137>80 .7), green-(E)-8-hydroxylinalool (135>10 6.8), purple-(E)-8-oxolinalool (151.2>92.8) and red-(E)-8carboxylinalool (167.2>92.8 ). Peak num bers refer to those in Figure 5.

Figu

  Figu re S17. Co n ve rs io n o f ge ran io l by grap e vin e cyto ch ro m e s P4 5 0 . The experim ental procedure and the evaluation of activity are the sam e as those used in Figure 4.

Figu re S2 0 .

 0 Figu re S2 0 . Co m p aris o n o f th e in v it r o activitie s o f Ge w u rztram in e r an d Mu s cat Otto n e l CYP76 F14 . UPLC-MS/ MS analysis of the reaction products of three enzym e candidates in m ultiple-reactionm onitoring (MRM) m ode. Each color represents one MRM channel for the specific detection of target m olecules: blue-linalool (137>8 0 .7), green-(E)-8-hydroxylinalool (135>10 6.8) and red-(E)-8-carboxylinalool (167.2>92.8). Peaks 4 and 5 were putatively identified on the basis of their m ass spectra (Figure S 9).

Figu re S2 1 .

 1 Figu re S2 1. Co m p aris o n o f th e activitie s o f Ge w u rztram in e r an d Mu s cat Otto n e l CYP76 F14 in N . b e n t h a m ia n a . Each color represents one MRM channel for specific detection of target m olecules: blue-linalool (137>80 .7), green-(E)-8-hydroxylinalool (135>10 6.8) and red-(E)-8-carboxylinalool (167.2>92.8 ). Conjugated products were putatively identified on the basis of their m ass spectra (TableS3).

Figu re S2 2 .

 2 Figu re S2 2 . Co m p aris o n o f ( E) -8 -carbo xylin alo o l glu co s e e s te r co n ce n tratio n in be rrie s o f w ild ( Vit is v in ife r a s u bs p . s y lv e s t r is ) an d cu ltivate d grap e vin e ( Vit is v in ife r a s u bs p . s y lv e s t r is cv.Ge w u rztram in e r) . (E)-8-carboxylinalool glucose ester was quantified in m ethanol extracts of grape berries via LC-MS/ MS and is expressed as a m ean (±SD, N=3). Data were norm alized relative to fresh weight. Student's t-test revealed significant differences between the two subspecies (p-value<0 .0 0 1 ***, <0 .0 0 1 **).

Figu re S2 3 . 7

 37 Figu re S2 3 . Co n ve rs io n o f tw o lin alo o l e n an tio m e rs by CYP76 F14 e xp re s s e d in ye as t. ( Mo= MuscatOttonel and Gw = Gewurztram iner). Sam ple preparation is identical to those used to generate data in Figure4. Conversion (± SD, N=3) is calculated relative to the residual concentration of substrate in negative control (m icrosom al fraction from yeast transform ed with an em pty vector -EV) incubated under the sam e conditions. Student's t-test showed no significant difference between the two substrates at Į=0 .0 5 for neither of the enzyme variants.

Figu re 3 4 .

 4 Figu re 3 4 . GC-MS p ro file s o f N . b e n t h a m ia n a le af d is cs e xp re s s in g grap e vin e P4 50 s afte r vacu u m in filtratio n o f s u bs trate s lin alo o l ( a ) an d ge ra n io l ( b) co m p are d to au th e n tic s tan d ard s . N . bentham iana leaves were infiltrated with Agrobacterium tum efaciens bearing P450 genes. GFP was used as a negative control. Leaf discs were incubated in a substrate solution for 4 h. Buffer containing substrate and products was extracted using solid phase extraction cartridges and analyzed using GC-MS. Chrom atogram s show relative abundance of selected m / z 71 (a) and 68 (b). 1a was identified as (E)-8-hydroxylinalool, 1b as (E)-8oxolinalool and 2 as (E)-8-hydroxygeraniol. Corresponding m ass spectra are displayed in Figu re 2 .

Figu re 3 5 .

 5 Figu re 3 5. Mas s s p e ctra o f CYP76 F14 an d CYP76 T2 1 p ro d u cts fo rm e d fro m lin alo o l ( a) an d ge ran io l ( b) in N . b e n t h a m ia n a le af d is cs ( Figu re 1) .

Figu re 3 7 .

 7 Figu re 3 7. Mas s s p e ctra o f CYP76 F14 an d CYP76 T2 1 p ro d u cts fo rm e d fro m n e ro l ( a) an d citro n e llo l ( b) by grap e vin e in N . b e n t h a m ia n a le af d is cs ( Figu re 3 ) . Product form ed from nerol ( a) was tentatively identified as (E)-8-hydroxynerol and the product form ed from citronellol ( b) was tentatively identified as (E)-8-hydroxycitronellol based on com parison of m ass spectra to those published in (1).

Figu re 3 9 .

 9 Figu re 3 9 . D e te rm in atio n o f th e co n figu ratio n o f th e lin alo o l p ro d u ce d by th e grap e vin e lin alo o l s yn th as e u s e d in o u r N . b e n t h a m ia n a e xp e rim e n ts . GC-MS chrom atogram s of the headspace volatiles collected from N . bentham iana leaves transiently expressing the grapevine linalool synthase (VvLIS). Negative control is N . bentham iana leaves infiltrated with A. tum efaciens bearing the GFP gene.

Figu re 4 0 .

 0 Figu re 4 0 . Kin e tics o f in v it r o ( S) -lin alo o l co n ve rs io n by CYP76 F14 . The reaction was stopped after 30 m in with addition of ethyl acetate. Sam ples were extracted and substrate conversion was m onitored by GC-FID.

  Other CYP76 genes only have one intron and are hom ologous to CYP76T12v2 in the 93-base pair region of the first intron of CYP76T12v1. The additional intron in CYP76T12v1 does not disrupt the open reading fram e of the sequence.

Figu re 4 1 .

 1 Figu re 4 1. Stru ctu re o f th e alte rn ative s p licin g varian ts o f th e CY P76 T12 ge n e . CYP76T12 was cloned from Gewurztram iner green berry cDNA. Two alternative splicing variants were present am ong the sequenced clones. Conserved P450 m otifs (I-helix m otif, the catalytic ERR triad and the hem e binding m otif) are highlighted.

Figu re 4 2 .

 2 Figu re 4 2 . Re lative e xp re s s io n o f CY P76 F10 an d CY P76 T12 d u rin g be rry d e ve lo p m e n t. Error bars represent standard deviation of three technical and three biological replicates. CYP71AT7 is added for com parison of relative gene expression levels to genes discussed in the previous chapter.

Figu re 4 3 .

 3 Figu re 4 3 . CO-d iffe re n ce s p e ctra o f re d u ce d m icro s o m e s o f ye as t e xp re s s in g CYP76 F10 an d th e tw o s p licin g varian ts o f CYP76 T12 .

  CYP76F10 and CYP76T12 were transiently expressed in N. bentham iana leaves with grapevine linalool synthase (LIS) to evaluate their activity in planta. Leaves were extracted with m ethanol and analyzed by UPLC-MS/ MS. Both enzym es produced (E)-8carboxylinalool, which accum ulated m ostly in glycosylated form . (Z)-8 -carboxylinalool, one of the putative products of CYP76T12 in vitro, could not be detected in extracts of N . bentham iana leaves expressing this gene. Neither could putative (Z)-8-carboxylinalool be detected in any of the wine sam ples analyzed in Ch a p te r 6 .Like other linalool-m etabolizing enzym es from the CYP76 fam ily presented in this work, CYP76F10 and CYP76T12 catalyze the oxidation of the term inal (E)-and (Z)-8 -carbon atom s of the linalool m olecule. However, due to their low expression they are unlikely to significantly contribute to the production of (E)-8-carboxylinalool in grapes.

Figu re 4 4 .Table 3 .

 43 Figu re 4 4 . GC-FID id e n tificatio n o f th e p ro d u cts o f lin alo o l ( a) an d ( E) -8 -h yd ro xylin alo o l ( b) in v it r o co n ve rs io n by th e re co m bin an t CYP76 F10 a n d CYP76 T12 e n zym e s Reactions were carried out using recom binant yeast m icrosom al m em branes. Microsom al m em branes from yeast transform ed with an em pty vector were used as a negative control. Top lanes are authentic standards of m ain reaction products. 1-(E)-8hydroxylinalool, 3 -(Z)-8-hydroxylinalool, 4 -(E)-8-oxo-6,7-dihydrolinalool, 5-(E)-8-hydroxy-6,7dihydrolinalool. 4 and 5 are side products of yeast metabolism of (E)-8-oxolinalool.

(

  Figure 14). Reactions were carried out in 50 0 NjL reaction volum e in closed glass vials in 20 m M Na + / K + phosphate buffer (pH 7.4) containing 20 0 NjM of substrate and 5 % vol. of the m icrosom al preparation. After 30 m in at 27°C, the reaction was stopped and extracted with a m ixture of pentane and ethyl acetate (1:1 vol.), and the extract analyzed using GC-MS.

Figu re 4 7 .

 7 Figu re 4 7. Mo n o te rp e n e o xid atio n catalyze d by CYP71AT7. Five olefinic m onoterpenes (top row) were tested as substrates of the yeast-expressed CYP71AT7. Two of them were oxygenated as shown.

Figu re 4 8 .Figu re 4 9 .

 89 Figu re 4 8 . GC-MS p ro file s o f th e p ro d u cts o f th e in v it r o co n ve rs io n o f Į-p in e n e ( a) an d p -cym e n e by ye as t-e xp re s s e d CYP71AT7 ( b) . Reaction in the absence of NADPH was used as a negative control. Chrom atogram s show the relative abundance of selected m / z 93+67 ( a) and 135 ( b) . Products were tentatively identified on the basis of their m ass spectra (Figu re 16 ). S -substrate, 7 -2,3-epoxypinane, 8p-cym en-8-ol , 9 -cum inol.

Figu re 50 .

 50 Figu re 50 . Cyto ch ro m e P4 5 0 ge n e s in d u ce d d u rin g rip e n in g o f Ge w u rztram in e r be rrie s . Gene expression of cytochrom e P450 genes was evaluated with RNAseq on green and m id ripe Gewurztram iner grape berries. Heatmap displays cytochrom e P450 genes with higher expression in m id ripe berries com pared to green berries and with expression exceeding 5 fragm ents per kilobase of transcript per m illion m apped reads (FPKM).

Figu re 5 1 .

 1 Figu re 5 1. Align m e n t o f tran s late d CYP8 2 D 13 s e qu e n ce s fro m d iffe re n t V. v in ife r a varie tie s . CYP82D13 was cloned from berry skin cDNA of different grapevine varieties and wild grapevine V. sy lvestris subsp. sy lvestris. The hem e-binding cysteine residue is labeled with an arrow.

Figu re 5 2 .

 2 Figu re 5 2 . CO-d iffe re n ce s p e ctra o f re d u ce d ye as t m icro s o m e s e xp re s s in g th e s e le cte d cyto ch ro m e P4 5 0 can d id ate s fro m CYP8 0 , CYP8 1, CYP8 2 an d CYP8 9 fam ilie s .

  une grande place tant culturelle et qu'économ ique en Europe. Alors que la qualité d'un vin dépend directem ent de ses arôm es, la com position et la form ation de ces derniers est encore m al connue. Une m éta-analyse des données bibliographiques nous a perm is de m ontrer que les arôm es du raisin et du vin sont riches en m onoterpènes, en particulier en dérivés du linalool. Les récentes avancées dans la com préhension du m étabolism e oxydatif du linalool et du géraniol ont ensuite été résum ées dans une revue bibliographique. L'annotation détaillée de la superfam ille des cytochrom es P450 dans le génom e de la vigne nous a perm is d'étudier sa structure génétique, sa phylogénie et son expression, m ais aussi d'identifier des gènes dont l'expression est activée dans le grain à m aturité, lors de la synthèse de nom breux com posés arom atiques. La lactone du vin est la m olécule dont le seuil de détection olfactive est le plus bas, ce qui en fait un com posant essentiel de l'arôm e du vin. Nous avons pu dém ontrer que cette lactone se form e au cours du vieillissem ent du vin par une réaction lente et non-enzym atique à partir du 8 -carboxylinalool. L'accum ulation de ce dernier dans la baie est concom itante à l'expression de plusieurs P450 s, dont CYP76F14 est le plus fortem ent exprim é. L'activité des candidats a été évaluée in vitro et in planta. Trois enzym es catalysent des étapes d'oxydation conduisant du linalool au (E)-8-carboxylinalool, m ais seul CYP76F14 catalyse efficacem ent la form ation de l'acide. Tant par son activité catalytique que son profil d'expression, CYP76F14 apparaît donc com m e le responsable le plus probable de la form ation du précurseur de la lactone du vin. Ce travail établit une base solide pour l'étude des autres P450 s im pliqués dans la form ation des arôm es du vin ainsi que dans d'autres processus biologiques tels que le développem ent du fruit ou les interactions plante-pathogène chez la vigne. Mots-clés : arô m e s , m o n o te rpe n o l, Vitis vin ife ra, cyto ch ro m e P4 5 0 , a n n o tatio n .

Keywords: a ro

  m a, m o n o te rpe n o l, Vitis vin ife ra, cyto ch ro m e P4 5 0 , ge n e an n o tatio n .

Table 1 . Pu blicatio n s s e le cte d fo r th e an alys is .

 1

	Publication	Sam ple type	Num ber of	Genotypes/ Varieties
			sam ples
	(58)	Wine	1	Karaoglan
	(59)	Wine	5	Verdejo
	(60 )	Wine	2	Narince
	(61)	Wine	2	Negroam aro
	(62)	Wine	5	Lourerio, Alvarinho
	(63)	Wine	1	Sangiovese
	(64)	Wine	5	Bobal
	(65)	Wine	5	Moravia Agria
	(66)	Wine	3	Moravia Dulce, Rojal, Tortosi
	(67)	Grape	1	Tannat
	(68)	Grape	6	F3P30 , IASMA ECO3, F3P63, Riesling,
				Gewürztram iner, Moscato Rosa*
	(69)	Grape	2	Muscat of Frontignan
	(70 )	Grape	5	Mencía, Espadeiro, Caíño Redondo, Pedral, Sousón
	(71)	Grape	2	Muscat Ham burg
	(72)	Grape	14	Alvarinho, Arinto, Avesso, Azal, Batoca, Lourerio,
				Trajadura
	(73)	Grape	1	Fiano
	(74)	Grape	5	Sauvignon Blanc, Chardonnay, Gewürztram iner,
				Grüner Veltliner, Sangiovese
	(75)	Grape	2	Superior Seedless
	(76)	Grape	15	Albarinho, Listan, Treixadura, Greanche,
				Parellada, Tempranillo, Viura, Xarel•lo
	(77)	Grape	(3)	Vitis rotundifolia (Muscadine) **
	*Non-vinifera genotypes (Nero, Isabella, Vitis arizonica and Vitis cinerea) were excuded from the quantitative
	analysis. **Excluded from the quantitative analysis.

Table S1 . Lis t o f grap e an d w in e vo latile s re p o rte d in at le as t th re e s tu d ie s w ith m e an re te n tio n in d ice s .

 S1

		Isovaleric acid	1659	Dodecanoic acid	2447
	Benzenoids	Pentanoic acid	170 5	Tetradecanoic acid (m yristic acid)	2669
		Hexanoic acid Benzaldehyde	1835 1519	Benzyl alcohol
		Phenylacetaldehyde	1639	2-phenylethanol
	Monoterpenes	Com pound Acetophenone	RI 1667	Com pound Benzenepropanol	RI 20
	Lim onene Ethylbenzaldehyde Methanol trans-linalool oxide (F) Benzyl acetate 1-propanol cis-linalool oxide (F) Ethyl benzene acetate 2-m ethyl-propanol (isobutanol) Aliphatic alcohols Linalool 2-phenyl ethyl acetate 1-butanol Terpinen-4-ol 2-m ethyl-1-butanol Hotrienol N itrogen-containing volatiles 3-m ethyl-1-butanol (isoam yl alcohol) alpha-terpineol N-ethyl-benzam ine 1-pentanol 3-Methyl-3-buten-1-ol 2-heptanol 4-m ethyl-1-pentanol trans-linalool oxide (P) Citral/ (E)-geranial Other volatiles Citronellol gam m a-butyrolactone cis-linalool oxide (P) Furaneol	120 2 1728 879 1447 1735 10 45 1476 1782 10 85 1547 180 9 1146 1599 1212 1624 1213 1697 1750 1249 1252 1318 1319 1727 1743 1764 1628 1766 20 23	Nerol Ethyl cinnam ate Geraniol Benzoic acid 1-hexanol exo-2-hydroxy-1,8-cineole Benzylacetic acid (E)-3-hexen-1-ol 7-hydroxylinalool (diendiol I) Cinnam ic acid 3-ethoxy-1-propanol 7-hydroxy-6,7-dihydrolinalool (Z)-3-hexen-1-ol (E)-2-hexen-1-ol 6-hydroxylinalool (diendiol II) 8-hydroxy-6,7-dihydrolinalool N-(2-phenylethyl)-acetam ide (Z)-2-hexen-1-ol 1-octen-3-ol 1-heptanol 2-ethyl-1-hexanol 2,3-butanediol (Z)-8-hydroxylinalool (E)-8-hydroxylinalool Geranic acid 2,3-dihydrobenzofuran p-m enth-1-ene-7,8-diol	1811 1846 1851 250 1950 30 1988 2152 140 2217 2274 230 4 2317 250 2
	N orisoprenoids	(Z)-2-penten-1-ol	1321	1-octanol
		3-m ethyl-1-pentanol 4-Oxo-isophorone	1326 170 3	Dehydrovom ifoliol	2554
	beta-damascenone 3-hydroxy-2-butanone (acetoin) Aliphatic ketones 3,4-dihydro-3-oxo-actinidol I 3,4-dihydro-3-oxo-actinidol III	180 4 2382 1284 2412	3-Oxo-alpha-ionol 3-Hydroxy-7,8-dihydro-beta-ionol 3-Hydroxy-7,8-dehydro-beta-ionol	2617 2684 2732
	Aliphatic aldehy des 3,4-Dihydro-3-oxo-actinidol II	2420	Vom ifoliol	3148
	Hexanal 3-Hydroxy-beta-dam ascone	10 95 2553	(E)-2-hexenal
	Sulfur-containing volatiles
	Aliphatic esters 3(2H)-2-m ethyldihydro-thiophenone	150 5	3-Methylthiopropanoic acid	1757
	Ethyl acetate 3-(m ethylthio)-1-propanol (m ethionol)	836 1719	Ethyl 3-hydroxybutanoate 1,2-Benzothiazole	150 1899
		Isobutyl acetate	10 12	Diethyl m alonate
	Volatile phenols	Ethyl butanoate	10 55	Methyl decanoate
		Ethyl 3-m ethylbutanoate Methyl salicylate	10 70 1779	Ethyl decanoate 4-Methoxyphenylethyl alcohol	230 2
	Butyl acetate Guaiacol (2-m ethoxyphenol)	10 70 1869	Diethyl succinate 4-vinylphenol	2388
		Isoam yl acetate Phenol	1134 1982	Ethyl m ethyl succinate (E)-4-Allylsyringol	2424
		Ethyl hexanoate 4-Ethylguaiacol	1218 20 55	Methyl 4-hydroxybutyrate Vanillin	2550
	Hexyl acetate 4-m ethyl phenol (p-cresol)	1285 20 87	Ethyl 4-hydroxybutyrate Methyl vanillate	2568
	Ethyl lactate 3-m ethyl phenol (m -cresol)	1336 2119	Ethyl dodecanoate Acetovanillone (Apocynin)	2654
		Ethyl octanoate Eugenol	1437 2181	Diethyl m alate Ethyl vanillate	20 2676
	2-Hydroxy 2-m ethylpropyl butyrate 4-ethylphenol	1461 2187	Ethyl succinate 3,4-dim ethoxyphenol	2756
		p-vinylguaiacol	220 0	Zingerone	2796
	Aliphatic acids 4-Hydroxy-2-m ethyl acetophenone	2212	Tyrosol	30 0 8
	Acetic acid Syringol (2,6-dim ethoxyphenol)	1440 2243	(E)-2-hexenoic acid Methyl vanillyl eter	30 30
		Propanoic acid Isoeugenol	1524 230 2	Octanoic acid 3,4,5-trim ethoxyphenol	20 30 32
	2-m ethylpropanoic acid (isobutyric acid) Methyl salicylate	1579 1779	Nonanoic acid 4-Methoxyphenylethyl alcohol	230 2
	Butanoic acid (butyric acid) Guaiacol (2-m ethoxyphenol)	1617 1869	Decanoic acid 4-vinylphenol	2388

Table S2 . Ke y w in e o d o ran ts .

 S2

Table S3 . Param e te rs o f lin e ar re gre s s io n d e s cribin g re latio n s h ip s be tw e e n lin alo o l an d e ach o f its o xyge n ate d d e rivative s ( ln [ D e rivative ] = a•ln [ Lin alo o l] + b) . Co m p o u n d Mw b a R 2 p -valu e

 S3

			Guaiacol		4-vinylphenol
	Chapter 2		p-vinylguaiacol		Isoeugenol
			Vanillin		Syringol
	Linalool		4-ethylphenol 154.25	0 .0 0	Ethyl phenylacetate 1.0 0
	Benzenoids trans-linalool oxide (F)		Ethyl cinnam ate 170 .25	-2.0 8	0 .55	0 .56	Ethyl dihydrocinnam ate 2.35E-12
	Included key wine odorants Phenethyl acetate trans-linalool oxide (P) 170 .25	Potential key wine odorants -1.69 0 .59	0 .57	Overlooked key wine 2.62E-11
	cis-linalool oxide (P)	Phenylethyl alcohol 170 .25	-2.28	0 .57	0 .48	odorants 1.48E-0 8
	Aliphatic 7-hydroxylinalool alcohols N itrogen-6-hydroxylinalool containing volatiles 8-hydroxy-6,7-dihydrolinalool Others (Z)-8-hydroxylinalool Aliphatic ketones (E)-8-hydroxylinalool Aliphatic aldehy des cis-linalool oxide (F) Hotrienol	(Z)-3-hexen-1-ol Phenylethanal 170 .25 1-hexanol 170 .25 172.26 Isobutanol Furaneol 170 .25 Isoam yl alcohol Acetoin 170 .25 170 .25 152.23	-0 .34 -1.84 -2.76 -0 .94 -0 .57 -2.16 -2.69	2-m ethyl-1-butanol 0 .82 0 .71 1-butanol 0 .44 0 .59 0 .49 (E)-2-hexenal 0 .42 Hexanal 0 .50	0 .60 0 .67 0 .45 0 .68 0 .51 0 .34 0 .34	8.86E-10 2-m ethoxy-3-(2-3.18E-0 7 m ethylpropyl)pyrazine 3.71E-0 5 1,1-diethoxyethane 3.72E-11 Sotolon 1.11E-0 7 Butane-2,3-dione gam m a-nonalactone 5.0 5E-0 7 Acetaldehyde gam m a-decalactone 7.10 E-0 4
	Aliphatic acids	2-m ethylpropanoic acid		Pentanoic acid		(Z)-oak lactone
			Decanoic acid				gam m a-dodecalactone
			Octanoic acid Propanoic acid				gam m a-(Z)-6-dodecenolactone
			Hexanoic acid
			Acetic acid
			Butanoic acid
			Isovaleric acid
	Aliphatic esters		Ethyl acetate		Butyl acetate		ethyl 2-and 3-
							m ethylbutanoate
			Ethyl decanoate		Hexyl acetate
			Ethyl butanoate
			Ethyl hexanoate
			Ethyl octanoate
			Isoam yl acetate
		Ethyl 3-m ethylbutanoate
	Monoterpenes		Geraniol		alpha-terpineol		Wine lactone
			Linalool		Lim onene		cis-rose oxide
	N orisoprenoids		beta-damascenone				beta-ionone
	Sulfur-		Methionol			Ethyl 2-m ethylpropanoate
	containing volatiles						Dim ethyl sulfide
							2-m ethylfuran-3-thiol
							3-sulfanyl-1-hexanol
							3-sulfanylhexyl acetate
						4-m ethyl-4-sulfanylpentan-
							2-one
							Benzenem ethanethiol
	Volatile phenols		4-Ethylguaiacol		m -cresol
			Eugenol		p-cresol

Table 1 . Lin alo o l d e rivative s in fru its an d o th e r a gricu ltu rally im

 1

	Plan t	5-hydroxylinalool	6-hydroxylinalool	7-hydroxylinalool	6,7-epoxylinalool	6,7-dihydroxylinalool	8-hydroxylinalool	9-hydroxylinalool	8-carboxylinalool	Wine lactone	trans-linalool oxide (5)	cis-linalool oxide (5)	trans linalool oxide (6)	cis -linalool oxide (6)	Anhydrolinalool oxide (5)	Hotrienol	7-hydroxydihydrolinalool	Rose oxide	Re f.
	Actinidia arguta (baby kiwi)			B			B B											(65)
	Cam ellia sinensis (tea)										F	F	F	F					(66-68)
											B	B	B	B
	Carica papay a (papaya)		F F	F F	F	F			F	F	F F		B			(69, 70 )
				B		B	B	B			B	B
	Chichorium endivia (endive)										F F							(71)
	Citrus paradisi (grapefruit)									F									(72)
	Coffea arabica (coffee)										F F			F				(73)
	Cy m bopogon (Citronelle)																	F (74)
	Ficus carica (fig tree)										F F F F					(75)
	Solanum ly copersicum (tom ato)										B B F			B			(76, 77)
	Mangifera indica (Mango)						F				B B							(78, 79)
							B
	Orange (Citrus sinensis)						F			F F								(80 , 81)
	Passiflora (Passionfruit)		B B			B B											(82)
	Prunus arm eniaca (Apricot)		B				B				B B B B			B		(83, 84)
	Prunus dom estica (Yellow Plum )						B												(83)
	Prunus persica (Peach)						B						B B					(83)
	Ribes nigrum (Blackcurrant)										F							F (85)
											B
	Vitis vinifera (Grapevine)	B F	F		F B		B		F	F	F	F		F		F (62, 86-
			B	B							B	B	B	B					90 )
	Zingiber officinale (ginger)						B												(91)

p o rtan t p lan t s p e cie s ( F: fre e , B: bo u n d ) .

  in the 1970 s, geraniol and nerol were shown to be alm ost exclusively oxidized at the position 8 by a m onooxygenase activity in Catharanthus roseus (Vinca rosea at that tim e)

	8-
	hydroxylation of geraniol (137). Other m em bers of the CYP76 fam ily were subsequently
	found to catalyze oxidation of geraniol or nerol such as CYP76C4 from A. thaliana, catalyzing
	both the geraniol to 8 -and 9-hydroxylations, and CYP76B1 from Helianthus tuberosus,
	catalyzing nerol hydroxylation (14). In the sam e study, other CYP76 m em bers from A.

extract fraction to form 8-hydroxygeraniol (also som etim es referred as 10 -hydroxygeraniol)

(130 , 131)

. The first characterized plant P450 showing the 2,3-or 6,7-epoxidation of geraniol and nerol in vitro, CYP71A1, was isolated from the avocado fruit (132), and a hom olog CYP71 from catm int was found to catalyze the 8-hydroxylation of geraniol and nerol (133), although in both cases no further experim ents confirm ed the physiological functions of the enzym es in planta. Later, investigations focused on the first oxidative step in the pathway leading from geraniol to MIAs led to the characterization of CYP76B6 as a geraniol hydroxylase in C. roseus, and this activity was found related to the accum ulation of alkaloids in Apocynaceae (134). Recently, a m ore thorough functional analysis of CYP76B6 catalytic properties dem onstrated that this P450 catalyzed two successive regio-specific oxidations at the C8 position of geraniol to form the derived aldehyde

(14)

. This activity is likely widespread in the plant kingdom , as sim ilar oxidized geraniol derivatives have been reported in tobacco (a plant that does not produce geraniol) upon expression of geraniol synthases

(14, 135)

. The strong induction of the CYP76B6 gene expression by the horm one m ethyl-jasm onate, as well as its co-expression with the other MIA genes in the phloem -associated parenchym a confirm ed that it was the best candidate for catalyzing the geraniol oxidation step toward iridoids and MIAs in C. roseus (136). Sim ilarly, in the iridoid producing plant Sw ertia m ussotii, the m ethyl jasm onate-induced CYP76B10 was found to catalyze the thaliana were shown to m etabolize other m onoterpenols. This suggested that other m em bers of the CYP76 fam ily m ight be involved in m onoterpenol m etabolism .

Table S4 . Lis t o f P4 50 fa m ilie s w ith m ajo rity o f its m e m be rs gro u p e d in p h ys ical clu s te rs .

 S4

	Fam ily	Fam ily size (total sequences)	Num ber m em bers of clustered	Exam ples of functions in other organism s
	CYP82	69	66	Biosynthesis of hom oterpenes in A. thaliana (20 ), opioids in Papaver
				som niferum (75-77).
	CYP71	51	45	Biosynthesis of m onoterpenoids in m int species Mentha x piperita and
				Mentha x spicata (78), cyanogenic glucosides in cassava (Manihot
				esculenta) (79), furanocoum arins in several species (80 ), artem isinin in
				Artem isia annua (81), flavon oids in soybean (Gly cine m ax). (40 )
	CYP81	50	42	Biosynthesis of indole glucosinolates in A. thaliana (82), isoflavonoid
				phytoalexins in Medicago truncatula, G. echinata and, Lotus japonicus
				(40 ), sesam in in Sesam um spp. (83)
	CYP76	42	42	Biosynthesis of m onoterpene volatiles in A. thaliana (84), m onoterpene
				indole alkaloids in Catharanthus roseus (28, 85), sesquiterpene
				volatiles in sandalwood (Santalum album ) (86), phytoalexins in rice
				(Ory za sativa) (25, 26), tanshinones in Chinese sage (Salvia
				m iltiorrhiza)(87), pigm ent betalain in beetroot (Beta vulgaris)
				(88).Metabolism of xenobiotics in A. thaliana (27).
	CYP72	36	33	Biosynthesis of m onoterpene indole alkaloids in C. roseus (28, 89),
				glycyrrhizin in licorice (Gly cy rrhiza) (90 ), saponins in M. trucantula
				(91).
	CYP79	26	25	Biosynthesis of glucosinolates in A. thaliana (22, 92, 93), cyanogenic
				glucosides in cassava (M. esculenta) (94).
	CYP89	25	21	Chlorophyll degradation in A. thaliana (95).
	CYP75	24	23	Biosynthesis of flavonoids in Petunia x hy brida, A. thaliana, Gentiana
				triflora, C. roseus, etc. (40 )
	CYP716	23	12	Biosynthesis of saponins in M. trucantula (96) and Maesa lanceolata
				(97).
	CYP70 6	21	19	Biosyntheis of sesquiterpenoids in cotton (Gossy pium arboreum ) (98).
	CYP8 7	20	15	Biosynthesis of saponins in Maesa lanceolata (97).
	CYP714	16	13	Degradation of horm ones (gibberelin) in rice (O. sativa) (99).
	CYP736	13	11	Unknown. Pathogen response in grapevine V. vinifera (43).
	CYP728	11	9	Unknown.
	CYP80	10	9	Alkaloid biosynthesis in barberry (Berberis stolonifera) (10 0 ) and
				California poppy (Eschscholzia californica) (10 1).
	CYP96	9	9	Biosynthesis of cuticular wax in A. thaliana (10 2).
	CYP721	8	8	Unknown.
	CYP74	7	7	Biosynthesis of horm ones (jasm onates) and C6 volatiles in A. thaliana
				and other plants (10 3-10 5).
	CYP92	7	5	Unknown.
	CYP93	7	6	Biosynthesis of flavonoids in soybean (G. m ax), Gly cy rrhiza echinata,
				Gerbera hy brid, Antirrhinum m ajus, Torrenia hy brid, etc. (40 )
	CYP712	6	5	Unknown.

Table S5 . D e s crip tio n o f RN A-Se q e xpe rim e n ts u s e d fo r an alys is o f ge n e e xp re s s io n .

 S5

	Ge n o typ e Chapter 6	Tis s u e	Co n d itio n s	Re fe re n ce
	Carignan CYP76 F14 catalyze Leaves	Infection with powdery m ildew pathogen Ery siphe	(70 )
			nectator
	Pinot noir	Leaves	Infection with downy m ildew pathogen	(52)
			Plasm opara viticola
	Touriga Nacional	Flowers	Developm ent	(71)
	Tannat	Berries	Developm ent	(42)
	Shiraz	Berries	Developm ent	(53)
	Corvina	Berries	Developm ent	(55)
	4 cultivars	Berries	Developm ent	Unpublished

s bio s yn th e s is o f w in e lacto n e p re cu rs o r fro m lin alo o l

  Tina Ilc 1 , Laurence Miesch 2 , Florian Lauvoisard 2 , Lucie Kriegshauser 1 , Andrea Ilg 3 , Eric Duchêne 3 , Philippe Hugueney 3 , Danièle Werck-Reichhart 1 , Nicolas Navrot 1 1 Institute of Plant Molecular Biology, Centre National de la Recherche Scientifique,

	University of Strasbourg, Strasbourg, France
	2 Laboratoire de Chim ie Organique Synthétique, Centre National de la Recherche Scientifique
	University of Strasbourg, Strasbourg, France

3

Laboratoire Métabolism e Secondaire de la Vigne, Institut National de la Recherche Agronom ique, University of Strasbourg, Colm ar, France

Table 4 . Prim e rs u s e d fo r am p lificatio n o f th e CYP8 0 , CYP8 1, CYP8 2 an d CYP9 8 can d id ate s .

 4

	Restriction sites are underlined.
	Gene	Forward prim er	Reverse prim er
	CYP8 0 E3	TAGGTACC ATGGTAGCCATGTTAGCAGAAG	TA GGTACC TCATTTTCGAACTTTAGGAATGAT
		GAAC	GAG
	CYP81Q19	ATAGGATCC ATGGAAGCTAGATGGCTATACT	TA GGTACC TCAGACACTGTTCAGAATTTTGGA
		CATC	TG
	CYP82D13	GGATCCATGGATTTCCTTCTCCAATGCCTAA	GGTACC CTACTTATAAAGATTATAAGAAGCTAA
		AC	GCGTGGG
	CYP89A41	ATAGGATCC ATGGAGATATGGGTCTTCTTCT	TA
		TCATC

Ackn o w le d ge m e n ts

We thank J ulien Denos for helpful discussions on data analysis.

Au th o rs ' co n tribu tio n s

Investigation, T.I.; Form al Analysis, T.I.; Writing -Original Draft, T.I.; Writing -Review & Editing, N.N and D.W-R.; Visualization, T.I.; Supervision, N.N.and D.W-R.; Project adm inistration, D.W-R.; Funding Acquisition, D.W-R.

Ackn o w le d ge m e n ts

We thank David R Nelson for assigning nam es to newly discovered sequences and updating the nam es for previously known sequences; and Adrian Arellano Davin for editing the CYP76 phylogenetic tree.

Ackn o w le d ge m e n ts

We thank Gisèle Butterlin (INRA, Colm ar) for help with grape berry sam pling and RNA preparation and Grégory Lem arquis (INRA, Colm ar) for providing Gewurztram iner win e sam ples.

Acce s s io n N u m be rs

Cytochrom e P450 annotation will be subm itted to public databases.

Au th o rs ' co n tribu tio n s

TI contributed to m anual curation and phylogenetic analysis, perform ed an expert check of the m anual curation and gene classification, and wrote the m anuscript. GA contributed to m anual curation, perform ed the analysis of gene expression and wrote the m anuscript. RT perform ed the phylogenetic analysis. NN contributed to m anual curation and wrote the m anuscript. FC and EP participated in the study design. MF contributed to m anual curation.

PH and DWR acquired funding, contributed to study design and supervision and edited the m anuscript. CR contributed to study design, perform ed the analysis of structural organization, coordinated the work and wrote the m anuscript.

Su p p le m e n tal in fo rm atio n

Kin e tics o f lin a lo o l o xid atio n by CYP76 F14

Kinetics of cytochrom e P450 reactions can usually be m odeled with the Michaelis-Menten equation (4). To evaluate the efficiency of the CYP76F14 enzym e we m onitored its reaction kinetics in vitro. Reaction m ixtures containing 0 .0 0 9 NjM of the CYP76F14 enzym e and 0 .5 m M NADPH were prepared in 20 m M K + / Na + phosphate buffer. Enzym e concentration was estim ated from its differential CO-spectrum . For the lowest substrate concentration the am ount of enzym e was halved to avoid excessive substrate consum ption. Reaction was started with addition of (S)-linalool at varying concentrations. Reaction m ixtures were incubated with agitation at 27°C for 30 m in in closed glass test tubes to avoid sam ple evaporation. Reaction was stopped with the addition of 50 0 NjL ethyl acetate and im m ediately extracted by vortexing. Ethyl acetate extracts were dried over Na 2 SO 4 and injected to GC-FID.

Experim ental data were fitted to Michaelis-Menten equation using the R software. Velocity was divided by enzym e concentration because the latter was not constant for all substrate concentrations. The kinetic param eters of linalool oxidation were k cat = (1.11 ± 0 .6) s -1 and K m = (22 ± 3) NjM. The K m is com parable to those obtained for the CYP76 enzym es from A. thaliana (5), whereas the k cat is lower. However, the value of k cat has to be considered with care due to the inaccurate determ ination of enzym e concentration, resulting from the low peak intensity in the CO-difference spectrum . Furtherm ore, because CYP76F14 catalyses three sequential oxidations of linalool to (E)-8-carboxylinalool we m onitored substrate depletion, and not product form ation. This com prom ised the reliability of results because it forced us to work at relatively long reaction tim es to reduce the error in substrate consum ption, whereas the Michaelis-Menten kinetic m odel best describes the initial reaction rate when substrate concentration rem ains approxim ately constant ([S] § [S] 0 ). In addition enzym e products com pete with linalool for binding to the enzym e.

Appendix I

Exp re s s io n o f V it is v in ife r a cyto ch ro m e P4 5 0 s e qu e n ce s

Gene expression data was retrieved from publically available RNA-Seq datasets and 8 unpublished datasets (Ch ap te r 5 ). Datasets were divided into 5 categories and average gene expression (FPKM) was calculated for each category. Expression clusters correspond to the heatm ap in Ch ap te r 5 , Figure 5. Abbreviations: "Chr"chrom osom e, "Or" -orientation, "par" -partial gene, "ps" -pseudogene, "ps?" -putative pseudogene.

Appendix II

D N A an d p ro te in s e qu e n ce align m e n ts o f ge n e can d id a te s fo r ( E) -8 -ca rbo xylin alo o l bio s yn th e s is

Gene candidates for (E)-8-carboxylinalool biosynthesis (CYP71AT7, CYP76F12, CYP76F14, CYP76T21, CYP76Y1 and CYP76Y2) were am plified from Gewurztram iner (Gw) and Muscat Ottonel (Mo) grape skin cDNA. Their sequences were aligned to the sequence from the reference grapevine genom e PN40 0 24 (Pn) using ClustalW.

Differences between the varieties are highlighted with light gray.