# New insights into the adaptation of yeast to wine making using comparative genomics

Jean-Luc Legras, Anna Lisa Coi, Frederic Bigey, Virginie Galeote, Souhir Marsit, Arnaud Couloux, Julie Guy, Ricardo Franco-Duarte, Dorit Schuller, José Paulo Sampaio, et al.

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## To cite this version:

Jean-Luc Legras, Anna Lisa Coi, Frederic Bigey, Virginie Galeote, Souhir Marsit, et al. New insights into the adaptation of yeast to wine making using comparative genomics. 10. Symposium International d'Œnologie de Bordeaux, Œno2015, 2015, Bordeaux, France. 2015. hal-01606120

HAL Id: hal-01606120

https://hal.science/hal-01606120

Submitted on 3 Jun 2020

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ŒNO2015

Actes de colloque
du 10e symposium international
d'œnologie de Bordeaux

université
de BORDEAUX

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ISVV
INSTITUT DES SCIENCES
DE LA VIGNE ET DU VIN
BORDEAUX AQUITAINE
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The opening of the 10th International Symposium of Enology took place on June 29, 2015 in the presence of :

M. Alain Rousset, President of the Regional Council of New Aquitaine

M. Pr Vincent Dousset, Vice-President of the University of Bordeaux
in charge of international affairs,

representing M. Pr Manuel Tunon de Lara, President of the University of Bordeaux

M. Yann Jestin, official in charge of the vine wine sector,

representing M. Pierre Goguet, President of the Bordeaux Chamber of Commerce and Industry (CCI)

symposium under the patronage of the OIV

L'ouverture du 10e Symposium International d'Œnologie s'est déroulée le 29 juin 2015 en présence de :

M. Alain Rousset, Président du Conseil régional de la Nouvelle Aquitaine

M. Pr Vincent Dousset, Vice Président de l'Université de Bordeaux
en charge des relations internationales,

représentant M. Pr Manuel Tunon de Lara, Président de l'Université de Bordeaux

M. Yann Jestin, élu en charge du secteur vigne vin,

représentant M. Pierre Goguet, Président de la Chambre de commerce et d'industrie (CCI) de Bordeaux

colloque placé sous le patronage de l'OIV
The 10 th International Symposium of Enology was supported by :
Le 10 e Symposium International d'Œnologie a bénéficié du soutien de :

Companies - Compagnies

Biolaffort

Thermoscientific

Bucher Vaslin

Cazaux Rotorflex

Bellot

Demptos

Amorim

AEB France

Lamothe Abiet

DIAM Bouchage

CHR HANSEN

Bayer

Innovin

Suntory Wine International

Lallemand

Public partners - Partenaires publics

IDEX - University of Bordeaux

University of Bordeaux Foundation

Bordeaux Chamber of Commerce and Industry

Regional Council of New Aquitaine

Bordeaux National Polytechnic Institute

Integrative Biology and Ecology Research Federation

Bordeaux City Hall

Bordeaux Interprofessional Wine Council

Bergerac Interprofessional Wine Council

ODG Saint-Émilion, Pomerol, Graves, Sauternes, Lalande de Pomerol

Domaines André Lurton

Champagnes Mumm

Champagne Louis Roederer

Château Le Bon Pasteur, Pomerol

Château Montrose, Saint-Estèphe

Château Pichon-Comtesse de Lalande, Saint-Julien

Château Fleur La Mothe, Médoc
**Scientific committee - Comité scientifique**

Hervé ALEXANDRE Pr, UMR PAM, IUVV, Université de Bourgogne

Jean-Christophe BARBE MC HDR, Bx Sci Agro, Unité de recherche Œnologie, USC INRA, Université de Bordeaux

Marina BELY MCU HDR, Unité de recherche Œnologie, USC INRA, ISVV, Université de Bordeaux

Christophe CLEMENT Pr, EA Vignes Vins de Champagne, Université Reims Champagne-Ardennes

Philippe DARRIET Pr, Unité de recherche Œnologie, EA 4577, USC 1366 INRA, ISVV, Université de Bordeaux, président

Serge DELROT Pr, UMR EGFV, ISVV, Université de Bordeaux

Gilles de REVEL Pr, Unité de recherche Œnologie, USC INRA, ISVV, Université de Bordeaux

Dr Hélène FULCRAND DR, INRA, UMR Sciences pour l'Œnologie, Montpellier SupAgro

Laurence GENY MCU HDR, Unité de recherche Œnologie, USC INRA, ISVV, Université de Bordeaux

Eric GIRAUD HERAUD DR, INRA, Unité de recherche Œnologie, USC INRA et UMR GREThA, ISVV,
Université de Bordeaux

Eric GOMES Pr, UMR EGFV, ISVV, Université de Bordeaux

Régis GOUGEON Pr, UMR PAM, IUVV, Université de Bourgogne

Claire Le HENAFF-Le MARREC Pr, ENSCBP, Unité de recherche Œnologie, USC INRA, Bx INP, Université de Bordeaux

Patrick LUCAS Pr, Unité de recherche Œnologie, ISVV, Université de Bordeaux

Isabelle MASNEUF-POMAREDE Pr, Bx Sci Agro, Unité de recherche Œnologie, USC INRA, ISVV, Université de Bordeaux

Jean-Michel MERILLON Pr, Unité de recherche Œnologie, ISVV, Université de Bordeaux

Martine MIETTON-PEUCHOT Pr, Unité de recherche Œnologie, USC INRA, ISVV, Université de Bordeaux

Jean-Marie SABLAYROLLES DR, INRA, UMR SPO, Montpellier SupAgro

Patricia TAILLANDIER Pr, UMR 5503, Laboratoire de Génie Chimique, Université de Toulouse III, INP Toulouse 3

Pierre-Louis TEISSEDRE Pr, Unité de recherche Œnologie, USC INRA, ISVV, Université de Bordeaux

Kees VAN LEEUWEN Pr, UMR EGFV, Bx Sci Agro, ISVV, Université de Bordeaux

Hélène VELASCO Pr, UMR ADESS, ISVV, Université de Bordeaux 3

**Editorial committee - Comité d'édition**

Warren ALBERTIN MC, ENSCBP, EA Œnologie, USC INRA, ISVV, Bordeaux INP, Université de Bordeaux

Patricia BALLESTRA MCU, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Jean-Christophe BARBE MC, Bx Sci Agro, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Marina BELY MCU HDR, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Philippe DARRIET Pr, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Laurence GENY Pr, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Rémy GHIDOSSI MCU, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Michael JOURDES MCU, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Claire Le HENAFF-Le MARREC Pr, Bordeaux INP, EA Œnologie, ISVV, USC INRA, ISVV, Université de Bordeaux

Patrick LUCAS Pr, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Axel MARCHAL MCU, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Stéphanie MARCHAND-MARION MCU, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Isabelle MASNEUF-POMAREDE Pr, Bx Sci Agro, EA Œnologie, USC INRA, Université de Bordeaux

Jean-Michel MERILLON Pr, EA Œnologie, ISVV, Université de Bordeaux

Pierre-Louis TEISSEDRE Pr, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Nathalie BOUCHER PhD, scientific copy editor, réviseuse scientifique

Marylène PERREAUD, ed. Vigne Vin Publications Internationales

**Organizing committee - Comité d'organisation**

Honorary President - Président Honoraire :
Professeur Denis DUBOURDIEU, Directeur Général, Institut des Sciences de la Vigne et du Vin,
Unité de recherche Œnologie, EA 4577, USC 1366 INRA, Université de Bordeaux
## Members - Membres

Emmanuel BAUGIER, ISVV, Université de Bordeaux, webmaster

Philippe DARRIET, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Laurence GENY, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Rémy GHIDOSSI, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Elodie GUITTARD, ISVV, Université de Bordeaux

Michael JOURDES, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Cécile LEFFET, ISVV, Université de Bordeaux

Patrick LÉGER, ISVV, Université de Bordeaux

Claire Le HENAFF-Le MARREC, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Joëlle LEROY, ISVV, Université de Bordeaux

Patrick LUCAS, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Axel MARCHAL, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Stéphanie MARCHAND-MARION, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Christine MARTY, ISVV, Université de Bordeaux

Pascaline REDON, EA Œnologie, USC INRA, ISVV, Université de Bordeaux

Cécile THIBON, EA Œnologie, USC INRA, ISVV, Université de Bordeaux
# Preface to the 10th International Symposium of Enology

Pr Philippe Darriet

*Director of Enology Research Unit*

EA 4577, USC 1366 Inra, Institute of Vine and Wine Sciences, University of Bordeaux

This edition of the International Symposium of Enology is the 10th meeting of an international event initiated by Pr Jean Ribéreau-Gayon and Dr Émile Peynaud more than 50 years ago, in 1963. At the time, the symposium aimed to celebrate the 100th anniversary of Louis Pasteur's work and to put forward a young science - enology - in order to show its importance in wine quality. After the first edition, the symposium continued in 1967, 1977, 1989 and then in 1995, 1999, 2003, 2007, 2011 under the lead of Pr Pascal Ribéreau-Gayon, then Prs Yves Glories, Aline Lonvaud and Bernard Donèche, with the same philosophy of a general-interest symposium covering the fields of enological science. The research questions have evolved: enological research initially focused on wine clarity and instability problems, then on fermentation control, and more recently on the qualitative potential of fruit and its evolution during vinification and aging, and the parameters of sensory perception.

The Œno2015 edition is consistent with the principle of a general-interest symposium allowing exchanges within a multidisciplinary scientific community dedicated to vine and wine sciences. Thus, the 4 traditional themes - Plant, environment and grape quality, Grape and wine microorganisms, Wine chemistry, Sensory analysis - were the foundation of the 10th Enology Symposium. However, enology, as Emile Peynaud said in « *Le vin et les jours* » (meaning « the wine and the days »), is a science in motion ; as an applied science, it is shaped by developments in basic sciences, as well as in human and social sciences. In order to open up new avenues of research for the next 10 years, we invited research experts from various fields including microbiology (Pr David Mills, University of California), chemistry (Pr Ludovic Julien, Ecole Nationale Supérieure Paris ; Dr Markus Herderich, AWRI), cognitive psychology (Dr Wendy Parr, University of Lincoln NZ), neurosciences (Dr Gabriel Lepousez, Institut Pasteur Paris) and experimental economics (Dr Pierre Combris, INRA Evry) ; enological research should focus not only on the product and its components, but also on the taster and his/her perception, and the consumer and his/her act of purchase.

The 10th Enology Symposium brought together nearly 200 presentations in the 4 traditional themes. This included specific workshops (short oral presentations) on grape and wine microorganisms, oxygen, sensory impact compounds, grape quality parameters and new processes, and more than 80 poster presentations.

Focusing on some of the highlights of the 10th Enology Symposium, it is worth mentioning the important proportion of interdisciplinary research :

- In the field of vine sciences, several studies have shown the essential contribution of genomic, transcriptomic, proteomic and metabolomic approaches to the study of vine ecophysiology parameters, and even sensory analysis.

- In the field of microbiology, inputs linked to the spectacular development of genomics in microbial ecosystem analysis and in the study of the metabolism of grape and wine microorganisms allow, along with directed evolution, to consider new perspectives for yeast and bacterial strains adapted to wine making requirements.

- In the field of chemistry and processes, several studies were carried out on the transfer of oxygen through stoppers and barrels and its impact on wine components. Research work in analytical chemistry using global (metabolomics) or targeted approaches and ever more sensitive and precise mass detectors (high resolution), often related to sensory aspects of wines, was strongly present during Œno2015, such as the interactions between tannins and macromolecules, sometimes revealing new health-promoting properties in wine.

- Finally, in the field of sensory analysis coupled to cognitive psychology, neuroscience and experimental economics, research now takes into account not only the characteristics of the product, but also the tasting conditions and the psychology and purchasing decisions of the taster.

The 10th International Symposium of Enology, Œno2015, was held thanks to financial support from public partners, private companies, wine interprofessional associations and wine producers who contributed to the sustainability of this scientific event and to whom the Enology research unit, within the ISVV, wishes to express its gratitude.

Sincere thanks also to :
- The members of the scientific committee, from the ISVV and various French research centers in Montpellier, Dijon, Reims and Toulouse, who helped us select the abstracts ;

- ISVV administrative staff, BIATSS professors/researchers and staff, in particular my colleagues Dr Marina Bely, Dr Stéphanie Marchand-Marion, Dr Michael Jourdes, Dr Axel Marchal, Dr Rémy Ghidossi, Dr Cécile Thibon and Pascaline Redon, who have invested a great deal of time and effort in the organization of Œno2015, as well as Patrick Leger, Elodie Guittard and our webmaster, Emmanuel Baugier. Thanks also to the doctoral students, post-doctoral researchers and BI-ATSS staff of the Enology research unit who participated in the organization of this symposium.

At the end, I would like to pay a brief tribute to Pr Denis Dubourdieu, founder and director general of the Institute of Vine and Wine Sciences Bordeaux-Aquitaine, on July 26, 2016, after a long illness. Pr Denis Dubourdieu, author of more than 200 scientific and technical publications, was a talented scientist. Guided by his empirical observations as winemaker and consultant, he was committed not only to advancing knowledge in enology, but above all to preserving and enhancing the quality of wines. « The aim of enology », he said, « is not only to determine the composition of wines. Its true role is to explain the natural phenomena involved in the production and aging of wine in order to better guide them ».

The scientific activity of Denis Dubourdieu was that of a researcher deeply involved in the various fields of enology, carrying out studies on the chemistry of aromas and taste compounds, the biochemistry of grapes, the definition of maturity criteria (aromas and aroma precursors, polysaccharides) and the enological impact of *Botrytis cinerea* as an agent of gray rot or noble rot. He also provided insights in yeast microbiology (characterization of strains and species of *Saccharomyces* by molecular biology techniques, role of microorganisms in the sensory quality of wine, and impact of spoilage microorganisms (*Brettanomyces* sp)) and contributed to a better definition of wine quality perceived by the taster. He wrote a beautiful page in the history of enology research and set a course for the future.

My colleagues and I wish you good reading and invite you in 2019 for the 11th International Symposium of Enology.
# Préface au 10e Symposium international d'œnologie

Pr Philippe Darriet

Directeur de l'Unité de recherche Œnologie

EA 4577, USC 1366 Inra Institut des Sciences de la Vigne et du Vin, Université de Bordeaux

Cette édition du Symposium International d'Œnologie est la 10e rencontre d'une manifestation internationale initiée par le Pr Jean Ribéreau-Gayon et le Dr Émile Peynaud, il y a plus de 50 ans, en 1963. Il s'agissait à l'époque de célébrer le centenaire des travaux de Louis Pasteur et de mettre en avant une science encore jeune - l'œnologie - afin de montrer son importance dans la valorisation de la qualité des vins. Après la première édition, le rassemblement s'est poursuivi en 1967, 1977, 1989 puis en 1995, 1999, 2003, 2007, 2011 sous l'impulsion du Pr Pascal Ribéreau-Gayon, puis des Professeurs Yves Glories, Aline Lonvaud et Bernard Donèche, toujours avec la même philosophie d'un colloque généraliste couvrant les champs de la science œnologique. Les questions de recherche ont évolué : au départ, la recherche œnologique s'est penchée sur des problèmes de limpidité et d'instabilité des vins, puis sur la maîtrise des fermentations, avant de s'intéresser au potentiel qualitatif contenu dans le fruit et à son évolution au cours de la vinification et du vieillissement, aux paramètres de la perception sensorielle.

L'édition Œno2015 se positionne toujours avec le principe d'un symposium généraliste permettant des échanges au sein d'une communauté scientifique multidisciplinaire au service des sciences de la vigne et du vin. Ainsi, les quatre thèmes traditionnels - Plante, environnement et qualité du raisin, Microorganismes du raisin et du vin, Chimie du vin, Analyse sensorielle - ont constitué le socle de l'organisation du 10e Symposium d'Œnologie. Cependant, l'œnologie, comme le dit Émile Peynaud dans « Le vin et les jours », est une science en mouvement ; en tant que science appliquée, elle a toujours vocation à être irriguée par le progrès des connaissances en sciences fondamentales, en intégrant aussi les sciences humaines et sociales. Aussi, dans le cadre de cette dixième édition, afin d'ouvrir de nouvelles pistes de recherche pour les dix prochaines années, ont été conviés des chercheurs spécialisés dans le domaine de la microbiologie (Pr David Mills, Université de Californie), de la chimie (Pr Ludovic Julien, École Nationale Supérieure, Paris ; Dr Markus Herderich, AWRI), et aussi dans les champs de la psychologie cognitive (Dr Wendy Parr, Université Lincoln, NZ), des neurosciences (Dr Gabriel Lepousez, Institut Pasteur, Paris), de l'économie expérimentale (Dr Pierre Combris, INRA Evry), la recherche œnologique devant non seulement porter sur le produit dégusté et ses composants, mais également sur le dégustateur et sa perception, le consommateur et son acte d'achat.

Le 10e Symposium d'œnologie a ainsi rassemblé près de deux cents communications dans les quatre thèmes traditionnels présentés. De plus, des séances de travail spécifiques ont été organisées sur les thématiques des microorganismes du raisin et du vin, sur l'oxygène, les composés d'impact sensoriel, les paramètres de qualité des raisins ou les nouveaux procédés dans le cadre de short com. Plus de quatre-vingts communications ont aussi été présentées sous forme d'affiches.

Dressant brièvement quelques faits marquants du 10e Symposium d'œnologie, il convient d'abord de souligner la proportion importante de travaux menés en interaction entre différents champs disciplinaires :

- Dans le domaine des sciences de la vigne, plusieurs travaux ont montré l'apport incontournable des approches de génomique, transcriptomique, protéomique, métabolomique en lien avec des paramètres d'écophysiologie de la vigne et parfois en interaction avec l'analyse sensorielle.

- Dans le domaine de la microbiologie, les apports liés aux développements spectaculaires de la génomique dans l'analyse des écosystèmes, dans l'étude du métabolisme des microorganismes du raisin et des vins permettent, avec l'évolution dirigée, d'envisager de nouvelles perspectives pour les souches de levures et bactéries adaptées aux exigences de l'élaboration du vin.

- Dans le domaine de la chimie et des procédés, plusieurs travaux portaient sur le transfert d'oxygène au travers des obturateurs et des barriques ainsi que sur son impact vis-à-vis des composants du vin. Les travaux en chimie analytique, avec des approches globales (métabolomique) ou ciblées, avec des détecteurs de masse sans cesse plus sensibles et précis (haute résolution), souvent en lien avec les aspects sensoriels dans les vins ont été très représentés au cours d'Œno2015,
comme ceux concernant les interactions entre tanins et macromolécules, révélant parfois dans le vin de nouvelles propriétés hygiéniques.

- Enfin, dans le domaine de l'analyse sensorielle en interaction avec les champs de la psychologie cognitive, des neurosciences et de l'économie expérimentale, les travaux ne prennent plus seulement en compte les caractéristiques du produit dégusté, mais également l'environnement de la perception et la psychologie du dégustateur, l'étude de son acte d'achat.

Le 10e Symposium international d'œnologie, Œno2015, a pu se dérouler grâce à de nombreux soutiens financiers de partenaires public, d'entreprises, interprofessions viticoles, crus, châteaux vinicoles qui ont contribué à pérenniser cette manifestation scientifique et auxquels l'unité de recherche Œnologie, au sein de l'ISVV, témoigne toute sa reconnaissance.

Sont aussi remerciés :

- Les membres du comité scientifique, venant de l'ISVV et de différents centres de recherche français à Montpellier, Dijon, Reims, Toulouse qui nous ont aidés pour la sélection des travaux ;

- Les personnels administratifs de l'ISVV, les enseignant-chercheurs et personnels BIATSS, en particulier mes collègues Dr Marina Bely, Dr Stéphanie Marchand-Marion, Dr Michael Jourdes, Dr Axel Marchal, Dr Rémy Ghidossi, Dr Cécile Thibon, Pascaline Redon qui se sont beaucoup investis dans l'organisation d'Œno2015 ainsi que Patrick Léger et Élodie Guittard et notre webmaster, Emmanuel Baugier. Merci aussi aux doctorants, post-doctorants et personnels BIATSS de l'unité de recherche Œnologie qui ont participé à l'organisation de ce symposium.

J'aimerais également rendre hommage au Professeur Denis Dubourdieu, fondateur et directeur général de l'Institut des Sciences de la Vigne et du Vin Bordeaux-Aquitaine, décédé, le 26 juillet 2016, des suites d'une longue maladie. Le Pr Denis Dubourdieu, auteur de plus de deux cents publications scientifiques et techniques, était un scientifique talentueux. Guidé par ses observations empiriques de vinificateur et de consultant, il était toujours désireux que ses travaux conduisent non seulement à une meilleure connaissance du sujet mais plus encore permettent de préserver et de valoriser la qualité des vins. « L'œnologie n'a pas pour seul but de déterminer la composition des vins. Son véritable rôle est, disait-il, d'expliquer pour mieux orienter les phénomènes naturels intervenant au cours de l'élaboration et du vieillissement des vins ».

L'activité scientifique de Denis Dubourdieu a bien illustré celle d'un chercheur profondément engagé dans les différents champs de l'œnologie ayant mené des travaux sur la chimie des arômes et des composés du goût, en biochimie du raisin autour de la définition de critères de maturité (arômes et leurs précurseurs, polysaccharides), concernant l'impact œnologique lié au développement de *Botrytis cinerea* comme agent de la pourriture grise ou la pourriture noble. Il a aussi apporté des éclairages en microbiologie des levures (définition des souches et espèces de *Saccharomyces* avec les moyens de la biologie moléculaire, rôle des microorganismes dans la qualité sensorielle des vins ou impact de microorganismes d'alération (*Brettanomyces* sp)) et a contribué à une meilleure compréhension de la perception de la qualité du vin dans la conscience du dégustateur. Il a écrit une belle page de l'histoire de la recherche en œnologie et il y a donné un cap.

Nous vous souhaitons une bonne lecture et avec mes collègues vous donnons rendez vous en 2019 pour le 11e symposium international d'Œnologie.
In tribute - En hommage

Honorary President of the 10th International Symposium of Enology
Président Honoraire du 10e Symposium International d'Œnologie

<figure><img src="image_9.png" /><figcaption>Pr Denis Dubourdieu (1er juillet 1949 - 26 juillet 2016)</figcaption></figure>

Director General of the Institute of Vine and Wine Sciences Bordeaux-Aquitaine

Knight of the Legion of Honor - Chevalier de la Légion d'Honneur

Knight of the Order of Agricultural Merit - Chevalier de l'Ordre du Mérite Agricole
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# TABLE DES MATIÈRES

## THÈME 1

### Plante, environnement et qualité du raisin

*Plant, environment and grape quality*

**Terroir effects on grape and wine aroma compounds**
M. Herderich 3

**Untargeted metabolomics to study the influence of scion x rootstock x nitrogen interactions on berry content**
A. Habran, P. Helwi, G. Hilbert, E. Gomès, C. van Leeuwen, S. Negri, M. Commisso, F. Guzzo, S. Delrot 8

**Adaptation to climate change : strategic issues to face for the French wine industry**
N. Ollat, H. Quénol, G. Barbeau, C. Van Leeuwen, P. Darriet, I. Garcia de Cortazar Atauri, B. Bois, H. Ojeda, E. Duchêne, E. Lebon, P. Vivin, P. This, J.-M. Sablayrolles, G. Teil, P. Lagacherie, E. Giraud-Héraud, P. Neveu, J.-M. Touzard 12

**Growing grapes in predicted climate change conditions :**
What can we learn from multiple stress in controlled conditions ?
J. Martinez-Lüscher, F. Morales, G. Hilbert, M. Sánchez-Diaz, J. Aguirreola, S. Delrot, I. Pascual, E. Gomes 17

**Pre- and post-flowering leaf removal as affecting methoxypyrazines, thiol precursors, glutathione and thiols in Sauvignon blanc grapes and wines**
P. Sivilotti, B. Škvarč, L. Butinar, M. Sternad Lemut, H. Baša Česnik, Š. Velikonja Bolta, A. Vanzo, K. Lisjak 20

**Micro-vinification for assessment of the effect of field spatial variability on wine quality**
Y. Kotseridis, E. Kallithraka, M. Kyraleou, S. Fountas, A. Balafoutis, E. Anastasiou, S. Koundouras 24

**Polyphenol transcriptional and metabolic profiling of the astringent grape variety Aglianico (Vitis vinifera L) during berry ripening**
A. Rinaldi, D. Carputo, L. Frusciante, C. Lanzillo, L. Moio, A.J. Tamburrino, C. Villano, R. Aversano 28

**Développement de méthodes sensorielles d'analyse du raisin pour un usage par les professionnels du vin : un outil d'aide à la décision pour le choix des itinéraires œnologiques**
R. Siret, C. Patron, P. Chrétien, L. Guerin, S. Beauchet, C. Coulon-Leroy, R. Symoneaux 33

**Effect of local temperature variability on vine phenology in the Saint-Emilion area (Bordeaux, France)**
L. de Resseguier, H. Quenol, R. Leroux, C. van Leeuwen 40
<table>
  <tbody>
    <tr>
      <td>Influence du Rapport Feuille/Fruit sur le profil sensoriel et la composition aromatique de vins issus du cépage Cabernet franc en Val de Loire<br>J. Béguin, E. Fortin, L. Guérin, R. Symoneaux</td>
      <td>44</td>
    </tr>
    <tr>
      <td>Impact du porte-greffe sur les composés phénoliques du cépage Marselan en climat méditerranéen<br>M. Nadal, A. Sanchez-Ortiz</td>
      <td>48</td>
    </tr>
    <tr>
      <td>Contribution to confirm the existence of a Secondary Domestication Centre of Vitis vinifera subsp. vinifera in the south-east of France (Languedoc area)<br>G. Marsal, J.-M. Mateo-Sanz, J.-M. Canals, F. Zamora, F. Fort</td>
      <td>51</td>
    </tr>
    <tr>
      <td>Effect of trellising system on volatile compounds of red wines in Mediterranean climate<br>M. Kyraleou, S. Kallithraka, S. Koundouras, S. Haroutounian, Y. Kotseridis</td>
      <td>55</td>
    </tr>
    <tr>
      <td>Multidisciplinary approach of the regulation of the biosynthetic pathway of tartrate in Vitis vinifera L. Ugni blanc grape berries<br>C. Cholet, O. Claisse, S. Claverol, A. Rabot, A. Li-Mallet, A. Osowski, V. Dumot, G. Ferrari, L. Lurton, L. Geny</td>
      <td>59</td>
    </tr>
    <tr>
      <td>Etude des facteurs influençant la synthèse de géosmine dans le raisin – Recensement des connaissances acquises et présentation sous la forme d'un modèle conceptuel<br>S. Dupin, L. Guerin, J. Wery</td>
      <td>63</td>
    </tr>
    <tr>
      <td>Application of UV-light in the vineyard to increase the content of phytochemicals in grapes<br>E. Elejalde, I. López-de-Armentia, S. Quintela, M.C. Villarán, S. Arina</td>
      <td>66</td>
    </tr>
    <tr>
      <td>Effect of rootstock on berry and wine composition of new red grapevine cultivars<br>M. Michlovský, A. Khafizova</td>
      <td>69</td>
    </tr>
    <tr>
      <td>Influence de la botrytisation dans la genèse des composés associés à des nuances typiques de l'arôme des vins liquoreux de pourriture noble<br>P. Stamatopoulos, C. Thibon, P. Redon, P. Darriet</td>
      <td>75</td>
    </tr>
    <tr>
      <td>Phenotyping berries from the grapevine Vitis vinifera L. by means of Fourier Transform Infrared Spectroscopy<br>A. Destrac Irvine, C. Renaud, E. Morin, L. Durand, S. Delrot, C. van Leeuwen</td>
      <td>80</td>
    </tr>
    <tr>
      <td>Microscopical and biochemical approaches of modifications induced by pulsed electric field treatments on cell-wall total tannins and polysaccharides of Cabernet Sauvignon grape berry skins<br>C. Cholet, C. Delsart, M. Petrel, E. Gontier, N. Grimi, R. Ghidossi, E. Vorobiev, M. Mieton-Peachot, L. Geny</td>
      <td>84</td>
    </tr>
    <tr>
      <td>Can wine aromas be manipulated by nitrogen applications in the vineyard ?<br>P. Helwi, A. Habran, S. Guillaumie, C. Thibon, G. Hilbert, C. Renaud, E. Gomes, P. Darriet, S. Delrot, C. van Leeuwen</td>
      <td>88</td>
    </tr>
    <tr>
      <td>Adaptation de méthodes d'immunomarquage aux pépins de raisin (Vitis vinifera L. cv. Cabernet-Sauvignon)<br>A. Li-Mallet, M. Petrel, E. Gontier, C. Cholet, A. Rabot, A. Osowski, L. Geny</td>
      <td>92</td>
    </tr>
    <tr>
      <td>Relationship between water and nitrogen status of the vine and aroma compounds potentially involved in the tertiary bouquet of red Bordeaux wines<br>N. Le Menn, M. Picard, C. van Leeuwen, G. de Revel, S. Marchand</td>
      <td>96</td>
    </tr>
    <tr>
      <td>Phenolic contents and antioxidant activity of grape pomace seed and skin edible extracts<br>I. Ky, G. Cros, A. Crozier, P.L. Teissedre</td>
      <td>100</td>
    </tr>
    <tr>
      <td>Tannin composition of Merlot and Cabernet-Sauvignon grapes from seven vineyards of Bordeaux in vintage 2014<br>W. Ma, M. Jourdes, P.L. Teissedre</td>
      <td>107</td>
    </tr>
  </tbody>
</table>
# THÈME 2

## Microorganismes du raisin et du vin

*Grape and wine microorganisms*

<table>
  <tbody>
    <tr>
      <td><strong>New insights into the adaptation of yeast to wine making using comparative genomics</strong><br>J.-L. Legras, A.L. Coi, F. Bigey, V. Galeote, S. Marsit, A. Couloux, J. Guy, R. Franco-Duarte, D. Schuller, J.-P. Sampaio, M. Budroni, S. Dequin</td>
      <td>115</td>
    </tr>
    <tr>
      <td><strong>Biodiversity of wine yeasts: new insights from population genetics</strong><br>W. Albertin, P. Marullo, E. Peltier, S. Windholtz, M. Bely, I. Masneuf-Pomarède</td>
      <td>120</td>
    </tr>
    <tr>
      <td><strong>Phylogenomic analysis of <em>Oenococcus oeni</em> reveals specific domestication of strains to cider and wines</strong><br>H. Campbell-Sills, M. El Khoury, M. Favier, A. Romano, F. Biasioli, G. Spano, D. Sherman, O. Bouchez, E. Coton, M. Coton, S. Okada, N. Tanaka, M. Dols-Lafargue, P. Lucas</td>
      <td>124</td>
    </tr>
    <tr>
      <td><strong>Influence of nutrients and temperature on the production of fermentative aromas in winemaking conditions by an evolved strain of <em>Saccharomyces cerevisiae</em></strong><br>S. Rollero, J.-R. Mouret, I. Sanchez, A. Ortiz-Julien, J.-M. Sablayrolles, S. Dequin</td>
      <td>129</td>
    </tr>
    <tr>
      <td><strong>Hybridization within <em>Saccharomyces</em> genus results in homoeostasis, heterosis and phenotypic novelty in winemaking conditions</strong><br>T. da Silva, W. Albertin, M. Blein-Nicolas, C. Dillmann, M. Bely, S. la Guerche, D. Sicard, I. Masneuf-Pomarede, M. Zivy, P. Marullo, D. de Vienne</td>
      <td>134</td>
    </tr>
    <tr>
      <td><strong>Genome-scale metabolic model of <em>Oenococcus oeni</em> and its application to the analysis of malolactic fermentation</strong><br>S. Mendoza, P. Cañón, E. Agosin</td>
      <td>138</td>
    </tr>
    <tr>
      <td><strong>Proteomic study of the adaptation of <em>Oenococcus oeni</em> to wine conditions</strong><br>M. Margalef, I. Araque, A. Bordons, C. Reguant</td>
      <td>143</td>
    </tr>
    <tr>
      <td><strong>Involvement of yeast-derived peptides in the inhibition of malolactic fermentation and impact of these peptides on malolactic enzyme activity</strong><br>Z. Rizk, Y. El Rayess, C. Ghanem, F. Mathieu, P. Taillandier, N. Nehme</td>
      <td>147</td>
    </tr>
    <tr>
      <td><strong>Candida pyralidae secretes killer toxins active against <em>Brettanomyces bruxellensis</em></strong><br>N.N. Mehlomakulu, M.E. Setati, B. Divol</td>
      <td>151</td>
    </tr>
    <tr>
      <td><strong>The “Terroir Yeast” does not exist !</strong><br>R. Tourdot-Maréchal, C. Longin, V. Cirillo, M. Sadoudi, A. Ducerf-Gaudin, J. Drinkine-Magneux</td>
      <td>155</td>
    </tr>
    <tr>
      <td><strong>Reducing alcohol levels in wines through adaptive evolution of wine yeast</strong><br>V. Tilloy, J. Noble, A. Ortiz-Julien, S. Dequin</td>
      <td>159</td>
    </tr>
    <tr>
      <td><strong>Generation and characterisation of improved wine microbes</strong><br>V. Jiranek, T. Liccioli, A. Betteridge, K. Sumby, M. Walker, J. Gardner, J. Sundstrom</td>
      <td>163</td>
    </tr>
    <tr>
      <td><strong>Increase of fruity aroma during mixed <em>T. delbrueckii</em> / <em>S. cerevisiae</em> wine fermentation is linked to specific ester enhancement</strong><br>P. Renault, J. Coulon, G. de Revel, J.-C. Barbe, M. Belly</td>
      <td>167</td>
    </tr>
    <tr>
      <td><strong>Effects of yeast cultures on sensory properties and volatile esters in white wines</strong><br>M.J. Ruiz-Moreno, B. Puertas, A. Marrufo, E. Cantos-Villar</td>
      <td>172</td>
    </tr>
    <tr>
      <td><strong>Importance of lactic acid bacteria strain on hydroxylated branched ester level variation</strong><br>M. Gammacurta, S. Marchand, G. Lytra, J.-C. Barbe, V. Moine, G. de Revel</td>
      <td>176</td>
    </tr>
    <tr>
      <td><strong>Residual fungicides compromise the beginning of alcoholic fermentation in grape juice with a considerable impact on wine quality and safety</strong><br>P. Russo, T. Nardi, N. Sieczkowski, G. Spano, V. Capozzi</td>
      <td>180</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>Effect of water activity on grape berry microbiota from vineyards subject to different microclimates<br>G. Martins, C. Casini, L. Geny, A. Lonvaud, I. Masneuf-Pomarède</td>
      <td>185</td>
    </tr>
    <tr>
      <td>Copper and sulfur in vitro tolerance of wine relevant strains: a focus on the genera Candida and Metschnikowia<br>C. Grangeteau, C. Voelcker, T. Boiron, H. Alexandre, M. Guilloux-Benatier, S. Rousseaux</td>
      <td>189</td>
    </tr>
    <tr>
      <td>Effect of grape must saturation with carbon dioxide at the beginning of alcoholic fermentation: valorisation of species of interest<br>L. Chasseriaud, J. Coulon, M. Bely</td>
      <td>192</td>
    </tr>
    <tr>
      <td>Metabolic impact of redox perturbations on the formation of aroma compounds during alcoholic fermentation<br>A. Bloem, I. Sanchez, S. Dequin, C. Camarasa</td>
      <td>196</td>
    </tr>
    <tr>
      <td>Mixed fermentations with Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae for the reduction of alcohol in wine<br>V. Englezos, K. Rantsiou, F. Torchio, A. Ortiz-Julien, F. Cravero, L. Rolle, L. Cocolin</td>
      <td>200</td>
    </tr>
    <tr>
      <td>Comparaison des phénomènes d'interactions directes/indirectes lors de co-cultures entre des levures Saccharomyces et non-Saccharomyces<br>S. Beaufort, N. Luyt, F. Bauer, C.L. Fernandez Lopez, C. Brandam, B. Divol, P. Taillandier</td>
      <td>202</td>
    </tr>
    <tr>
      <td>Metabolomics pipeline for monitoring alcoholic fermentation: a tool to reveal the “wine-like aroma” volatile feature<br>A.C. da Silva Ferreira, A.R. Monforte, C.S. Silva, R. Martins, S. Fairbairn, F. Bauer</td>
      <td>206</td>
    </tr>
    <tr>
      <td>Enzymatic characterization of an oenological relevant protease isolated from Metschnikowia pulcherrima<br>L.W. Theron, M. Bely, B. Divol</td>
      <td>211</td>
    </tr>
    <tr>
      <td>Network reconstruction: a tool for yeast strain selection (preliminary results)<br>A.R. Monforte, J. Domingues, C.S. Silva, S. Fairbairn, M.J. Sousa, A.C. Silva Ferreira</td>
      <td>215</td>
    </tr>
    <tr>
      <td>Genetic background for second fermentation in sparkling wine production: QTL detection and validation<br>A. Mas, M. Marti-Raga, G. Beltran, P. Marullo</td>
      <td>218</td>
    </tr>
    <tr>
      <td>Impact of Oenococcus oeni esterase EstA7 on wine aromatic compounds<br>M. Darsonval, C. Grandvalet, H. Alexandre</td>
      <td>222</td>
    </tr>
    <tr>
      <td>The exopolysaccharides (EPS) of Oenococcus oeni and their implication in Champagne wine<br>M. Dimopoulou, Y. Vasserot, R. Marchal, M. Dols-Lafargue</td>
      <td>225</td>
    </tr>
    <tr>
      <td>Wine bacteria to control volatile phenols and Brettanomyces<br>M. Deleris-Bou, S. Krieger-Weber, J.-P. Osborne, V. Gerbaux, C. Briffox, A. Dumont</td>
      <td>230</td>
    </tr>
    <tr>
      <td>Caractérisation des phénomènes de bio-adhésion à l'origine des altérations des vins<br>P. Poupault</td>
      <td>234</td>
    </tr>
    <tr>
      <td>Effect of wine phenolic compounds on the lytic activity of oenophages<br>P. Ballestra, F. Jaomanjaka, M. Cayssials, C. Philippe, A. Lagarde, M. Blanche-Barbat, A. Decendit, E. Renouf, C. Le Marrec, S. Cluzet</td>
      <td>238</td>
    </tr>
    <tr>
      <td>La bio-protection du moût par l'utilisation de levures non-Saccharomyces<br>A. Delaherche, A. Immele, C. Pagagni, D. Maurel, W. Mulinazzi</td>
      <td>242</td>
    </tr>
    <tr>
      <td>Selection characterization and evaluation of autochthonous Saccharomyces cerevisiae strains as wine starters for industrial fermentations of Agiorgitiko and Moschofilero grape musts<br>D. Kontogiannatos, A. Belka, V. Troianou, Y. Kotseridis</td>
      <td>245</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>Acetic acid bacteria detection and quantification in red wine by qPCR<br/>C. Longin, M. Guilloux-Benatier, H. Alexandre</td>
      <td>249</td>
    </tr>
    <tr>
      <td>Carbohydrate metabolism of <i>Brettanomyces bruxellensis</i><br/>A. Cibrario, E. Canac, M. Dols-Lafargue, P. Ballestra</td>
      <td>252</td>
    </tr>
    <tr>
      <td>Comparative metabolomics in yeast cells after dehydration stress<br/>G. López-Martínez, A. Borrull, M. Poblet, N. Rozès, R. Cordero-Otero</td>
      <td>256</td>
    </tr>
    <tr>
      <td>Distribution of <i>Oenococcus oeni</i> strains during malolactic fermentation<br/>over regions and products in France and Lebanon<br/>M. El Khoury, O. Claisse, H. Campbell-Sills, P.M. Lucas</td>
      <td>261</td>
    </tr>
    <tr>
      <td>Genomics of <i>Oenococcus oeni</i> : a new strategy for the selection of<br/>efficient malolactic starters<br/>M. Favier, H. Campbell-Sills, J. Coulon, V. Moine, P. Lucas</td>
      <td>265</td>
    </tr>
    <tr>
      <td>Influence of selected wine lactic acid bacteria on the sensory profile of wines<br/>M. Deleris-Bou, A. Silvano, S. Krieger-Weber</td>
      <td>269</td>
    </tr>
    <tr>
      <td>Inter-laboratory comparison of available <i>Brettanomyces</i> quantification kits<br/>C. Longin, C. Petitgonnet, V. Serpaggi, J. Maupeu, A. Vallet- Courbin, S. Rousseaux,<br/>M. Guilloux-Bennatier, H. Alexandre</td>
      <td>273</td>
    </tr>
    <tr>
      <td>Low environmental impact sanitization treatments : effect of electrolyzed<br/>water and ozone treatments on grape and wine microbiota<br/>F. Cravero, K. Rantsiou, V. Englezos, F. Torchio, S. Giacosa,<br/>S. Río Segade, L. Rolle, L. Cocolin</td>
      <td>276</td>
    </tr>
    <tr>
      <td>Managing wild yeast potential as an added <i>terroir</i> layer for<br/>wines' regional distinctiveness<br/>N. Fontes, F. Centeno, M.F. Teixeira, R. Tenreiro, B. Cabral Almeida, A. Graça</td>
      <td>279</td>
    </tr>
    <tr>
      <td>Monitoring extracellular proteolytic activity of wine yeasts during<br/>alcoholic fermentation<br/>L. Chasseriaud, C. Miot-Sertier, J. Coulon, W. Albertin, M. Bely</td>
      <td>284</td>
    </tr>
    <tr>
      <td>New insights into yeast acclimation for sparkling wine elaboration<br/>A. Borrull, E. Miró, G. López-Martínez, M. Poblet, R. Cordero-Otero, N. Rozès</td>
      <td>288</td>
    </tr>
    <tr>
      <td>Non-<i>Saccharomyces</i> biodiversity in Nero di Troia wine<br/>C. Garofalo, L. Beneduce, S. Massa, P. Russo, G. Spano, V. Capozzi</td>
      <td>292</td>
    </tr>
    <tr>
      <td>Observations macroscopiques et microscopiques de préparations à base<br/>de chitosane sur le développement de <i>Brettanomyces bruxellensis</i> :<br/>application à usage curatif et préventif<br/>B. Nazaris, E. Gontier, L. Geay, C. Mansour, V. Moine, J. Coulon</td>
      <td>297</td>
    </tr>
    <tr>
      <td>Selection and characterization of native lactic acid bacteria from ecological<br/>grapes and wines of Priorat in South Catalonia<br/>J. Franquès, B. Palahí, I. Araque, C. Reguant, A. Bordons</td>
      <td>301</td>
    </tr>
    <tr>
      <td>Selection of indigenous non-<i>Saccharomyces</i> yeast strains through<br/>fermentive potential via GC analysis<br/>J. Calzada, I. Baselga, D.J. García Martínez, E.P. Lago, R. Francisco-Álvarez,<br/>L. Rodríguez, A.M. Prieto, C. Santos</td>
      <td>305</td>
    </tr>
    <tr>
      <td>Sequential inoculation with native <i>Saccharomyces cerevisiae</i> and non-<br/><i>Saccharomyces</i> strains for the production of Grenache and Carignan red wines<br/>A. Mas, B. Padilla, À. Ferreres, R. Pastor, B. Esteve-Zarzoso, G. Beltran</td>
      <td>309</td>
    </tr>
    <tr>
      <td>The impact of oxygen on the behaviour and interaction of<br/>S. cerevisiae and L. Thermotolerans<br/>B. Divol, N.A. Luyt, S. Beaufort, P. Taillandier, F.F. Bauer</td>
      <td>313</td>
    </tr>
  </tbody>
</table>
Diversity and involvement of the cellar yeast in the fermentation of grape must : example of *Hanseniaspora* genus 317

C. Grangeteau, D. Gerhards, S. Rousseaux, C. von Wallbrunn, H. Alexandre, M. Guilloux-Benatier

Adaptive advantage of oligopeptide transporters acquired by horizontal transfer during wine fermentation 320

S. Marsit, F. Bigey, J.-L. Legras, S. Dequin, V. Galeote

## THÈME 3

### Chimie du vin, procédés et composants du vin à effets physiologiques

*Wine chemistry, processes, and physiologically active wine components*

Grape and wine polyphenomics : progresses in flavanol and stilbenoid metabolite profiling 329

C. Saucier, R. Moss, Q. Mao, D. Taylor, K. Pedneault

Study of polymeric pigments in red wine : strategy for quantification via phloroglucinolysis and identification by high resolution mass spectrometry (Q-TOF) 332

L. Zeng, P.-L. Teissèdre, M. Jourdes

Development of a multi-way approach to non-targeted GC-MS analysis of wine volatiles 336

J. Vestner, S. Krieger-Weber, D. Rauhut, G. de Revel, M. du Toit, A. de Villiers

DNA and type of grain : which factor better explain sensory differences between sessile and pedunculate oaks ? 339

M. Mirabel, R. Teissier du Cros, V. Renouf

Développement d'une méthode chimique pour discriminer les bois de chêne sessile et pédonculé sur la base de leur composition triterpénique 343

A. Marchal, D. Dubourdieu

Internal structure and gas transport through cork 348

Oxygen transfer through oak wood using an innovative permeameter 351

Identification of new S-conjugate precursors of 3SH in Sauvignon juice 356

Le « potentiel thiol » : un nouveau concept de mesure de la qualité aromatique du vin 359

A. Roland, S. Delpech, F. Cavelier, L. Dagan, M.A. Ducasse, R. Schneider

Recherche des marqueurs moléculaires de l'arôme de fruits cuits des raisins et des vins rouges de Merlot et de Cabernet Sauvignon : incidence de la date de récolte sur la massoia lactone et le furanéol 363

L. Allamy, P. Darriet, D. Dubourdieu, A. Pons

Investigation and sensory characterisation of 1,4-cineole : a potential marker of Australian Cabernet Sauvignon wine aroma 367

G. Antalick, S. Tempere, K. Šuklje, J.W. Blackman, A. Deloire, G. de Revel, L.M. Schmidtke

The direct contribution of several volatile sulfur compounds in the aromatic expression of the wine ageing bouquet 371

M. Picard, C. Thibon, M.C. Perello, P. Redon, P. Darriet, G. de Revel, S. Marchand

Identification of new trimeric anthocyanins occurring in Merlot red wine 374
<table>
  <tbody>
    <tr>
      <td>Does stereochemistry influence the taste of wine? Purification, characterization and sensory evaluation of lyoniresinol isomers<br>B.N. Cretin, Q. Sallembien, L. Sindt, N. Daugey, T. Buffeteau, P. Waffo-Téguo, D. Dubourdieu, A. Marchal</td>
      <td>378</td>
    </tr>
    <tr>
      <td>How does chirality of substituted esters affect red wine fruity aroma?<br>G. Lytra, S. Tempere, M. Cameleyre, G. de Revel, J.-C. Barbe</td>
      <td>383</td>
    </tr>
    <tr>
      <td>Importance de la forme énantiomère ou diastéréoisomère dans les phénomènes d'interactions perceptives participant à des nuances aromatiques typiques de vins liquoreux de pourriture noble<br>P. Stamatopoulos, E. Frérot, E. Brohan, C. Provost, T. Siebert, M. Herderich, P. Darriet</td>
      <td>387</td>
    </tr>
    <tr>
      <td>Impact of grape processing and yeast strain selection on glycosylated aroma compounds and flavor of sparkling wine<br>D. Schober, M. Wacker, S. Klink, H.-G. Schmarr, U. Fischer</td>
      <td>392</td>
    </tr>
    <tr>
      <td>Interactions of grape proanthocyanidins and wine polyphenols with yeast protein extract, mannoproteins and β-glucans : isothermal titration calorimetry and dynamic light scattering studies<br>J. Mekoue Nguela, A. Vernhet, N. Sieczkowski, C. Poncet-Legrand</td>
      <td>396</td>
    </tr>
    <tr>
      <td>Red wine adsorption on functionalised surfaces<br>A. Mierczynska-Vasilev, P. Smith</td>
      <td>402</td>
    </tr>
    <tr>
      <td>The concentration, composition and structure of oligosaccharides in wine are affected by grape cultivar<br>R. Apolinar-Valiente, I. Romero-Cascales, P. Williams, E. Gómez-Plaza, J.M. López-Roca, J.-M. Ros-García, T. Doco</td>
      <td>406</td>
    </tr>
    <tr>
      <td>Impact of grape juice press fractioning on polysaccharide and oligosaccharide composition of Pinot meunier Champagne base wines<br>S. Jégou, D.A. Hoang, T. Salmon, P. Williams, C. Vrigneau, T. Doco, R. Marchal</td>
      <td>410</td>
    </tr>
    <tr>
      <td>Stilbenes as inhibitors of α-synuclein aggregation and cytotoxicity<br>H. Temsamani, S. Krisa, M. Decossas, O. Lambert, J.-M. Merillon, T. Richard</td>
      <td>414</td>
    </tr>
    <tr>
      <td>Centrifugal partition chromatography (CPC) gradient elution applied to grapevine stilbenoids purification<br>J. Bisson, A. Palos-Pinto, M. Brunel, J.-M. Mérillon, P. Waffo-Téguo</td>
      <td>418</td>
    </tr>
    <tr>
      <td>Caractérisation électrochimique des polyphénols dans les vins rouges<br>F. Garcia, E. Newair, C. Saucier, H. Fulcrand</td>
      <td>422</td>
    </tr>
    <tr>
      <td>Oak wood ellagitannin changes during toasting<br>K. Chira, M. Jourdes, P.L. Teissedre</td>
      <td>425</td>
    </tr>
    <tr>
      <td>Influence of closure type and post-bottling oxygen exposure on the evolution of reductive and varietal volatile sulfur compounds in Carmenere wine<br>M. Ugliano, G. Casaubon, M.C. Zuñiga, M.I. Espinoza, J.-B. Dieval, S. Vidal, E. Agosin</td>
      <td>429</td>
    </tr>
    <tr>
      <td>Polysaccharide and lignin composition in oak wood used in cooperage<br>A. Le Floch, M. Jourdes, T. Giordanengo, N. Mourey, P.-L. Teissedre</td>
      <td>431</td>
    </tr>
    <tr>
      <td>Ellagitannin content, volatile composition and sensory profile of Cabernet Sauvignon wine from three different countries matured in oak barrels subjected to different toasting methods<br>M.R. González-Centeno, K. Chira, P.-L. Teissedre</td>
      <td>435</td>
    </tr>
    <tr>
      <td>Identification of six new odor active alkylpyrazines in oak wood<br>M. Floch, S. Shinkaruk, B. Benneteau, P. Darriet, A. Pons</td>
      <td>441</td>
    </tr>
    <tr>
      <td>A novel method for evaluation of white wine aging potential<br>M. Nikolantonaki, C. Coelho, R.D. Gougeon</td>
      <td>445</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>Comparison of precipitates from Bordeaux red and white wines by solution NMR<br>S. Prakash, N. Iturmendi, A. Grélard, V. Moine, E.J. Dufourc</td>
      <td>448</td>
    </tr>
    <tr>
      <td>A comparative study of the bioconversion kinetic profiles of both p-coumaric acid isomers (trans and cis) by strains of Brettanomyces bruxellensis<br>J. Kheir, D. Salameh, P. Strehaiano, C. Brandam, R. Lteif</td>
      <td>452</td>
    </tr>
    <tr>
      <td>Non-invasive gas monitoring in sparkling wines<br>D. Goffette, M. Valade</td>
      <td>456</td>
    </tr>
    <tr>
      <td>Role of natural cork stoppers concerning oxygen transmission range (OTR) vs cork parameters<br>A.C. Lopes Cardoso, A.C. Pereira, C.M. Oliveira, A.C. Silva Ferreira</td>
      <td>460</td>
    </tr>
    <tr>
      <td>Étude globale de la pénétration en oxygène durant le remplissage et la conservation des BIB® : Mesure de l'impact sur la qualité des vins<br>J. Ducruet, B. Bach, M. Sarrazin, M. Blind, E. Martine, D. Louvier</td>
      <td>464</td>
    </tr>
    <tr>
      <td>Effet d'un ajout de glutathion au conditionnement sur la qualité aromatique des vins blancs et rosés<br>F. Charrier, F. Davaux, G. Masson, L. Cayla, R. Schneider</td>
      <td>467</td>
    </tr>
    <tr>
      <td>Repetitive oxidation of Sauvignon blanc wine : comparison of chemical and sensory data<br>C. Coetzee, A.C. Silva Ferreira, W.J. du Toit</td>
      <td>470</td>
    </tr>
    <tr>
      <td>Nano-oxygenation of three Southern Italy red wines : evolution of sensory active phenolics<br>A. Gambuti, T. Siani, L. Picariello, A. Rinaldi, M. Ugliano, L. Moio</td>
      <td>474</td>
    </tr>
    <tr>
      <td>Modified carbon paste screen printed electrodes for rapid fingerprinting of white wine oxidizable fraction<br>A. Gonzalez Zavala, J. Wirth, J.-B. Dieval, S. Vidal, M. Ugliano</td>
      <td>479</td>
    </tr>
    <tr>
      <td>A voltammetric approach for rapid characterization of oxidation patterns of white wines<br>A. Gonzalez Zavala, J.-B. Dieval, S. Vidal, M. Ugliano</td>
      <td>481</td>
    </tr>
    <tr>
      <td>Study of quinone reactions with wine nucleophiles in wine-model solutions by cyclic voltammetry<br>C.M. Oliveira, A. Barros, A.C. Silva Ferreira, A.M.S. Silva</td>
      <td>484</td>
    </tr>
    <tr>
      <td>Network Reconstruction : a tool to unravel wine aging<br>A.R. Monforte, D. Jacobson, A.C. Silva Ferreira</td>
      <td>488</td>
    </tr>
    <tr>
      <td>Long term effects of pre-fermentative maceration on the phenolic characteristics and sensory attributes of Pinot noir<br>P. Wegmann-Herr, D. Golitko, P. Nickolaus, D. Durner, U. Fischer</td>
      <td>491</td>
    </tr>
    <tr>
      <td>Pre-fermentation heat treatment of grapes at 50 °C : a promising alternative to produce red wines with fruity varietal character<br>O. Geffroy, R. Lopez, F. Violleau, D. Kleiber, J.-L. Favarel, V. Ferreira</td>
      <td>497</td>
    </tr>
    <tr>
      <td>Réduction du SO₂ dans les vins rouges<br>J. Coulon, B. Nazaris, C. Mansour, V. Moine</td>
      <td>501</td>
    </tr>
    <tr>
      <td>A combination of biotechnology tools and cooper elements for an alternative to post-MLF SO₂ addition<br>V. Renouf, M. Mirabel, A.-C. Monteau</td>
      <td>505</td>
    </tr>
    <tr>
      <td>Procédé innovant de régénération de barriques par Ultrasons Haute Puissance : impact sur la qualité du vin<br>P. Renault, A. Trillaud, F. Meunier, A. Massot, M. Mietton-Peachot, R. Ghidossy</td>
      <td>509</td>
    </tr>
    <tr>
      <td>Effect of potassium polyaspartate on the tartaric stability of white and red wines as a function of their physicochemical composition<br>A. Asproudi, M. Guaita, L. Panero, S. Motta, M. Petrozziello, A. Bosso</td>
      <td>515</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>Recent developments of membrane technologies in the wine industry<br/>Y. EL Rayess, M. Mietton-Peuchot</td>
      <td>519</td>
    </tr>
    <tr>
      <td>Subcritical water extraction of polyphenols from red grape pomace<br/>coupled with membrane filtration<br/>S. Yammine, R. Rabagliato, X. Vitrac, R. Ghidossi, M. Mietton Peuchot</td>
      <td>523</td>
    </tr>
    <tr>
      <td>Alcoholic fermentation in new oak tanks : effects on the colour, chemical<br/>composition and sensory profile of Shiraz wine, before and after barrel ageing<br/>A.C. Monteau, V. Renouf, N. Scrimgeour, N. Tiquet-Lavandier</td>
      <td>527</td>
    </tr>
    <tr>
      <td>Prevention of wine oxidation during barrel aging : an innovative method<br/>to measure antioxidant compounds released by oak<br/>M. Mirabel, V. Renouf</td>
      <td>530</td>
    </tr>
    <tr>
      <td>A protein extracted from potato as non-allergenic fining agent for juices and wines<br/>N. Iturmendi, A. Rinaldi, A. Gambutti, V. Moine, L. Moio</td>
      <td>533</td>
    </tr>
    <tr>
      <td>Anthocyanin-derived pigments and tannins of red wines from Rioja and Bordeaux<br/>N. Prieto-Perea, C. Quaglieri, L.A. Berrueta, B. Gallo, M. Jourdes, P.L. Teissedre</td>
      <td>538</td>
    </tr>
    <tr>
      <td>Bee pollen application as fermentative activator in red winemaking<br/>A. Amores-Arrocha, A.M. Roldán, R. Ghidossi, I. Caro, M. Mietton-Peuchot,<br/>V.M. Palacios</td>
      <td>543</td>
    </tr>
    <tr>
      <td>Characterization and prognosis of the oxidative behavior of wine using chemiluminescence,<br/>fluorescence, and the colorimetric reducing power method<br/>P. Schonenberger, B. Bach, J.-P. Bourgeois, J.-M. Segura, J. Heritier, J. Ducruet</td>
      <td>549</td>
    </tr>
    <tr>
      <td>Colour evolution of wines aged in different containers by CIELAB :<br/>discrimination between type of container and contact time<br/>M. Basalekou, E. Kontaxakis, A. Strataridaki, D. Lydakis, S. Kallithraka</td>
      <td>552</td>
    </tr>
    <tr>
      <td>Determination of pesticide residues from vine leaves and grapes to the wine<br/>C. Franc, L. Riquier, G. Jabs, V. Peltier, G. de Revel</td>
      <td>556</td>
    </tr>
    <tr>
      <td>Effect of ageing and variety on dimethyl sulfide levels<br/>M. Kyraleou, S. Kallithraka, P. Tarantilis, T. Garde-Cerdan, Y. Kotseridis</td>
      <td>560</td>
    </tr>
    <tr>
      <td>Grape marcs : could their composition be affected by the grape cultivar<br/>or the enological technique ?<br/>R. Apolinar-Valiente, I. Romero-Cascales, E. Gómez-Plaza, J.-M. López-Roca, J.-M. Ros-García</td>
      <td>563</td>
    </tr>
    <tr>
      <td>Use of innovative tools to select and toast oak wood and<br/>the impact on wine quality<br/>M.-L. Badet-Murat, J.-C. Vicard, B. Leaute, A.A. Watrelot, J.A. Kennedy</td>
      <td>567</td>
    </tr>
    <tr>
      <td>Effect of grape maturity on carbohydrate composition of red sparkling wines<br/>L. Martínez-Lapuente, R. Apolinar-Valiente, Z. Guadalupe, B. Ayestarán,<br/>S. Pérez-Magariño, T. Doco, P. Williams</td>
      <td>572</td>
    </tr>
    <tr>
      <td>L'amertume des vins blancs secs<br/>S. Caillé, C. Begue, C. Leriche, F. Dellova, A. Samson</td>
      <td>576</td>
    </tr>
    <tr>
      <td>Fractionation and determination method of proanthocyanidol mass from<br/>grapes and wine. Validation of proanthocyanidol standards used for<br/>the calibration of size exclusion chromatography columns<br/>M.F. Nonier, N. Vivas de Gaulejac, N. Vivas, I. Pianet, C. Absalon</td>
      <td>580</td>
    </tr>
    <tr>
      <td>Variations in ageing bouquet typicality of red Bordeaux wines and<br/>associated aromatic marker levels during ageing in three vineyards<br/>M. Picard, C. Thibon, M.C. Perello, P. Redon, P. Darriet, G. de Revel, S. Marchand</td>
      <td>584</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>Characterization of precipitate formation in Bordeaux red wine with solid-state and solution NMR<br>S. Prakash, N. Iturmendi, A. Grélard, V. Moine, E.J. Dufourc</td>
      <td>587</td>
    </tr>
    <tr>
      <td>Impact des thiols volatils dans l'arôme typique des vins de Vitis vinifera var. Chardonnay<br>J. Gros, F. Thibaud, A. Marchal, V. Lavigne, V. Moine, P. Darriet</td>
      <td>592</td>
    </tr>
    <tr>
      <td>Origine de notes olfactives « mastic » dans les eaux-de-vie nouvelles de cognac<br>L. Urruty, D. Laurichesse, L. Mornet, B. Loiseau, J.-C. Mathurin</td>
      <td>596</td>
    </tr>
    <tr>
      <td>Purification and characterization of three new condensed tannin oligomers in red wine<br>L. Zeng, T. Richard, P.-L. Teissèdre, M. Jourdes</td>
      <td>599</td>
    </tr>
    <tr>
      <td>Rosé wines: impact of storage conditions in tank on polyphenol composition and color<br>M.-A. Ducasse, L. Cayla, G. Masson, C. Anneraud, P. Chrétien, F. Davaux, J.-C. Boulet, E. Meudec, A. Verbaere, N. Sommerer, V. Cheynier</td>
      <td>603</td>
    </tr>
    <tr>
      <td>Identification and quantification of stilbenes in some Tunisian red wines using UPLC-MS and HPLC-DAD<br>K. Arraki, E. Renouf, P. Waffo-Téguo, J.-M. Mérillon, T. Richard, A. Decendit</td>
      <td>608</td>
    </tr>
    <tr>
      <td>Study of the colour and phenolic compounds in grapes and wines elaborated from two canopy treatments of Vitis vinifera L. cv Shiraz<br>G. Garrido-Bañuelos, A. Buica, W.J. du Toit</td>
      <td>612</td>
    </tr>
    <tr>
      <td>Investigations on the green aromas in French red wines related to grape ripening<br>X. Poitou, P. Darriet</td>
      <td>616</td>
    </tr>
    <tr>
      <td>The effect of supplementation with three commercial inactive dry yeasts on the chemical composition and astringency of model wine solution and red wine<br>E. González-Royo, M. Esteruelas, N. Kontoudakis, F. Fort, J.-M. Canals, F. Zamora</td>
      <td>620</td>
    </tr>
    <tr>
      <td>Volatile aroma compounds related to 'stone fruit' aroma in Viognier and Chardonnay wine<br>T.E. Siebert, W.P. Pearson, A. Barker, M.A. De Barros Lopes, P. Darriet, M.J. Herderich, I.L. Francis</td>
      <td>623</td>
    </tr>
    <tr>
      <td>Wine oxygen and organoleptic property evolutions in classified near infra red oak wood barrel during aging<br>J. Michel, M. Jourdes, T. Giordanengo, N. Mourey, P.L. Teissedre</td>
      <td>627</td>
    </tr>
    <tr>
      <td>Antioxidant activities and presence of bioactive micronutrients in new South Korean wines: effect of winemaking processes and chemical composition<br>H.W. Choi, J.-P. Souchard</td>
      <td>633</td>
    </tr>
    <tr>
      <td>Impact of maceration time and temperature on the extraction and evolution of polyphenols in Lebanese musts<br>C. Ghanem, P. Taillandier, L.H. Wakim, Z. Rizk, N. Nehme, J.-P. Souchard, Y. El Rayess</td>
      <td>637</td>
    </tr>
    <tr>
      <td>The effect of different oak treatments on the phenolic composition of red wines<br>B. Öztürk, R.E. Anli</td>
      <td>641</td>
    </tr>
    <tr>
      <td>Phenolic oxidation in red Port wine<br>C.M. Oliveira, A.C. Lopes Cardoso, A. Barros, A.C. Silva Ferreira, A.M.S. Silva</td>
      <td>645</td>
    </tr>
    <tr>
      <td>On-line monitoring of oak wood volatiles by active capillary plasma ionisation mass spectrometry<br>R. Farrell, J.-C. Wolf, R. Zenobi</td>
      <td>649</td>
    </tr>
    <tr>
      <td>Grape cluster zone defoliation and choice of yeast strain – Impact on TDN potential in Riesling wines<br>A. Schüttler, C. Guthier, M. Stoll, P. Darriet, D. Rauhut</td>
      <td>651</td>
    </tr>
  </tbody>
</table>
Tentative identification of a fruity aroma compound, recalling blackcurrant, released from grape skins of *Vitis vinifera* grape var. Cabernet Sauvignon during alcoholic fermentation using MDGC-MS-Olfactometry
L. Boutegrabet, R. Sakurai, M.J. Ruiz-Moreno, Ph. Darriet

654

# THÈME 4

## Analyse sensorielle et interface consommateur

*Sensory analysis and consumer interface*

Mieux comprendre les goûts des consommateurs grâce à l'économie expérimentale
P. Combris

661

Le réchauffement climatique est-il souhaité par les consommateurs de vin ?

666

Approches croisées d'économie expérimentale et d'analyse sensorielle

A. Fuentes Espinoza, S. Pérès, A. Pons, S. Tempère, P. Darriet, E. Giraud-Héraud

Making sense of wine : Cognitive Psychology's contribution to understanding wine tasting and wine tasters
W.V. Parr

669

Comment le cerveau perçoit-il le monde des odeurs ?

675

De nouvelles perspectives pour la perception sensorielle du vin

G. Lepousez, G. Gheusi, P.-M. Lledo

Minerality in wine : what do consumers and wine professionals think about ?

P. Deneulin, Y. Le Fur, G. Le Bras, L. Gautier, F. Bavaud

678

Effet du vieillissement sur la perception sensorielle de la minéralité des vins Chablis 1er Cru

H. Rodrigues, D. Valentin, J. Ballester

Varietal typicality of Riesling wines – unravelling universal and regional aspects

A. Schüttler, K. Sirén, J.-F. Harbertson, R. Jung, C.-D. Patz, T. Henick-Kling, P. Darriet, D. Rauhut

681

685

Holistic approach to the characterization of three South African

Chenin blanc wine styles. Comprehensive chemical volatile composition and sensory evaluation using Polarized Projective Mapping method

J. Brand, C. Wilson, A. Buica

690

Perceptive interactions among fruity esters involved in red wine fruity aromas

G. Lytra, S. Tempere, A. Le Floch, G. de Revel, J.-C. Barbe

2-Methylisoborneol in wine, is it responsible for off-odours ?

A. Lopez Pinar, R. Ghadiriasli, P. Darriet, A. Buettner

693

697

Development of a picture book : a new tool for description of wine aromas

S. Marchand, M. Dufoir, S. Tempere, G. de Revel

700

Sensory evaluation of vescalagin, castalagin and acutissimin A in model solutions

Z. Rasines-Perea, R. Jaquet, M. Jourdes, S. Quideau, P.-L. Teissedre

Sensory profiling of Chenin Blanc wines matured in different oak wood products

A. Botha, J. Brand, W.J. Du Toit

704

706

Study of sensory interactions among fruity and non-fruity compounds in various mixtures

M. Cameleyre, G. Lytra, S. Tempère, G. De Revel, J.-C. Barbe

709

Etude sensorielle, analytique et consommateur de la typicité aromatique poivrée des vins de Gamay N de l'AOP Côtes d'Auvergne

O. Geffroy, C. Buissière, T. Dufourcq, E. Serrano

714
Unveiling the “wine-like” feature - a sensory driven approach 717

C.S. Teixeira, A.R. Monforte, J. Domingues, S. Fairbairn, M.J. Sousa, A.C. da Silva Ferreira

Characterization of the “Bretty” character masking effect 721

S. Tempere, M.H. Schaaper, E. Cuzange, R. de Lescar, G. Sicard, G. de Revel
Tentative identification of a fruity aroma compound, recalling blackcurrant, released from grape skins of *Vitis vinifera* grape var. Cabernet Sauvignon during alcoholic fermentation using MDGC-MS-Olfactometry

L. Boutegrabet, R. Sakurai, M.J. Ruiz-Moreno, Ph. Darriet

654

# THÈME 4

## Analyse sensorielle et interface consommateur

*Sensory analysis and consumer interface*

Mieux comprendre les goûts des consommateurs grâce à l'économie expérimentale

P. Combris

661

Le réchauffement climatique est-il souhaité par les consommateurs de vin ?

666

Approches croisées d'économie expérimentale et d'analyse sensorielle

A. Fuentes Espinoza, S. Pérès, A. Pons, S. Tempère, P. Darriet, E. Giraud-Héraud

Making sense of wine : Cognitive Psychology's contribution to understanding wine tasting and wine tasters

W.V. Parr

669

Comment le cerveau perçoit-il le monde des odeurs ?

675

De nouvelles perspectives pour la perception sensorielle du vin

G. Lepousez, G. Gheusi, P.-M. Lledo

Minerality in wine : what do consumers and wine professionals think about ?

P. Deneulin, Y. Le Fur, G. Le Bras, L. Gautier, F. Bavaud

678

Effet du vieillissement sur la perception sensorielle de la minéralité des vins Chablis 1er Cru

H. Rodrigues, D. Valentin, J. Ballester

Varietal typicality of Riesling wines – unravelling universal and regional aspects

A. Schüttler, K. Sirén, J.-F. Harbertson, R. Jung, C.-D. Patz, T. Henick-Kling, P. Darriet, D. Rauhut

681

685

Holistic approach to the characterization of three South African

Chenin blanc wine styles. Comprehensive chemical volatile composition and sensory evaluation using Polarized Projective Mapping method

J. Brand, C. Wilson, A. Buica

690

Perceptive interactions among fruity esters involved in red wine fruity aromas

G. Lytra, S. Tempere, A. Le Floch, G. de Revel, J.-C. Barbe

693

2-Methylisoborneol in wine, is it responsible for off-odours ?

A. Lopez Pinar, R. Ghadiriasli, P. Darriet, A. Buettner

697

Development of a picture book : a new tool for description of wine aromas

S. Marchand, M. Dufoir, S. Tempere, G. de Revel

700

Sensory evaluation of vescalagin, castalagin and acutissimin A in model solutions

Z. Rasines-Perea, R. Jaquet, M. Jourdes, S. Quideau, P.-L. Teissedre

704

Sensory profiling of Chenin Blanc wines matured in different oak wood products

A. Botha, J. Brand, W.J. Du Toit

706

Study of sensory interactions among fruity and non-fruity compounds in various mixtures

M. Cameleyre, G. Lytra, S. Tempère, G. De Revel, J.-C. Barbe

709

Etude sensorielle, analytique et consommateur de la typicité aromatique poivrée des vins de Gamay N de l'AOP Côtes d'Auvergne

O. Geffroy, C. Buissière, T. Dufourcq, E. Serrano

714
Unveiling the “wine-like” feature - a sensory driven approach 717

C.S. Teixeira, A.R. Monforte, J. Domingues, S. Fairbairn, M.J. Sousa, A.C. da Silva Ferreira

Characterization of the “Bretty” character masking effect 721

S. Tempere, M.H. Schaaper, E. Cuzange, R. de Lescar, G. Sicard, G. de Revel
# THÈME 1

## Plante, environnement et qualité du raisin

*Plant, environment and grape quality*
<figure><img src="image_14.png" /></figure>
# Terroir effects on grape and wine aroma compounds

M. HERDERICH*

*The Australian Wine Research Institute, Hartley Grove cnr Paratoo Road, Urrbrae, SA 5064, Australia*

Terroir – grape aroma – 1,8-cineole – rotundone – TDN

## Introduction

Terroir is a holistic concept which refers to interactions between the identifiable physical and biological environment and applied vitivinicultural practices that provide distinctive characteristics for the products originating from the area. From a linguistic perspective terroir stems from the Latin word *terra*, or land. Hence at a conceptual level terroir may be defined through characterising distinctive geologies and soils in individual vineyards. Yet the inorganic chemical profile of a grape berry has only a distant and indirect relationship with vineyard geochemistry, concentrations of minerals in wine bear little relationship to geological minerals in vineyard, and so far no evidence supports a direct link between geology, soil composition and wine flavour. To further explore the assumption that specific grape and wine flavours can be attributed to a vineyard or delimited geographical area where grapes have been produced, we have recently studied the spatial and temporal variability of key wine aroma compounds, 1,8-cineole, TDN and rotundone. Taken together, these studies suggest that environmental and potentially biological factors – together with viticultural management and winemaking practices – are key to shaping wine flavour and to creating distinctive wine styles.

## Materials and methods

Vineyard and fermentation studies conducted to elucidate the origin of 1,8-cineole in red wine have been summarised in [1, 2]. The TDN field experiment was conducted during the 2014/15 growing season with established Riesling vines within two commercial vineyards separated by 13.7 km. Riesling vineyard sites in the Barossa and the Eden Valley, Australia (elevation ~280m and ~450m, respectively), were selected based on their 2°C difference in mean January temperature (MJT). Leaf removal (33% leaf removal) was conducted by hand at 30 days post-fruit set. As control, light exclusion polypropylene boxes over one grape bunch per vine were applied to vines with otherwise normal bunch sunlight exposure and to bunches on vines with leaf removal [3]. The analysis of total C13-norisoprenoids was based on a solid-phase extraction (SPE) protocol described in [4] with modifications to improve TDN precursor hydrolysis. Rotundone analysis was carried out following [5] with the modifications described in [6]. Geostatistical analysis and mapping are described in [6] and [7].

## Results and discussion

A significant amount of research over many years has aimed at characterising the links between climate, soil and cultivar, grape composition, and sometimes wine composition and sensory. For example, it was found that the impacts of climate and soil were greater than that of cultivar and that the effects of climate and soil on fruit composition were likely mediated through their influence on vine water status, that is a reflection of vine water stress in an un-irrigated vineyard [8]. In a study of Pinot Noir the authors observed a strong association between soil depth and corresponding water-holding capacity and vine vigour which was thought to have an indirect effect on bunch sunlight exposure and vine microclimate, leading to the observed variations in grape and wine anthocyanins and tannins [9]. Recently, we have explored the impact of viticultural practices on the concentration of TDN precursors in Riesling at Barossa and Eden Valley vineyards. In 2014/15, removing 33% of leaves from around the bunch-zone resulted at both sites in greater sun exposure and a substantial increase in TDN precursors (6-fold compared to shaded controls). When grapes were grown under fully shaded conditions in light excluding boxes, a small increase of ~20% in TDN precursors was observed for grapes from the warmer Barossa Valley vineyard compared to the Eden
Valley grapes, which may be attributed to the many significant differences between the soils, management practices and meso-climates [3]. Taken together, these studies point out that vine water status and stress, which may vary according to seasonal weather conditions and irrigation practices, as well as vigour and light exposure, are more likely to influence grape and wine composition than soils or sites per se. While this in no way suggests that soil properties are unimportant, it is much more likely that it is the diversity in weather and climatic conditions, soil fertility, vigour, light exposure and vineyard management practices that leads to a broad range of wine styles, flavours and unique wines of distinction, rather than the diversity of soils as such.

*The origin of 'eucalypt' and 'minty' aromas in wine*

This has been the subject of discussions between scientists, grape producers and winemakers and provides an interesting case study into how a vineyard's location and surrounding vegetation affect grape and wine aroma. Some researchers believed that 'eucalypt' characters were associated with the proximity of vineyards to *Eucalyptus* trees [10] and hence typical expressions of the terroir of some wines; others proposed that grape-derived monoterpenes acted as precursors for the 'minty' aroma compound 1,8-cineole in berries [11]. Further investigations revealed, however, that grape monoterpenes are unable to generate high enough levels of 1,8-cineole to reach sensory threshold concentrations [1]. These results shifted the focus onto non-grape sources of 1,8-cineole, specifically eucalypt trees that are native to Australia, widely planted throughout the world, and are known for their essential oils rich in 1,8-cineole. In a detailed study the relationship between grape composition and the proximity of vines to *Eucalyptus* trees was investigated. Indeed, the results demonstrated that the greatest amount of 1,8-cineole was found in wine made from grapes that were closest to the *Eucalyptus* trees. Winemaking experiments revealed a continuous increase in the concentration of 1,8-cineole during fermentation that stopped once a wine was drained from skins. This indicated that 1,8-cineole was extracted from the grape skins and/or matter other than grapes (MOG). Importantly, it was not airborne transfer of eucalypt essential oil volatiles that was the main source of 1,8-cineole, but the presence of eucalypt leaves (and to a lesser extent grapevine leaves and stems) in harvested grapes that were the key factors responsible for elevated concentrations of 1,8-cineole in wine [2]. Overall, the proximity of grapevines to *Eucalyptus* trees has a major effect on 1,8-cineole concentrations in wine, and the presence of MOG can significantly influence 1,8-cineole levels. Hence the unique 'minty' aroma and terroir effects from 1,8-cineole can be enhanced or reduced to create balanced wines that express their terroir through harvesting practices that consider the proximity of native or planted eucalypt trees, the species of Eucalypt trees and typical wind direction. In addition to capturing aromas from vegetation growing amongst grapevines or nearby, wine grapes also host a wide range of microbes originating from the surrounding environment. Recently, it has been shown that regional and site-specific factors and grape variety shape the fungal and bacterial consortia in vineyards and on grape surfaces [12, 13]. This provides evidence for the existence of a distinctive 'microbial terroir' as a key factor that contributes to regional variation among wine grapes, potentially through influencing the secondary metabolism of the grapevines and grape composition. In addition, such distinctive 'microbial terroir' on grapes might also be able to influence the microbial populations during fermentation and the diversity of flavours in wine made by spontaneous fermentations.

*Rotundone in cool climate Shiraz wine*

Shiraz is one of the most important grape varieties and grown across all regions in Australia. Prominent Australian Shiraz styles include elegant, peppery cool-climate wines (for example from the Adelaide Hills, or the Grampians); more intensely flavoured, spicy and sometimes minty styles of Margaret River, Coonawarra or Clare Valley; sweet chocolaty and ripe-fruited wines (Barossa Valley, McLaren Vale), and leathery and rich wines (Hunter Valley). Notably, only a few vineyards consistently produce peppery wines, especially in cooler years, and this can be predicted from analysing the concentration of a sesquiterpene, α-ylangene, as a biomarker in grapes [14]. Subsequent studies resulted in the identification of the sesquiterpene rotundone that is found in the skins of Shiraz berries [15, 16] and responsible for the distinctive peppery aroma in grapes and wine [15, 17].

*Key factors associated with the variability of rotundone and peppery aromas in grapes and wine*

Recent research [6] demonstrated that, for a Shiraz vineyard in the Grampians region, variation in the concentration of grape berry rotundone was spatially structured at the within-vineyard scale and was related to variation in the land underlying the vineyard. There appeared to be a clear soil effect, but of particular note, was the apparent control exerted by topography through its effects on slope and aspect [6], with the inference being that variation in temperature and/or incident solar radiation was driving variation in berry
rotundone concentration. Also, results from another study [18] working at vineyard, vine and bunch scales in the same vineyard as that used by Scarlett et al. [6], have highlighted the likely importance of temperature and light interception on rotundone accumulation. In addition, the concentration of a number of additional grape aroma compounds was also clearly spatially structured and for some compounds from presumably closely related biosynthetic pathways - such as β-damascenone and theaspirane - distinctive differences in their spatial concentration distributions were observed [19]. Notably, the spatially structured differences in aroma compounds were not related to differences in maturity as no significant differences in commonly used grape compositional parameters, including total soluble solids, pH, titratable acidity, total phenolics and total anthocyanins were observed among low, medium and high rotundone zones in 2012 [18].

When the original 2012 study was repeated in 2013 and 2015 [7], a 40-fold difference in mean rotundone concentration was observed between 2012 and 2013, a 13-fold difference between 2012 and 2015, and a 3-fold difference between 2013 and 2015, yet the patterns of within-vineyard variation in berry rotundone concentration were strikingly similar. That is spatial analysis of maps of rotundone variation produced for each year demonstrated that the distinctive patterns of spatial variation in grape rotundone were stable between the seasons [7]. This remarkable observation provides a starting point for future research aimed at differentiating between the influences of seasonal climatic conditions and the contributions of other terroir-related environmental, genetic and/or biological factors that could independently or in combination contribute to higher rotundone concentrations and distinctively peppery red wine. While other work has started to unravel the genetic elements of the biosynthesis of rotundone [20, 21], the question of which factors cause the observed spatial and temporal variability in grape rotundone concentration remains uncertain. As 'high-rotundone' vineyards are found across regions with rather diverse and distinct geological terroirs it is unlikely that soil chemistry or geology are playing a causative role in rotundone's formation. In addition, the observed seasonal and spatial variability in a key aroma compound between vineyards, within a vineyard, and within grapes from the same vine within a single vineyard point towards combinations of genetic and environmental factors that result in enhanced rotundone concentrations in grapes. Arguably, the presence of the sesquiterpene rotundone in specific varieties such as Shiraz or Duras [22], while it is absent in other varieties that are planted in the same region and similar iso-climates, indicate a genetic basis for sesquiterpene biosynthesis that is specific to high-rotundone varietals. The observation that specific vineyards within cool climate regions such as Adelaide Hills and Grampians (Australia), or Hawke's Bay (New Zealand), have a historical record of consistently yielding grapes high in pepper aroma and rotundone might also point towards the potential presence of a genetic and/or epigenetic determinant, although such a suggestion would need further investigation to corroborate it. However, the observed differences at a grapevine scale, that is the differences between rotundone concentrations of individual grapes within a bunch and between bunches grown on the same grapevine but on exposed or shaded sides of a row, cannot be explained by genetic effects. Rather, the observed within-vine variability points towards a major role for environmental factors such as light exposure and/or temperature of the bunch zone in regulating the formation of rotundone. Similarly the propensity of 'high-rotundone' Shiraz vineyards to be located across cool-climate regions and the significant seasonal differences in rotundone's concentration point towards weather conditions, especially temperature and perhaps rain or water availability, as playing key roles in influencing the formation of rotundone. When reconciling environmental factors that may play a key role in the formation of rotundone in grapes and would explain the observed spatial and temporal variability in its concentration, it is important to recognise that biosynthesis of many volatile organic plant metabolites, including sesquiterpenes, can be induced by a herbivore attack [23, 24]. With this in mind it is intriguing to speculate that the presence of rotundone in grapes, or of one of its close precursors such as α-guaiene, could potentially be the consequence of a herbivore attack onto a vine above or below ground. As the final step in the formation of rotundone, an oxidation of a sesquiterpene precursor, most likely α-guaiene, is required. Indeed, the oxidation of α-guaiene to rotundone has been shown to occur in the presence of the enzyme laccase [25], by autoxidation in air [26], and through a P450 oxidase in grapevine [21]. Recently, the role of the light-catalysed aerial oxidation of α-guaiene was described, with highest rotundone concentrations being formed under shaded/moderate light exposure [27]. This observation lends further support to the hypothesis that interaction(s) of a grapevine with its biological environment are key for achieving elevated concentrations of direct rotundone precursors such as α-guaiene in certain vineyards, vines, bunches and grapes, and that the final concentration of rotundone is limited by the concentration of its precursors and not by the final oxidation step.
## Conclusion

The results obtained from studying spatial and temporal variability of TDN precursors, 1,8-cineol and rotundone highlight the important contributions of environmental conditions, biological factors and viticultural management practices to shaping distinctive profiles of flavour compounds in grapes and wines. The observed variability among aroma compounds indicates that the scale at which terroir can be considered a useful construct for explaining distinctive profiles of grape and wine aroma compounds might differ from approaches relying on geology, geomorphology or geochemistry-based definitions of terroir.

## Acknowledgements

This work was jointly funded by the Australian Wine Research Institute, CSIRO, the Rathbone Wine Group and by Australia's grapegrowers and winemakers through their investment body, Wine Australia with matching funds from the Australian Government. The essential contributions over many years by all colleagues and collaborators who co-authored publications listed under references are acknowledged. The support and discussions with Damien Sheehan and the late Nathan Scarlett (Rathbone Wine Group) have been essential for establishing the studies into the spatial variability of rotundone at Mt Langi, Victoria.

## References

1. Capone DL, van Leeuwen K, Taylor DK et al (2011) Evolution and occurrence of 1,8-cineole (Eucalyptol) in Australian Wine. *J Agric Food Chem* **59**: 953-959.

2. Capone DL, Jeffery DW, Sefton MA (2012) Vineyard and fermentation studies to elucidate the origin of 1,8-cineole in Australian red wine. *J Agric Food Chem* **60**: 2281-2287.

3. Grebneva Y (2015) The effect of sunlight exposure on TDN development in South Australian Riesling. Wissenschaftliche Abschlussarbeit im Studiengang Lebensmittelchemie, Technische University Dresden, Germany.

4. Kwasniewski MT, Vanden Heuvel JE, Pan BS et al (2010) Timing of cluster light environment manipulation during grape development affects C13 norisoprenoid and carotenoid concentrations in Riesling. *J Agric Food Chem* **58**: 6841-6849.

5. Siebert TE, Wood C, Elsey GM et al (2008) Determination of rotundone, the pepper aroma impact compound, in grapes and wine. *J Agric Food Chem* **56**: 3745-3748.

6. Scarlett NJ, Bramley RGV, Siebert TE (2014) Within-vineyard variation in the ‘pepper’ compound rotundone is spatially structured and related to variation in the land underlying the vineyard. *Aust J Grape Wine Res* **20**: 214-222.

7. Bramley RGV et al (2016) Patterns of within-vineyard spatial variation in the ‘pepper’ compound rotundone are temporally stable from year to year. *Aust J Grape Wine Res*, accepted.

8. Van Leeuwen C, Friant P, Choné X et al (2004) Influence of climate, soil, and cultivar on terroir. *Am J Enol Vitic* **55**: 207-216.

9. Cortell JM, Halbleib M, Gallagher AV et al (2005) Influence of vine vigor on grape (*Vitis vinifera* L. cv. Pinot Noir) and wine proanthocyanidins. *J Agric Food Chem* **53**: 5798-5808.

10. Herve E, Price S, Burns G (2003) Eucalyptol in wines showing a "eucalyptus" aroma, pp 598-600. In *Proceedings of the VIIème Symposium International d'Œnologie*, Bordeaux, France; Lonvaud A, De Revel G, Darriet P (Eds); Lavoisier: Paris, France.

11. Farina L, Boido E, Carrau F et al (2005) Terpene compounds as possible precursors of 1,8-cineole in red grapes and wines. *J Agric Food Chem* **53**: 1633-1636.

12. Bokulich NA, Thorngate JH, Richardson PM et al (2014) Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. *PNAS* **111**: E139-E148.

13. Zarraonaindia I, Owens SM, Weisenhorn P et al (2015) The soil microbiome influences grapevine-associated microbiota. *mBio* **6**: e02527-14.

14. Parker M, Pollnitz AP, Cozzolino D et al (2007) Identification and quantification of a marker compound for ‘pepper’ aroma and flavor in Shiraz grape berries by combination of chemometrics and gas chromatography-mass spectrometry. *J Agric Food Chem* **55**: 5948-5955.

15. Wood C, Siebert TE, Parker M et al (2008) From wine to pepper: rotundone, an obscure sesquiterpene, is a potent spicy aroma compound. *J Agric Food Chem* **56**: 3738-3744.

16. Siebert TE, Solomon MR (2011) Rotundone: development in the grape and extraction during fermentation, pp 307-308. In: Blair RJ, Lee TH, Pretorius IS (Eds); Australian Wine Industry Technical Conference Inc., Adelaide, SA, Australia.
17. Herderich MJ et al (2012) Spice up your life: analysis of key aroma compounds in Shiraz, pp 3-13. In *Flavor chemistry of wine and other alcoholic beverages*; ACS Symposium Series 1104; Qian MC, Shellhammer TH (Eds); American Chemical Society, Washington, DC, USA.

18. Zhang P, Barlow S, Krstic M et al (2015) Within-vineyard, within-vine, and within-bunch variability of the rotundone concentration in berries of *Vitis vinifera* L. cv. Shiraz. *J Agric Food Chem* **63**: 4276-4283.

19. Herderich M et al (2015) Terroir effects on grape and wine aroma compounds, pp 131-146. In *Advances in wine research*; ACS Symposium Series 1203; Ebeler SB et al (Eds); American Chemical Society, Washington, DC, USA.

20. Drew DP, Andersen TB, Sweetman C et al (2016) Two key polymorphisms in a newly discovered allele of the *Vitis vinifera* TPS24 gene are responsible for the production of the rotundone precursor α-guaiene. *J Exp Bot* **67**: 799-808.

21. Takase H, Sasaki K, Shinmori H et al (2016) Cytochrome P450 CYP71BE5 in grapevine (*Vitis vinifera*) catalyzes the formation of the spicy aroma compound (−)-rotundone. *J Exp Bot* **67**: 787-798.

22. Geffroy O, Dufourcq T, Carcenac D et al (2014) Effect of ripeness and viticultural techniques on the rotundone concentration in red wine made from *Vitis vinifera* L. cv. Duras. *Aust J Grape Wine Res* **20**: 401-408.

23. Dicke M, Baldwin IT (2010) The evolutionary context for herbivore-induced plant volatiles: beyond the ‘cry for help’. *Trends Plant Sci* **15**: 167-175.

24. Suckling DM, Twidle AM, Gibb AR et al (2012) Volatiles from apple trees infested with light brown apple moth larvae attract the parasitoid *Dolichogenidia tasmanica*. *J Agric Food Chem* **60**: 9562-9566.

25. Schilling B, Granier T, Locher F (2012) 1-Hydroxy-octahydroazulenes as fragrances? Patent WO 2012/001018 A1.

26. Huang A-C, Burrett S, Sefton MA et al (2014) Production of the pepper aroma compound, (−)-rotundone, by aerial oxidation of α-guaiene. *J Agric Food Chem* **62**: 10809-10815.

27. Zibi N (2015) Investigating α-guaiene, a potential sesquiterpene precursor, in the formation of (−)-rotundone, an oxygenated bicyclic sesquiterpene. Thesis as part of the State examination for food chemists. Friedrich-Alexander University, Erlangen-Nürnberg, Germany.
# Untargeted metabolomics to study the influence of scion x rootstock x nitrogen interactions on berry content

A. HABRAN<fnref n="1" />, P. HELWI<fnref n="1" />, G. HILBERT<fnref n="1" />, E. GOMÈS<fnref n="1" />, C. VAN LEEUWEN<fnref n="1" />, S. NEGRI<fnref n="2" />, M. COMMISSO<fnref n="2" />, F. GUZZO<fnref n="2" />, S. DELROT<fnref n="1" />*

Berry content – nitrogen – systems biology – untargeted metabolomics

## Introduction

The oenological potential of grape berries depends on an adequate balance between sugar content, acidity, concentration of polyphenols and aroma precursors. Due to climate change and modifications of vineyard management, berry sugar content at harvest has increased over the last three decades, while acidity has decreased, and phenology was accelerated, with potential detrimental effects on aroma profiles. It is therefore important to understand the mechanisms controlling the balance between sugars/organic acids/polyphenols and aromas in order to maintain the present typicity of the wines through changes in viticultural practices and/or genetic improvement. Preliminary results obtained in our laboratory [1, 2] suggest that water stress indirectly affects the sugar/polyphenol balance through the nitrogen status.

Nitrogen deficiency not only affects the amino acid content of berries, but also their sugar content [3]. Nitrogen deficiency promotes anthocyanin biosynthesis in grapevine [4], tomato fruit [5] and apple [6]. However, the positive effects of nitrogen deficiency on polyphenol synthesis may be in part indirect, through a decreased vigour and an increase of berry exposure to sunlight [7]. Various results obtained in Bordeaux also suggest interactions between water stress, nitrogen deficiency and metabolism of aroma compounds [8-12].

Wine quality largely depends on the berry content at harvest. Berry content at harvest is made up of several hundreds of primary and secondary metabolites that will be transformed totally or in part during wine making, and finally interact with the sensory organs of the consumer in a complex way. The sensory properties are then integrated in the brain to determine the willingness of the consumer to pay a given price for an expected quality and tasting pleasure.

Berry composition at harvest depends on complex interactions between the genotype of the rootstock, the genotype of the scion, and their respective environment. In addition to choosing the genotypes used for vineyard plantation, the vine grower affects the environment sensed by the canopy and the fruit through plantation density, row orientation, partial leaf removal, and cluster thinning. The environment sensed by the root also depends in part of vineyard management, through irrigation (where it is authorized) and fertilizer supply. Among the different components provided by mineral nutrition, nitrogen plays an indirect role in the control of canopy vigour and sensitivity to diseases. It is also a component of many molecules contributing to berry composition and oenological potential, e.g. amino acids and aroma precursors. The nitrogen status of the cell directly or indirectly controls many different processes, which has led to the concept of nitrogen signalling.

While targeted approaches allow to identify isolated key compounds and parameters that affect precise steps affecting berry growth, ripening and composition, integrative approaches are definitely needed to identify interaction networks between climate, viticultural treatments, berry composition, wine making and wine quality.

<footnote n="1">¹Univ. Bordeaux, Ecophysiologie et Génomique Fonctionnelle de la Vigne, UMR 1287, ISVV, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²Univ. Verona, Biotechnology Department, Verona, Italy</footnote>
The present work uses this type of approach to investigate the effects of nitrogen treatment on the berry content of several variety/rootstock combinations.

# Materials and methods

## Plant material and extract preparation

Grapevines (cv. Cabernet sauvignon and Pinot noir) grafted on either RGM or 110R rootstocks were grown in 10-L pots and supplied three times per day (1.2 L/day/plant) by drip irrigation with three nitrogen levels (0.8, 1.4 and 3.6 mM). Except for nitrogen, all solutions had the same non-limiting concentrations of other mineral elements. Berries were collected at two stages: mid-ripening and ripening in 2013 and 2014. The skins were separated from the pulp, frozen at -80°C and freeze-dried. The dried skins and pulp were powdered, and skin metabolites were extracted in methanol containing 10% water and 0.1% formic acid, while pulp metabolites were extracted in methanol containing 10% water.

## Data analysis

An untargeted metabolomics LC-ESI-MS approach was developed for the simultaneous analysis of many compounds in the berries. The MS data matrix was imported into Simca software and unsupervised Principal Component (PCA) and supervised Orthogonal Partial Least Square Discriminant (OPLS-DA) analyses were performed. The unsupervised PCA was carried out to observe homogeneous sample clusters that were used as Y classes in the supervised OPLS-DA. As final output, molecules responsible for cluster separation in OPLS-DA were identified by plotting the pq Nature of the data (based on the correlation between *p* and *q*). The statistical analyses were validated by performing a) permutation test (200 permutations); b) CV-ANOVA (*p*<0.05); and c) *t*-test (*p*<0.05) for a cluster characterizing molecule.

# Results and discussion

The metabolomics analysis made on berry skin and pulp in negative ionization mode, and the following extraction of relevant information by MZmine software allowed to detect about 500 m/z features in the pulp and 700 m/z features in the skin; about 150 of them were putatively identified as molecular ion and their isotopes and adducts. The identified metabolites mainly belonged to the classes of anthocyanins, flavonoids, hydroxycinnamic and hydroxybenzoic acids, procyanidins and stilbenes. Since the more abundant metabolites were all putatively identified, the identified total signal (sum of the individual signal intensity of all the putatively identified metabolites) corresponded to the 61-80% of the total signal (sum of the signals of all the individual m/z features) in the skin, depending on the individual samples, and to 64-83% in the pulp. Skin and pulp were analysed separately; since they showed similar pattern, but were more pronounced in the skin samples, here only the skin sample results are shown.

As a first approach, data matrix were preliminary explored by the unsupervised PCA, showing, as expected, that the samples grouped primarily according to the cultivars and, secondly, also by years (fig. 1A,B).

The two cultivars were then analysed separately using O2PLS-DA, a supervised approach. The models generated by discriminant analysis were validated through ANOVA and through a permutation test (200 permutations). When the cultivars were analysed separately in more details, using the above supervised approach, they clustered both according to the nitrogen status (fig. 2A,B) and rootstock (fig.2 C,D).

<figure><img src="image_15.png" /><figcaption>**Fig. 1** - PCA score scatter plot. (A) The PCA shows two groups of samples: CS (Cabernet sauvignon) and PN (Pinot noir). (B) The PCA also shows two groups of samples according to years.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Fig. 2** - OPLS-DA score plot for nitrogen status (A and B) or rootstocks (C and D) dataset in CS and PN. Each symbol represents a single chromatography experiment. The analysis was carried out using three independent biological replicates for each treatment.</figcaption></figure>

Moreover, the inspections of the loadings showed that the behaviour of the two cultivars depended on the specific cultivar/rootstock combination: in the Cabernet sauvignon cultivar, the graft on the 110R rootstock yielded berries positively characterized by many metabolites, while, on the other hand, the berries that came from the Cabernet sauvignon-RGM combination did not show any characterizing metabolites (fig. 3A). At the opposite, the RGM-Pinot noir combination generates berries more rich in discriminating metabolites than the 110R-Pinot noir combination (fig. 3B).

<figure><img src="image_17.png" /><figcaption>**Fig. 3** - S-plot for the rootstock dataset, obtained by mapping p against pq Nature in OPLS-DA. Each point represents a compound detected and quantified by LC-MS. p is an indicator of the discriminant power of each molecule in the model, which directly depends on its abundance; pq Nature indicates the Correlation variable/rootstocks.</figcaption></figure>

## Conclusion

The metabolome of grape berry skin was analysed in order to investigate the impact of rootstocks and nitrogen on berry composition. This study revealed that the multivariate OPLS technique can be a useful approach for the analysis of metabolomics data. This technique allowed us to identify metabolites which
characterize specific cultivar/rootstock combinations. For each cultivar (CS or PN), the type of rootstock affects the berry metabolome. The CS grafted onto 110R positively correlates with the accumulation of anthocyanin 3-O-glucoside and anthocyanin (acetyl)-3-O-glucoside. On the other hand, no specific secondary metabolites are correlated with the grafting of CS on RGM. Likewise, in Pinot noir, only one metabolite (quercetin (acetyl)-3-O-glucoside) is highly positively correlated with the 110R rootstock. On the other hand, PN grafted onto RGM positively correlates with two resveratrol-O-glucosides and with some hydroxycinnamic acid derivatives. Our result confirms that the genotypes of both rootstock and cultivar affect berry composition and oenological potential. In particular, some cultivar/rootstock combinations are characterized by the accumulation of secondary metabolites such as anthocyanins or stilbenes.

A better knowledge of the accumulation of berry metabolites in response to nitrogen, rootstock genotype and variety genotype could be an asset in viticultural practices to maintain or improve the quality of the wines. Nitrogen status affects the grape berry metabolome depending on cultivar/rootstock combination (fig. 2 and data not shown). This large metabolomic dataset can serve as a basic framework to integrate omics data and understand the rootstock-scion relationship with nitrogen status at the berry level.

## Abbreviations

CS, Cabernet sauvignon; PN, Pinot noir; RGM, Riparia Gloire de Montpellier; 110R, 110 Richter; PCA, principal component analysis; OPLS-DA, orthogonal projection to latent structures-discriminant analysis.

## References

1. Hilbert G (2002) Effets de la nutrition azotée et du stress hydrique sur la maturation et la composition en anthocyanes des baies de Vitis vinifera L. au vignoble et en conditions contrôlées. *Thèse de doctorat*, Université de Bordeaux.

2. Peccoux A (2011) Molecular and physiological characterization of grapevine rootstock adaptation to drought. *Thèse de doctorat*, Université de Bordeaux.

3. Barbier H (2004) Recherche de marqueurs associés à la contrainte en azote et en carbone chez la vigne (Vitis vinifera var. Cabernet sauvignon). *Thèse de doctorat*, Université de Bordeaux.

4. Do CB, Cormier F (1991) Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension. *Plant Cell Rep* **9**: 500-504.

5. Tan SC (1980) Phenylalanine ammonia-lyase and the phenyl-alanine ammonia-lyase inactivating system: effects of light, temperature and mineral deficiencies. *Aust J Plant Physiol* **7**: 159-167.

6. Bongue-Bartelsman M, Phillips DA (1995) Nitrogen stress regulates gene expression of enzymes in the flavonoid biosynthetic pathway of tomato. *Plant Physiol Biochem* **33**: 539-546.

7. Maigre D (2001) Enherbement permanent et fumure azotée sur cv. Gamay dans le Valais central. 3: microclimat lumineux et thermique des grappes. *Rev Suisse Vitic Arboric Hortic* **33**: 305-308.

8. Van Leeuwen C, Friant P, Soyer JP et al (2000) L'intérêt du dosage de l'azote total et de l'azote assimilable dans le moût comme indicateur de la nutrition azotée de la vigne. *J Int Sci Vigne Vin* **34**: 75-82.

9. Chone X, Van Leeuwen C, Chéry P et al (2001) Terroir influence on water status and nitrogen status of non-irrigated Cabernet sauvignon (*Vitis vinifera*): vegetative development, must and wine composition (example of a Medoc top estate vineyard, Saint Julien area, Bordeaux, 1997). *S Afr J Enol Vitic* **22**: 8-15.

10. Chone X, Lavigne-Cruege V, Tominaga T et al (2006) Effect of vine nitrogen status on grape aromatic potential: flavor precursors (S-cysteine conjugates), glutathione and phenolic content in *Vitis vinifera* L. cv. Sauvignon blanc grape juice. *J Int Sci Vigne Vin* **40**: 1-6.

11. Tregoat O, Van Leeuwen C, Chone X et al (2002) Etude du régime hydrique et de la nutrition azotée de la vigne par des indicateurs physiologiques. Influence sur le comportement de la vigne et la maturation du raisin (Vitis vinifera L. cv Merlot, 2000, Bordeaux). *J Int Sci Vigne Vin* **36**: 133-142.

12. Peyrot des Gachons C, Van Leeuwen C, Tominaga T et al (2005) Influence of water and nitrogen deficit on fruit ripening and aroma potential of Vitis vinifera L. cv Sauvignon blanc in field conditions. *J Sci Food Agric* **85**: 73-85.
# Adaptation to climate change: strategic issues to face for the French wine industry

N. OLLAT¹*, H. QUÉNOL², G. BARBEAU³, C. VAN LEEUWEN¹, P. DARRIET⁴, I. GARCIA DE CORTAZAR ATAURI⁵,
B. BOIS⁶, H. OJEDA⁷, E. DUCHÊNE⁸, E. LEBON⁹, P. VIVIN¹, P. THIS¹⁰, J.M. SABLAYROLLES¹¹, G. TEIL¹²,
P. LAGACHERIE¹³, E. GIRAUD-HÉRAUD¹⁴, P. NEVEU¹⁵, J.M. TOUZARD¹⁶

¹EGFV, Bordeaux Sciences Agro, INRA, Université de Bordeaux, 33140 Villenave d'Ornon, France

²UMR Costel, CNRS-Univ de Rennes, Place du Recteur Henri Le Moal, 35043 Rennes

³UVV, INRA, 42 rue Georges Morel BP 60057, 49 071 Beaucouzé, France

⁴Laboratoire d'Œnologie, ISVV-Univ de Bordeaux, 33140 Villenave d'Ornon, France

⁵AgroClim, INRA, Domaine Saint Paul, Site Agroparc, CS 40 509 - 84914 Avignon, France

⁶Institut Universitaire de la Vigne et du Vin - Univ de Bourgogne, 21000 Dijon

⁷Experimental Unit of Pech Rouge, INRA, 11430 Gruissan, France

⁸UMR SVQV, INRA-Univ de Strasbourg, 28, rue de Herrlisheim, 68021 Colmar, France

⁹UMR LEPSE, INRA-Supagro, 2, place Viala, 34060 Montpellier, France

¹⁰UMR AGAP, INRA-Supagro-CIRAD, 2, place Viala, 34060 Montpellier, France

¹¹UMR SPO, INRA-Supagro, 2, place Viala, 34060 Montpellier, France

¹²UMR SAD APT, INRA-Agroparistech, 16 rue Claude Bernard, 75005 Paris, France

¹³UMR LISAH, INRA-IRD-Supagro, 2, place Viala, 34060 Montpellier, France

¹⁴UMR ALISS, INRA, 65 Boulevard Brandebourg, 94205 Ivry Sur Seine, France

¹⁵UMR MISTEA, INRA-Supagro, 2, place Viala, 34060 Montpellier, France

¹⁶UMR Innovation, INRA-Supagro, 2, place Viala, 34060 Montpellier, France

## Introduction

## Introduction

According to the Vth Intergovernmental Panel on Climate Change report, climate change over the last decades is unequivocal and will continue for the following century [1]. As stated by several authors [2-4], these changes will impact vine growing and the quality of wines produced all over the world. Consequently, adaptation defined as “the set of organization, localization and technical changes that societies will have to implement to limit the negative effects of climate change and to maximize the beneficial ones” is a necessity [5, 6]. The main goal of adaptation strategies, as expressed by representatives of the French wine industry, is to maintain yield potential and wine typical quality – i.e. typicity – at a regional level [7]. Facing increasing socio-economic and scientific questions, at regional and national levels, the Institut National de la Recherche Agronomique (INRA) decided to launch a multidisciplinary research project to explore not only the impacts of climate change on grapevines and wines, but also the current and future adaptation strategies. The LACCAVE project brings together the expertise of 23 research laboratories, aiming at developing a common knowledge base on climate change issues, as well as defining and assessing adaptation strategies at local, regional and national levels. The aim of this paper is to give an overview of the predicted impacts of climate change on the wine industry, especially in France, and of the possible adaptation strategies that are explored in the frame of the LACCAVE project.

## Climatic scenario for French vineyards

All over the world, the increase in average surface temperature by the end of the 21st century is predicted to be likely over 1.5°C in all the scenarios and up to 4.8°C in the most extreme one corresponding to an atmospheric [CO₂] of about 1400 ppm. Precipitations will be affected with a larger range between dry and
wet regions, as well as between dry and wet seasons [1]. However, changes will be uneven all over the world and adaptation will occur at local levels. Consequently, there is a crucial need for downscaling climatic scenario at regional or local levels. For France, regional simulations were established from two climatic models (Aladin-Climat and WRF) at 64 km² resolution (SAFRAN grid 8 km x 8 km) for 3 of the 4 general RCP scenarios of GIEC. The reference period was defined as 1976-2005 and climatic conditions were simulated for the near future (2021-2050) and the far future (2071-2100). Data are available on the DRIAS platform (www.drias-climat.fr). Computations exhibited an increase of average temperature of 0.6 to 1.3°C regardless of the season and the scenario for the 2021-2050 period, with a more pronounced increase of 1.5-2.0°C in the South East of France during summer. Heat waves during summer might increase from 0 to 5 days in average and 5 to 10 days in the South East of France. Extreme cold day number will decrease by 1 to 4 days in average, and by up to 6 days in the North East of France (currently the coldest region). Precipitations might slightly increase regardless of the season but uncertainties are very high. For the 2071-2100 period, a strong increase of temperature is predicted, reaching 0.9°C in winter and 1.3°C in summer for the optimistic scenario, and 3.4-3.6°C increase in winter and 2.6-5.3°C increase in summer for the more pessimistic scenario, with a rise in heat waves during summer (> 20 days) and a decrease in extreme cold days during winter. Uncertainties remain for precipitations with a simulated increase in winter (up to 76 mm over the whole winter) and a decrease (Aladin-Climat model) or increase (WRF) in summer for the two medium and pessimistic scenarios. Extreme precipitation events might occur more frequently all over France but with a large variability among regions. An increase of drought periods are simulated in the South East of France, but this situation could be extended all over the country [8]. For each of six selected grape growing regions in France, climatic data were extracted and analyzed for three SAFRAN grid 64 km² areas located in zones actually planted with vine (class #221 of the Corine Land Cover 2006 geodatabase; [9]). In that case, the more pessimistic SRES A2 (equivalent to RCP8.5) scenario was used. Focusing on the temperature during ripening, it was shown that maximal and minimal temperatures are expected to overlap between vineyards and region over the 21st century. This means that vineyards from the Northern part of France would progressively face situations already experienced by French Mediterranean vineyards. In addition, projected thermic range between regions decreases, with the Northern vineyards warming up more than the Southern vineyards. In Languedoc-Roussillon (southern France), Lebon and Garcia de Cortazar-Atauri have shown a rise of average temperature from 1.2 to 3.2°C for the near and far future periods, respectively [10]. This leads to an increase of the evaporative demand of 53 mm (+6%) and 148 mm (+18%) for the corresponding periods. Meanwhile, the amount of annual rain is expected to decrease of 100 mm as early as 2021-2050 period with an increase of inter-annual variability for the near future, and a decrease for the far future.

## Impacts on grapevine production

Impacts of climate change on grapevine biology and production has been reviewed recently [7]; and references therein. The timing of all the phenological stages is expected to be anticipated in the future. Compared to the last 30 years, budbreak and the ripening period should occur 3 to 18 days and 20 to 40 days earlier, respectively, in the second half of the 21st century. The effects of extreme temperatures on grapevine development must also be taken into account. The shift towards earlier, warmer ripening periods will increase the impact of temperature on the ripening process. Based on changes in grapevine phenology, a temperature increase of 4-6°C in Southern France and 6-8°C in Northern France may be observed during the ripening period. Yield is under the control of several climatic drivers such as atmospheric [CO₂], temperature, water supply and nutrient availability. These drivers and their effects will interact in a complex manner. For example, leaf area and total vegetative dry weight increase to a greater extent than fruit dry weight and yield for vines growing in a free-air CO₂ enrichment system without stress. Water deficit is expected to increase in duration and intensity after 2050, with negative impacts on yield mainly in the South of France [10]. An increase in berry sugar content has already been reported for the last decades of the 20th century. Fruit composition will be further modified towards higher sugar content, lower acidity, and modified polyphenol and aroma content, with major impacts on the suitability to elaborate the current types of wines. High temperature induces a greater decrease of malic acid concentration compared to that of tartaric acid concentration. Combined with an increase in potassium uptake, grape juice pH is strongly increased. Large varietal differences have been observed in the balance between malate and tartrate concentrations and in the response of titratable acidity and pH to heating. Polyphenolic and aroma compounds will be affected quantitatively and qualitatively as well. The interactions between various environmental parameters such as extreme temperatures, light intensity and quality are critical for this kind
of compounds. The disequilibrium between primary and secondary metabolisms may increase, which should make the decision about harvest dates more difficult [11]. Changes in the epidemiological pattern and geographical distribution of pests and pathogens are expected [12]. Beyond microbial, microflora and pathogen activity, the whole ecosystems surrounding the vineyards will also be impacted by climate change. Considering the high speed of climate change when compared to geological climate changes, the probability for these ecosystems to survive unscathed is very low [13]. The disappearance of certain species may set off a chain reaction among other species. These changes may be very wide-ranging and are extremely difficult to predict [1]. They will affect microclimate, landscape, local competition for resource, composition and structure of soils, and consequently grapevine development [3, 14]. The wine growers must therefore adapt to the change in the climate itself, and the modification of the environment due to climate change [15].

## Adaptation strategies

Adaptation will likely occur through the combination of technical innovations, some growing area migration and institutional changes, at both organizational and regulatory levels [7].

Many oenological innovations are already available and can offset some effects of climate change. In addition to physical and chemical techniques, the selection of yeast reducing the alcohol content is an option [16]. Blends of musts with high and low (from early picking) sugar content may also be efficient. Although oenological practices can be considered as the most flexible short-term adaptation strategy with a short life span, long-term costs and acceptance have to be considered [11]. The beneficial effects of existing viticultural practices have to be evaluated in regards of the intensity of forecasted climate changes and spatial scales of adaptation [17, 18]. Among techniques, irrigation becomes a critical option, raising issues related to cost, quality and access to water, regulation and quality objectives [19]. Even if it appears as the most straightforward way to cope with drought, investing in costly irrigation systems could not be a sustainable option on a long-term scale [15]. It should only be promoted after a comprehensive assessment of water resources and competing needs at the territory scale. In addition to precision viticulture, innovative techniques of mechanization and micro-climate management must also be dreamed-up. In the long term, breeding and the introduction of varieties and rootstocks more resilient to higher temperatures or drought are probably unavoidable [20]. Spatial management of the implementation of technical innovations is important. Large variations in climatic conditions exist within the same wine regions due to geomorphology, land use or distance to large water bodies and urban areas [14]. These variations bring many opportunities for adaptation at a local scale. Modeling the spatial repartition of practices, taking into account human and environmental components, may help to define practical solutions to the growers and policy makers [21]. Relocation of vineyards and wineries at higher altitude or to cooler parts of a wine region also has to be considered [7]. In the context of adaptation, institutional changes appear inevitable. The ability of the AOC system to integrate climatic, technical and zoning changes is a major issue. Insurance systems may also be considered. At another level, the development of innovative regional clusters is an important lever to reinforce the capacity of growers to cope with climate change [22].

## Challenges for the wine industry

The adaptability of the wine sector is influenced by economic, sociological and legal factors. French northern regions have relatively more freedom with respect to adaptation in comparison to southern regions where, in the long term, bifurcation towards new systems may be more likely [15]. Climate change will impact production costs and the relationship between quality and geographical origins. These factors are critical for the competitiveness of the wine industry at local, national but also international scale.

The perception of changes and of the associated challenges is a condition for adaptation [23]. The awareness of growers, wine-makers and professional organizations and the strategies implemented or planned have to be evaluated [21, 24] with a particular focus on the concept of “terroir” [25, 26]. Finally, the perception of consumers and their willingness to purchase new types of wines are also key issues [27]. Our research, based on sensory analysis frameworks and the implementation of experimental markets, shows how the stability of preferences cannot be guaranteed. Instead, consumers could less enjoy wines whose characteristics are particularly influenced by global warming. To maintain a wine type as it is defined today will become a big challenge for producers and researchers. To project ourselves – actors of
the wine industry and scientists – into an uncertain future is a complicated task. A foresight exercise is a comprehensive approach which proposes to define adaptation scenarios combining different possible levers depending on the intensity of the changes planned or contemplated. For France, five scenarios have been drawn in the frame of the LACCAVE project [7]: i) the “static scenario” where the industry does not change to adapt; ii) the “conservative scenario” relies on incremental innovations and relocations; iii) the “innovative scenario” focuses on radical technical changes (new varieties, practices, wines, etc.); iv) the “nomad scenario” promotes vine relocation, including in new regions; and finally v) the “liberal scenario” expresses a wide range of adaptation opportunities. These scenarios will serve as a basis of debate with the actors of the wine industry in the different producing regions and should provide a framework to guide and prioritize actions whether in research, innovation transfer agencies or the institutional device of the French wine industry.

## References

1. IPCC (2013) *Climate Change 2013 - The Physical Science Basis. Summary for Policymakers*. http://www.ipcc.ch/report/ar5/wg1/#.Ulu4Tz8udZi.

2. Schultz HR (2000) Climate change and viticulture: a European perspective on climatology, carbon dioxide and IV-B effects. *Aust J Grape Wine Res* **6**: 2-12.

3. Seguin B (2010) *Coup de Chaud sur l'Agriculture*. Ed. Delachaux et Niestlé, Paris.

4. Hannah L, Roehrdanz PR, Ikegami M et al (2013) Climate change, wine, and conservation. *Proc Natl Acad Sci USA* doi/10.1073/pnas.1210127110.

5. Hallegatte S, Lecocq F, de Perthuis C (2011) *Designing Climate Change Adaptation Policies - An Economic Framework*. Policy Research Working Paper 5568, The World Bank.

6. Van Leeuwen C, Schultz HR, Garcia de Cortazar-Atauri I et al (2013) Why climate change will not dramatically decrease viticultural suitability in main wine-producing areas by 2050. *Proc Natl Acad Sci USA* doi/10.1073/pnas.1307927110.

7. Ollat N, Touzard JM (2014) Impacts and adaptation to climate change: new challenges for the French wine industry. *J Int Sci Vigne Vin*, Laccave project (special issue): 75-78.

8. Ouzeau G, Déqué M, Jouini M et al (2014) *Le Climat de la France au XXIe Siècle. Vol 4. Scénarios Régionalisés : Édition 2014 pour la Métropole et les Régions d'Outre-Mer*. Ministère de l'écologie, du développement durable et de l'énergie.

9. European Environment Agency (2007) *CLC2006 Technical Guidelines EEA Technical Report 17/2007*. Luxembourg: Office for Official Publications of the European Communities.

10. Lebon E, Garcia de Cortazar-Atauri I (2014) CIAG, Montpellier, INRA, 38: 1-12.

11. Zamora F (2014) Adapting red winemaking to climate change conditions. *J Int Sci Vigne Vin*, Laccave project (special issue): 69-74.

12. Caubel J, Launay M, Garcia de Cortazar-Atauri I et al (2014) A new integrated approach to assess the impacts of climate change on grapevine fungal diseases: the coupled MILA-STICS model. *J Int Sci Vigne Vin*, Laccave project (special issue): 43-52.

13. Soussana JF (2013) *S'Adapter au Changement Climatique : Agriculture, Écosystèmes et Territoires*. Ed. QUAE.

14. Quénot H, Bonnardot V (2014) A multi-scale climatic analysis of viticultural terroirs in the context of climate change: the “TERADCLIM” project. *J Int Sci Vigne Vin*, Laccave project (special issue): 23-32.

15. Viguié V, Lecocq F, Touzard JM (2014) Viticulture and adaptation to climate change. *J Int Sci Vigne Vin*, Laccave project (special issue): 53-58.

16. Tilloy V, Ortiz-Julien A, Dequin S (2014) Reducing ethanol and improving glycerol yield by adaptive evolution of *Saccharomyces cerevisiae* wine yeast under hyperosmotic conditions. *Appl Environ Microbiol* doi/10.1128/AEM.03710-13.

17. Barbeau G, Goulet E, Neethling E et al (2014) Les méthodes d'adaptation au changement climatique. In: *Changement Climatique et Terroirs Viticoles*, Paris, France, Editions Tec & Doc, Lavoisier.

18. Parker AK, Hofmann RW, van Leeuwen C et al (2014) Leaf area to fruit mass ratio determines the time of veraison in Sauvignon Blanc and Pinot Noir grapevines. *Aust J Grape Wine Res* 20: 422-431.

19. Ojeda H, Saurin N (2014) CIAG, Montpellier, INRA, 38: 97-108.

20. Duchêne E, Huard F, Pieri P (2014) Grapevine and climate change: what adaptations of plant material and training systems should we anticipate? *J Int Sci Vigne Vin*, Laccave project (special issue): 59-67.

21. Neethling E, Barbeau G, Quénot H et al (2013) *International Conference: Climate Change and Regional Response*, May 27-29, 2013, Dresden, Germany.

22. Touzard JM (2010) *Innovation and Sustainable Development in Agriculture and Food*, Montpellier, CIRAD.
23. Holland T, Smit B (2010) Climate change and the wine industry: current research themes and new directions. *J Wine Res* **21**: 125-136.

24. Boyer J, Ollat N, Touzard JM et al (2014) *XXI Enometrics Conference*, Lyon, France.

25. Van Leeuwen C, Seguin G (2006). The concept of terroir in viticulture. *J Wine Res* **17**: 1-10.

26. Teil G (2014) Nature, the co-author of its products? *J World Intell Prop* **17**: 96-113.

27. Fuentes A, Giraud-Heraud E, Peres S et al (2014) *XXI Enometrics Conference*, Lyon, France.
# Growing grapes in predicted climate change conditions: what can we learn from multiple stress in controlled conditions?

J. MARTINEZ-LÜSCHER<fnref n="1" />,<fnref n="2" />, F. MORALES<fnref n="3" />, G. HILBERT<fnref n="4" />, M. SÁNCHEZ-DIAZ<fnref n="2" />, J. AGUIRREOLA<fnref n="2" />, S. DELROT<fnref n="1" />, I. PASCUAL<fnref n="2" />, E. GOMES<fnref n="1" />*

Grape physiology – berry quality – abiotic stress – climate change

## Introduction

Environmental factors such as average atmospheric temperature and carbon dioxide (CO₂) levels are predicted to increase by the end of the century [1]. In addition, changes in climate (more clear days) as well as stratospheric ozone may condition the exposure of viticultural areas to UV-B. This is likely to impact leaf carbon metabolism, which in turn will affect fruit ripening rates and berry quality at harvest [2]. Indeed, vine cultivation is already affected by global climate change, and a greater impact is expected in the coming decades. Comparing harvest dates from previous decades with current ones revealed an advance of 2-3 weeks. Thus, there is a widespread phenomenon whereby the accumulation of sugar in the grapes occurs at an accelerated rate, and this in turn may affect other quality parameters, such as acidity or berry color [3]. Factors framed within climate predictions (CO₂ levels, temperature, water availability and solar radiation) have individually shown impact on both physiology and composition of grapes [3]. The aim of this work was to study how the combination of UV-B radiation and various environmental factors affected by climate change would impact the most important physiological processes in vine leaf and how it might affect ripening and fruit quality.

## Materials and methods

Grapevine (Vitis vinifera cv. Tempranillo) fruit-bearing cuttings were grown in controlled conditions (glasshouse) as previously described [4]. Controlled stress conditions were applied on the grapevines in glasshouses at the University of Navarra, Pamplona, Spain (42°48'N; 1º40'W). Three separate experiments were performed:

- experiment 1, in which three doses of UV-B were tested (0, 5.98 and 9.66 kJ.m⁻<fnref n="2" />.d⁻<fnref n="1" /> of biologically effective UV-B) in two different developmental stages (from fruit set to maturity and from the onset of *véraison* to maturity);

- experiment 2, in which the same three doses of UV-B were applied, and combined with two water availability regimes (cyclic drought and optimum irrigation);

- experiment 3, in which the three doses of UV-B, combined with two CO₂ and temperature regimes, were tested: 390 ppm CO₂–24/14°C day/night (designated -CO₂/-T) and 700 ppm CO₂–28/18°C day/night (designated +CO₂/+T).

<footnote n="1">¹Univ. Bordeaux, Ecophysiologie et Génomique Fonctionnelle de la Vigne, UMR 1287, ISVV, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²Universidad de Navarra, Grupo de Fisiología del Estrés en Plantas (Dpto. Biología Ambiental), Unidad Asociada al CSIC, EEAD, Zaragoza e ICVV, Logroño, Irunlarrea 1, 31008 Pamplona, Spain</footnote>

<footnote n="3">³Estación Experimental de Aula Dei (EEAD), CSIC, Dpto. Nutrición Vegetal, Apdo. 13034, 50080 Zaragoza, Spain</footnote>

<footnote n="4">⁴INRA Bordeaux, Ecophysiologie et Génomique Fonctionnelle de la Vigne, UMR 1287, ISVV, F-33140 Villenave d'Ornon, France</footnote>

<footnote>*Corresponding author: eric.gomes@bordeaux.inra.fr</footnote>
Leaf gas exchange, chlorophyll fluorescence, lipid peroxidation, antioxidant enzyme activity, level of UV-absorbing compounds, and concentration of chlorophylls and carotenoids were determined according to Martínez-Lüscher et al. [4]. Berry phenology, skin anthocyanin concentration and must pH were analyzed as described previously [5].

## Results and discussion

### Impact on leaf physiology

Leaf exposure to UV-B radiation induced a reduction in photosynthetic rates, mainly in the short term (20 days). This was caused largely by a reduction in leaf stomatal conductance, although the efficiency of photosystem II (ΦPSII and Fv/Fm) was also significantly affected. After an acclimation period, photosynthetic rates were restored to initial values with a sharp increase in antioxidant levels in leaves and berries [4].

<table><caption>**Table 1** - Time to reach technological maturity (in days) for the various stress conditions. Values are the mean ± standard error of 10-12 replicates. Letters indicate the results of an ANOVA and LSD post-hoc test (p< 0.05).</caption><thead><tr><th>UV-B doses (kJ.m<sup>-2</sup>.d<sup>-1</sup>)</th><th>0</th><th>5.98</th><th>9.66</th></tr></thead><tbody><tr><td><strong>Experiment 1</strong> UV from fruit set</td><td>84.3±1.5<sup>b</sup></td><td>82.5±0.7<sup>b</sup></td><td>84.0±2.2<sup>b</sup></td></tr><tr><td>UV from <em>véraison</em></td><td>nd</td><td>83.7±2.0<sup>b</sup></td><td>85.5±4.2<sup>b</sup></td></tr><tr><td><strong>Experiment 2</strong> Optimal irrigation</td><td>64.5±1.0<sup>d</sup></td><td>76.0±5.7<sup>bc</sup></td><td>70.6±3.5<sup>c</sup></td></tr><tr><td>Cyclic drought</td><td>77.7±3.4<sup>b</sup></td><td>82.4±2.4<sup>b</sup></td><td>91.6±2.8<sup>a</sup></td></tr><tr><td><strong>Experiment 3</strong> -CO<sub>2</sub>/-T</td><td>101.0±6.9<sup>a</sup></td><td>103.2±6.8<sup>a</sup></td><td>108.8±2.5<sup>a</sup></td></tr><tr><td>+CO<sub>2</sub>/+T</td><td>78.0±2.7<sup>b</sup></td><td>83.0±5.5<sup>b</sup></td><td>82.6±5.2<sup>b</sup></td></tr></tbody></table>

### Impact on grape phenology

The time to reach the onset of véraison was not affected by UV-B radiation doses. Conversely, drought stress and the combination of high temperature and CO₂ did significantly advanced the onset of ripening. Indeed, numerous studies have shown that early véraison is a general response to water stress in grape, through an increase in abscissic acid, a ripening promoting hormone in this non-climacteric fruit [3]. The time to reach technological maturity (22 °Brix) was affected by all the applied stress factors (Table 1). It was increased in plants exposed to UV-B radiation combined with optimal irrigation and further increased in plant submitted to a combination of high UV-B dose and cyclic drought. Elevated CO₂ and high temperature hastened berry ripening, a trend mildly attenuated by high UV-B doses [6].

<table><caption>**Table 2** - Skin anthocyanin concentrations in berry skins at maturity, expressed in mg.g⁻¹ of dry mass. Values are the mean ± standard error of 10-12 replicates. Letters indicate the results of an ANOVA and LSD post-hoc test (p< 0.05).</caption><thead><tr><th>UV-B doses (kJ.m<sup>-2</sup>.d<sup>-1</sup>)</th><th>0</th><th>5.98</th><th>9.66</th></tr></thead><tbody><tr><td><strong>Experiment 1</strong> UV from fruit set</td><td>15.4±1.1<sup>ab</sup></td><td>18.0±2.3<sup>a</sup></td><td>15.9±1.9<sup>ab</sup></td></tr><tr><td>UV from <em>véraison</em></td><td>nd</td><td>17.6±0.8<sup>a</sup></td><td>13.2±0.6<sup>b</sup></td></tr><tr><td><strong>Experiment 2</strong> Optimal irrigation</td><td>13.5±0.6<sup>b</sup></td><td>14.1±0.5<sup>ab</sup></td><td>16.2±0.7<sup>a</sup></td></tr><tr><td>Cyclic drought</td><td>13.4±0.6<sup>b</sup></td><td>14.9±1.3<sup>ab</sup></td><td>15.3±0.9<sup>ab</sup></td></tr><tr><td><strong>Experiment 3</strong> -CO<sub>2</sub>/-T</td><td>21.6±0.4<sup>b</sup></td><td>28.1±0.5<sup>ab</sup></td><td>29.9±0.6<sup>a</sup></td></tr><tr><td>+CO<sub>2</sub>/+T</td><td>18.6±0.2<sup>d</sup></td><td>22.9±1.6<sup>c</sup></td><td>23.5±0.5<sup>c</sup></td></tr></tbody></table>

### Impact on must pH values

Neither UV-B radiation, applied from fruit set or from véraison, nor elevated CO₂ and temperature conditions did affect must pH (Table 2, experiments 1 and 3). Conversely, water deficit significantly increased must pH, whatever the UV-B dose applied (Table 2, experiment 2).
<table><caption>**Table 3** - Must pH values at maturity. Values are the mean ± standard error of 10-12 replicates.
Letters indicate the results of an ANOVA and LSD post-hoc test (p< 0.05).</caption><thead><tr><th>UV-B doses (kJ.m<sup>-2</sup>.d<sup>-1</sup>)</th><th>0</th><th>5.98</th><th>9.66</th></tr></thead><tbody><tr><td><strong>Experiment 1</strong> UV from fruit set</td><td>3.99±0.04<sup>a</sup></td><td>3.91±0.10<sup>a</sup></td><td>3.91±0.10<sup>a</sup></td></tr><tr><td>UV from véraison</td><td>nd</td><td>3.88±0.01<sup>a</sup></td><td>3.98±0.10<sup>a</sup></td></tr><tr><td><strong>Experiment 2</strong> Optimal irrigation</td><td>3.38±0.03<sup>d</sup></td><td>3.48±0.06<sup>d</sup></td><td>3.59±0.07<sup>c</sup></td></tr><tr><td>Cyclic drought</td><td>3.65±0.04<sup>c</sup></td><td>3.89±0.07<sup>b</sup></td><td>3.59±0.07<sup>c</sup></td></tr><tr><td><strong>Experiment 3</strong> -CO<sub>2</sub>/-T</td><td>3.60±0.01<sup>b</sup></td><td>3.63±0.03<sup>b</sup></td><td>3.70±0.01<sup>a</sup></td></tr><tr><td>+CO<sub>2</sub>/+T</td><td>3.63±0.03<sup>b</sup></td><td>3.63±0.03<sup>b</sup></td><td>3.70±0.01<sup>a</sup></td></tr></tbody></table>

## Conclusion

In this study, grapevine, and more specifically cv. Tempranillo, showed great resilience to high levels of UV-B radiation. Despite inducing a transient reduction in photosynthetic activity, the contribution of UV-B radiation (delaying maturation, increasing the proportion of skins and the synthesis of anthocyanins and flavonols in the berry) resulted in a positive balance in terms of grape quality. The water deficit in this case did not produce an increase in the synthesis of phenolic compounds in the skin, but increased their proportion in the skins. The conditions of elevated CO₂ and temperature did not negatively and directly affect the leaf physiology and grape composition, but severely affected the rate of maturation, which largely decoupled technological-phenolic maturation. The result was a lower concentration of anthocyanins for the same degree Brix. This study raises the need to (i) further investigate the effects of climate change on plants and (ii) develop cultivation techniques adapted to the new conditions that are to come, in order to preserve not only crop yields, but also the enological potential of the berries.

## References

1. IPCC (2014) *Climate Change 2014: Mitigation of Climate Change*. Working group III contribution to the fifth assessment report of the Intergovernmental Panel on Climate Change.

2. Chuine I, Yiou P, Viovy N et al (2004) Historical phenology: grape ripening as a past climate indicator. *Nature* **432**: 289-290.

3. Kuhn N, Guan L, Dai ZW et al (2014) Berry ripening: recently heard through the grapevine. *J Exp Bot* **65**: 4543-4559.

4. Martínez-Lüscher J, Morales F, Delrot S et al (2013) Short- and long-term physiological responses of grapevine leaves to UV-B radiation. *Plant Sci* **213**: 144-122.

5. Martínez-Lüscher J, Torres N, Hilbert G et al (2014) Ultraviolet-B radiation modifies the quantitative and qualitative profile of flavonoids and amino acids in grape berries. *Phytochemistry* **102**: 106-114.

6. Martínez-Lüscher J, Morales F, Sánchez-Díaz M et al (2015) Climate change conditions (elevated CO₂ and temperature) and UV-B radiation affect grapevine (*Vitis vinifera* cv Tempranillo) leaf carbon assimilation, altering fruit ripening rates. *Plant Sci* **236**: 168-175.

7. Martínez-Lüscher J, Sánchez-Díaz M, Delrot S et al (2014) Ultraviolet-B radiation and water deficit interact to alter flavonol and anthocyanin profiles in grapevine berries through transcriptomic regulation. *Plant Cell Physiol* **55**: 1925-1936.
# Pre- and post-flowering leaf removal as affecting methoxypyrazines, thiol precursors, glutathione and thiols in Sauvignon blanc grapes and wines

P. SIVILOTTI<fnref n="1" />*, B. ŠKVARČ<fnref n="1" />, L. BUTINAR<fnref n="1" />, M. STERNAD LEMUT<fnref n="1" />, H. BAŠA ČESNIK<fnref n="2" />,
Š. VELIKONJA BOLTA<fnref n="2" />, A. VANZO<fnref n="2" />, K. LISJAK<fnref n="2" />

Sauvignon blanc – early leaf removal – thiols – methoxypyrazines – glutathione

## Introduction

The removal of leaves has important effects on yield components and secondary metabolism in grapes. In general, leaf removal improves the microclimate of the cluster area, and the application of the technique at different timings also has a different impact on composition and concentration of secondary metabolites in grapes at harvest [1]. Caspari et al. already reported that leaf removal around flowering has important impact on flower fertility, leading to smaller and looser clusters [2]. Among secondary metabolites, a lot of results were published in the last years as regard polyphenols, whereas little information is available for aroma compounds [3-5], and only one study reported the positive effect of leaf removal on thiol occurrence [6].

Volatile thiols have a low perception threshold and their concentrations in wines will determine the aroma perceived [7]. 4-Mercapto-4-methylpentan-2-one (4MMP) is a volatile thiol that accounts for a reminiscence of box tree, passion fruit, and elderberry (olfactory threshold of 0.8 ng/L), while 3-mercaptohexan-1-ol (3MH) impacts wine aroma with notes of passion fruit, grapefruit, gooseberry and guava (olfactory threshold 60 ng/L) and 3-mercaptohexyl acetate (3MHA) with notes of passion fruit, box tree, and gooseberry (olfactory threshold of 4.2 ng/L) [8, 9]. Since there is a lack of knowledge of the effect of leaf removal on the occurrence of thiol precursors in grapes, an experiment was carried out in a Sauvignon blanc vineyard in the seasons 2013 and 2014.

## Materials and methods

### Leaf removal treatments

An experimental trial was carried out in a Sauvignon blanc vineyard located in Oslavia (North-Eastern Italy) during the seasons 2013 and 2014. A completely randomized experimental design was set up with three treatments and three replicates. The treatments compared were: UNT, untreated (no leaves removed); ELR, pre-flowering leaf removal (10 days before flowering 5-6 basal leaves/shoot were removed); and LLR, post-flowering leaf removal (10 days after flowering 2-3 basal leaves/shoot were removed).

### Metabolite analysis

For each plot, 10 plants were marked and yield parameters at harvest were collected. From veraison to harvest, grapes were collected for the analysis of both basic parameters and secondary metabolites. 50-Berry samples were collected in plastic bags, hand-squeezed and analyzed with a WineScan™ FT120 Basic (FOSS, Hillerød, Danimarca). Additional 50-berry samples were collected and immediately frozen in liquid nitrogen in the vineyard, transferred in dry ice and stored at -80°C until thiol precursor and methoxypyrazine analyses. Frozen grapes were cryogenically ground and the powder was stored back at -80°C. For the analysis of thiol precursors, a slightly modified method as described by Lisjak et al. was used [10]. Pulverized grapes were rapidly transferred into ice-cold, deoxygenated methanol (1:4, w/v). The extract was vortexed, spiked by deuterium-labelled internal standards and centrifuged. Part of the extract was directly injected for the determination of glutathione and oxidized glutathione. Extracts were cleaned-

<footnote n="1">¹University of Nova Gorica, Wine Research Centre, Slovenia</footnote>

<footnote n="2">²Agricultural Institute of Slovenia</footnote>
up and concentrated by ion exchange and styrene-divinylbenzene columns for determination of thiol precursors. Analyses were performed by liquid chromatography coupled to tandem mass spectrometric detector (Agilent Technologies, Palo Alto, USA).

For the determination of methoxypyrazines in grapes, calibration standards were prepared using water (purified by a Milli-Q system) working solutions of 3-isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP) and 2-isobutyl-3-methoxy-d3-pyrazine ([2H3]-IBMP). 3 g of NaCl was placed into a 20-mL SPME vial along with a stir bar, followed by 6 mL of Milli-Q-purified water, 2 mL of working solution of IBMP and IPMP (IBMP = 50 ng/L + IPMP = 56 ng/L), 2 mL of 4 M NaOH and 100 µL of working solution of [2H3]-IBMP (0.5 µg/L). The vial was closed and placed onto a magnetic stir plate to dissolve the NaCl.

The samples were prepared by placing 3 g of NaCl into a 20-mL SPME vial along with a stir bar, followed by 2 g of grape powder, 6 mL of Milli-Q-purified water, 2 mL of 4 M NaOH and 100 µL of working solution of [2H3]-IBMP (0.5 µg/L). The vial was closed and placed onto a magnetic stir plate to dissolve the NaCl. The samples were analyzed using a gas chromatograph (Agilent Technologies 7890A) equipped with a Gerstel MPS2 multipurpose sampler and connected to a mass spectrometer (Agilent Technologies 5975C, upgraded with triple axis detector) as described by Šuklje et al. [11].

Wines were made from the same treatments under reductive conditions in order to prevent oxidation. After fermentation with Alchemy II yeast (Anchor wine yeast, South Africa) at 15°C, wine samples were added with 50 ppm SO2 and frozen until thiol analyses. Frozen wine samples were unthawed in nitrogen atmosphere and submitted to thiol analyses. Determination of volatile thiols 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexylacetate (3MHA) was performed using a modified method of Tominaga et al. [8] and Šuklje et al. [12] by using a gas chromatograph (Agilent Technologies 7890A) coupled to a mass spectrometric detector (Agilent Technologies 5975C, upgraded with triple axis detector).

The data were analyzed through ANOVA and when the differences were significant, Tukey's HSD test was applied to separate the averages (P<0.05).

## Results and discussion

The timing of leaf removal impacted in a different way the physiology of the grapevine as compared with the control where no leaves were removed. At harvest, no significant differences between treatments were found in the yield and basic maturation parameters of grapes in both years, even if a trend of lower yield in the case of ELR was shown (data not reported). The IBMP content decreased during maturation in all treatments. At harvest, both ELR and LLR grapes had slightly but not significantly higher IBMP contents than UNT grapes in both years (figure 1). Opposite to the results on grapes, in 2014 the wines showed significantly higher IBMP content in UNT (data not reported).

<figure><img src="image_18.png" /><figcaption>**Fig. 1** - IBMP content in Sauvignon blanc grapes at harvest. UNT, untreated (no leaf removal); ELR, early (pre-flowering) leaf removal; LLR, late (post-flowering) leaf removal. Bars represent standard error. Means were separated with Tukey's HSD test (P<0.05; same letters for not significant differences).</figcaption></figure>
The application of leaf removal resulted in a change in the concentration and relative proportion between thiol precursors, with a particular behavior in the case of ELR and LLR as compared with UNT (figure 2). No significant differences were found as regard the content of 4-S-cysteinyl-4-methylpentan-2-one (Cys-4MMP) and 4-S-glutathionyl-4-methylpentan-2-one (G-4MMP) in both seasons, but there were higher values in ELR grapes and lower values in LLR grapes at harvest in 2013. The 3-S-cysteinyl hexan-1-ol (Cys-3MH) content was not significantly different between treatments in both years, whereas the 3-S-glutathionyl hexan-1-ol (G-3MH) content was significantly higher in ELR and LLR grapes than in UNT grapes in 2013. The total (Cys+G) 3MH precursors were found at higher levels in LLR in 2013 as compared with UNT, while ELR was intermediate. Regarding GSH, no significant differences were found, but there was a trend towards a higher content in the case of LLR (data not reported).

<figure><img src="image_19.png" /><figcaption>**Fig. 2** - Effect of the timing of leaf removal on thiol precursor concentration in Sauvignon blanc grapes at harvest. A, cys-4MMP; B, cys-3MH; C, G-4MMP; D, G-3MH. Means were separated with Tukey's HSD test (P<0.05; same letters for not significant differences).</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>**Fig. 3** - Correlation between A. cys-4MMP, B. cys-3MH, C. G-4MMP and D. G-3MH in grapes and the related 4MMP and 3MH thiols in wines for the season 2013 (●) and 2014 (▲).</figcaption></figure>

The 4MMP content in the wines produced from the same treatments was higher in UNT and ELR wines than in LLR wines in 2013, and in 2014 as a trend. On the opposite, the concentration of 3MH was
significantly higher in UNT and LLR wines than in ELR wines in 2013, whereas in 2014 it was the highest in UNT wines. The concentration of thiols in the wines was closely related with the concentration of cysteinylated precursors in the grapes in both seasons, while no good relationship was found with glutathionylated precursors (figure 3). There were, however, dramatic changes between the two seasons. In 2014 the concentration of 4MMP and 3MH was much higher than in 2013, and the obtained result was opposite to that reported above for thiol precursors. In the second season (2014), the severe *Botrytis cinerea* infection accounted for the differences between grapes and wines [13]. During grape collection, only healthy grapes were sampled for the analysis, while for wine production all grapes were harvested; even if grapes were cleaned, a lot of infected berries still remained.

Conclusion

Leaf removal did not have a profound effect on yield components and basic maturation parameters in grapes, but important changes in the composition of thiol precursors in grapes and thiols in wines were ascertained. It can be speculated that the accumulation of thiol precursors was affected by the timing of leaf removal; thus, in the case of post-flowering application, a faster aromatic maturation resulted in higher 3MH contents, while with pre-flowering leaf removal the maturation was delayed, obtaining a higher concentration of 4MMP and a lower concentration of 3MH. In the second season (2014), the concentration of thiol precursors in grapes and that of thiols in wines was probably significantly affected also by the occurrence of *Botrytis cinerea* and other rots. Thus, the relative contribution of pre- and post-flowering leaf removal was somehow masked and therefore unclear.

References

1. Sternad Lemut M, Sivilotti P, Franceschi P et al (2013) Use of metabolic profiling to study grape skin polyphenol behavior as a result of canopy microclimate manipulation in a "Pinot noir" vineyard. *J Agric Food Chem* **61**: 8976-8986.

2. Caspari HW, Lang A, Alspach P (1998) Effects of girdling and leaf removal on fruit set and vegetative growth in grape. *Am J Enol Vitic* **49**: 359-366.

3. Feng H, Yuan F, Skinkis PA et al (2015) Influence of cluster zone leaf removal on Pinot noir grape chemical and volatile composition. *Food Chem* **173**: 414-423.

4. Scafidi P, Pisciotta A, Patti D et al (2013) Effect of artificial shading on the tannin accumulation and aromatic composition of the Grillo cultivar (*Vitis vinifera* L.). *BMC Plant Biol* **13**: 175.

5. Verzera A, Tripodi G, Dima G et al (2015) Leaf removal and wine composition of *Vitis vinifera* L. cv. Nero d'Avola: the volatile aroma constituents. *J Sci Food Agric*, doi/10.1002/jsfa.7075.

6. Šuklje K, Antalick G, Coetzee Z et al (2014) Effect of leaf removal and ultraviolet radiation on the composition and sensory perception of *Vitis vinifera* L. cv. Sauvignon Blanc wine. *Aust J Grape Wine Res* **20**: 223-233.

7. Swiegers JH, Francis IL, Herderich M et al (2006) Meeting consumer expectations through management in vineyard and winery: the choice of yeast for fermentation offers great potential to adjust the aroma of Sauvignon blanc wine. *Aust NZ Wine Ind J* **21**: 34-42.

8. Tominaga T, Murat ML, Dubourdieu D (1998) Development of a method for analyzing the volatile thiols involved in the characteristic aroma of wines made from *Vitis vinifera* L. cv. Sauvignon Blanc. *J Agric Food Chem* **46**: 1044-1048.

9. Dubourdieu D, Tominaga T, Masneuf I et al (2006) The role of yeasts in grape flavor development during fermentation: the example of Sauvignon blanc. *Am J Enol Vitic* **57**: 81-88.

10. Lisjak K, Janeš L et al (2013) Determination of thiol precursors in Sauvignon Blanc grapes. In: Biasioli F (ed.) *Book of Abstracts*. San Michele al Adige - Italy: Research and Innovation Centre Fondazione Edmund Mach, pp. 86-88.

11. Šuklje K, Lisjak K, Baša Česnik H et al (2012) Classification of grape berries according to diameter and total soluble solids to study the effect of light and temperature on methoxypyrazine, glutathione, and hydroxycinnamate evolution during ripening of Sauvignon blanc (*Vitis vinifera* L.). *J Agric Food Chem* **60**: 9454-9461.

12. Šuklje K, Baša Česnik H, Janeš L et al (2013) The effect of leaf area to yield ratio on secondary metabolites in grapes and wines of *Vitis vinifera* L. cv. Sauvignon blanc. *J Int Sci Vigne Vin* **47**: 83-97.

13. Thibon C, Cluzet S, Merillon JM et al (2011) 3-Sulfanylhexanol precursor biogenesis in grapevine cells: the stimulating effect of *Botrytis cinerea*. *J Agric Food Chem* **59**: 1344-1351.
# Micro-vinification for assessment of the effect of field spatial variability on wine quality

Y. KOTSERIDIS<fnref n="1" />*, E. KALLITHRAKA<fnref n="1" />, M. KYRALEOU<fnref n="1" />, S. FOUNTAS<fnref n="2" />, A. BALAFOUTIS<fnref n="2" />, E. ANASTASIOU<fnref n="2" />, S. KOUNDOURAS<fnref n="3" />

Precision viticulture – micro-vinification – berry ripening – wine composition

## Introduction

Precision Viticulture (PV) is the implementation of Precision Agriculture (PA) practices in viticulture [1]. Soil attributes commonly measured in the framework of PV are soil structure, texture, water and nutrient content, depth, and electrical conductivity (ECa). Previous studies have reported significant correlations between soil ECa and vine properties [2, 3]. The majority of PV studies in winegrapes have focused on the relation of soil and vine-related spatial data with grape composition at harvest. However, the inclusion of site-specific wine quality data are very rare in literature, even though grape quality is ultimately judged upon wine properties. This study aimed to apply PV on both grape composition and wine quality. The experimental field was set up in the region of Nemea, northern Peloponnesus, Greece. This region is planted with Agiorgitiko, a red variety producing PDO (Protected Designation of Origin) Nemea wines.

## Materials and methods

### Experimental field

The study was conducted in a non-irrigated vineyard (0.83 ha) at Asprokampos, Peloponnese, Greece (latitude 37.54°, longitude 22.33°), during the 2013 season. The vineyard was situated in the Nemea PDO area, which is the biggest in Greece, covering about 3000 ha. The vineyard was planted with *Vitis vinifera* L. cv. Agiorgitiko, a Greek red winegrape variety, grafted onto 1103P rootstock. Vines were spaced 1.0 x 2.5 m and trained to a Lyre system. The vineyard was situated on a slope (≈12%) and was sectioned in a regular grid of 18 cells of similar size (400 – 550 m<fnref n="2" />) in order to analyze the spatial variability in grape and wine components.

### Data collection

Elevation and slope data were acquired from SRTM satellite in Global Mapper 14 (Blue Marble Geographics, Maine, USA). Soil variability was assessed by ECa measurements using an EM-38 probe (EM38 RT, Geonics LTD, Ontario Canada). Ground conductivity was measured within 1.5 m of effective depth range, using vertical dipole mode. The mean ECa value of every cell was calculated in ArcMap 10.1 (ArcGIS 10.1, ESRI Inc., California, USA) through the join data menu between cells and measurement points. Maps of the above measurements were produced with the Surfer 11 (Golden Software Inc., Colorado, USA) software.

### Grape analysis

Harvesting was performed manually and yield was measured by counting and weighing the total number of plastic bins per cell, when completely full. 300-Berry samples were taken from each vineyard cell five times during the ripening period, respectively, on 17th of August, 25th of August, 5th of September, and 15th and 25th of September (harvest time) 2014. Individual berry fresh weight was determined on a sub-

<footnote n="1">¹Department of Food Science and Human Nutrition, Laboratory of Enology, Agricultural University of Athens, Athens, Greece</footnote>

<footnote n="2">²Department of Natural Resources Management & Agricultural Engineering, Laboratory of Agricultural Mechanization, Agricultural University of Athens, Athens, Greece</footnote>

<footnote n="3">³Department of Agriculture, Laboratory of Viticulture, Aristotle University of Thessaloniki, Thessaloniki, Greece</footnote>
sample of 50 berries per vineyard cell, while a second sub-sample of 200 berries per cell was pressed and the must analyzed for total soluble solids (°Brix), total acidity and pH. Total anthocyanins and total phenolics were analyzed as previously indicated [4, 5]. Grape tannins were estimated using the MCP (methyl cellulose precipitable) tannin assay method [5].

### Micro-vinifications

20 kg of berries from each of the 18 vineyard cells were transferred to the Oenology Laboratory of the Agricultural University of Athens to be micro-vinified separately in duplicate. For the winemaking trials, grapes of each cell were crushed and destemmed and 60 mg/L SO₂ (as potassium metabisulfite) were added. Pectolytic enzymes (Safizym Colour, Fermentis, France) at 3 g/hL as well as lyophilized yeasts of the commercial strain SC 22 (Fermentis, France) at 20 g/hL, previously hydrated in water (15 min, 38°C), were also added. Beginning on the second day of fermentation, and for the following days, three punching downs per day were conducted to extract phenolic compounds. After 7 days of maceration, the wines were drained and transferred to other tanks and were inoculated with Viniflora Oenos, CHR Hansen malolactic bacteria and malolactic fermentation was completed after approximately 1 week. The wines were racked, supplemented with 50 mg/L SO₂ (as potassium metabisulfite), filtered and bottled until the time of analysis.

### Wine analysis

In the final wines, alcoholic degree and titratable acidity were determined according to the OIV methods [6]. Color intensity and hue were assessed by measurement of the absorbances at 420, 520 and 620 nm under 1 mm optical way [7]. Total anthocyanins were determined using the SO₂ bleaching method at 520 nm optical density in HCl media [8]. Tannin concentration was measured both after heating in acid medium [8] and after precipitation with methyl cellulose [5]. Total wine phenolics were determined using the Folin-Ciocalteu reagent [9].

Statistical analysis included correlation matrices and descriptive statistics for all measured parameters by SPSS 20 (IBM Corp., New York, USA).

## Results and discussion

Initial maps of elevation and ECₐ provided a visual representation of the variability across the vineyard (Fig. 1). Elevation varied from 785 to 803 m above sea level with a slope of 11.82%. Yield and ECₐ showed the highest within-field variability with higher levels at the lower-east side of the field. ECₐ ranged from 70.40 to 106.91 (Table 1).

<figure><img src="image_21.png" /><figcaption>Fig. 1 - Elevation (with sampling grid), electrical conductivity (ECₐ) and yield maps of the experimental vineyard.</figcaption></figure>

ECₐ presented significant negative correlations with both elevation and slope (r = -0.638 and r = -0.701 respectively, p < 0.01; Table 2). This result shows that the effect of landscape on soil properties was probably due to erosion, soil from the elevated part migrating towards the leveler part of the field. In general, increased slope indicates that the soil root zone in the leveler part of the field is deeper, thus increasing access to soil water reserves [10]. Besides, yield presented a positive correlation with ECₐ (r = 0.508, p < 0.05; Table 2) and a significant negative correlation with elevation and slope (r = -0.523 and r = -0.602 respectively, p < 0.05; Table 2), which confirms that higher depth of the soil leads to higher vine development and yield.
<table><caption>**Table 1** - Descriptive statistics of field parameters</caption><thead><tr><th></th><th>N</th><th>Minimum</th><th>Maximum</th><th>Range</th><th>Mean</th><th>Std. Deviation</th><th>CV (%)</th></tr></thead><tbody><tr><td>Elevation</td><td>18</td><td>801.252</td><td>811.629</td><td>10.38</td><td>805.863</td><td>3.734</td><td>0.5%</td></tr><tr><td>Slope (%)</td><td>18</td><td>10.930</td><td>12.710</td><td>1.78</td><td>11.827</td><td>.533</td><td>5%</td></tr><tr><td>Yield (t/ha)</td><td>18</td><td>4.98</td><td>12.74</td><td>7.76</td><td>8.08</td><td>2.36</td><td>29%</td></tr><tr><td>ECa</td><td>18</td><td>70.402</td><td>106.914</td><td>36.51</td><td>89.620</td><td>11.667</td><td>13%</td></tr></tbody></table>

<table><caption>**Table 2** - Pearson's correlation matrix among field parameters</caption><thead><tr><th></th><th>Elevation</th><th>Slope (%)</th><th>Yield (t/ha)</th><th>ECa</th></tr></thead><tbody><tr><td>Elevation</td><td>1</td><td></td><td></td><td></td></tr><tr><td>Slope (%)</td><td><strong>0.977**</strong></td><td>1</td><td></td><td></td></tr><tr><td>Yield (t/ha)</td><td><strong>-0.523*</strong></td><td><strong>-0.602*</strong></td><td>1</td><td></td></tr><tr><td>ECa</td><td><strong>-0.638**</strong></td><td><strong>-0.701**</strong></td><td><strong>0.508*</strong></td><td>1</td></tr></tbody></table>

Berry weight presented a consistent spatial pattern through ripening, linked to soil variability, with consistently higher values at the lowest-east part of the vineyard (data not shown). Low variability within the vineyard was observed for total solids (Brix), acidity and pH, which are directly related to must chemical composition. Grape phenolic compounds (anthocyanins and total phenols) showed the highest (2-3 fold) within-field variability (data not shown). However, this variability in phenolic compounds was reduced in the wines, although remaining the highest among wine components (Table 3).

<table><caption>**Table 3** - Descriptive statistics of wine composition parameters</caption><thead><tr><th></th><th>N</th><th>Min</th><th>Max</th><th>Range</th><th>Mean</th><th>Std. Deviation</th><th>CV (%)</th></tr></thead><tbody><tr><td>Colour density</td><td>18</td><td>8.19</td><td>12.52</td><td>4.33</td><td>10.16</td><td>1.43</td><td>14</td></tr><tr><td>Colour tint</td><td>18</td><td>0.45</td><td>0.58</td><td>0.14</td><td>0.51</td><td>0.04</td><td>8</td></tr><tr><td>Total anthocyanins (mg/L)</td><td>18</td><td>150</td><td>389</td><td>239</td><td>214</td><td>57</td><td>26</td></tr><tr><td>Total phenolics (mg/L of catechin)</td><td>18</td><td>1.07</td><td>1.61</td><td>0.54</td><td>1.41</td><td>0.17</td><td>12</td></tr><tr><td>Total tannins (g/L)</td><td>18</td><td>2.12</td><td>3.39</td><td>1.27</td><td>2.83</td><td>0.40</td><td>14</td></tr><tr><td>Astringency (mg/L)</td><td>18</td><td>110</td><td>266</td><td>156</td><td>165</td><td>45</td><td>27</td></tr><tr><td>Alcohol (v/v, %)</td><td>18</td><td>12.35</td><td>13.37</td><td>1.02</td><td>13.03</td><td>0.34</td><td>3</td></tr><tr><td>pH</td><td>18</td><td>3.30</td><td>3.70</td><td>0.40</td><td>3.49</td><td>0.12</td><td>3</td></tr><tr><td>Titratable acidity (g tartaric acid/L)</td><td>18</td><td>4.50</td><td>7.20</td><td>2.70</td><td>6.06</td><td>0.72</td><td>12</td></tr></tbody></table>

<table><caption>**Table 4** - Pearson's correlations among field and wine composition parameters</caption><thead><tr><th></th><th>Elevation</th><th>Slope</th><th>Yield (t/ha)</th><th>ECa</th></tr></thead><tbody><tr><td>Colour density</td><td>0.008</td><td>0.025</td><td>-0.061</td><td>-0.244</td></tr><tr><td>Colour tint</td><td><strong>0.601**</strong></td><td><strong>0.627**</strong></td><td><strong>-0.741**</strong></td><td>-0.426</td></tr><tr><td>Total anthocyanins (mg/L)</td><td>0.116</td><td>0.037</td><td>0.134</td><td>0.109</td></tr><tr><td>Total phenolics (mg/L of catechin)</td><td>-0.380</td><td>-0.353</td><td>0.411</td><td>0.315</td></tr><tr><td>Total tannins (g/L)</td><td>0.166</td><td>0.195</td><td>-0.114</td><td>-0.468</td></tr><tr><td>Astringency (mg/L)</td><td>0.207</td><td>0.198</td><td>0.113</td><td>-0.252</td></tr><tr><td>Alcohol (v/v, %)</td><td>-0.240</td><td>-0.222</td><td>0.001</td><td>0.132</td></tr><tr><td>pH</td><td><strong>0.646**</strong></td><td><strong>0.726**</strong></td><td><strong>-0.540*</strong></td><td><strong>-0.495*</strong></td></tr><tr><td>Titratable acidity (g/L tart. acid)</td><td><strong>-0.492*</strong></td><td><strong>-0.481*</strong></td><td>0.284</td><td>0.362</td></tr></tbody></table>

<footnote>*Correlation is significant at the 0.05 level (2-tailed).</footnote>

<footnote>**Correlation is significant at the 0.01 level (2-tailed).</footnote>

<footnote>*Correlation is significant at the 0.05 level (2-tailed).</footnote>

<footnote>**Correlation is significant at the 0.01 level (2-tailed).</footnote>
Wine composition parameters presented limited significant correlations with field characteristics. A positive correlation of colour tint with elevation and slope (r = 0.601 and r = 0.627 respectively, p < 0.05; Table 4) was recorded, while the correlation with yield was negative (r = -0.741, p < 0.05; Table 4). Besides, a negative correlation was found for titratable acidity and elevation and slope (r = -0.492 and r = -0.481 respectively, p < 0.01; Table 4), while a positive correlation for both these parameters was found for pH. No correlation was found for tannins and astringency with field characteristics.

Conclusion

Most Greek vineyards have small areas but present significant variation in soil properties, mainly due to variations in topography. According to our results, grape and wine composition was not strongly spatially structured with the exception of berry weight, which was significantly linked to the variation in soil and topography and colour tint and wine acidity. Both these last parameters are very important for wine quality and can help the selection of fields according to the quality of the produced wine. Thus, this study indicated the necessity to evaluate wine quality spatial data alongside grape yield and composition to increase the profitability of PA implementation in vineyards.

References

1. Bramley RGV (2003) Viticulture: smarter thinking on soil survey. *Aust NZ Wine Ind J* **18**: 88-94.

2. Imre SP, Mauk JL, Bell S et al (2013) Mapping grapevine vigour, topographic changes and lateral variation in soils. *J Wine Res* **24**: 1-18.

3. Trought M (2005) Fruitset - possible implications on wine quality. *In Transforming Flowers to Fruit*. Australian Society of Viticulture and Oenology, Adelaide, SA.

4. Iland P, Ewart A, Markides A et al (2000) *Techniques for Chemical Analysis and Quality Monitoring during Winemaking*. Patrick Iland Wine Promotions Campbelltown, Adelaide, Australia.

5. Sarneckis CJ, Dambergs RG, Jones P et al (2006) Quantification of condensed tannins by precipitation with methyl cellulose: development and validation of an optimised tool for grape and wine analysis. *Aust J Grape Wine Res* **12**: 39-49.

6. OIV (1990) *Recueil des Méthodes Internationales d'Analyse des Vins et des Moûts*. Office International de la Vigne et du Vin, Paris.

7. Glories Y (1984) La couleur des vins rouges. II. Mesure, origine et interprétation. *Conn Vigne Vin* **18**: 253-271.

8. Ribereau-Gayon P, Glories Y, Maujean A et al (2006) Phenolic compounds, pp. 141-203. *In Handbook of Enology*. John Wiley & sons, Chichester.

9. Singleton VL, Rossi Jr JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. *Am J Enol Vitic* **16**: 144-158.

10. Tardaguila J, Baluja J, Arpon L et al (2011) Variations of soil properties affect the vegetative growth and yield components of “Tempranillo” grapevines. *Precision Agric* **12**: 762-773.
# Polyphenol transcriptional and metabolic profiling of the astringent grape variety Aglianico (*Vitis vinifera* L) during berry ripening

A. RINALDI<fnref n="1" />,<fnref n="2" />*, D. CARPUTO<fnref n="3" />, L. FRUSCIANTE<fnref n="3" />, C. LANZILLO<fnref n="3" />, L. MOIO<fnref n="1" />,
A.J TAMBURRINO<fnref n="3" />, C. VILLANO<fnref n="3" />, R. AVERSANO<fnref n="1" />

Aglianico – berry ripening – proanthocyanidins – anthocyanins

## Introduction

Aglianico is a high-quality and late-ripening grape variety of Southern Italy. Aglianico grapes can accumulate a high content of polyphenols, mainly tannins which, depending on the vinification condition, may confer different astringent qualities to wines. Astringency is a tactile sensation felt as drying, puckering and roughing in the epithelium [1], and is mainly associated with the precipitation of salivary proteins by tannins [2]. Structural characteristics of Aglianico grape tannins have been recently characterized both in seeds and skins, and compared to international cultivars such as Merlot and Cabernet Sauvignon [3]. The high content of proanthocyanidins and flavans as well as the elevated degree of galloylation of tannin fractions make this cultivar a model for astringency. The accumulation of polyphenols through berry development has been widely studied for many grape varieties [4, 5], both at chemical and molecular levels. However, it has never investigated in Aglianico grapes until now. Towards this goal, we carried out a combined chemical and transcriptional profiling of Aglianico cultivar over five stages of fruit development and in different berry tissues.

## Materials and methods

### Plant material

Aglianico del Taburno berries (clone Ampelos TEA 22 grafted on rootstock 1103 Paulsen – *V. berlandieri* x *V. rupestris* – clone ISV 1) were collected at the following developmental stages: fruit set (15 days after flowering [DAF]); post-fruit set (35 DAF); véraison (V, 70 DAF); mid-ripening stage (R, 84 DAF); and technological ripening (TR, 115 DAF). Starting from the véraison stage, berries were manually dissected into skin, pulp and seeds starting from frozen berries. Three biological replicates were sampled.

### RNA extraction and cDNA synthesis

Total RNA was isolated from 40 mg of ground material using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich, St. Louis, USA) following the manufacturer's protocol with some modifications. Quantity and quality of the isolated RNA was measured using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA). For cDNA synthesis, 100 ng of each RNA sample was reverse transcripted using the SuperScript® III cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocol.

### Gene expression analysis

Expression analysis was conducted by real-time PCR using a SYBR Green method on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

<footnote n="1">¹Università degli Studi di Napoli Federico II, Department of Agricultural Sciences,
Division of Grape and Wine Sciences, Viale Italia, angolo via Perrottelli, 83100, Avellino, Italy</footnote>

<footnote n="2">²Biolaffort, 126 Quai de la Souys, 33100, Bordeaux, France</footnote>

<footnote n="3">³Università degli Studi di Napoli Federico II, Department of Agricultural Sciences,
Division of Plant Genetics and Biotechnology, Via Univeristà, 100, 80055, Portici, Italy</footnote>
*Preparation of extracts*

Extracts from Aglianico skins and seeds were prepared in triplicate as follows. Seeds and skins were removed by hand from 20 berries and treated with liquid nitrogen. The frozen seeds and skins were ground in a ball grinder. The obtained powder was weighted and extracted using acetone/water (80:20, v/v) in a ratio 1:10 (g:mL) for 4 h and methanol/water (60:40, v/v) for 2.5 h in the same ratio. The extracts were fractionated into monomers, oligomers and polymers according to Sun et al. [6].

*Phenolic analyses*

Tannins were evaluated according to the Bate-Smith method [7]. Vanillin reactive flavans were determined according to Di Stefano and Guidoni [8]. Anthocyanins were analysed using the method by Harbertson et al. [9].

*The Saliva Precipitation Index (SPI) analysis*

The SPI was obtained by the chip electrophoresis of human saliva before and after the binding assay with grape fractions [3].

# Results and discussion

To investigate the genetic regulation of anthocyanins and tannins in Aglianico del Taburno, the transcript abundance of genes involved in the flavonoid pathway was monitored using a quantitative assay in whole berry, berry skin and seeds. Based on their key roles, the 15 key-genes selected for the present study were grouped into five groups as follows: *PAL*, *C4H* and *4CL* genes in the core phenyl-propanoid-related group; *F3'H*, *F3'5'H*, *FLS4*, *FLS5*, *DFR* and *LDOX* genes in the flavonoid-related group; *ANR*, *LAR1* and *LAR2* in the proanthocyanidins-related group; and *UFGT* in the anthocyanins-related group. The transcriptional factors *MybPA1* and *MybPA2* were taken separately. The selection of reference genes to normalize the cDNA represents a critical step in any quantitative RT-PCR analysis. In this analysis, β-tubulin (*TUB*), β-actin and elongation factor were tested as housekeeping genes. The expression of β-tubulin was the most stable among the three candidates in different tissues and therefore was selected as reference gene.

The pattern of expression of the phenyl-propanoid-related genes suggested not significant variations between the two tissues, except for the *PAL* gene, which showed both a decrease and an increase of the expression at mid-ripening in seeds and skins, respectively. As reported by Ali et al. [10], the increasing levels of *PAL* transcripts at post-véraison stages led to higher accumulation of the substrate 4-coumaryl-CoA for the downstream pathways. The expression pattern of *F3'H*, *FLS4*, *LDOX* and *LAR2* genes in skins increased at the post-véraison step, consistently with the knowledge that this stage is the starting point for the transcriptional regulation of the flavonoid pathway. Véraison, the onset of ripening in red cultivars, represents the major switch in the flavonoid biosynthetic pathway from proanthocyanidins to anthocyanins and is associated with an increased gene expression of the anthocyanin-specific gene *UFGT*. The regulation of *UFGT* and *F3'5'H* genes have been demonstrated to be similar [11] and indeed our data are consistent as *UFGT* and *F3'5'H* showed the same expression pattern in whole berry and skins. In skins, the expression increased at ripening, which is correlated to the higher production of anthocyanins.

<figure><img src="image_22.png" /><figcaption>**Fig. 1** - Skin anthocyanin accumulation through Aglianico berry development (V véraison, R ripening, TR technological ripening).</figcaption></figure>
As shown in Figure 1, the production of anthocyanins starts after véraison in ripening skins. From R to TR, an increase of anthocyanin accumulation was observed.

Regarding proanthocyanidin production across the three stages of berry development, véraison represents the maximum point (Figure 2). After this, for skins and seeds a decline can be observed when grape ripened. While berry skins are characterized only by polymers and oligomers, monomers are still abundant in seeds. An important reduction of both polymeric and oligomeric proanthocyanidins occurred in skins. A slight diminution of monomers and oligomers was observed in seeds, while polymers decreased at R, and then increased at the last point TR.

<figure><img src="image_23.png" /><figcaption>**Fig. 2** - Proanthocyanidin (polymers, oligomers and monomers) concentration of Aglianico skin and seed fractions during development (V véraison, R ripening, TR technological ripening).</figcaption></figure>

Considering the tannins reactive towards salivary proteins, the SPI showed a progressive reduction from V to TR in skins and seeds, meaning that during grape ripening the tannins causing astringency decreased in both tissues (Figure 3). Maximum astringency matched with the maximum accumulation of proanthocyanidins at V. At this point, astringent tannins act as a defense mechanism of the plant in order to defend the fruit from predators before seed maturation. In skins, the polymeric fraction at V was highly astringent, as shown by the high value of SPI, at R the SPI decreased but remained unchanged at TR despite the drastic reduction in proanthocyanidin amount. Indeed, during ripening stages the concentration of skin tannins may decline because of the interaction with pectins, polysaccharides, and anthocyanins [12]. However, astringency of skins, which is due to the high degree of polymerization, seemed to be only slightly affected. The astringency of seed fractions also changed, but similarly with proanthocyanidin concentration.

Proanthocyanidin synthesis is catalyzed by the action of *ANR*, *LAR1* and *LAR2*. These genes have been found to be tissue specific [13], since they showed opposite expression levels in skins and seeds. The higher expression in the proanthocyanidin pathway has been recorded with *ANR* at the post-véraison stage in seeds, with *LAR2* at post-véraison time points in skins, and with *LAR1* at ripening in seeds. These results demonstrated the seed specificity of *ANR* and *LAR1* and the skin specificity of *LAR2*.

A different behavior has been identified in the transcriptional factors *VvMybPA1* and *VvMybPA2*. These genes are extremely specific to the polyphenol pathway and positively regulate proanthocyanidin synthesis in grape tissues [14]. The spatiotemporal expression of *VvMybPA2* is compatible with a putative role in the regulation of proanthocyanidin synthesis, because its expression increased exponentially in the whole berry analysis and is comparable with the *LAR2* expression in the seed and skin analyses. Previous studies demonstrated that the majority of enzymes encoding for polyphenolic compounds can be expressed
differentially in different grape varieties [10] and tissues, especially in berry skin development [15, 16]. The reason for this could be that the enzymes encoded by the investigated genes are controlled by post-transcriptional or post-translational modifications, or that their levels are sufficient to allow the pathway to operate without coordinated regulation; it would explain the different transcript abundance in skin and seeds.

<figure><img src="image_24.png" /><figcaption>**Fig. 3** - The SPI (astringent tannins) of Aglianico skin and seed fractions during development (V véraison, R ripening, TR technological ripening).</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>**Fig. 4** - A Principal Component Analysis (PCA) performed on Aglianico skins and seeds at the three stages of development (SK-V, SK-R, SK-TR; Sd-V, Sd-R, Sd-TR), according to analytical data in monomers (m), oligomers (o), polymers (p) [SPI<sub>m,o,p</sub>; T(tannins)<sub>o,p</sub>; FL(flavans)<sub>m,o,p</sub>], and proanthocyanidins key-genes and transcriptional factors (MybPA1, MybPA2) expression.</figcaption></figure>

A Principal Component Analysis (PCA) was carried out in order to assess relationships between Aglianico skins (SK) and seeds (Sd) at the three stages of development (SK-V, SK-R, SK-TR; Sd-V, Sd-R, Sd-TR), and analytical data in monomers (m), oligomers (o), polymers (p) [SPI<sub>m,o,p</sub>; T(tannins)<sub>o,p</sub>; FL(flavans)<sub>m,o,p</sub>], and proanthocyanidins key-genes (*LAR1*, *LAR2*, *ANR*) and transcriptional factors (*MybPA1*, *MybPA2*) expression.
In Figure 4, the first two principal components accounted for 75.94% of the total variation (F1+F2). In the first axis, representing 45.44% of the total variance, were distributed the skin samples, which were mainly characterized by polymers and oligomers. Skins at ripening (SK-R; SK-TR) significantly differed from samples at véraison (SK-V), and were mainly associated to the expression of the *LAR2* gene. The *LAR1* gene was highly correlated with flavan concentration in monomeric fraction. In the second principal component (F2), accounting for 30.50% of variance, were loaded seed samples, with no difference between developmental stages. Seeds were characterized by high astringency in monomers and oligomers, which was positively correlated with the expression of the *ANR* gene in this tissue.

Conclusion

In Aglianico grapes, the expression levels of genes involved in the first steps of the phenyl-propanoid pathway appear to be similar to other red grape cultivars, both in skins and seeds of developing fruit. The overall high expression levels of such genes from véraison to technological ripening (harvest) correlate to the accumulation of anthocyanins and the decrease in tannins. The total astringency of skin and seed proanthocyanidins also decreased as berry ripened, but the astringency of tannin fractions (monomers, oligomers and polymers) differed depending on the fruit tissue. These tissues contain proanthocyanidins of different polymer length and at different concentrations. No correlation of gene expression with proanthocyanidin accumulation was found, while the transcript level of some genes positively correlated with the reactivity of tannins towards salivary proteins (SPI).

References

1. ASTM (2004) Standard definitions of terms relating to sensory evaluation of materials and products. Annual book of ASTM standards, Philadelphia: American Society for Testing and Materials.

2. Rinaldi A, Gambuti A, Moio L (2012) Application of the SPI (Saliva Precipitation Index) to the evaluation of red wine astringency. *Food Chem* **135**: 2498-2504.

3. Rinaldi A, Iturmendi N, Gambuti A et al (2014) Chip electrophoresis as a novel approach to measure the polyphenols reactivity toward human saliva. *Electrophoresis* **35**: 1735-1741.

4. Kennedy JA, Hayasaka Y, Vidal S et al (2001) Composition of grape skin proanthocyanidins at different stages of berry development. *J Agric Food Chem* **49**: 5348-5355.

5. Downey MO, Harvey JS, Robinson SP (2003) Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development. *Aust J Grape Wine Res* **9**: 15-27.

6. Sun B, Belchior GP, Ricardo-da-Silva JM et al (1999) Isolation and purification of dimeric and trimeric procyanidins from grape seeds. *J Chromatogr A* **841**: 115-121.

7. Bate-Smith EC (1954) Astringency in foods. *Food Process Packag* **23**: 124-127.

8. Di Stefano R, Guidoni S (1989) La determinazione dei polifenoli totali nei mosti e nei vini. *Vignevini* **16**: 47-52.

9. Harbertson JF, Picciotto EA, Adams DO (2003) Measurement of polymeric pigments in grape berry extract sand wines using a protein precipitation assay combined with bisulfite bleaching. *Am J Enol Vitic* **54**: 301-306.

10. Ali MB, Howard S, Chen S et al (2011) Berry skin development in Norton grape: distinct patterns of transcriptional regulation and flavonoid biosynthesis. *BMC Plant Biol* **11**: 7.

11. Bogs J, Ebadi A, McDavid D et al (2006) Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development. *Plant Physiol* **140**: 279-291.

12. Bindon KA, Smith PA, Holt H et al (2010) Interaction between grape-derived proanthocyanidins and cell wall material. 2. Implications for vinification. *J Agric Food Chem* **58**: 10736-10746.

13. Bogs J, Downey MO, Harvey JS et al (2005) Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. *Plant Physiol* **139**: 652-663.

14. Terrier N, Torregrosa L, Ageorges A et al (2009) Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. *Plant Physiol* **149**: 1028-1041.

15. Grimplet J, Wheatley MD, Jouira HB et al (2009) Proteomic and selected metabolite analysis of grape berry tissues under well-watered and water-deficit stress conditions. *Proteomics* **9**: 2503-2528.

16. Fujita M, Fujita Y, Noutoshi Y et al (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. *Curr Opin Plant Biol* **9**: 436-442.
# Développement de méthodes sensorielles d'analyse du raisin pour un usage par les professionnels du vin :
un outil d'aide à la décision pour le choix des itinéraires œnologiques

R. SIRET<fnref n="1" />*, C. PATRON<fnref n="1" />, P. CHRETIEN<fnref n="2" />, L. GUERIN<fnref n="2" />, S. BEAUCHET<fnref n="1" />,<fnref n="3" />, C. COULON-LEROY<fnref n="1" />, R. SYMONEAUX<fnref n="1" />

Analyse sensorielle – dégustation – raisin – qualité – outil d'aide à la décision

## Introduction

La dégustation et l'analyse sensorielle des raisins sont aujourd'hui des outils reconnus d'aide à la décision pour déterminer la date optimale des vendanges [1-4]. Ces outils permettent en effet d'appréhender le niveau de maturité des baies, d'y associer une caractérisation descriptive de la matière première mais aussi une aide au choix de l'itinéraire œnologique le mieux adapté à la qualité de raisin évaluée par dégustation.

Ces aspects ont été développés depuis 2010 en Anjou-Saumur sur les raisins de Cabernet Franc. Ces travaux, avec un panel entraîné à l'évaluation sensorielle, ont permis la mise au point d'une fiche de dégustation des raisins à destination des vignerons et des professionnels de terrain. L'utilisation de panels entraînés peu adaptés à une dégustation quotidienne d'un nombre important de parcelles a été substituée par cette méthode rapide, efficace et spécifique aux besoins des professionnels. Suite à ces premiers travaux, avec la publication d'une fiche technique [5], les professionnels sont aujourd'hui demandeurs de la mise en œuvre de ces techniques sur d'autres variétés et pour d'autres types de produits. Pour cela, une adaptation aux particularités de chaque cépage a été nécessaire. Après les travaux sur le Cabernet Franc, une méthodologie permettant de passer du profil sensoriel mis en place en laboratoire avec un panel entraîné à une méthode d'évaluation pour les professionnels a été aussi développée pour le cépage blanc Chenin [6] et transférée au cépage blanc Melon de Bourgogne.

Ce travail a ainsi permis de proposer depuis 2010 des méthodes sensorielles rigoureuses d'analyse sur le terrain de différentes variétés de raisin du Val de Loire (France).

Dans ce contexte, nous présenterons ainsi la mise en place du processus global qui a permis de générer puis de valider une méthode rapide d'évaluation de la qualité sensorielle des raisins adaptée aux attentes des professionnels qui souhaitent caractériser leurs produits dans un temps de dégustation limitée et d'avoir un outil d'aide à la décision leur permettant d'orienter leurs itinéraires œnologiques. Ce travail sera également, pour les cépages étudiés, un support qui nous permettra de définir différentes typologies de raisins correspondantes à différents types de vins.

## La co-construction d'une fiche de dégustation pour les professionnels

La démarche globale a été décomposée en grandes 2 phases. Une première période (millésimes 2010 et 2011) avec l'identification des descripteurs sensoriels qui caractérisent les raisins de Chenin au cours de la maturité et ceci de façon spécifique en lien avec les différents objectifs de production du vigneron et

<footnote n="1">¹LUNAM Université, SFR 4207 QUASAV, UMT VINITERA, UPSP GRAPPE, Groupe ESA,
55 Rue Rabelais, ANGERS 49007, France</footnote>

<footnote n="2">²Institut Français de la Vigne et du Vin, UMT VINITERA, Pôle Val de Loire, 42 Rue Georges Morel,
BP 60057, 49071 BEAUCOUZE Cedex, France</footnote>

<footnote n="3">³ADEME, SAF, 20 Avenue du Grésillé, 49 000 Angers, France</footnote>
l'origine parcellaire. Suite au travail réalisé sur Cabernet Franc, le panel entraîné constitué d'une quinzaine de juges qualifiés à l'analyse sensorielle et à la dégustation de baies de raisin a suivi les recommandations de Le Moigne et al. [1]. La deuxième période (millésime 2012), qui a permis la validation d'une fiche réduite par la mise en place d'un protocole commun de dégustation des raisins du cépage Chenin [4] sera par conséquent présentée en détail dans cette article.

<table><caption>**Tableau 1** - Attributs sensoriels de la baie de Chenin évalués par le panel entraîné et le panel professionnel</caption><thead><tr><th></th><th>Panel entraîné</th><th>Panel professionnel</th></tr></thead><tbody><tr><td rowspan="12">Baie n°1</td><td>Elasticité</td><td>Fermeté</td></tr><tr><td>Force pour détacher le pédicelle</td><td></td></tr><tr><td>Quantité de chair autour du pédicelle</td><td>Quantité de chair autour du pédicelle</td></tr><tr><td>Croquant</td><td>Croquant</td></tr><tr><td>Jutosité</td><td>Jutosité</td></tr><tr><td>Sucre</td><td></td></tr><tr><td>Acide</td><td></td></tr><tr><td>Intensité globale aromatique</td><td rowspan="12">Baie n°1</td><td rowspan="12">Arômes de la baie (de 1 à 5) :<br/>Végétal (1)<br/>Floral (2)<br/>Floral-Fruité (3)<br/>Fruité (4)<br/>Confit (5)</td></tr><tr><td>Végétal</td></tr><tr><td>Floral</td></tr><tr><td>Note confite</td></tr><tr><td>Fruité blanc</td></tr><tr><td>Fruité jaune</td></tr><tr><td>Fruité exotique</td></tr><tr><td>Fruité agrumes</td></tr><tr><td>Moisi</td></tr><tr><td>Champignon</td></tr><tr><td>Astringence de la pellicule</td></tr><tr><td rowspan="4">Baie n°2</td><td>Teinte</td><td>Agressivité de la pellicule (sécheresse, acidité, amertume)</td></tr><tr><td>Quantité de chair collée à la pellicule</td><td>Teinte</td></tr><tr><td>Epaisseur de la pellicule en bouche</td><td></td></tr><tr><td>Dilacération</td><td>Dilacération de la pellicule</td></tr><tr><td rowspan="2">Baie n°2</td><td>Couleur des pépins</td><td>Couleur des pépins</td></tr><tr><td></td><td>Conclusion sur la maturité (1 à 4)</td></tr></tbody></table>

# Validation d'une fiche réduite de dégustation des raisins du cépage Chenin

## Les parcelles de Chenin

Des prélèvements de raisin de Chenin ont été effectués en 2012 à trois maturités différentes en Anjou-Saumur (As) et en Touraine (To). Pour chacune des 2 zones, des prélèvements ont été effectués sur deux parcelles dont le raisin est destiné à élaborer un vin de base correspondant à une maturité plus précoce (VB), sur deux parcelles correspondant à un vin blanc frais dit « de printemps » (VP) et sur deux parcelles correspondant à un vin blanc de caractère (VC). Au total, douze parcelles (6 en Anjou-Saumur et 6 en Touraine) ont donc été sélectionnées en fonction de leur potentiel qualitatif (évalué par les vignerons) pour un objectif de production d'un des trois types de vins précédemment cités. Sur ces 12 parcelles, huit ont été
évaluées par l'ensemble des dégustateurs (les deux jurys de professionnels à Angers et à Tours et le panel entraîné de l'ESA) et feront l'objet de la validation méthodologique.

### Les panels

Le panel entraîné est constitué de 13 juges « qualifiés », tous salariés du groupe ESA, entraînés à la dégustation des baies de Chenin depuis 3 années [4]. Le panel professionnel est constitué de 17 professionnels du monde viticole (techniciens, chercheurs, viticulteurs) et/ou salariés de l'IFV (9 à Tours) et (8 à Angers). Les données des deux panels professionnels ont été concatenées en un seul jeu pour les traitements statistiques des résultats [4].

### Méthode de dégustation des baies

Des différences fondamentales de protocole de dégustation caractérisent les deux groupes de dégustateurs: liste des attributs, échelle de notation et nombre de baie par lot dégustés. La liste des attributs sensoriels utilisés pour la caractérisation des baies pour chacun des 2 panels (23 attributs pour le panel entraîné et 10 pour le panel professionnel) est présentée au **Tableau 1**. L'évaluation des 23 attributs par le panel entraîné se fait à partir de 2 baies par lot (répétée 4 fois) et par juge présentées dans un flacon teinté. En revanche l'évaluation des 10 attributs par le panel professionnel se fait avec une seule baie par lot et par juge.

### Méthode de dégustation des jus

Afin de statuer sur le lien sensoriel qui existe entre baies et jus d'une même parcelle, les jus de chacune de ces parcelles ont également été caractérisés (2 répétitions par jus) par le panel professionnel et le panel entraîné. La liste des attributs sensoriels utilisés pour la caractérisation des jus pour chacun des panels (17 attributs pour le panel entraîné et 5 pour le panel professionnel) est présentée au **Tableau 2**. Les échelles de notation utilisées sont les mêmes que celle des baies. Le dernier attribut « Conclusion sur la maturité » de la liste du panel professionnel permet à nouveau aux juges de noter globalement le niveau de maturité du jus sur 4 niveaux comme pour la dégustation des baies de Chenin.

<table><caption>**Tableau 2** - Attributs sensoriels des jus de raisins de Chenin évalués par le panel entraîné et le panel professionnel</caption><thead><tr><th></th><th>Panel entraîné</th><th>Panel professionnel</th></tr></thead><tbody><tr><td></td><td>Teinte</td><td>Teinte</td></tr><tr><td></td><td>Limpidité</td><td>Sucre</td></tr><tr><td></td><td>Sucre</td><td>Acide</td></tr><tr><td></td><td>Acide</td><td>Acide</td></tr><tr><td></td><td>Astringent</td><td></td></tr><tr><td></td><td>Intensité globale aromatique</td><td></td></tr><tr><td></td><td>Végétal</td><td></td></tr><tr><td></td><td>Floral</td><td></td></tr><tr><td rowspan="10">Moût</td><td>Note confite</td><td rowspan="10">Moût</td><td rowspan="10">Arômes de la baie (de 1 à 5) :<br/>Végétal (1)<br/>Floral (2)<br/>Floral-Fruité (3)<br/>Fruité (4)<br/>Confit (5)</td></tr><tr><td>Fruité blanc</td></tr><tr><td>Fruité jaune</td></tr><tr><td>Fruité exotique</td></tr><tr><td>Fruité agrumes</td></tr><tr><td>Moisi</td></tr><tr><td>Champignon</td></tr><tr><td>Terreux</td></tr><tr><td>Pruneau</td></tr><tr><td></td><td>Conclusion sur la maturité (de 1 à 4)</td></tr></tbody></table>

## Résultats de la dégustation des baies

### Des panels très discriminants

Une analyse de variance (ANOVA), à 2 facteurs (parcelle et sujet) avec interaction, a été réalisée pour chaque attribut par panel. Tous les attributs évalués par le panel professionnel sont significativement discriminants à un seuil de 5%. Sur 23 attributs évalués par le panel entraîné, 7 ne sont pas discriminants à
5% (quantité de chair autour du pédicelle, force pour détacher le pédicelle, quantité de chair collée à la pellicule et les arômes « fruité jaune, moisi, champignon, fruité exotique »). Les tableaux des ANOVA par panel sont présentés au **Tableau 3**. Parmi les 6 attributs « communs » aux deux méthodes de dégustations, 5 sont discriminants à 5% « teinte des baies, couleur des pépins, croquant de la baie, dilacération de la pellicule et jutosité de la baie ». Le descripteur « quantité de chair autour du pédicelle » est non discriminant pour le panel entraîné.

<table><caption>**Tableau 3** - P values des ANOVA par panels [Var(description) = Parcelle + Sujet + Parcelle*Sujet avec Sujet en aléatoire]. **En gras : attributs discriminants au seuil de 5%**.</caption><thead><tr><th>Anova Baie<br>Panel professionnel</th><th>Parcelle</th><th>Sujet</th><th>Parcelle:Sujet</th></tr></thead><tbody><tr><td>Teinte</td><td>4,09E-35</td><td>1,35E-16</td><td>5,44E-06</td></tr><tr><td>Couleur des pépins</td><td>5,96E-22</td><td>1,42E-30</td><td>3,43E-06</td></tr><tr><td>Arôme</td><td>1,50E-21</td><td>1,02E-15</td><td>9,45E-04</td></tr><tr><td>Maturité</td><td>2,91E-21</td><td>4,14E-36</td><td>2,22E-28</td></tr><tr><td>Dilacération</td><td>2,99E-18</td><td>5,44E-35</td><td>1,88E-07</td></tr><tr><td>Agressivité</td><td>2,74E-14</td><td>1,44E-08</td><td>1,98E-07</td></tr><tr><td>Croquant</td><td>5,27E-07</td><td>1,37E-20</td><td>8,58E-04</td></tr><tr><td>Fermeté</td><td>6,63E-07</td><td>1,87E-41</td><td>1,49E-03</td></tr><tr><td>Quantité de chair autour du pédicelle</td><td>1,72E-06</td><td>1,23E-40</td><td>6,01E-05</td></tr><tr><td>Jutosité</td><td>5,41E-06</td><td>1,74E-20</td><td>3,59E-09</td></tr></tbody></table>

<table><thead><tr><th>Anova Baie<br>Panel entraîné</th><th>Parcelle</th><th>Sujet</th><th>Parcelle:Sujet</th></tr></thead><tbody><tr><td>Teinte</td><td>1,38E-18</td><td>1,27E-22</td><td>1,31E-03</td></tr><tr><td>Couleur des pépins</td><td>1,83E-10</td><td>9,04E-10</td><td>3,40E-01</td></tr><tr><td>Croquant</td><td>9,00E-06</td><td>3,19E-25</td><td>2,42E-02</td></tr><tr><td>Astringence de la pellicule</td><td>1,44E-05</td><td>7,92E-55</td><td>1,97E-04</td></tr><tr><td>Acidité de la baie</td><td>1,81E-05</td><td>3,39E-56</td><td>3,43E-05</td></tr><tr><td>Dilacération</td><td>3,58E-05</td><td>1,63E-52</td><td>3,50E-08</td></tr><tr><td>Arôme Végétal</td><td>8,60E-05</td><td>1,75E-106</td><td>2,99E-09</td></tr><tr><td>Arôme Confit</td><td>9,54E-05</td><td>7,65E-28</td><td>6,83E-08</td></tr><tr><td>Sucrosité de la baie</td><td>1,71E-04</td><td>6,38E-58</td><td>4,00E-07</td></tr><tr><td>Elasticité de la baie</td><td>1,58E-03</td><td>1,48E-34</td><td>3,77E-06</td></tr><tr><td>Epaisseur de la pellicule</td><td>2,37E-03</td><td>1,77E-27</td><td>1,24E-04</td></tr><tr><td>Arôme Fruité blanc</td><td>5,99E-03</td><td>1,71E-99</td><td>6,10E-10</td></tr><tr><td>Intensité Globale Aromatique</td><td>1,32E-02</td><td>1,42E-88</td><td>5,88E-05</td></tr><tr><td>Arôme Fruité agrumes</td><td>1,45E-02</td><td>2,88E-77</td><td>2,73E-08</td></tr><tr><td>Jutosité</td><td>2,37E-02</td><td>3,21E-51</td><td>2,56E-07</td></tr><tr><td>Arôme Floral</td><td>4,24E-02</td><td>6,71E-136</td><td>2,61E-04</td></tr><tr><td>Arôme Fruité jaune</td><td>7,31E-02</td><td>2,30E-69</td><td>3,47E-12</td></tr><tr><td>Arôme Moisi</td><td>2,13E-01</td><td>6,07E-03</td><td>1,71E-05</td></tr><tr><td>Arôme Champignon</td><td>2,80E-01</td><td>1,22E-54</td><td>1,39E-12</td></tr><tr><td>Force pour détacher le pédicelle</td><td>3,98E-01</td><td>2,17E-21</td><td>2,08E-04</td></tr><tr><td>Quantité de chair collée à la pellicule</td><td>4,52E-01</td><td>2,13E-53</td><td>1,70E-02</td></tr><tr><td>Quantité de chair autour du pédicelle</td><td>6,60E-01</td><td>6,68E-78</td><td>3,52E-06</td></tr><tr><td>Arôme Fruité exotique</td><td>9,38E-01</td><td>2,11E-47</td><td>2,23E-11</td></tr></tbody></table>
Une forte proximité entre les panels

Le calcul du R² montre qu'il y a un accord entre les deux panels pour les descripteurs « couleur des pépins, teinte des baies, dilacération de la pellicule et croquant de la baie » (Figure 1). « L'agressivité de la baie » évaluée par le panel professionnel est davantage corrélée à « l'acidité » qu'à « l'astringence » notées par le panel entraîné. Concernant « la jutosité », le désaccord observé entre les deux panels peut être lié à une compréhension différente de ce descripteur, toutefois il reste consensuel lorsqu'il est pris individuellement au sein de chacun des groupes.

L'analyse factorielle multiple (AFM) réalisée sur les données sensorielles des baies moyennées des 8 parcelles issues du panel entraîné et du panel professionnel (Figure 2) montre (i) une bonne discrimination des différentes maturités et (ii) confirme l'accord entre les deux panels pour les descripteurs « couleur des pépins, teinte des baies, dilacération de la pellicule et croquant de la baie ».

<figure><img src="image_26.png" /><figcaption>Fig. 1 - Accords entre les deux panels (professionnel en abscisse et entraîné en ordonné).</figcaption></figure>

Résultats de la dégustation des jus

L'acidité des jus évalués par le panel professionnel n'a pas été un facteur discriminant en lien avec un très fort désaccord entre les juges (IFV Angers et Tours) pour ce descripteur. Sur les 17 attributs évalués par le panel entraîné ESA, 10 sont significativement discriminants acide, sucre et astringence, teinte, les aromes (vert, fruité blanc, fruité agrumes, pruneau, moisi, terreux). L'analyse globale des résultats conjoints des 2 panels (professionnel IFV et entraîné ESA) montre cependant des panels qui sont très discriminants en ce qui concerne l'analyse des jus. L'AFM (non montrée) réalisée sur l'ensemble des données des deux panels confirme une forte proximité entre les deux groupes pour la dégustation des jus. Cette analyse montre également un arôme fruité blanc qui caractérise la maturité des raisins à destination de l'élaboration d'un vin de printemps.
<figure><img src="image_27.png" /><figcaption>**Fig. 2** - AFM réalisée sur les données moyennées des baies issues du panel expert et du panel professionnel, attributs à 20% (en rouge descripteurs méthode professionnelle, en vert descripteurs méthode panel entraîné).</figcaption></figure>

## Discussion et conclusion

Les résultats obtenus lors du millésime 2012 par les 2 panels (entraîné ESA et professionnel IFV) sont en accord avec une forte corrélation entre les attributs sensoriels obtenus sur baie et sur jus. Néanmoins les résultats du panel entraîné montrent un meilleur niveau de précision sur les différences aromatiques (fruité blanc) validant sa pertinence quand la problématique nécessite un focus aromatique. La dégustation des jus qui permet d'assurer une homogénéité des lots dégustés par juge (dégustation d'un plus grand nombre de baies) et une meilleure représentativité de l'état général de la parcelle ressort comme indispensable et complémentaire à la dégustation des baies. Le processus consistant à mettre au point une liste exhaustive d'attributs pertinents par l'analyse sensorielle avec un panel entraîné avant de les soumettre à des professionnels pour en réduire le nombre en fonction de leurs pratique sur le terrain et de leur expertise, est par conséquent une démarche qui fonctionne. La méthodologie d'analyse sensorielle des baies et des jus de
Chenin par les professionnels a ainsi été validée. Un transfert de cette méthode de terrain a été réalisé pour les millésimes 2013 et 2014 pour une caractérisation sensorielle de la qualité des raisins d'autres cépages blancs tels que le Melon de Bourgogne et le Riesling.

Références

1. Le Moigne M, Symoneaux R, Jourjon F (2006) Évaluation sensorielle de baies de raisin de cabernet franc : suivi de maturité. *Rev Œnol* **122**: 49-52.

2. Le Moigne M, Symoneaux R, Jourjon F (2008) How to follow grape maturity for wine professionals with a seasonal judge training? *Food Qual Prefer* **19**: 672-681.

3. Siret R, Symoneaux R, Jourjon F et al (2013) Analyse sensorielle des baies de raisin : un outil d'aide à la décision pour les professionnels. *Rev Suisse Vitic Arboric Hortic* **45**: 368-374.

4. Patron C, Symoneaux R, Chretien P et al (2014) Transfert de méthodes sensorielles du raisin pour un usage par les professionnels du vin. Application au cépage Chenin. *Rev Œnol* **151**: 14-19.

5. Techniloire.com (2010) Comment déguster des baies de raisin : application au Cabernet Franc de Loire. *Site internet de l'Interprofession des Vins de Loire*, Edition InterLoire.

6. Techniloire.com (2013) Comment déguster des baies de raisin : application au Chenin. *Site internet de l'Interprofession des Vins de Loire*, Edition InterLoire.