Grapevine (Vitis spp.) is one of the most widely cultivated perennial fruit crops in the world and its economic relevance is mainly related to wine production. In recent years, the increased frequency of extreme phenomena such as heat waves has been acknowledged as one of the most significant climate variables negatively affecting grape yield and berry composition, with consequences also on wine quality. Thus, studying the physiological, metabolic and genetic factors that are involved in grapevine response to high temperatures is essential to improve the knowledge of mechanisms underlying thermotolerance, aiming to support plant breeding innovation and the development of new management strategies in viticulture. In this work, a segregating population obtained from the crossing of ‘Rhine Riesling’ and ‘Cabernet Sauvignon’ was studied in the field with a multidisciplinary approach. The progeny (around 120 genotypes) was evaluated for phenological traits affected by changing temperatures, in particular bud burst, flowering and véraison, while physiological response to heat stress was assessed in various hot summer days by measuring chlorophyll fluorescence kinetics and stomatal conductance. Measures were collected in the early morning as control and in the afternoon during hot hours. Phenotypic data were then used in combination with a high-density linkage map (average distance between adjacent markers 0.78 cM), previously developed using genotypic information from 139 individuals, to perform QTL analysis. Based on physiological responses to high temperatures, selected individuals showing contrasting behaviour, together with parental lines, were further studied in controlled conditions. In the field, in fact, plants may be subjected to combined stresses and changes in environmental conditions may heavily influence plants response. With the experiment in controlled condition, on the other hand, plants were stressed at higher temperatures, compared to the ones registered in the field, by maintaining all the other sources of variability constant. In the growth chamber plants were studied for their physiological response to heat stress by using the same approach adopted in the field. To better understand mechanisms involved in grapevine adaptation to heat stress conditions, individuals with contrasting behaviour were studied also for their metabolome modifications, both in the field and in controlled conditions. Volatile organic compounds (VOCs) were investigated with an untargeted approach applying conventional methods of analysis. Accumulation of VOCs in grapevine leaves was analysed using gas chromatography coupled with mass spectrometry (GC-MS) after a pre-concentration with a solid-phase micro-extraction (SPME) approach. On the other hand, VOCs emission during stress was investigated in controlled conditions thanks to the use of the Closed-Loop Stripping Analysis (CLSA) which allows the collection of VOCs directly emitted by plants. Analysis was then performed with GC-MS. Metabolic alterations of non-volatile compounds were examined with an untargeted analysis using high-performance liquid chromatography coupled with a high-resolution mass spectrometer equipped with an electrospray soft ionization (HPLC-HR-ESI-MS). In this work a metabolomic workflow was developed, starting from sample collection and extraction to sample analysis and data interpretation. The analytical method developed allowed the preliminary evaluation of leaf metabolome alterations due to stress factors. In fact, the use of a weak cation-exchange mixed mode column, in combination with a data dependent acquisition mode, allowed a first wide screening of both primary and secondary metabolites resulting in a good compromise for metabolic fingerprinting. QTL analysis on the segregating population allowed the identification of several QTLs, related to both phenological and physiological traits, with the discovery of interesting putative candidate genes for grapevine resilience to changing temperatures. This is the first time that a similar approach has been applied to a perennial fruit crop by analysing chlorophyll fluorescence and leaf transpiration traits related to heat stress. On the other hand, the multidisciplinary approach allowed the fine characterization of Rhine Riesling and Cabernet Sauvignon response to high temperatures, both in controlled and field conditions, a tentative classification of ‘tolerant’ and ‘susceptible’ progeny individuals and the identification of metabolic pathways altered during heat stress in the susceptible plants. Together with the implementation of a novel metabolomic workflow based on HPLC-HR-ESI-MS, this work represents a novelty in studies on grapevine response to changing temperatures, as it considered not only the berry metabolism but the resilience of the plants itself, paving the way for future studies on thermotolerance.

Grape root borer, Vitacea polistiformis (Harris), is an oligophagous pest of grapevines in the eastern USA. Neonates must burrow into the soil to find grape roots. In Virginia, larvae feed on roots for ~2 years, then pupate just beneath the soil surface. Emerging adults leave an empty pupal exuviae at the soil surface around the vine base. There was no relationship between weekly captures in pheromone traps and pupal exuviae counts, indicating that exuviae sampling is most appropriate to assess infestations. Exuviae sampling in Virginia vineyards revealed infestations that ranged from light to very heavy. Eighteen biotic and abiotic variables were measured and used in analyses that assessed their relative contributions to differences in exuviae density. Water holding capacity and clay/sand ratio were most strongly associated with pupal exuviae density; these variables were used to develop a model for predicting the extent of infestation of individual vineyards. The spatial distribution of pupal exuviae was characterized using non-spatial and geospatial techniques. Although the non-spatial method (Taylor's Power Law) indicated that exuviae showed an aggregated distribution in all blocks, spatial methods (variograms, SADIE) revealed aggregated distributions only in blocks with ≥ 0.5 pupal exuviae per vine. Independent pupal exuviae samples for population assessment in vineyards can be achieved using sampling points separated by >8.8 m. Combined results from geospatial analyses and the temporal distribution of pupal exuviae within years enabled the development of a practical and quantitative sampling protocol. Bioassays used to measure the behavioral response of larvae to host stimuli revealed that neonates were attracted to grape root volatiles. In soil column bioassays, larvae moved vertically and horizontally over distances of up to 120 cm and apparently perceived the presence of grape roots from a distance of 5 cm in soil. Results are discussed in relation to their potential implications for monitoring and managing grape root borer. / Ph. D.

Tesis por compendio / Técnicas de cultivo para adaptar la viticultura mediterránea al cambio climático La viticultura mediterránea se está viendo afectada por el cambio climático, lo que amenaza la tipicidad del vino y, por tanto, la sostenibilidad de la vitivinicultura. Los principales factores que se prevé que afecten al cultivo de la vid son la escasez de agua, los incrementos térmicos y el aumento de la concentración de dióxido de carbono en el aire, lo que influirá en la vid: i) adelantando su fenología, disociando así la maduración tecnológica y fenólica de la uva y ii) incrementando las necesidades hídricas del viñedo. Éste trabajo pretende evaluar los efectos de diferentes técnicas de cultivo para la adaptación de la viticultura mediterránea al cambio climático. Se realizaron tres experimentos en Valencia (España) estudiando los efectos de: 1) el riego deficitario controlado; 2) la arquitectura de los sistemas de conducción y 3) el manejo del dosel vegetal. Las principales respuestas evaluadas fueron la eficiencia en el uso del agua de los viñedos (EUA) y la composición de la uva. El experimento 1 se llevó a cabo durante tres campañas en un viñedo de Moscatel de Alejandría/161-49C regado por goteo con el objetivo de definir la estrategia de riego más sostenible para uva blanca en el levante español. Para ello, se ensayaron cuatro tratamientos: (i) Testigo, regado al 100% de la evapotranspiración del cultivo (ETc) durante toda la campaña; (ii) riego deficitario sostenido (SDI), regado al 50% del Testigo; (iii) déficit temprano (ED), donde se restringió el riego antes del envero, seguido de un 100% de ETc; y (iv) déficit tardío (LD), regado como el Testigo hasta el envero y posteriormente al 25% de la ETc hasta la vendimia. La producción en el ED y el LD se redujo en un 25 y 15% respectivamente en comparación a la del Testigo, mientras que la producción del SDI no difirió significativamente del Testigo ni de los tratamientos de ED y LD. Todos los tratamientos con riego deficitario mostraron efectos acumulativos en la productividad de las cepas como consecuencia de la reducción del número de racimos por sarmiento y del tamaño del racimo debido al menor tamaño de la baya. El experimento 2 pone de manifiesto que la respuesta a la minimización de la radiación interceptada por el dosel vegetal es dependiente de la variedad y que reducir la carga de radiación en viñedos de clima mediterráneo podría tener efectos positivos en la EUA de la vid. La efectividad de la técnica de LLR parece depender de sus efectos sobre la relación área foliar/producción y el estado hídrico de la vid, la capacidad fotosintética compensatoria de la variedad y de las condiciones ambientales. Por consiguiente, en viñedos de baja vigorosidad, una defoliación severa puede no ser aconsejable para adaptar las variedades Bobal y Tempranillo al calentamiento global debido a los efectos perjudiciales sobre la calidad del vino tinto. Por el contrario, los resultados de los experimentos de riego deficitario y de orientación de las filas de espaldera proporcionan nuevos hallazgos en la EUA como posibles técnicas para paliar la escasez de agua en el viñedo. Por lo tanto, las técnicas de campo estudiadas resultan eficaces en adaptar la viticultura mediterránea al cambio climático en cuanto al estrés hídrico. Sin embargo, sus efectos en términos de mitigación del estrés térmico no fueron del todo positivos. Por tanto, es necesario realizar más investigaciones para determinar la viabilidad de emplear otras prácticas de campo, distintas al LLR, para equilibrar la madurez glúcida y fenólica de la uva. / Field practices for adapting Mediterranean viticulture to climate change Mediterranean viticulture is being affected by climate change, which threatens wine typicity and, therefore, the whole viticulture sustainability. The main factors expected to affect grapevine cultivation are water scarcity, warm air temperature and the increase in the air carbon dioxide concentration, and impact of these factors will be mainly the: i) advancing of grapevine phenology, thereby decoupling berry technological and phenolic composition and ii) increasing vine water requirements. This PhD Thesis aimed at evaluating the effects of different field practices to adapt Mediterranean grapevine cultivation to climate change. Three experiments were performed in Valencia (Spain) to assess the effects of 1) regulated deficit irrigation; 2) training systems architecture and; 3) canopy management. The main responses evaluated were vineyard water-use efficiency (WUE) and grape composition. Experiment 1 was undertaken for three seasons in a drip-irrigated, cv. Muscat of Alexandria/161-49C vineyard to define the most sustainable irrigation strategy for white winegrapes in eastern Spain. For this, four treatments were tested: (i) Control, irrigated at 100% of estimated crop evapotranspiration (ETc) for the entire season; (ii) sustained deficit irrigation (SDI), irrigated at 50% of Control; (iii) early deficit (ED), where pre-veraison irrigation was withheld, followed by 100% ETc; and (iv) late deficit (LD), irrigated as in the Control until veraison and thereafter at 25% ETc until harvest. Results showed that yield in ED and LD was reduced 25 and 15%, respectively, compared to that of the Control, while yield under SDI did not differ significantly from that of the Control and was similar to that of the ED and LD treatments. All the deficit irrigation treatments had some carry-over effects on vine performance due to a significant decrease in shoot fruitfulness and in cluster mass due to smaller berries. Experiment 2 highlights that the response to the minimizing of the radiation load intercepted by the canopy is cultivar-dependent and that decreasing the radiation load in the Mediterranean vineyards could have positive effects on the whole vine's WUE. The effectiveness of the LLR technique seemed to depend on its final impacts on leaf area-to-fruit ratio and vine water status, the cultivar's photosynthetic compensation capacity and the environmental conditions. Therefore, under low vigour vine conditions, severe defoliation might not be advisable for adapting Bobal and Tempranillo to global warming due to the detrimental effects on red wine quality. On the contrary, the results of deficit irrigation and row orientation experiments provided novel insights on WUE as possible techniques to alleviate vineyard water scarcity. Therefore, the field practices studied are effective in adapting Mediterranean viticulture to climate change in terms of water stress. On the other hand, the impacts of the studied techniques in terms of thermal stress alleviation were not fully positive. Hence, further research is needed to determine the possibility of using other field practices, besides LLR, to couple berry sugars and phenolic maturity. / Tècniques de cultiu per adaptar la viticultura mediterrània al canvi climàtic La viticultura mediterrània s'està veient afectada pel canvi climàtic, el que amenaça la tipicitat del vi i, per tant, la sostenibilitat de la vitivinicultura. Els principals factors que es preveu que afecten el cultiu de la vinya són l'escassetat d'aigua, els increments tèrmics i l'augment de la concentració de diòxid de carboni en l'aire, el que influirà en la vinya: i) avançant la seva fenologia, dissociant així la maduració tecnològica i fenòlica del raïm i ii) incrementant les necessitats hídriques de la vinya. Aquest treball pretén avaluar els efectes de diferents tècniques de cultiu per a l'adaptació de la viticultura mediterrània al canvi climàtic. Es van realitzar tres experiments a València (Espanya) estudiant els efectes de: 1) el reg deficitari controlat; 2) l'arquitectura dels sistemes de conducció i 3) el maneig del dosser vegetal. Les principals respostes avaluades van ser l'eficiència en l'ús de l'aigua de la vinya (EUA) i la composició del raïm. L'experiment 1 es va dur a terme durant tres campanyes en una vinya de Moscatell d'Alexandria/161-49C regat per goteig amb l'objectiu de definir l'estratègia de reg més sostenible per al raïm blanc en el llevant espanyol. A tal efecte, es van assajar quatre tractaments: (i) Control, regat al 100% de l'evapotranspiració del cultiu (ETc) durant tota la campanya; (ii) reg deficitari sostingut (SDI), regat al 50% del Contol; (iii) dèficit primerenc (ED), on es va restringir el reg abans del verol, seguit d'un 100% d'ETc; i (iv) dèficit tardà (LD), regat com el Control fins al verol i posteriorment al 25% de l'ETc fins a la verema. La producció en l'ED i el LD es va reduir en un 25 i 15% respectivament en comparació a la del Control, mentre que la producció del SDI no va diferir significativament del Control ni dels tractaments d'ED i LD. Tots els tractaments amb reg deficitari mostraren efectes acumulatius en la productivitat dels ceps, com a conseqüència de la reducció del nombre de raïms per sarment i del tamany del pomell a causa de la menor grandària de la baia. L'experiment 2 posa de manifest que la resposta a la minimització de la radiació interceptada pel dosser vegetal és dependent de la varietat i que reduir la càrrega de radiació en ceps de clima mediterrani podria tindre efectes positius en l'EUA de la vinya. L'efectivitat de la tècnica de LLR pareix dependre dels seus efectes sobre la relació àrea foliar/producció i l'estat hídric de la vinya, de la capacitat fotosintètica compensatòria de la varietat i de les condicions ambientals. Per consegüent, en vinyes de baixa vigorositat, una defoliació severa pot no ser recomanable per a adaptar les varietats Boval i Ull de llebre al calfament global, tenint en compte els efectes perjudicials sobre la qualitat del vi negre. Per contra, els resultats dels experiments de reg deficitari i d'orientació de les files d'espatlera proporcionen noves troballes en l'EUA com a possibles tècniques per pal·liar l'escassetat d'aigua a la vinya. Per tant, les tècniques de camp estudiades resulten eficaces a adaptar la viticultura mediterrània al canvi climàtic pel que fa a l'estrés hídric. No obstant, els seus efectes en termes de mitigació de l'estrés tèrmic no van ser del tot positius. Per tant, cal realitzar més investigacions per a determinar la viabilitat d'emprar altres pràctiques de camp, distintes al LLR, per a equilibrar la maduresa glúcida i fenòlica del raïm. / Buesa Pueyo, I. (2018). Field practices for adapting Mediterranean viticulture to climate change [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/107365 / Compendio

Plants can produce a wide variety of volatile organic compounds (VOCs), which are mediators of plant responses against stresses. Abiotic and biotic factors can modulate VOC emission dynamics from plants, suggesting a key role of these compounds in plant defense processes. Although grapevine VOCs responsible for wine aroma and plant-insect communications are well characterized, the functional properties of VOCs produced in response to phytopathogens were less studied. The aims of this work were i) to summarize the information available in the literature on emission patterns and possible biological functions of VOCs produced by grapevine plants in response to abiotic and biotic factors, ii) to investigate the defense mechanisms activated by volatile linalool in grapevine leaves against downy mildew caused by Plasmopara viticola, and iii) to identify metabolic changes associated with VOC-induced resistance activated by 2-phenylethanol and β-cyclocitral against downy mildew. The first chapter includes an introduction to VOCs in plant communication and analytical methods for metabolic detections, while the second chapter defines the aims of the project. In the third chapter, the literature on grapevine VOCs was reviewed and volatile molecules emitted in response to the exogenous stimuli were discussed. The key findings of this chapter are that specific VOC emission patterns can be stimulated by phytopathogens, resistance inducers, beneficial microorganisms, and abiotic factors, suggesting the precise involvement of airborne signals in grapevine responses to abiotic and biotic stresses. In particular, VOCs with inhibitory activities against grapevine pathogens are known to be emitted mainly by resistant grapevine genotypes upon pathogen inoculation, and some of these VOCs were proposed as biomarkers of grapevine resistance traits. In the fourth chapter, defense responses activated by volatile linalool in grapevine leaves were analyzed. Linalool treatment reduced downy mildew severity on leaf disks of susceptible grapevines (cultivar Pinot noir) and stimulated callose deposition at the sites of P. viticola infection. Moreover, the upregulation of defense-related genes was found in linalool-treated leaf disks, indicating the activation of grapevine defense mechanisms related to salicylic acid and jasmonic acid pathways. By ultra-high pressure liquid chromatography-electrospray ionization-high-resolution quadrupole time of flight-mass spectrometry it was demonstrated that linalool treatment caused changes in the leaf metabolome of mock-inoculated and P. viticola-inoculated samples at one and six days post inoculation (dpi). In the fifth chapter, a deep metabolomic analysis of VOC-induced grapevine resistance was carried out by ultra-high pressure liquid chromatography-heated electrospray ionization-high-resolution Orbitrap-mass spectrometry, which allowed accurate mass detection, fragment and isotope pattern matching, and metabolite annotation. In this study, 2-phenylethanol and β-cyclocitral treatment reduced downy mildew severity on leaf disks of susceptible grapevines (cultivar Pinot noir) causing complex changes in the leaf metabolome of mock-inoculated and P. viticola-inoculated samples at 1 dpi and 6 dpi. Metabolic features with significant increases in abundance in 2-phenylethanol- and β-cyclocitral-treated samples mainly belonged to putative carbohydrates and conjugates, carboxylic acids and derivates, carbonyl compounds, phenylpropanoids, and terpenoids, including molecules possibly associated with plant defense against pathogens. Thus, the chemical classification of annotated compounds revealed that VOCs treatments can activate specific metabolic responses in grapevine leaves that include the accumulation of defense-related compounds. These results demonstrated that linalool, 2-phenylethanol, and β-cyclocitral induce grapevine resistance against downy mildew, acting as volatile signaling molecules for plant resistance induction. Although further studies on formulation and efficacy under controlled and field conditions are required, these VOCs have great potential for the further development of sustainable products for downy mildew control.

Maîtriser l’impact des maladies sur les cultures est un défi majeur de l’agriculture moderne. Cette préoccupation est un aspect important de l’optimisation de la productivité, notamment en viticulture. En France, le mildiou de la vigne causé par Plasmopara viticola est une des maladies cryptogamiques responsable des épidémies les plus dévastatrices et les plus redoutées. Les traitements reposent sur l’utilisation préventive, systématique et onéreuse de composés chimiques antifongiques dont l’utilisation massive constitue un risque à la fois pour l’homme et l’environnement. La réduction de l’utilisation de fongicide implique le développement d’outils de diagnostic au champ, qui requiert la compréhension des interactions entre la plante et les agents pathogènes. Les travaux de cette thèse pluridisciplinaire ont porté sur le pathosystème Plasmopara viticola - Vitis vinifera, notamment pour répondre à l’intérêt croissant pour un outil de diagnostic en temps réel de la maladie utilisable au vignoble. Les stilbènes sont des phytoalexines impliqués dans la défense de certaines plantes supérieures vis-à-vis de stress biotiques et abiotiques. L’autofluorescence de ces composés phénoliques, dont la biosynthèse est induite dans les feuilles de vigne par P. viticola, en fait un potentiel marqueur naturel de l’infection. En effet, la faible autofluorescence bleu-verte des feuilles de vigne saines est considérablement renforcée par l’autofluorescence violet-bleue des stilbènes à la surface de feuilles de vigne infectée par P. viticola. Cette étude a montré que quelque soit le niveau de résistance du génotype, l’autofluorescence violet-bleue des stilbènes induit par l’infection est présente au niveau des parois des cellules de l’épiderme. En dehors de la concentration, la viscosité s’est révélé être la principale variable physico-chimique influençant l’intensité de l’autofluorescence des stilbènes dans les différents compartiments cellulaires des feuilles de vigne. Ceci explique la fluorescence intense des parois, particulièrement rigides, des cellules de garde (stomates) des feuilles infectées. Le suivi cinétique journalier a révélé la nature transitoire de l’autofluorescence des stilbènes lors de l’infection. La robustesse et l’intérêt de ce signal a également été validée par la mesure à différentes échelles (de la cellule à la feuille entière) et avec différentes méthodes fluorimétriques. Les résultats de ce travail ont permis des avancées sur la connaissance du rôle de composés phénoliques induits et constitutifs dans la défense contre P. viticola. En plus de la localisation de l’autofluorescence des stilbènes en surface des feuilles, la microscopie confocale couplée à la microspectrofluorimetrie a révélé différentes localisations de ces phytoalexines dans la profondeur des tissus en corrélation avec le niveau de résistance des génotypes. L’utilisation de l’autofluorescence des stilbènes comme marqueur de l’infection a permis de mettre en évidence : 1) le fait que les flavonols constitutifs des feuilles de V. vinifera retardent le développement de l’infection par P. viticola; et 2) le fait que les acides hydroxycinnamiques constitutifs ne semble pas participer à la défense contre P. viticola. Enfin, une nouvelle méthode de diagnostic non-destructive du mildiou sur feuille basée sur l’autofluorescence des stilbènes a été développée. Elle a montré une détection pré-symptomatique du mildiou sur les feuilles de vigne entières dès le premier jour après l’infection sur la face abaxiale et le troisième jour sur la face adaxiale. Cette méthode de diagnostic du mildiou a été validée au laboratoire notamment grâce à un prototype de capteur proximal développé en collaboration avec la société Force-A. La validation de la méthode au vignoble dans le cadre d’infection naturelle est la prochaine étape pour une utilisation de ce capteur optique dans le cadre de l’agriculture durable et de la sélection variétale. / Controlling the impact of diseases on crops is a major challenge of modern agriculture. This concern is an important aspect of optimizing productivity, notably in viticulture. In France, downy mildew caused by Plasmopara viticola is a fungal disease responsible for the most devastating epidemics. The preventive and systematic treatments are expensive, while the massive use of antifungal chemicals is a risk to both humans and the environment. Reducing the use of fungicide involves the development of diagnostic tools in the field, which requires understanding the interactions between plants and pathogens. The work of this multidisciplinary thesis focused on the pathosystem Plasmopara viticola - Vitis vinifera, especially to meet the growing interest in a real-time diagnostic tool of disease applicable in the vineyard. Stilbenes are phytoalexins involved in the defense of certain higher plants against biotic and abiotic stresses. The autofluorescence of these phenolic compounds, whose biosynthesis is induced in grapevine leaves by P. viticola, makes it a potential marker of natural infection. Indeed, the low blue-green autofluorescence of grapevine leaves is greatly enhanced by the violet-blue autofluorescence of stilbenes on the surface of leaves infected by P. viticola. This study showed that whatever the level of resistance in various genotypes, violet-blue autofluorescence induced by stilbene is present in the walls of epidermal cells. In addition to their concentration, viscosity proved the main physico-chemical variable affecting the intensity of the autofluorescence of stilbenes in different compartments of vine leaves. This explains the intense fluorescence of the walls, particularly rigid, of guard cells (stomata) of infected leaves. Daily monitoring revealed a kinetic with a transient rise of the autofluorescence of stilbenes during infection. The robustness and value of this signal was also validated by measuring at different levels (cellular to whole leaf) and with various fluorimetric methods (imaging, spectroscopy, proximal sensing). These results advance our understanding of the role of constitutive and induced phenolic compounds in plant defence against P. viticola. In addition to a common location of the autofluorescence of stilbenes on the leaf surface, confocal microscopy coupled with microspectrofluorometry revealed distinctive localizations of these phytoalexins in the deep tissue correlated with the level of resistance in genotypes. This aspect no doubt needs broader testing. The use of autofluorescence of stilbene as a marker of infection allowed us to ascertain that: 1) constitutive flavonols of the leaves of V. vinifera retard the development of infection by P. viticola and 2) the constitutive hydroxycinnamic acids do not seem to participate in the defence against P. viticola. Finally, a new method for the non-destructive diagnosis of leaf infection based on the autofluorescence of stilbenes has been developed. We have demonstrated a pre-symptomatic detection of downy mildew on whole grape leaves from the first day after infection on the abaxial surface and from the third day on the adaxial surface. This method of diagnosis has been validated in the laboratory thanks to a proximal sensor prototype developed in collaboration with the company Force-A. The validation of the method in the vineyard in a context of natural infections is the next step for use of this optical sensor as a tool for sustainable agriculture and for genetic screening.

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La masse de la baie est le résultat de l’effet intégré de plusieurs facteurs. La recherche a été dessinée afin d’étudier l’effet simultané des facteurs majeurs influençant la masse et la composition de la baie, de les hiérarchiser selon leur degré d’impact à des échelles différentes, de séparer leur effet direct et indirect sur la composition du raisin et de comparer le profil de vins élaborés à partir de petites et grosses baies. L’étude a été conduite sur deux sites expérimentaux, localisés dans les régions de Saint-Emilion (France) et Alcamo (Italie), pendant les années 2014 et 2015. Sur le premier site, les vignes sont plantées sur deux types de sols, tandis que sur le deuxième, deux traitements hydriques étaient appliqués. A l’échelle intra-parcellaire, l’état hydrique de la vigne représente le facteur le plus important, tandis que l’effet du nombre de pépins par baie n’est pas significatif. Des résultats opposés sont obtenus lorsque les relations sont étudiées à l’échelle de la grappe et de la plante. A large échelle, les facteurs impactent directement et indirectement la composition du raisin et les petites baies produisent des moûts et des vins plus concentrés. A l’inverse, à l’échelle de la grappe et de la plante, la masse de la baie n’influence pas la composition du raisin. Seule la concentration en anthocyanes est significativement liée à la masse à toutes les échelles. Cette relation est particulièrement évidente sous conditions hydriques limitantes. Un déficit hydrique augmente le ratio pellicule/pulpe, indépendamment de la masse de la baie. Petites et grosses baies d’une parcelle ayant une condition hydrique homogène, tendent à avoir un profil similaire. / Final berry mass is the result of the integrated effect of several factors. They also influence berry composition. The present work was designed to study the simultaneous effect of major factors influencing berry mass and composition, to hierarchize their impact at different scales, to distinguish their direct and indirect effect on berry composition and to compare the profile of wines made from large and small berries. The study was carried out simultaneously on two vineyards located in the Saint Emil ion (France) and Alcamo (Sicily) areas, during 2014 and 2015. On the first site, vines were planted on two soil types, while on the second site two different irrigation treatments were applied. Depending on the scale, some factors homogeneously impacted the berry mass and composition. At the intra-parcel scale, vine water status represented the most impacting factor, while berry seed number did not have significant effect. Opposite results were obtained when the investigation was carried out at the intra-bunch and intra-plant scales. At large scale, factors impacted directly and indirectly berry compounds and grape juices and wines produced from smaller berries were more concentrated. Neither at intra-bunch, nor at intra-plant scales, berry size effect on juice composition was significant. Only anthocyanin concentration was related to berry size at all scales. This fact was particularly obvious in berries produced under limited water conditions. Water deficit increased the skin to flesh ratio, independently of berry size. This means that small and large berries, produced from a single parcel with homogenous water uptake conditions, tend to have similar enological profiles.

Les flavan-3-ols et leurs polymères, les proanthocyanidines, appartiennent à une famille de flavonoïdes appelée les tannins condensés. Ces composés polyphénoliques jouent un rôle essentiel dans la qualité phytosanitaire des baies de raisin ainsi que dans les propriétés organoleptiques du vin (flaveur, astringence et couleur). La connaissance des mécanismes qui régissent leur biosynthèse est donc primordiale afin de mieux comprendre la mise en place de la typicité d’un vin. Ce mémoire est consacré à l’étude de l’une des enzymes responsables de la synthèse des flavan-3-ols : la leucoanthocyanidine réductase 1 de Vitis vinifera (VvLAR1). Dans une première partie, nous décrivons les conditions d’expression, de purification et de stabilité de l’enzyme recombinante. L’activité enzymatique est démontrée et la configuration 2R,3S des produits réactionnels caractérisée. La seconde partie de ce manuscrit décrit l’étude structurale de l’enzyme. Des monocristaux d’apoenzyme, de complexes binaires (VvLAR1 / NADPH) et d’un complexe ternaire (VvLAR1 / NADPH / (+)-catéchine) ont été obtenus. Les différentes structures permettent de décrire les modifications structurales associées à la fixation du coenzyme puis du produit. Un mécanisme catalytique basé sur les interactions intermoléculaires observées au sein du complexe ternaire est proposé. La troisième partie de ce mémoire est consacrée à la recherche de conditions expérimentales permettant la production et la stabilisation de la leucocyanidine en solution, un des substrats naturels de l’enzyme. Les résultats obtenus permettent une première approche de l’étude des propriétés cinétiques de la VvLAR1. / Flavan-3-ols and their polymerisation products, proanthocyanidins, belong to a flavonoid family named condensed tannins. These polyphenolic compounds play a major role in the protection of grape berries to intruders and in the organoleptic properties of wine (flavour, astringency and colour). Knowledge of the mechanisms which govern their biosynthesis is thus essential to better understand wine typical composition. The present thesis is devoted to the investigation of one of the two enzymes which catalyse the flavan-3-ols formation: leucoanthocyanidin reductase 1 from Vitis vinifera (VvLAR1). In the first part of the manuscript, we describe the expression, purification and stability conditions of the recombinant enzyme. The enzyme activity is demonstrated and the reaction product 2R,3S configuration is characterised. The second part of this report describes the structural studies of the enzyme. Monocrystals of the apoenzyme and of binaries (VvLAR1 / NADPH) and a ternary (VvLAR1 / NADPH / (+)-catechin) complexes were obtained. These different structures allow the description of the structural changes associated with the coenzyme and then the substrate binding. A catalytic mechanism, based on the intermolecular interactions within the ternary complex, is proposed. The last part of the work is devoted to the investigation of the experimental conditions leading to the stability of leucocyanidin in solution, one of the natural substrates of the enzyme. The results obtained allow a first study of the VvLAR1 kinetic properties.

A seguito degli scenari imposti dal cambio climatico il miglioramento dell’efficienza di uso dell’acqua (WUE) delle colture sono divenuti obiettivi fondamentali da perseguire. Il seguente studio pone al centro la WUE come parametro fisiologico cardine nella definizione del comportamento della vite in caso di riduzione della disponibilità idrica e si pone come obiettivo di valutarne le diverse forme di espressione, per individuare quale di queste sia meglio correlata alle performance delle varietà considerate, al fine di ottenere dati oggettivi che consentano di derivare consigli per la messa a punto di una più idonea tecnica irrigua. Nel triennio è stata valutata la risposta fisiologica di Sangiovese e Montepulciano, sottoposti a stress idrico di differente intensità in fasi fenologiche diverse. Inoltre, sono state saggiate le potenzialità genetiche della vite in condizioni di limitata disponibilità idrica attraverso il confronto delle performance di viti di Sangiovese innestate su due diversi portinnesti, ovvero confrontando un genotipo tradizionalmente sensibile, SO4, con uno selezionato in funzione della resistenza allo stress idrico, ovvero M4. Tutte le ricerche sono state condotte implementando apparati di misura innovativi che hanno permesso di ottenere la stima dell’efficienza di uso dell’acqua dell’intera chioma per la durata complessiva di ciascuna prova, poi confrontata con le forme più usuali di espressione di questo parametro fisiologico. Il lavoro rimette in discussione la metodologia e le deduzioni che si possono trarre da determinazioni di WUE condotte su singola foglia che, nella grande maggioranza dei casi, non hanno trovato corrispondenza nelle misure che hanno interessato l’intera chioma. / Within the current climate change scenario, the optimization of irrigation water use in vineyards by improving its water use efficiency (WUE) is a core subject of interest to assure sustainability in viticulture. The following research focuses on the physiological parameter WUE as pivotal in defining the behavior of grapevine under water stress. The aim is to assess the various forms of expression of the parameter, to identify which of these better correlates to the performance of the considered varieties, in order to obtain objective data and to derive recommendations for a more suitable irrigation technique. Over three years we evaluated physiological responses of Sangiovese and Montepulciano vines subjected to progressive water deficit either pre and post-veraison. Moreover, the genetic potential of grapevine under conditions of limited water availability was tested through the comparison of the performance of Sangiovese vines grafted on two different rootstocks; SO4, a traditional susceptible genotype and M4, a recently selected scion supposed to be tolerant to water stress. During the research we also designed, implemented and tested an innovative apparatus able to run automated water deficit experiments and to concurrently monitor whole canopy even for a season long time frame. These measurements were compared to traditional single leaf readings and a general conclusion was that, in most cases, single leaf WUE assessment did not match with the measures obtained from the whole canopy enclosure system.

Il cambio climatico sta inducendo una precocità negli fasi fenologiche della vite, tra cui la maturazione, un fenomeno che si verifica spesso nelle stagioni più calde e che porta a vini sbilanciati. La composizione della bacca consiste principalmente di metaboliti primari e secondari. Entrambi sono sensibili all'ambiente e alla gestione del vigneto. Di conseguenza, il cambiamento climatico può influenzare la composizione delle bacche e modificare la qualità del vino e la tipicità. La defogliazione può influenzare la composizione della bacca, modulando il bilancio vegeto-produttivo. Durante i tre anni del dottorato di ricerca, diversi rapporti tra superficie fogliare e uva sono state studiate per chiarire i meccanismi d'azione contro il processo di maturazione. In questa ricerca è stato studiato l'effetto della severità, tempismo e la posizione di defogliazione su due vitigni, Sangiovese e Cabernet Sauvignon. / Global warming is inducing a general earliness in the onset of grapevine phenological stages including ripening, a phenomenon that occurs often in the hottest seasons and which leads to unbalanced wines. Grape berry composition mainly consists of primary and secondary metabolites. Both are sensitive to environment and viticultural management. As a consequence, climate change can affect berry composition and modify wine quality and typicity. Leaf removal techniques can impact berry composition by modulating the source-to-sink balance. During the three years of PhD, different leaf-to-fruit ratios were studied to clarify action mechanisms vs the ripening process. In this research was studied the effect of severity, timing and position of leaf removal on two cultivars, Sangiovese and Cabernet Sauvignon.