Dissertations / Theses on the topic 'Vitis vinifera'

Il massiccio utilizzo di fungicidi in Viticoltura e i loro effetti negativi sulla salute dell’uomo e dell’ambiente sollecitano lo sviluppo di strategie agronomiche e di sistemi agrari innovativi. Il Lavoro di Tesi ha investigato l’influenza della cv Isabella su cultivars di Vitis vinifera in campo, in Fari Agroecologici, sistemi agrari frutto di una gestione partecipativa. L'esperimento ha dimostrato, per la prima volta, che la cultivar Isabella, nota per la sua tolleranza ai principali patogeni fungini, può contenere l’Incidenza della peronospora in cultivars di Vitis vinifera consociate e contigue. Studi precedenti dimostrano che i VOCs emessi dalla cv Isabella sono in grado di ridurre la presenza di patogeni fungini. La ricerca pioneristica ha evidenziato il ruolo chiave della biodiversità nella gestione dell’ecosistema vigneto, nella prospettiva della progettazione di sistemi viticoli agroecologici.

Mestrado em Engenharia Agronómica - Instituto Superior de Agronomia
This work describes the leaf morphoanatomy of 11 grapevine cultivars, grown at Tapada Ajuda, Lisbon. The white cultivars studied were ‘Alvarinho’, ‘Arinto’, ‘Encruzado’, ‘Macabeu’, ‘Moscatel Galego’, ‘Moscatel de Setúbal’ and ‘Viosinho’. The red ones were ‘Cabernet Sauvignon’, ‘Syrah’, ‘Touriga Nacional’ and ‘Trindadeira’. The leaf area was determined by scanning and under Light Microscopy the thickness of the cuticule, epidermis, total mesophyll as well as palisade and spongy parenchyma. Under Scanning Electron Microscopy, stomata type, their length and width and density were observed, as the existence of indumentum. Significant differences were observed among the white and red cultivars for all the parameters studied. In general, leaves with lower specific weight showed ticker spongy parenchyma. In all cultivars, three types of stomata were observed – at the same level, raised above and sunken regarding the other epidermal cells, showing differences among their relative proportions. ‘Moscatel de Setúbal’ and ‘Moscatel Galego’ showed the highest stomata density value, for the white cultivars. Among the red ones, differences in the sunken and raised above stomata were observed, but not for the same level stomata. ‘Trincadeira’ presented the highest value for the leaf area, the greater stomata density and the highest sunken stomata percentage, with 38.1%.

Mestrado em Bioquímica Clínic
A videira é uma das espécies frutíferas mais importantes a nível mundial. Dentro do género Vitis, a Vitis vinifera representa a espécie mais importante e mais utilizada para a produção de vinho, e apresenta muitas variedades, quer a nível de castas, quer ao nível dos clones. A sua grande variabilidade morfológica, metabólica e genética por um lado apresentam-se como uma vantagem, como um fator diferenciador, mas por outro lado dificulta a identificação de castas e clones de videira. Deste modo, a diferenciação inter e intra castas é de extrema importância para os vários agentes económico: o viveirista, o agricultor, as adegas a até consumidores, dado que a qualidade do vinho, entre outros fatores, depende da casta/clone utilizada. Desta forma, a identificação molecular de V. vinifera apresenta-se como uma ferramenta de extrema utilidade para a identificação inequívoca das videiras. Atualmente, esta é feita com microssatélites (SSRs). Neste trabalho procurou-se explorar o potencial dos Single Nucleotide Polymorphisms (SNPs) como marcadores moleculares. Para tal, selecionaram-se 32 regiões do genoma de V. vinifera que foram testados em 10 castas distintas, com 2 clones de cada casta. Neste trabalho, identificaram-se um total de 103 SNPs que permitem discriminar as castas estudadas, muito embora não tenham a capacidade de diferenciar os clones. Contudo, dos 103 SNPs, verificou-se que bastariam 12 SNPs para discriminar as 10 castas estudadas. Em conclusão, este estudo confirma o potencial de utilização dos SNPs como ferramenta moleculares para a discriminação de castas de V. vinifera.
Grapevine is one of the world’s most important fruit species. Within the genus Vitis, Vitis vinifera is the species that represents the most important and used for the production of wine. This specie includes many varieties and clones. The morphological, metabolic and genetic variability on one hand appear as an advantage, as a differentiating factor, but on the other hand, it is difficult to identify varieties and clones of the grapevine. Thus, the differentiation within and between varieties is extremely important for various economic agents: the nursery, the farmer, the wineries and ultimately the consumers, since the quality of wine, among other factors, depends on the variety/clone used. Therefore, the molecular identification of V. vinifera is of utmost importance for the unequivocally identification of the grape varieties. Currently this is done with microsatellites (SSRs). In this study we investigate the potential of the Single Nucleotide Polymorphisms (SNPs) as molecular markers. For this purpose, we selected regions of the genome of 32 V. vinifera that were tested on 10 different varieties, 2 clones from each variety. In this study, we identified a total of 103 SNPs that allow us to discriminate the varieties studied, although it does not have the ability to differentiate clones. From the 103 SNPs that were identified, it was found that 12 of them were enough to discriminate all the 10 cultivars studied. In conclusion, this study confirms the potential use of SNPs as molecular tool for the discrimination of varieties of V. vinifera.

Both the physiological and biochemical control of budburst in the grapevine, Vitis Vinifera L. were investigated. It was found that the accuracy of a predictive model for grapevine budburst based on ambient temperature was limited under the experimental conditions. There was a significant correlation of 4.7 ± 0.3 days between the days of maximal xylem exudation and budburst over the 3 years of investigation. The co-relationships between daily xylem exudate volume and a range of environmental parameters were considered. It was found that soil temperature was highly correlated against daily xylem exudation. Ambient temperature and soil moisture were significantly correlated with xylem exudation, however the coefficients of correlation were much lower than that of soil temperature. Rainfall showed only a very limited correlation with daily xylem exudate flow. Seasonal variations in the pH and the carbohydrate and inorganic nutrient concentrations of xylem exudate were investigated. Exudate carbohydrate concentrations fell from 660 µM before the day of maximal xylem exudation to zero levels within 4 weeks. Xylem exudate pH was found to consistently fall to a minimum at the time of maximal exudate flow. Exudate concentrations of the metallic cofactors Ca, K, Mg, Mn and Zn varied directly with daily exudate flow, suggesting some sort of flow-dependent mobilisation of these nutrients. A growth promontory oligosaccharide fraction was prepared by partial acid hydrolysis of grapevine primary cell wall material. This fraction significantly increased control growth of the Lemna minor L. bioassay over a limited ‘window’ of bioactivity. A growth inhibitory oligosaccharide fraction, similar in activity to abscisic acid was isolated from grapevine xylem exudate prior to budburst. The exudate concentration or efficacy of this substance declined after budburst such that there was no apparent growth inhibition. A model is proposed for grapevine budburst whereby an oligosaccharide growth inhibitor is gradually removed from the xylematic stream under the effects of soil temperature, allowing the surge of metabolic activity and vegetative growth that constitute budburst.

L'ambition de cette thèse était de proposer un nouvel élan pour la création variétale chez la vigne, incluant les connaissances et les derniers outils de la recherche. En effet, la viticulture française comme d'autres filières agricoles doit aujourd'hui faire face à 3 grands défis: la réduction des intrants phytosanitaires (plan Ecophyto 2018), les changements climatiques, et de nouveaux concurrents, notamment les pays du Nouveau Monde. La création variétale, qui a été peu exploitée chez la vigne, peut être une des solutions pour répondre à ces défis. S'appuyant sur un génotypage dense, plusieurs outils et concepts innovants - réunis sous le terme de sélection génomique (GS) - ont vu le jour ces dernières années en sélection animale, qui permettent de prédire les phénotypes des individus seulement génotypés. Afin d'atteindre notre but, nous avons évalué et comparé l'efficacité de la GS et de la sélection assistée par marqueur (SAM) " classique ", basée sur la génétique d'association " genome-wide " (GWAS) chez la vigne. Le potentiel théorique des deux méthodes a été évalué dans une étude de simulation, puis sur des données réelles. Nous montrons que la GS est plus pertinente que la SAM " classique " pour prédire les phénotypes et ce pour des caractères complexes et / ou structurés. Cependant la GS couplée aux résultats issus de la GWAS semble être une méthode intéressante lorsque le marquage moléculaire est non limitant. Finalement, nous discutons des conditions d'utilisation de la GS en termes économiques et d'efficacité au cours du temps. Nous proposons trois scénarios fonction de l'investissement de départ et des besoins en termes de création variétale.

Dissertation presented in fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology (Molecular Genetics) at the Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa
Anthocyanin content of grape berry skin determines the colour of grapes and wine. This trait has been widely studied due to its importance for grape and wine marketing and also due to the antioxidant properties of anthocyanins. In this thesis the variation of this trait was investigated within and between cultivars. DNA sequence variation and differential gene expression were studied among clones of the cultivars Aragonez and Negra Mole. Grape colour phenotyping was explored using different phenotyping approaches. Association mapping was performed for a sample of 149 cultivars and association mapping methodologies considering structure and relatedness in the sample were discussed. It was observed that no DNA sequence variation was present in the studied genomic regions between different clones of the same cultivar. Differential expression between Aragonez clones with contrasting values of skin total anthocyanin concentration was found to be very subtle not showing any significant results after correction for multiple testing and with two fold-change. However, relaxing statistical stringency and focusing on functional groups of interest (flavonoid metabolism and transcription factors) a list of 24 genes of interest was identified. This included two genes involved in the flavonoid metabolism, coding enzymes related with the glucosylation of flavonoids and transcription factors of the following families: Myb, Myc, zinc fingers, WRKY, DOF, GRAS, homeobox domain, YABBY, basic-leucine zipper, pathogenesisrelated and plant homeodomain finger.(...)
Financial support from Fundação para a Ciência e a Tecnologia, grant number SFRH / BD / 29379 / 2006 and ERA-PG 074B GRASP GRAPE WINE.

L'ambition de cette thèse était de proposer un nouvel élan pour la création variétale chez la vigne, incluant les connaissances et les derniers outils de la recherche. En effet, la viticulture française comme d'autres filières agricoles doit aujourd'hui faire face à 3 grands défis: la réduction des intrants phytosanitaires (plan Ecophyto 2018), les changements climatiques, et de nouveaux concurrents, notamment les pays du Nouveau Monde. La création variétale, qui a été peu exploitée chez la vigne, peut être une des solutions pour répondre à ces défis.S'appuyant sur un génotypage dense, plusieurs outils et concepts innovants – réunis sous le terme de sélection génomique (GS) – ont vu le jour ces dernières années en sélection animale, qui permettent de prédire les phénotypes des individus seulement génotypés.Afin d'atteindre notre but, nous avons évalué et comparé l'efficacité de la GS et de la sélection assistée par marqueur (SAM) « classique », basée sur la génétique d'association « genome-wide » (GWAS) chez la vigne. Le potentiel théorique des deux méthodes a été évalué dans une étude de simulation, puis sur des données réelles.Nous montrons que la GS est plus pertinente que la SAM « classique » pour prédire les phénotypes et ce pour des caractères complexes et / ou structurés. Cependant la GS couplée aux résultats issus de la GWAS semble être une méthode intéressante lorsque le marquage moléculaire est non limitant. Finalement, nous discutons des conditions d'utilisation de la GS en termes économiques et d'efficacité au cours du temps. Nous proposons trois scénarios fonction de l'investissement de départ et des besoins en termes de création variétale
The aim of this PhD project was to provide a new impulse for grapevine breeding, applying the latest knowledge and research tools on this species. Indeed, French viticulture, as well as various other agricultural sectors, faces today three major challenges: how to reduce phytosanitary inputs (Ecophyto 2018), impact of climatic changes and new competitors on the market, especially New World countries. Plant breeding in grapevine has not been much exploited until today, but could be a solution to these challenges.Several innovative tools and concepts have seen the light in animal breeding in the last decade. Using high density genome-wide marker information and advanced statistical models, phenotypes can be predicted for individuals that were merely genotyped. The method termed genomic selection (GS) is implementing this type of approach.To achieve our aim, we evaluated and compared the efficiency of GS and “classical” marker-assisted selection (MAS), based on genome-wide association study (GWAS) for grapevine. The theoretical potential of the two methods was evaluated in a simulation study but also on real data.We show that GS is more relevant than “classical” MAS to predict phenotypes of complex and / or structured traits. However, the combination of GS with results from GWAS seems to be of particular interest if the number of molecular markers available is adequate. Finally, we discuss GS implementation in terms of economic aspects and efficiency over time; we propose three scenarios differing by the initial investment required and the breeding objectives to be reached

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The viticulture has been expanding its cultivation in many regions of the country. Considering his success in the Northeast, in the Vale do São Francisco, research must be done to stimulate the development of this culture in new regions. The objective of this research was to evaluate the development of two grapevine cultivars produced in three rootstocks in Mossoró/RN, assessing maturity, quality and postharvest life of fruit. The experiment was conducted at the Experimental Farm Rafael Fernandesfrom 2013 to 2015. We used the design of randomized blocks with split plots for the three experiments. The first experiment evaluated the quality of the fruit in four crop cycles, with the rootstock (IAC 766, IAC 572 and IAC 313) in the plots and crop cycles subplots. In the second experiment, the treatments were three rootstocks (IAC 766, IAC 572 and IAC 313) and four maturity stages (14, 19, 24 and 28 days after the start of the color change of the berry) with five repetitions for Isabel Precoce. In the third experiment, we evaluated the shelflife of vine cultivars stored under refrigeration (4 ± 2 ° C and 95 ± 5% RH), where plots were assigned to rootstocks (IAC 766, IAC 572 and IAC 313) and time storage were assigned to the subplots. In third experiment for cultivar Isabel Precoce, storage times were 10, 20, 30, 40 and 50 days after harvest; to cultivar Itália Melhorada, were 8, 16, 24 and 32 days of storage after harvest. The characteristics evaluated for the three experiments were: mass of the bunch, number and berry mass, peel mass, berry length, berry diameter, color Berry (L, A * and B *), firmness (N) mass loss, wilting index stalk and injuries, soluble solids (SS), titratable acidity (TA), pH, SS/AT ratio, total and reducing sugars, anthocyanins, flavonoids, phenolic compounds and antioxidant activity. For the cycles of Isabel cultivar, the bunch weight and the number of berries were higher in the 3rd and 4th cycles; but the physical and chemical characteristics, as soluble solids, SS/AT ratio and sugar, were higher in the 1st cycle; as the bioactive compounds, phenolic and antioxidant activity was higher in the 4th harvest (first semester 2015). For cultivar Itália Melhorada, the 1st cycle gave higher soluble solids and SS/AT ratio, and the rootstock 766 shown superiority; in the second cycle, the mass of the bunch and number of berry was greater, and the rootstock 572 most massive result of the bunch. The ideal point of Isabel Precoce grape harvest is 28 days after the beginning of the maturation. The rootstock had a significant effect only for the SS/AT ratio, and highlight the IAC 766; for bioactive compounds, there was an increase with the maturation only for the phenolic content in the peel and pulp and anthocyanins, the other compounds did not vary throughout the maturation. Regarding storage the cultivar Isabel Precoce had a Shelflife relatively long, 50 days of storage at 4 ° C ± ± 2ºC and 90 ± 5% RH; the cultivar Itália Melhorada had a limited shelf life to 24 days of storage (4 ° C ± 2ºC and 90 ± 5% RH). As for the rootstocks used, the 766 and 572 had higher soluble sugar content for Isabel Precoce cultivar and higher soluble solids and total sugars to cultivar Itália Melhorada
A viticultura vem expandindo seu cultivo por várias regiões do país. Diante do seu sucesso na região nordeste, no Vale do São Francisco, pesquisas devem ser feitas visando estimular o desenvolvimento desta cultura em novas regiões. O objetivo do trabalho foi avaliar a qualidade, maturação e potencial de conservação dos frutos de duas cultivares de videira, produzidas sob três porta-enxertos no município de Mossoró/RN. O experimento foi realizado na Fazenda Experimental Rafael Fernandes, da UFERSA, no período de 2013 a 2015. Utilizou-se para os três experimentos o delineamento em blocos ao acaso com parcelas subdivididas. O primeiro experimento avaliou a qualidade dos frutos em quatro ciclos da cultura, com os porta-enxertos (IAC 766, IAC 572 e IAC 313) nas parcelas e os ciclos da cultura nas subparcelas. No segundo experimento, os tratamentos foram três porta-enxertos (IAC 766, IAC 572 e IAC 313) e quatro estádios de maturação (14, 19, 24 e 28 dias após o início da mudança de cor das bagas) com cinco repetições para Isabel Precoce. No terceiro experimento, foi avaliada a vida útil das cultivares de videira armazenadas sob refrigeração (4 ± 2º C e 95 ± 5% UR), onde as parcelas foram atribuídas aos porta-enxertos (IAC 766, IAC 572 e IAC 313) e tempo de armazenamento, às subparcelas. As características avaliadas para os três experimentos foram: massa do cacho, número de bagas, massa das bagas, massa da casca, comprimento da baga, diâmetro da baga, cor da baga (L, a* e b*), firmeza (N), perda de massa, índice de murchamento do engaço e injurias, sólidos solúveis (SS), acidez titulável (AT), pH, relação SS/AT, açúcares totais e redutores, antocianinas, flavonoides, compostos fenólicos e atividade antioxidante. Para os ciclos da cultivar Isabel, o peso do cacho e número de bagas foram superiores no 3º e 4º ciclos, mas as características físico-químicas como sólidos solúveis, relação SS/AT e açúcar foram superiores no 1º ciclo; quanto aos compostos bioativos, os fenólicos e a atividade antioxidante foram maiores no 4º ciclo. Para a cultivar Itália Melhorada, o 1º ciclo proporcionou maiores teores de sólidos solúveis e relação SS/AT, tendo o porta-enxerto IAC-766 mostrado superioridade; já no segundo ciclo, foram maiores a massa do cacho e número de bagas, tendo o porta-enxerto IAC-572 maior resultado de massa do cacho. O ponto ideal de colheita de uvas Isabel Precoce é aos 28 dias após o início da maturação. O porta-enxerto teve efeito significativo apenas para a relação SS/AT, tendo destaque o IAC-766; para os compostos bioativos, houve aumento com a maturação apenas para os teores de fenólicos na casca e polpa e para as antocianinas, os demais compostos não variaram ao longo da maturação. Com relação ao armazenamento, a cultivar Isabel Precoce teve vida útil relativamente longa, 50 dias de armazenamento a 4º C ± 2ºC e 90 ± 5% UR; a cultivar Itália Melhorada teve sua vida útil limitada aos 24 dias de armazenamento (4º C ± 2ºC e 90 ± 5% UR). Quanto aos porta-enxertos utilizados, o IAC-766 e IAC-572 proporcionaram maiores teores de açúcares solúveis para a cultivar Isabel Precoce e maiores teores de sólidos solúveis e açúcares totais para a cultivar Itália Melhorada
2017-02-20

Binderless fiberboard production from Cynara cardunculus and Vitis vinifera
Two lignocellulosic materials, Cynara cardunculus and Vitis vinifera, were pretreated and used to produce fiberboards without synthetic adhesives. The lignocellulosic materials were steam exploded through a thermo-mechanical vapor process in a batch reactor. After pretreatment the materials were dried, ground and pressed to produce the boards. The effects of pretreatment factors and pressing conditions on the chemical and physicomechanical properties of the fiberboards were evaluated and the conditions that optimize these properties were found. Response surface methodology based on a central composite design and multiple response optimization were used. The variables studied were: pretreatment temperature, pretreatment time, pressing temperature, pressing pressure, and pressing time.
Binderless fiberboards produced from Cynara cardunculus stalks at the optimum conditions found fulfilled the European standards for boards of internal use. Nevertheless, binderless fiberboards produced from Vitis vinifera prunings at the optimum conditions found for this material did not completely met the European standards; modulus of rupture and internal bond values for these boards were lower than required minimums.
Simultaneously, commercial Kraft lignin was reacted in an alkaline medium to enhance its adhesive properties. Chemical changes in reacted Kraft lignins that include ash content, Klason lignin, acid-soluble lignin and sugars were determined, as well as, structural characteristics of these lignins in terms of phenolic hydroxyl, aliphatic hydroxyl, methoxyl, carbonyl, Mw, Mn and polydispersity. The effects of reaction temperature and reaction time on lignin properties were studied using response surface methodology, and optimal reaction conditions were found.
Two different types of Kraft lignin were used, alkali treated Kraft lignin and crude acid-washed Kraft lignin, as additives to enhance the physicomechanical properties of binderless fiberboards produced from Vitis vinifera to reach and overcome the European standards completely. At the end fiberboards produced with 20% of Vitis vinifera fibers replaced by crude acid-washed Kraft lignin were able to meet the European standards completely.
This research work was an effort to reduce our dependency upon petroleum derivates, to diminish deforestation and to increase the use of renewable and biodegradable materials with the intention of preserving the environment and to encourage a sustainable development of our society.
Producción de Tableros de Fibras a partir de Cynara cardunculus y Vitis vinifera
En el presente estudio trozos Cynara cardunculus y Vitis vinifera fueron pretratados, y usados para producir tableros de fibras sin adhesivos sintéticos. Estos materiales lignocelulósicos se explotaron con vapor a través de un proceso termomecánico de vapor en un reactor por lotes. Después del pretratamiento el material fue secado, molido y prensado en caliente para producir los tableros. Se evaluaron los efectos de los factores del pretratamiento (temperatura de reacción y tiempo de reacción) y las condiciones de prensado (presión de prensado, temperatura y tiempo) sobre las propiedades químicas y físico-mecánicas de los tableros de fibras y se establecieron las condiciones que optimizan dichas propiedades. Las propiedades físico-mecánicas de los tableros de fibras que fueron estudiadas son: densidad, módulo de elasticidad (MOE), módulo de ruptura (MOR), enlace interno (IB), absorción de agua (WA) y hinchazón en hinchazón (TS) y las propiedades químicas estudiadas de la materia prima y el material pretratado fueron las siguientes: Cenizas, contenido de lignina Klason, contenido de celulosa y contenido de hemicelulosas. Se uso una metodología de superficie de respuesta basada en un diseño de experimentos del tipo central compuesto y una metodología de optimización de respuesta múltiple.
Los tableros de fibras sin adhesivos sintéticos producidos a partir de tallos de Cynara cardunculus a las condiciones óptimas encontradas cumplieron con las normas europeas para los tableros de uso interno. Sin embargo, los tableros de fibras sin adhesivos sintéticos producidos a partir de podas de Vitis vinifera a las condiciones óptimas encontradas para este material no cumplieron totalmente las normas europeas; los valores del módulo de ruptura y del enlace interno para estos tableros fueron inferiores a los mínimos requeridos.
Una lignina Kraft comercial fue sometida a reacción en un medio alcalino para mejorar sus propiedades adhesivas. Se determinaron los cambios químicos en las ligninas Kraft tratadas, las propiedades medidas fueron: contenido en cenizas, lignina Klason, lignina soluble en ácido y azúcares, también se determinaron las características estructurales de estas ligninas en términos de hidroxilos fenólicos, hidroxilos alifáticos, metóxilos, carbonilos, Mw, Mn y polidispersidad. Se estudiaron los efectos de la temperatura de reacción y el tiempo de reacción sobre las propiedades de la lignina con una metodología de superficie de respuesta, y se encontraron la condiciones óptimas de reacción.
Se usaron dos tipos diferentes de lignina Kraft, lignina Kraft tratada en medio alcalino y lignina Kraft cruda lavada con ácido, como aditivos para mejorar las propiedades físico-mecánicas de los tableros de fibras sin adhesivos sintéticos producidos a partir de Vitis vinifera, para alcanzar y superar las normas europeas completamente. Al final los tableros de fibras producidos con una substitución del 20% de fibras de Vitis vinifera por lignina Kraft cruda lavada con ácido fueron capaces de satisfacer las normas europeas por completo.Este trabajo de investigación fue un esfuerzo para reducir nuestra dependencia de los derivados del petróleo, para disminuir la deforestación y para aumentar el uso de materiales renovables y biodegradables con la intención de preservar el medio ambiente y fomentar un desarrollo sostenible de nuestra sociedad.

L'objectif principal de cette thèse de doctorat est d'étudier l'activité anti-inflammatoire et anti-tumorale potentielle du resvératrol et d’autres stilbènes obtenus à partir d’extraits de V. vinifera. Pour atteindre cet objectif, l'effet anti-inflammatoire des stilbènes est d'abord abordé sur un modèle de macrophage murin activé par les lipopolysaccharides (LPS), puis l'activité antitumorale est évaluée sur un modèle de carcinome hépatocellulaire humain.Les stilbènes utilisés pour l'analyse des activités biologiques comprennent 8 monomères (resvératrol, picéide, piceatannol, astringine A, pterostilbene, oxyresveratrol, isorhapontine et isorhapontigenine), 7 dimères (e-viniférine, d-viniférine, w-viniférine, ampélopsine A, scirpusine A, pallidol et vitisinol C), un trimère (miyabenol C) et 4 tétramères (hopeaphenol, isohopeaphenol, R2-viniférine et R-viniférine).Concernant les effets anti-inflammatoires, tous les stilbènes ont été testés sur une lignée de macrophages murins activés par du LPS, en analysant à la fois leur cytotoxicité et leur capacité à inhiber la production de NO. Leurs CI30 ou CI50 ont été calculées. A partir de ces travaux, 6 composés ont été sélectionnés : piceatannol, e-viniférine, δ-viniférine, hopéaphenol et l'isohopéaphenol. Sur ces composés, la libération de cytokines inflammatoires et la production de ROS a été étudié. L’hopeaphenol et le piceatannol inhibent la production de ROS. La δ-viniférine ne diminue pas la libération des cytokines IL-1β et TNF-α. Il a été montré que le piceatannol, l’e-viniférine, l’hopeaphenol et l’isohopeaphenol ont un effet sur la réponse inflammatoire produite par l'activation que le LPS exerce sur les macrophages murins. Cette observation justifie de poursuivre l'étude dans des modèles plus complexes étant donné son utilité possible dans le traitement des maladies à base inflammatoire.Pour l'étude de l'activité antitumorale, la CI50 obtenue après traitement par 6 stilbènes (monomère : resvératrol ; dimères : ampélopsine A et e-viniférine ; tétramères : hopeaphenol, isohopeaphenol, R2-viniférine et R-viniférine) de 2 lignées d'hépatomes a été comparée à une lignée d'hépatocytes non transformés. La R2-viniférine présente l'activité cytotoxique la plus élevée sur la lignée HepG2 (CI50 <10 µM), sans être toxique à cette concentration pour la lignée HH4. Les cellules Hep3B sont nettement plus résistantes à la R2-viniférine (CI50 48 µM). Le stilbène a produit une augmentation des cellules HepG2 en phase SubG0, sans altération des cellules Hep3B. La réponse différenciée des lignées cellulaires à l’action de ce stilbène peut être due au statut différent de p53. En effet, l’extinction de p53 dans les cellules HepG2 a augmenté leur résistance au traitement, tandis que la transfection de p53 dans les cellules Hep3B les a rendues plus sensibles. La R2-viniférine provoque la phosphorylation de p53 sur les lignées HepG2. L'activation des caspases 3 et 9, l'augmentation du rapport des protéines Bax/Bcl2 et la libération de LDH intracellulaire ont mis en évidence la mort cellulaire par apoptose et nécrose provoquée par l’action de la R2-viniférine. Dans les lignée HepG2, Cette action est modulable par PI3K/Akt et ERK1/2. De plus, l'expression de l'histone ɣ-H2AX a montré les dommages que le traitement par la R2-viniférine produit sur l'ADN. Le rôle clé que jouent les radicaux libres dans tout ce processus a été observé en raison de l'augmentation de la production de ROS et de H2O2, et de l'augmentation de l'activité Mn-SOD. Il a également été démontré que la R2-viniférine diminue la capacité de migration et de formation de colonies de cellules HepG2.Il est conclu que la R2-viniférine, un tétramère du resvératrol, exerce une activité antitumorale par le biais de mécanismes d'apoptose liés à p53. Ce composé est un agent prometteur avec des effets potentiels pour la chimioprévention et le traitement du cancer du foie
The main objective of this Doctoral Thesis is to study the potential anti-inflammatory and anti-tumor activity of resveratrol and other stilbenes present in Vitis vinifera. To achieve this goal, the anti-inflammatory effect of stilbenes is firstly addressed in a lipopolysaccharide (LPS) -activated murine macrophage model, and secondly, antitumor activity in a model of human hepatocarcinoma is analyzed.The stilbenes used for the analysis of antitumor activity comprise 8 monomers (resveratrol, piceide, piceatannol, astringin A, pterostilbene, oxyresveratrol, isorhapontine and isorhapontigenine), 7 dimers (e-viniferin, w-viniferin, d-viniferin, ampelopsin A escirpusin A, pallidol and vitisinol C), a trimer (miyabenol C) and 4 tetramers (hopeaphenol, isohopeaphenol, R2-viniferin and R-viniferin). All were tested in the LPS-activated murine macrophage line, analyzing both their cytotoxicity and their ability to inhibit NO production. From these experiments, their IC30 or IC50, respectively, were calculated. The stilbenes selected based on the results obtained were piceatanol, e-viniferin, δ-viniferin, hopeaphenol and isohopeaphenol, with which the effect that they exert on the release of inflammatory cytokines and the production of ROS was studied. Hopeaphenol and piceatannol inhibit ROS production, and only δ-viniferin does not decrease the release of cytokines IL-1β and TNF-α. It is concluded that the derivatives of resveratrol piceatanol, -viniferin, hopeaphenol and isohopeaphenol have interesting effects on the inflammatory response produced by the activation that LPS exerts on murine macrophages. This finding justifies continuing the study in more complex models given its possible usefulness in the treatment of diseases with an inflammatory basis.For the study of antitumor activity, the IC50 obtained after treatment with 6 stilbenes from the 2 hepatoma lines were compared with the line of non-transformed hepatocytes. Resveratrol (monomer), ampelopsin A and e-viniferin (dimers) and the tetramers hopeaphenol, isohopeaphenol, R2-viniferin and R-viniferin were analyzed. The R2-viniferin tetramer shows the highest cytotoxic activity in HepG2 (IC50 <10 µM), without being toxic at this concentration for HH4. Hep3B cells are notably more resistant to R2-viniferin (IC50 48 µM). Stilbene produced increased HepG2 cells in SubG0, without alteration in Hep3B. The different response to this stilbene turned out to be due to the different status of p53, since silencing p53 in HepG2 cells increased their resistance to treatment, while transfection of p53 into Hep3B made them more sensitive. R2-viniferin causes phosphorylation of p53 in HepG2.The activation of caspase 3 and 9, the increase in the ratio of Bax / Bcl2 proteins and the release of intracellular LDH evidenced cell death by apoptosis and necrosis caused by that stilbene, which can be modulated by the PI3K/Akt and ERK1/2 on HepG2. Furthermore, the expression of histone ɣ-H2AX showed the damage that treatment with R2-viniferin produces in DNA. The key role that free radicals play in this entire process was elucidated due to the increased production of ROS and H2O2 and the increase in Mn-SOD activity. It was also shown that R2-viniferin decreases the migration and colony formation capacity of HepG2.It is concluded that the R2-viniferin tetramer exerts its anti-tumor activity through apoptosis mechanisms linked to p53 and is a promising compound with potential effects for chemoprevention and treatment of liver cancer

Cette étude visait à établir un protocole de cryoconservation pour des apex de vigne et à tester l'efficacité de la cryoconservation pour éliminer des virus de la vigne sélectionnés. Des cultures in vitro de génotypes sains de huit cultivars croates autochtones de vigne, Plavac mali, Maraština, Pošip, Debit, Grk, Lasina, Plavina et Vugava et de génotypes infectés de Plavac mali ont été établies. Les différences de survie, de reprise et des paramètres de croissance observés se sont avérées spécifique des génotypes. Les cultivars infectés se sont montrés moins réactifs que les cultivars sains. Un protocole de cryoconservation basé sur la vitrification avec PVS2 a été établi. Les modifications de la préculture avec le saccharose et l'utilisation de solutions alternatives de cryoconservation n'ont pas amélioré la reprise. A l'opposé, l'état physiologique du matériel végétal a joué un rôle essentiel pour la cryoconservation. Des bourgeons en croissance active prélevés sur des microboutures mononodales ont montré une reprise plus élevée que des bourgeons prélevés directement sur les vitroplants. La position des bourgeons sur la tige des plantes-mères in vitro a affecté la reprise après cryoconservation. L'addition de benzylaminopurine au milieu de culture des microboutures a eu un effet positif sur la reprise après immersion dans l'azote liquide, alors qu'aucun effet positif n'a été observé sur la reprise avec la zéatine riboside ou la proline. Le protocole de cryoconservation établi a permis d'obtenir environ 50% de reprise avec le cultivar Portan et avec trois des quatre cultivars internationaux utilisés. Par contre, aucune reprise ou une reprise très faible a été observée avec les cultivars croates testés. En se basant sur les tests ELISA réalisés, le virus GFLV a été éliminé de 82,4% des apex non cryoconservés et de 77,8% des apex cryoconservés chez le cultivar Chardonnay et le GLRaV-3 a été éliminé de 100% des apex non cryoconservés et cryoconservés chez le cultivar Cabernet Sauvignon. Ces résultats sont à relier à nos études d'immunolocalisation, qui ont montré que le GFLV était présent dans le dôme apical et les tissus méristématiques chez le cultivar Pinot Noir and que le GLRaV-3 était présent dans les tubes criblés chez le cultivar Merlot. La stabilité génétique des plantes régénérées à partir des apex cryoconservés a été étudiée en utilisant les marqueurs AFLP. Avec les huit combinaisons d'amorces utilisées sur les 43 plantes testées, aucun polymorphisme n'a été observé après la préculture au saccharose, le traitement avec la solution de loading et la solution de PVS2 diluée de moitié. Par contre, des fragments polymorphes ont été observés sur des explants non cryoconservés et cryoconservés traités avec la solution PVS2, dont le nombre augmentait avec l'augmentation de la durée d'exposition à la solution PVS2
This study aimed at establishing a cryopreservation protocol for grapevine shoot tips and at testing the efficiency of cryopreservation in eliminating selected grapevine viruses. In vitro cultures of healthy genotypes of eight Croatian autochthonous grapevine cultivars Plavac mali, Maraština, Pošip, Debit, Grk, Lasina, Plavina and Vugava and of virus-infected genotypes of Plavac mali were successfully established. Differences in survival, regrowth and growth parameters were genotype-specific. Infected cultivars were less reactive compared to healthy ones. A PVS2-based cryopreservation protocol was successfully established. Modifications in sucrose preculture conditions and use of PVS2-derived alternative vitrification solutions did not improve growth recovery. By contrast, the physiological state of the plant material played a critical role in cryopreservation. Actively growing buds sampled from single-node microcuttings displayed higher regrowth compared to buds sampled directly on in vitro plantlets. The position of buds on the stem of in vitro mother-plants affected regrowth after cryopreservation. The addition benzylaminopurine in the shooting medium had a positive effect on regrowth after liquid nitrogen exposure, while no such positive effect was observed with zeatine riboside or proline. The cryopreservation protocol established led to approximately 50% recovery with cultivar Portan and three of the four international cultivars tested. By contrast, no or very low recovery was noted with the Croatian cultivars tested. Based on ELISA tests, the GFLV virus was eliminated from 82.4% of non-cryopreserved samples and from 77.78% of cryopreserved samples in cultivar Chardonnay and the GLRaV-3 virus was eliminated from 100% of both non-cryopreserved and cryopreserved samples in cultivar Cabernet Sauvignon. These results may be related with our immunolocalisation studies, which showed that GFLV was found in the apical dome and meristematic tissues in cultivar Pinot Noir and GLRaV-3 in sieve elements of cultivar Merlot. Genetic stability of plants regenerated from cryopreserved shoot tips was studied using AFLP markers. With the eight AFLP primer combinations employed on the 43 plants tested, no polymorphism was observed after sucrose preculture, treatment with the loading solution and half-strengthPVS2. However, polymorphic fragments were observed in non-cryopreserved and cryopreserved samples treated with PVS2 solution, the number of which increased with increasing durations of exposure to PVS2 solution

Evapotranspiration measured from two Vitis vinifera L. c.v. 'Cabernet Sauvignon' by the water budget method totaled 136.1 1 d⁻¹, and steady state leaf transpiration rates averaged 57 ± 47 mg m⁻² s⁻¹ from March to August 1986 in Oracle, Arizona. Rooted cuttings were subjected to four levels of water stress in the greenhouse by withholding water. Pot weight variation was correlated to soil water potential by thermocouple psychrometry. Mesophyll conductance and CO₂ assimilation, measured with an open gas exchange system, were decreased by 80 and 70%, respectively, indicating severe limitations imposed by water stress on photosynthesis for this cultivar.

Thesis (MScAgric)--Stellenbosch University, 2008.
ENGLISH ABSTRACT: Fruit quality is the most important factor that determines prices for the fruit in the international markets. Although different consumers perceive quality differently there are quality variables that are always associated with poor quality by all consumers. In table grapes (Vitis vinifera L.) these variables may include overall appearance, stem condition, SO2 damage, decay, berry browning and shatter. The presence of these quality defects negatively affects prices and most often results in quality claims. Cooling is the most widely used method to reduce the postharvest loss of fruit quality. In South Africa, most deciduous fruits including table grapes are forced air cooled to a statutory pulp temperature of –0.5°C prior to shipping in an effort to preserve quality, thus ensuring good market prices for the fruits. Despite these efforts, there are still quality claims from the markets and this reduces the returns to the growers. The objectives of this research were to: (i) see if cooling time can be reduced by cooling to higher pulp temperatures of 1.5°C and 3°C without causing quality losses, thus improving the throughput of the cold rooms; (ii) see if the problem of berry browning can be alleviated by cooling grapes to higher pulp temperature, and (iii) see whether pallet positioning in the cooling tunnels and reefer container affect quality. The trends showed better quality when ‘Victoria’ and ‘Regal Seedless’ were forced air cooled (FAC) to pulp temperatures of 1.5 °C and 3 °C as opposed to –0.5 °C. There were no economic losses associated with pre-cooling grapes to pulp temperatures of 1.5 °C and 3 °C. There were no significant differences in berry browning related to pre-cooling treatments. However, cooling time was reduced significantly. In most of the cooling tunnels and reefer containers used in this trial, grape quality results showed no significant differences between the positions in the stack and in reefer containers. However, in cases where there were significant differences, the middle and the rear positions showed better grape quality in terms of stem condition (dry and brown stems) than the front position (near fan) in both the pre-cooling stack and reefer containers. The trends showed that the front is cooler than the back of the pre-cooling stack. The pulp temperature differences between the front and rear positions in the reefer container were as high as 1.23 °C. The trends also showed that the bottom layers of the pallets were cooler than the top layers in the reefer container. FAC to 3°C resulted in a constant reduction in percentage electrolyte leakage after 4 weeks of storage at –0.5°C, while FAC to 1.5°C, -0.5°C and static room cooling (control) in some cases showed an initially low electrolyte leakage followed by an increase in leakage after 4 weeks of storage. FAC grapes to higher pulp temperatures of 3°C and 1.5°C could reduce the cooling time, thereby improving the throughput of cold rooms. There was no clear evidence to suggest that browning was due to pre-cooling practices. Both preharvest and postharvest conditions need to be further investigated to better understand the problems of browning in white table grapes.
AFRIKAANSE OPSOMMING: Vrugkwaliteit is ʼn kritiese faktor in die bepaling van pryse op die internasionale markte. Alhoewel daar variasie voorkom tussen verbruikers in wat vrugkwaliteit is, bly sekere aspekte altyd onveranderd. Ononderhandelbare kwaliteit aspekte in tafeldruiwe (Vitis vinifera L.) sluit die algemene voorkoms, toestand van die trosstingels, SO2 skade, bederf, korrel verbruining en los-korrels in. Indien enige van die kwaliteit-defekte voorkom het dit ʼn negatiewe impak op die prys en lei gewoonlik tot gehalte eise. Verkoeling word algemeen gebruik om die verlies van na-oes kwaliteit te verminder. Die meeste sagtevrugte geproduseer in Suid Afrika (insluitend tafeldruiwe) ondergaan geforseerde verkoeling tot ʼn statutêre pulptemperatuur van -0.5°C, voor verskeping. Ondanks hierdie maatreëls om hoë pryse te verseker, is daar steeds kwaliteiteise in die mark wat lei tot ‘n laer inkomste vir produsente. Die navorsing het dus ten doel gehad om : (i) te bepaal of die tyd van verkoeling verminder kan word, indien na hoër pulptemperature van 1.5°C en 3°C verkoel kan word, sonder ‘n verlies in kwaliteit en sodoende die deurvloeitempo van die koelkamers verhoog; (ii) om te bepaal of die voorkoms van korrelverbruining verlaag kan word indien tot hoër pulp-temperature verkoel word, en (iii) laastens om te bepaal of posisie van die palet in die verkoelingstonnel en verskepingshouer ʼn invloed het op vrugkwaliteit. Tendense toon dat ‘Victoria’ en ‘Regal Seedless’ kwaliteit beter was indien verkoel tot pulptemperature van 1.5°C en 3°C in vergelyking met -0.5°C. Daar was geen ekonomiese verliese waargeneem indien die hoër verkoelingstemperature gebruik is nie. Alhoewel daar geen betekenisvolle verskille in korrelverbruining voorgekom het tussen temperatuur behandelings nie is die verkoelingsperiode verkort. In die meeste van die verskepingshouers, asook in posisies tydens geforseerde verkoeling is daar geen betekenisvolle verskille waargeneem nie. In die gevalle waar daar egter wel betekenisvolle verskille voorgekom het, het die middel en agter posisies beter vrugkwaliteit gehad as die voorste posisie tydens verkoeling asook houerverskeping. Die palette aan die voorkant (naby die waaier) het as ʼn algemene tendens laer temperature as in die agterkant van die verkoelingstonnel. Verskille in pulptemperature tussen palette in die voor en agterkant van verskepingshouers was so hoog as 1.23°C. Die temperatuurdata het uitgewys dat die onderste laag kartonne neig om by ‘n laer temperatuur te wees as die boonste lae kartonne tydens houerverskeping. Geforseerde verkoeling teen 3°C het gelei tot ‘n afname in persentasie elektrolietlekkasie na 4 weke van verkoeling teen -0.5°C. Terselfdertyd het geforseerde verkoeling tot 1.5°C en -0.5°C asook statiese verkoeling (kontrole) in sekere gevalle gelei tot ‘n laer aanvanklike uitlek van elektrolietlekkasie, gevolg deur ʼn verhoging na 4 weke opberging. Geforseerde verkoeling van tafeldruiwe tot pulptemperature van 1.5°C en 3°C verkort die verkoelingstyd en verhoog dus die deurvloeitempo in die verkoelingskamers. Daar was gedurende die studie geen duidelike bewyse gevind dat korrelverbruining voorkom as gevolg van verkoelingspraktyke nie. Beide voor en na-oes praktyke sal verder ondersoek moet word om die invloed daarvan te bepaal op die verbruining van wit tafeldruiwe.

In the last years the increasing number of sequencing projects and the availability of completely sequenced genomes pose the problem of searching for gene sequences in a rapid and reliable way. Bioinformatics is playing a fundamental role in this research field. In fact, many bioinformatic tools and software that consider multiple and heterogeneous evidence sources have been developed in order to improve the genome annotation. Genome annotation can be divided in two distinct phases: gene prediction and functional annotation. The prediction phase is the process to identify the exact gene structure, delimiting the exon-intron boundaries and the localization of genes on the genome. Otherwise, the functional annotation is the action of characterizing predicted genes, assigning them a biological function, a metabolic role or describing structural features. This PhD project focuses on the development of computational methods for the management of data coming from a genome sequencing project. The work consists on the implementation of a bioinformatic platform for gene prediction and functional annotation of the Vitis vinifera genome. This work has been carried out in collaboration with CRIBI bioinformatic group, that is member of the Grape sequencing project. The annotation platform consists of two distinct modules. The first module regards gene prediction. Different computational methods showed a great reliability to discover molecular signals and to reconstruct gene boundaries, becoming fundamental in the annotation at genome-level. These methods are represented by ab-initio predictors, genome alignments of ESTs or proteins or comparative genomics. Otherwise, in the second module of annotation platform, the predicted genes are functionally characterized, adopting mainly a similarity approach. This approach bases on the assumption that regions highly conserved maintain the original functions or roles also in different species. This project includes also the development of databases and tools to store and retrieve genome data. In particular, the PhD work focused on the implementation of a XML-based query system that permits the information retrieval through web page access and, in the next future, also through web-services workflows.
Negli ultimi anni il crescente numero di progetti di sequenziamento e la disponibilità di genomi completamente sequenziati hanno posto il problema della ricerca di sequenze geniche in modo rapido e affidabile. La Bioinformatica sta giocando un ruolo fondamentale in questo campo di ricerca. Infatti, sono stati sviluppati molti strumenti informatici che utilizzano dati molteplici ed eterogenei al fine di migliorare l’annotazione genomica. L’annotazione genomica può essere suddivisa in due fasi distinte: la predizione genica e l’annotazione funzionale. La predizione genica consiste nell’individuazione dell’esatta struttura del gene, determinando il confine esone-introne e la localizzazione dei geni sul genoma. Invece, l’annotazione funzionale è il processo di caratterizzazione dei geni, che assegna loro una funzione biologica, un ruolo metabolico o che descrive le loro caratteristiche strutturali. Questo progetto di dottorato prevede lo sviluppo di metodi computazionali per la gestione dei dati provenienti da progetti di sequenziamento genomico. Il lavoro consiste nella realizzazione di una piattaforma bioinformatica per la predizione genica e l’annotazione funzionale del genoma di Vitis vinifera. Questo lavoro è stato svolto in collaborazione con il gruppo di bioinformatica del CRIBI, membro del progetto internazionale di sequenziamento del genoma di vite. La piattaforma di annotazione è suddivisa in due moduli. Il primo modulo riguarda la predizione genica. Diverse metodiche computazionali hanno mostrato una grande affidabilità nella ricerca di segnali molecolari e nella ricostruzione della struttura genica, diventando strumenti fondamentali per l’annotazione genomica. Questi metodi sono rappresentati da predittori ab-initio, da allineamenti di EST o proteine sul genoma o dalla genomica comparata. Invece, nel secondo modulo della piattaforma di annotazione, i geni predetti sono caratterizzati funzionalmente attraverso l’utilizzo di un approccio di similarità. Questo approccio si basa sul presupposto che le regioni altamente conservate mantengono le funzioni e i ruoli originali anche in specie diverse. Questo progetto prevede anche lo sviluppo di banche dati e strumenti per immagazzinare e recuperare i dati di annotazione. In particolare, il lavoro di dottorato si è concentrato sulla realizzazione di un sistema di query basato su XML che permette il recupero delle informazioni attraverso pagine web e, nel prossimo futuro, anche attraverso l’utilizzo di workflow basati sui web services.

No estado de São Paulo são produzidas uvas de mesa de origem europeia (Vitis vinifera) e de origem americana (Vitis labrusca). Devido as condições climáticas, doenças fúngicas foliares são recorrentes e causam danos e prejuízos aos viticultores paulistas. Entre essas doenças se destaca a ferrugem da videira (Phakopsora euvitis), por causar desfolha intensa, depreciar cachos e reduzir a produção. Entretanto, pouco se sabe sobre a interação desse patógeno e espécies de videiras. Portanto, o objetivo deste trabalho foi avaliar a severidade e eficiência de doença da ferrugem em V. labrusca; e comparar a resistência à ferrugem entre as espécies de videira V. vinifera cv. Moscato Giallo e V. labrusca cv. Niágara Rosada, por meio de componentes epidemiológicos da doença e alterações anatômicas, sintomatológicas e fisiológicas dos hospedeiros. O período de molhamento foliar e a temperatura no processo de infecção, assim como a concentração de inóculo de P. euvitis influenciam na eficiência de doença e consequentemente na severidade de ferrugem em Vitis labrusca. A maioria dos componentes epidemiológicos avaliados mostrou que a V. labrusca é mais suscetível à ferrugem do que a V. vinifera. O período de latência e período infeccioso da ferrugem da videira são similares entre as espécies de videiras e folhas mais velhas (com 60 dias após a brotação) são mais suscetíveis à ferrugem que folhas mais jovens (34 e 24 dias após a brotação). As folhas mais velhas tem maior capacidade de esporulação. Em V. labrusca foi observado maior severidade da doença durante o período infeccioso. Em folhas de V. labrusca são observadas urédias satélites oriundas da primeira infecção que caracterizam o crescimento da pústula. Em folhas de V. vinifera há a presença de um halo encharcado ao redor da lesão, onde há acúmulo de substâncias pécticas e celulose que funcionam como mecanismos de defesa do hospedeiro para a ferrugem da videira. Em folhas de V. vinifera assim como de V. labrusca a ferrugem da videira reduz a fotossíntese tanto na região da lesão quanto na região assintomática ao redor da pústula. A ferrugem da videira causa danos no processo fotoquímico, pela redução do transporte aparente de elétrons. Em ambas as espécies também pode ser observada o acúmulo de espécies reativas de oxigênio (ROS) no sítio de infecção horas após a inoculação, relacionando a resposta de hipersensibilidade a uma reação pós-formada do hospedeiro como estratégia de reduzir a área lesionada.
Grapevines cultivar of Vitis vinifera and Vitis labrusca are cultivated in Sao Paulo state for producing table grapes. Foliar fungal diseases are recurrent and cause yield losses when climatic conditions are favourable to disease development. Grapevine rust (Phakopsora euvitis) causes intense defoliation, fruit depreciation and yield losses. However, little is known about the interaction between P. euvitis and Vitis spp. Therefore, the objective of this work was to evaluate the severity and disease efficiency of rust in V. labrusca; and compare the resistance of the species V. vinifera cv. Moscato Giallo and V. labrusca cv. Niagara Rosada measuring of epidemiological components of the disease and anatomical, symptomatological and physiological alterations in the hosts. The leaf wetness period and the temperature during the infection process, as well as the inoculum concentration of P. euvitis influence the disease efficiency and, consequently the rust severity in V. labrusca. Most of the evaluated epidemiological components showed that V. labrusca is more susceptible to rust than V. vinifera. The latent period and the infectious period of rust are similar between the species and older leaves (60 days after bud break) are more susceptible to rust than younger leaves (34 and 24 days after bud break). Older leaves have greater sporulation capacity. During infectious period of rust in V. labrusca was observed a higher severity of disease than in V. vinifera. Satellite uredias originary from the first infection are observed in V. labrusca leaves which characterize the pustule growth. V. vinifera leaves showed a soaked halo surround the lesion, and accumulation of pectic substances and cellulose was observed in this region. These substances accumulation may are defense mechanisms of the host against the rust. Rust reduces photosynthesis in the region of lesion, and also in the asymptomatic region around the pustule in both grapevine species. Rust causes damage in photochemical process as a result of reduction from apparent transport of electrons. In both species, one day after inoculation it was observed accumulation of reactive oxygen species (ROS). Therefore, hypersensitivity response is related to a post-formed host reaction such as a strategy to reduce the injured area.

Ce memoire est consacre a l'obtention d'un materiel vegetal susceptible de fournir des protoplastes vegetaux, dans des conditions parfaites. Des feuilles, cotyledons, hypocotyles, racines et cals sont obtenus a la suite de microbouturages ou de germination de pepins in vitro sur 5 cultivars de la vigne: grenache, cabernet-sauvignon, clairette, carignan, rupestris du lot et un hybride de plantet (5455). L'isolement des protoplastes a ete realise a partir de ces explants eleves dans des conditions d'axenie rigoureuse et rendus plus reactifs que les memes organes preleves en vignoble sur les plantes-meres par cette acclimatation prealable a la culture in vitro. Pour l'isolement des protoplastes foliaires, nous avons utilise un melange enzymatique de maceration associant 1% de cellulase et 1,0% de driselase. Pour les autres organes 2% de cellulase et 0,5-1,0% de macerozyme. Plusieurs facteurs pouvant influencer l'isolement des protoplastes ont ete etudies en particulier pour les protoplastes issus de cals cotyledonnaires. Les protoplastes isoles sont purifies par la technique de flottaison sur une solution de 10% de ficoll centrifugee a 250 g pendant 20 min. Au cours de la culture des protoplastes: la regeneration d'une nouvelle paroi cellulaire, des phenomenes de "bourgeonnement" et de declenchement de trois divisions cellulaires successives, ont ete observes chez des protoplastes isoles a partir d'hypocotyles de grenache cultives dans le milieu liquide de kao & michayluk (8p) modifie par l'addition de 2,0 mg/l 2,4-d, 1,0 mg/l bap et 0,6 mannitol

Este trabalho desenvolveu-se em duas partes. A primeira parte consistiu em definir metodologias de micropropagação, cultura de calli e organogénese experimental em duas castas de videira - Baga e Maria Gomes - características da região da Bairrada. A segunda parte consistiu no estudo do comportamento da casta Baga, relativamente ao stress osmótico induzido pelo osmótico sorbitol, em microrrebentos de videira em cultura In vitro. O estudo da micropropagação através da cultura de gomos axilares, permitiu seleccionar o meio MA1 (meio MS com 4.4 μM de BAP) para o desenvolvimento dos gomos axilares, e os meios MA2 (0.4 macro MS, 1 micro MS, vit. RG) e MR2 (1/2 MS com 0.1 μM de IBA) para o enraizamento dos microrrebentos e alongamento das plântulas. A aclimatação das plântulas destas duas castas foi sempre bem sucedida, com a metodologia adaptada. A indução e cultura de tecido caloso foram obtidas, utilizando como explantes primários folhas jovens de microrrebentos de videira, e os meios MC1 (1/2 MS com 1 μM de 2,4-D) e MC6 (1/2 MS com 0.44pMde BAP e 0.45 IAM de 2,4-D). A indução e cultura de tecido caloso foram ainda obtidas, através da cultura de segmentos de caule e pecíolo no meio MC9 (112 MS com 1.6 μM de BAP e 8.5 μM de IAA). O meio MC9 possibilitou as taxas de crescimento de calli mais elevadas. A organogénese experimental em folhas jovens de microrrebentos de videira, deu origem à formação de folhas adventícias nos segmentos de pecíolo não se verificando, com a metodologia utilizada, o desenvolvimento de rebentos adventícios. Este tipo de desenvolvimento só foi possível na casta Maria Gomes embora com percentagens relativamente baixas, 20% no meio MO1 (meio NN com 8.8 μM de BAP) e 6.25% no meio MO3 (meio NN com 8.8 μM de BAP e 0.1 μM de NAA). O estudo do efeito do stress osmótico no crescimento de microrrebentos de videira cv. Baga, realizou-se utilizando como tratamentos as concentrações 0.1 M, 0.2 M e 0.4 M de sorbitol. O aumento deste osmótico induziu uma redução da percentagem de enraizamento e da taxa de crescimento no genótipo Baga. Os tratamentos mais severos (0.2 M e 0.4 M de sorbitol), conduziram a uma elevada necrose dos tecidos dos microrrebentos, caracterizada por uma diminuição significativa de proteínas totais solúveis nas porções aéreas, diminuição significativa do teor em clorofilas nas folhas e diminuição significativa na acumulação dos elementos P, B, Ca, Fe, Mn, Mg e K nas porções aéreas. Este tipo de estudo é importante para a caracterização de genótipos tolerantes ao stress hídrico.

Doutoramento em Biologia / Instituto Superior de Agronomia
Vitis vinifera vinifera is a domesticated hermaphrodite plant subspecies, and one of the most important crops in the world, required for wine making among other products. Vitis vinifera sylvestris is a dioecious subspecies considered the wild ancestral of V. v. vinifera. The molecular mechanisms responsible for the shift between hermaphroditism to dioecy are largely unknown. Male flowers of V. v. sylvestris show a reduced non-functional pistil while female plants exhibit reflexed stamens with infertile pollen. Hermaphrodite flowers develop functional male and female organs. In this work, the transcriptome of the three flower types were sequenced, assembled and de-novo assembled in order to map specific V. v. sylvestris transcripts, therefore understanding the primary players and mechanisms which contribute to the shift in flower types. This approach allowed for the detection of specific transcripts that might be responsible for the arrest of sexual organs in V. v. sylvestris flower type. Additionally, through transcription levels and RNA location, it seems that the ABCDE model genes are not responsible for the shift from hermaphroditism to dioecism and therefore sex determination mechanism most likely occurs downstream of the onset of flowering genes. The chromosome 2 was also specifically screened since it could contain a putative sex locus responsible for plant flower type determination. This analysis resulted in the development of two marker genes able to early differentiate between flower types, one of those genes specifically expressed only in the male flower carpels. Several crosses between both Vitis subspecies showed that the gene markers can predict the segregation based on flower type. Also, the genomic region where these genetic markers are located seem to be responsible for flower determination. It was possible to observe, that for VviAPRT3 and VviFSEX male plants are heterozygous dominant while female plants are homozygous recessive
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Los compuestos fenólicos son responsables de características sensoriales de las bayas y del vino tales como el color, astringencia y amargor. Además, determinan el potencial de envejecimiento de los vinos. Estos compuestos se sintetizan a través de la ruta fenilpropanoide y su formación sería determinada por múltiples factores. A pesar de la abundante información que evalúa los diversos factores que afectan la biosíntesis de compuestos fenólicos, ésta es a veces contradictoria. En este estudio se realizó una descripción y análisis de los principales factores que influyen en la biosíntesis de compuestos fenólicos de bayas de Vitis vinifera. Para ello, se realizó una recopilación y clasificación de diferentes estudios, para luego realizar un análisis, discusión y conclusión a partir de la información disponible. Los factores analizados en este estudio fueron la luz, temperatura, manejos agronómicos (orientación de las hileras, deshojes, vigor de la planta y raleo de racimos), etileno, ácido abscísico, déficit hídrico, fertilidad, altitud y cultivar. Los resultados demostraron que los diferentes factores estudiados afectan la composición fenólica de las bayas. En algunos casos, determinados factores generan efectos diferenciales sobre taninos de la piel, taninos de semillas y antocianos. Es importante señalar que los resultados de este estudio, permitieron analizar los principales factores individualmente. Sin embargo, investigaciones posteriores deberían profundizar en el estudio de efectos sinérgicos de los distintos factores, que probablemente afectarán diferencialmente la composición fenólica al compararse con los efectos individuales.
Phenolic compounds are responsible for sensory characteristics of the berries and wine such as color, astringency and bitterness. In addition, determine the aging potential of wines. These compounds are synthesized through the phenylpropanoid path and training would be determined by many factors. Despite the wealth of information that evaluates the various factors that affect the biosynthesis of phenolic compounds, it is sometimes contradictory. In this study was performed a description and analysis of key factors influencing the biosynthesis of phenolic compounds in berries of Vitis vinifera. This was achieved by a collection and classification of different studies, then analysis, discussion and conclusion from the information available. The factors analyzed in this study were light, temperature, agricultural management (row orientation, leaf plucking, plant vigor and cluster thinning), ethylene, abscisic acid, water deficit, fertility, altitude and cultivate. The results showed that the different factors affecting the phenolic composition of berries. In some cases, certain factors generate differential effects on the skin tannins, tannins and anthocyanins seed. Important to note that the results of this study, helped analyze the main factors individually. However, further research should further develop the study of synergistic effects of various factors likely to affect differentially the phenolic composition when compared to individual effects.

Thesis (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole.
AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm wanneer lig 'n versadigingspunt bereik vir fotosintese. Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel. Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer. Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1- deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s. die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad (d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van karotenoiëde ). Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase (IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED twee en drie kopieë, repektiewelik, beslaan. Die transkipsionele aktiwiteit van die verwagte promotors van ses van die geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die promotors van hierdie gene kan dus as transkipsioneel aktief beskou word. Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in 'n bakteriese funksionele kleurkomplementasiesisteem. In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies getoon. Gekonserveerde domeine is ook in die proteïensekwense van die geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante). Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY, BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken, terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die lokalisering van die biosintetiese paaie in die selooreen. Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant, Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense" orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig. Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01 proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei. Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om streskondisies te weerstaan sal die plant se algemene gesondheid en dus produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.

A Lesão Renal Aguda (LRA) é a complicação mais grave da rabdomiólise. Nessa síndrome, a liberação do pigmento heme desencadeia uma lesão que se caracteriza por vasoconstrição glomerular e toxicidade celular direta com componente oxidativo. A lesão oxidativa desencadeada é uma das linhas fisiopatológicas mais intrigantes. A renoproteção com antioxidantes tem demonstrado efeito satisfatório. As proantocianidinas são antioxidantes naturais encontrados no extrato da semente da uva. O objetivo deste estudo foi avaliar o efeito antioxidante da Vitis vinifera (extrato da semente de uva) sobre a função renal de ratos submetidos à lesão por rabdomiólise. Foram utilizados ratos Wistar, adultos machos, pesando entre 250-300 gramas. A LRA foi induzida pela administração de glicerol 50% i.m (intramuscular). Os animais foram distribuídos em 4 grupos: grupo Salina (6ml/Kg de NaCl 0,9% via intraperitoneal (i.p) 1 vez ao dia), grupo Glicerol (6ml/Kg de glicerol i.m, cada região femoral recebeu 3ml/Kg de glicerol, 1 vez ao dia), grupo Vitis vinifera (3mg/Kg v.o por 5 dias) e grupo Glicerol+Vitis vinifera que recebeu Vitis vinifera por 5 dias antes do glicerol. Foram avaliados o marcador de lesão muscular (CK), a função renal (FR), a função tubular (FENa e FEK), o perfil oxidativo (peróxidos urinários-FOX-2 e MDA-TBARS) , a histologia e morfometria renal. O grupo Glicerol tratado com Vitis vinifera apresentou melhora da FR e tubular, redução dos níveis da peroxidação lipídica e melhora da histologia renal. Os resultados deste estudo confirmaram a proteção antioxidante, com repercussão histológica, da Vitis vinifera na LRA induzida por glicerol.
The Acute Kidney Injury (AKI) is the worst complication of rhabdomyolysis. In this syndrome, the delivery of heme pigment induces an injury that distinguishes itself by glomerular vasoconstriction and direct cellular toxicity with oxidative component. The oxidative injury is an intriguing one of the pathophysiological mechanism. The renoprotection with antioxidants has demonstrated satisfactory effect. The proanthocyanidins are natural antioxidants found in grape seed extract. The aim of this study was to evaluate the antioxidant effect of Vitis vinifera (grape seed extract) on the renal function of rats submitted to the injury by rhabdomyolysis. Wistar rats, male, adults, weight ranging from 250-300 g were used. The AKI was induced intramuscular (i.m.) administration of glycerol 50%. The animals were distributed in 4 groups: Saline group (6ml/Kg of NaCl 0,9% intraperitoneal (i.p) once a day), Glycerol group (6ml/Kg of glycerol i.m, each femoral region received 3ml/Kg of glycerol, once a day), Vitis vinifera group (3mg/Kg v.o by 5 days) and Glycerol+Vitis vinifera group that has received Vitis vinifera by 5 days before glycerol. Marker of muscular injury (CK), renal function (RF), the tubular function (FENa and FEK), the oxidative profile (urinary peroxides -FOX-2 and MDA-TBARS), the histological and kidney morphometric were evaluated. The Glycerol group treated with Vitis vinifera has shown improvements in RF and tubular, reduction of levels of lipid peroxidation and amelioration of kidney histology. The results of this study have confirmed the antioxidant protection, with histological repercussion of Vitis vinifera in AKI induced by glycerol.

Water is a scarce resource worldwide and a particular problem for producers of wine grapes in Australia and Chile where periodic drought severely limits vine growth. Most vineyards in these countries are irrigated and the development of efficient water management practices for vineyards is required. Strategies such as regulated deficit irrigation (RDI) and partial root zone drying (PRD) have been introduced with the objective of maximising the efficiency of water use relative to yield while ensuring that grape quality is not compromised. The problem is that the advantages of these strategies can only be realised if there is accurate information about weather parameters, spatial distribution of soil moisture and vine water use and status. This information can then be used to provide answers for growers to the key irrigation questions of when to irrigate and how much water to apply. The overarching hypothesis of this thesis was than an integrated approach to irrigation scheduling incorporating data on weather, physiological status of the vines and soil wetting patterns is essential to accurately target the narrow range of soil moisture and vine stress thresholds required for realising the advantages of RDI and PRD. The hypothesis was tested through a series of field experiments. The results supported that an integrated approach to irrigation produces the most desirable outcomes in terms of water use and berry quality irrespective of irrigation strategies. The most appropriate way to answer the question of when to irrigate is to measure stem rather than leaf water potential. The question of how much water to apply can readily be answered using a new technique and software developed in this study. It was concluded that PRD is more difficult to manage than RDI because of the complexity pf PRD and the financial and logistic requirements of a double irrigation line. PRD had no effect on crop water use efficiency, berry quality or yield when the same amount of water was applied in the PRD and conventional drip treatments.

Thesis (Ph.D.) -- University of Western Sydney, 2005.
"Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy, Centre for Plant and Food Sciences, University of Western Sydney, Australia, November 2005." Includes bibliographical references and appendices.

Seeds of 41 white and 22 blue vine varieties cultivated on six vine-growing areas were assessed as a by-product after winemaking on the contents of total polyphenols (TP) and phosphorus (P) spectrophotometrically, total tocols (TC) including individual tocols by HPLC-FLD, and metals (Ca, Mg, Na, K, Fe, Zn, Cu, and Mn) by FAAS. Remaining TP and TC levels were mainly affected by the variety, while levels of microelements (Cu, Mn, and Zn) and P or K by the vine-growing area. The highest TC and TP levels were found in the seeds of white varieties. Varieties of grape seeds have significant impact on the gamma-tocotrienol content. The color of grape varieties has significant impact on alpha-tocotrienol content. Grape seeds from grape cultivated on the Czech growing area contained higher levels of macroelements except P, however no significant differences between growing areas have been found. The crop year has significant influence on TP, Fe and Cu content. Results herein revealed the considerable potential of grape seeds, a by-product of the vinification process, as a valuable inexpensive source of high added value of nutritionally beneficial compounds - polyphenol and tocol antioxidants and macro- and microelements for use as feed additives in animal nutrition.

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Crimson Seedless Grapes (Vitis vinifera L.) is one of the most important seedless variety, due to its attractive bunch medium, and large dark pink berries. It features excellent sensory characteristics due to its firm texture and crisp flavor that varies from sweet to neutral and uniform color. Stands out as the second most important seedless variety of the São Francisco. Osmotic dehydration is presented as a good alternative to reduce the water activity of this grape, allowing its storage for long periods and improving its stability and quality. This study aimed to relate the influences of different parameters for efficient osmotic dehydration of fruit, in order to reduce post-harvest losses as well as offer new alternatives for the producer. To optimize the osmotic dehydration preliminary test was conducted with independent variables: temperature (T) (20ºC to 40ºC), immersion time (t) (0.5 to 4.0 hours, with breaks every 30 minutes) and concentration of osmotic solution (C) (35ºBrix, 50ºBrix and 60ºBrix) was the dependent variable moisture loss (ML). Then 26-2 fractional design was used, having as independent variables: T (30ºC and 50ºC), t (1.0 and 4.0 hours), NaOH (0% and 2%), bleach (0 and 1 minute), perforated in the fruit (0 and 16 holes/cm2) and C (30ºBrix and 60ºBrix) and the dependent variables and solids ML and incorporation of solid (IS). Proceeding was conducted a factorial design 23, with independent variables: T (30ºC to 50ºC), t (1 to 4 hours) and C (40ºBrix to 50ºBrix), being constant bleaching (30 seconds) and perforated (8 holes/cm2); the dependent variables were ML, IS and DEI (Dehydration Efficiency Index). The best conditions for osmotic dehydration using DEI as a parameter was the application of bleach for 30 seconds, 8 holes/cm2, osmotic solution at 42ºBrix, immersion time of 1.6 hours and temperature of 46 ° C. The response surface models obtained were predictive of ML and IS, except for DEI. The product selected best set Page's equation (R2 = 0.995).
A uva Crimson Seedless (Vitis vinifera L.) é uma das mais importantes variedades sem sementes, devido ao seu atraente cacho médio, e grande bagas rosadas escuras. Apresenta característica sensorial excelente devido a sua textura firme e crocante, sabor que varia do doce ao neutro, e coloração uniforme. Destaca-se como a segunda variedade sem semente mais importante do Vale do São Francisco. À desidratação osmótica apresenta-se como boa alternativa para reduzir a atividade de água desta uva, permitindo o seu armazenamento por períodos longos e melhorando a sua estabilidade e qualidade. Esta pesquisa teve como objetivo relacionar as influências de diferentes parâmetros para um eficiente processo de desidratação osmótica deste fruto, com a finalidade de reduzir as perdas pós-colheita como também oferecer novas alternativas para o produtor. Para otimizar a desidratação osmótica foi realizado ensaio preliminar com as variáveis independentes: temperatura (T) (20ºC a 40ºC), tempo de imersão (t) (0,5 a 4,0 horas, com intervalos a cada 30 minutos) e concentração da solução osmótica (C) (35ºBrix, 50ºBrix e 60ºBrix); sendo a variável dependente perda de umidade (PU). Em seguida foi aplicado planejamento fracionado 26-2, tendo como variáveis independentes: T (30ºC e 50ºC), t (1,0 e 4,0 horas), NaOH (0% e 2%), branqueamento (0 e 1 minuto), perfurações no fruto (0 e 16 perfurações/cm2) e C (30ºBrix e 60ºBrix); e as variáveis dependentes PU e incorporação de sólidos (IS). Prosseguindo foi realizado um planejamento fatorial 23, com variáveis independentes: T (30ºC a 50ºC), t (1 a 4 horas) e C (40ºBrix a 50ºBrix), sendo constante o branqueamento (30 segundos) e perfurações (8 perfurações/cm2); as variáveis dependentes foram PU, IS e IED (Índice de Eficiência de Desidratação). As melhores condições para a desidratação osmótica utilizando o IED como parâmetro foi a aplicação de branqueamento por 30 segundos, 8 perfurações/cm2, solução osmótica com 42ºBrix, tempo de imersão de 1,6 horas e temperatura de 46ºC. Os modelos de superfície de resposta obtidos foram preditivos para PU e IS, exceto para o IED. O produto selecionado ajustou melhor a equação de Page (R2 = 0,995).

Il recupero e valorizzazione delle risorse genetiche tipiche di una specifica area è importante non solo per salvaguardare la biodiversità vegetale, ma anche per promuovere l’immagine territoriale e la diversificazione dell’offerta dei prodotti alimentari. La Puglia è un’antica regione viticola, con un ricco patrimonio di varietà di vite. L'area della Daunia, in provincia di Foggia (Nord della Puglia), è la principale area viticola pugliese in termini di superficie e di produzione. Un totale di 35 genotipi di vite reperiti in tre diverse aree delle provincia dauna sono stati caratterizzati utilizzando quattordici marcatori microsatelliti (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, VVMD32, VVMD6, VVMD17, VVMD21, VVMD24, VMC1b11) per valutare la loro diversità genetica e analizzando le principali caratteristiche qualitative delle uve dal punto di vista tecnologico e fenolico, al fine di valutare il potenziale enologico di questi genotipi. Dalle analisi genetiche, sono stati trovati 30 diversi profili genetici e 3 sovrapposizioni. Confrontando i 30 profili genetici con quelli inclusi nei database internazionali e con quelli individuati da altre Istituzioni scientifiche, sono stati identificati 23 genotipi. La maggior parte di loro (87%) corrispondono a cultivar iscritte al Catalogo Nazionale delle Varietà di Vite (RNVV); i restanti genotipi (13%) non sono iscritti nel RNVV. Il profilo genetico degli altri 7 genotipi non è stato trovato in nessun database; pertanto, ciascuna di queste accessioni può essere considerata “genotipo unico”. Per quanto riguarda il potenziale enologico di queste accessioni, la maggior parte di esse ha mostrato caratteristiche qualitative interessanti. In particolare, tra i genotipi considerati “unici”, quattro accessioni, due a bacca bianca e due a bacca nera, hanno mostrato una buona attitudine per la produzione di vini monovarietali dotati di un buon grado alcolico, una buona stabilità, struttura, colore e sapore, ma, anche, per la produzione di uvaggi con altri vitigni. In conclusione, questo studio ha evidenziato la ricchezza di vecchi genotipi di vite coltivate nella provincia di Foggia e le capacità enologiche dell'uva prodotta da questi genotipi, analizzando gli aspetti tecnologici e le caratteristiche fenoliche utili per sostenere la realizzazione di vini monovarietali o di vini ottenuti dalla miscelazione di diverse varietà locali.
The recovery and valorization of genetic resources typical of a specific growing area is fundamental to preserve the specie genetic pool, and presently it is thought as a strategy to promote the territorial identity and the diversification of the local food products. Apulia is an ancient grapevine-growing region, having a rich heritage of grapevine varieties. The Daunia area, in the Foggia province (Northern Apulia), is the main Apulian viticultural area in terms of surface and production. A total of 35 grapevine genotypes found in three different areas of the province dauna were characterized using fourteen microsatellite markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, VVMD32, VVMD6, VVMD17, VVMD21, VVMD24, VMC1b11) to evaluate genetic diversity and assessing the main qualitative characteristics of their grapes from a technological and phenolic point of view, in order to evaluate the potential interest of these genotypes for the oenological use. According to their genetic profiles at SSR loci, 30 different genetic profiles and 3 overlays were found. Comparing the 30 genetic profiles with those included in international databases or with those detected by other scientific Institutions, 23 genotypes have been identified. Most of them (87%) were found to match cultivars enrolled in National Catalogue of Grapevine Varieties (RNVV); the remaining genotypes (13%) are not enrolled in RNVV. The genetic profile of the other 7 genotypes was not found in any database; thus, by now, each of these accessions can be considered as being a “unique genotype”. As concerns the oenological potential of the accessions, all of them showed interesting traits. In particular, among the genotypes considered “unique”, four accessions, two white-berry accession and two black berry-accessions, showed a good attitude for the production of mono-varietal wines with a good level of alcohol, stability, structure, color and flavor, but, also for the production of blended wines. In conclusion, this study has highlighted the richness of old grapevine genotypes grown in the Foggia province and the oenological skills of the grape produced by these genotypes, analyzing the technological and the phenolic traits that may be useful to support the making of mono-varietal wines or that of wines obtained by blending more local varieties

Plants contain a heterogeneous and complex population of small RNAs (sRNAs) with regulatory function at the transcriptional and post-transcriptional level. sRNAs are involved in regulation of endogenous genes, in defence response and in maintaining genome integrity. Their biosynthetic pathways require different combinations of RNA silencing proteins such as DICER-LIKE protein (DCL), ARGONAUTE (AGO) as well as other components such as RNA dependent RNA polymerase (RDR). The aim of this Ph.D. research was to study the role of the RNA silencing proteins in the generation of sRNAs classes and to understand their biological function in plant development and viral resistance in grape (Vitis vinifera). A total of 4 DCLs, 6 RDRs and 10 AGO proteins were identified in grape. In order to identify sRNAs classes according to their specific requirement, hairpin-RNA interference (hpRNAi) technology was applied to silence VvDCL4 and VvRDR6. These two genes cooperate in the generation pathway of the trans-acting short interfering RNAs (tasiRNAs) and in viral defence. Transgenic plants carrying hpRNAi constructs were affected in growth and development and showed alteration in leaf development compare to control plants. Deep sequencing of small RNAs of the most highly silenced plants revealed the reduction in the accumulation of specific tasiRNAs involved in leaf development Furthermore, RNA silencing mechanism in grape upon viral infection were better characterized. Small RNAs deriving from plants infected with Grape leaf roll virus (GRLaV) and Grape fleck virus (GFKV) were sequenced using the Illumina next generation sequencing technology and their expression profile studied. Our results suggest an inter-complementation of DCLs in grape upon infection with GfKV and possibly the involvement of only VvDCL4 upon infection with GLRaV1. Our work provides a better understanding of the role of the RNA silencing machinery in grape during development and in response to viral infection. In the future, our results will help to develop novel cultivars resistant to GLRaV1 to prevent heavy losses in grape cultures worldwide.

Plasmopara viticola (Berk. et Curt.) Berl. and De Toni is an obligate biotrophic oomycete which causes grapevine downy mildew, one of the most important grapevine disease, affecting particularly the European grapevine Vitis vinifera L. The main objective of the study is to find possible sources of resistance to P. viticola in the less common V. vinifera germplasm (Caucasian origin) through the screening of an ample collection of cultivated and wild Caucasian accessions, grown in greenhouse and in open field. This could really simplify the genetic improvement programs. The level of susceptibility towards P. viticola has been assessed by experimental inoculations on 148 cultivated Caucasian (mainly Georgian) and Iranian accessions (V. vinifera var. vinifera) and 35 wild Georgian accessions (V. vinifera var. sylvestris) grown in pots in greenhouse. Moreover, natural downy mildew occurrence has been evaluated on 94 Georgian accessions cultivated in vineyard. These activities have been carried out for three consecutive years. Several accessions were characterized by an interesting resistance level, and in particular, the Georgian cv Mgaloblishvili N. was characterized by the most stable behavior. For this reason, further investigations, aiming at more deeply investigating the plant-pathogen interaction at the genetic and histological levels, were carried out on this cultivar. In order to get insights on the resistance mechanism at the genetic level, 272 Mgaloblishvili progenies were obtained by self pollination, open pollination and pollinated with ‘Pinot noir’ N and experimental inoculations have been carried out over a three-year period. The characterization of the interaction between P. viticola and Mgaloblishvili by histological analysis at different time points was assessed, in order to evaluate the type and the timing of the plant response and the development of the pathogen. To thoroughly analyze the effective level of resistance in Mgaloblishvili, experimental inoculation with different P. viticola strains characterized by different aggressiveness levels were carried out. Finally, the genetic variability of P. viticola in Italy was investigated to get insights on the possible durability of resistance in open field. The genetic diversity of P. viticola populations, collected from 13 different geographic Italian regions, has been investigated during one-month period at INRA, Institute de la vigne et du vin, Villeneuve d’Ornon (Bordeaux) in France to evaluate if the genetic differences could modulate the aggressiveness level of the pathogen.

AFE presentada como parte de los requisitos para optar al Título Profesional de Ingeniero Agrónomo y al Grado de Magíster en Ciencias Agropecuarias
El vino es una solución hidroalcohólica ácida que contiene diversos compuestos, tales como, azúcares, ácidos orgánicos, polifenoles, compuestos aromáticos, minerales y polisacáridos, los cuales provienen del fruto, el proceso de vinificación y de la guarda o crianza (Moreno y Peinado, 2012a). Los compuestos fenólicos y polisacarídicos presentes en el fruto juegan un rol muy importante en las características del vino (Guadalupe et al., 2007). Durante la vinificación estos se transforman e interactúan, otorgando diversas características de estructura, color y propiedades sensoriales del vino, las cuales resultan benéficas para el producto final (Ojeda, 2007).

A obtenção de novas cultivares de uvas de mesa e a compreensão da regulação da expressão gênica, associada à formação do fruto de uma cultivar de uva sem semente, constituem pré-requisitos essenciais para o desenvolvimento de ferramentas aplicadas ao melhoramento genético para essas espécies. Com este objetivo, dois trabalhos foram conduzidos a partir do estudo de transcritos derivados de frutos da cultivar (cv) Sultanina de videira. No primeiro trabalho, três bibliotecas subtraídas foram construídas a partir de RNA total de frutos em diferentes estádios de formação, utilizando-se uma metodologia modificada da análise representacional diferencial (RDA), denominada análise representacional de transcritos em grupos (BRAT). Um total de 2.500 fragmentos derivados de transcritos (TDFs) foram identificados e clonados a partir de estádios específicos do desenvolvimento do fruto. Após sequenciamento e análise in silico, 1.554 (62,16%) dos transcritos foram validados de acordo com a qualidade da sequência. A montagem das sequências derivadas de genes expressos (ESTs) resultou em 726 singletos e 69 clusters, com aproximadamente 76% de redundância. Entre os TDFs identificados, onze genes candidatos e dois genes-referência foram selecionados e submetidos a uma análise aprofundada dos seus perfis de expressão temporal por RT-qPCR. A expressão de sete genes foi concordante com os estádios específicos do desenvolvimento dos frutos. Entre os candidatos selecionados os genes a seguir listados são possivelmente os mais promissores quando se considera o desenvolvimento do fruto da cv. Sultanina: (i) VvUBP1 (proteína de ligação a oligouridilatos), (ii) VvRIP1 (proteínas induzidas pelo amadurecimento), (iii) VvP450 (citocromo P450), (iv) VvDOF1 (proteína do tipo Dof); (v) VvERF1 (fator de transcrição responsivo ao etileno), e (vi) VvGID1L1 (receptor de ácido giberélico). Num segundo momento, foi realizado um estudo mais detalhado dos genes Dof de videira. Tais genes correspondem a um grupo de fatores de transcrição específicos de plantas, os quais estão envolvidos na regulação de diferentes funções, tais como, resposta ao estresse, hormônios e luz, sinalização de fitocromo, germinação de sementes e expressão gênica tecido-específica. A família de genes Dof de videira compreende 26 membros, sendo que as sequências de aminoácidos de todos os domínios Dof alinham perfeitamente, incluindo resíduos de cisteína que são críticos para a ligação de zinco e outros resíduos conservados em fatores de transcrição Dof de A. thaliana, O. sativa e outras plantas. Baseado na análise de domínios Dof, sugere-se que estes domínios sejam possivelmente funcionais em videira. A localização física dos genes Dof nos cromossomos de videira foi realizada. Além disso, foi conduzida uma análise filogenética comparando o grupo de genes Dof em videira com os seus homólogos em outras duas eudicotiledôneas completamente sequenciadas, A. thaliana e Populus, permitindo identificar claramente clusters de genes parálogos e ortólogos. Por fim, os perfis de expressão de todos os 26 genes Dof foram estudados por RT-qPCR em nove órgãos de videira.
Table grapes production and kwnolegment about the gene expression regulation associated with the formation of seedless grape cultivar, constitute essential prerequisites for the development of tools applied to genetic improvement of this plant species. To this end, two studies were conducted using the analysis of transcripts derived from fruits of the Sultanina cultivar (cv.) grapevine. In the first study, three subtracted libraries were constructed from total RNA from fruit different developmental stages using a modified methodology of Representational Difference Analysis (RDA), named Bulk Representational Analysis of Transcripts (BRAT). A total of 2,500 transcripts-derived fragments (TDFs) were identified and cloned from specific stages of fruit development. After sequencing and analysis in silico, 1,554 (62.16%) transcripts were validated in accordance with the sequence quality. The assembly of sequences derived from expressed genes (ESTs) resulted in 726 singletons and 69 clusters, with overall EST redundancy of approximately 76%. Among the TDFs identified eleven candidate genes and two reference genes were selected and subjected to a thorough analysis of their temporal expression profiles by RT-qPCR. Among them, seven genes proved to be in agreement with the stage-specific expression. Among candidates selected from those differentially expressed, the genes listed below were considered the most promising when considering the fruit development in grapevine cv. Sultanina: (i) VvUBP1(oligouridylate binding protein), (ii)VvRIP1 (ripening induced protein), (iii) VvP450 (cytochrome P450), (iv) VvDOF1 (Dof-like protein), (v) VvERF1 (ethylene-responsive transcription factor), and (vi) VvGID1L1 (gibberellins receptor). The second study detailed the grapevine Dof gene family. Dof proteins are involved in the regulation of different functions such as plant response to stress, hormones and light phytochrome signaling, seed germination and tissue-specific gene expression. The Dof gene grapevine family includes 26 members. The amino acid sequences deduced from Dof domains matched perfectly including cysteine residues critical for zinc binding and other residues known to be conserved in Dof transcription factors from A. thaliana, O. sativa and other plants. Based on analysis of Dof domains, it is suggested that all grapevine Dof domains are possibly functional. The physical location of Dof genes in grapevine chromosome was determined. A phylogenetic study comparing grapevine Dof genes with their counterparts in two other eudicots completely sequenced, A. thaliana and Populus, allowed us to identify clear clusters of paralogous and orthologs genes. Finally, the expression profiles of all 26 Dof genes was studied by RT-qPCR in nine vegetative and reproductive grapevine organs.

Les stilbènes sont les métabolites de défense majeurs de la vigne, qui sont également connus pour leurs nombreuses propriétés pharmacologiques. Tirant parti du récent séquençage du génome de la vigne, l'objectif de ce travail est de caractériser les familles de gènes impliqués dans la biosynthèse des stilbènes chez la vigne, et de préciser leur rôle dans les défenses contre le mildiou (Plasmopara viticola). La première étape de la biosynthèse des stilbènes est catalysée par la stilbène synthase (STS), pour former le resvératrol. L'analyse détaillée du génome de la vigne a permis d'identifier 48 gènes STS, dont 32 gènes potentiellement fonctionnels. La caractérisation fonctionnelle d'une sélection de gènes représentatifs de la diversité de la famille suggère que l'ensemble des 32 gènes STS code pour des protéines ayant réellement une activité stilbène synthase. L'analyse évolutive des gènes STS montre que la famille est très contrainte, sans trace de néo-fonctionnalisation. La famille des STS de la vigne représente donc un exemple unique d'une famille de plus de 30 gènes codant pour des protéines de fonction identique, et la signification biologique de cette expansion est discutée. Une seconde enzyme importante du métabolisme des stilbènes est la resvératrol O-méthyltransférase (ROMT). La ROMT est impliquée dans la méthylation du resvératrol pour former le ptérostilbène, un composé hautement fongitoxique qui pourrait jouer un rôle important dans les mécanismes de défenses de la vigne. Notre analyse de la famille ROMT montre qu'elle est constituée de 17 gènes, dont deux seulement (VvROMT1 et VvROMT2) semblent impliqués dans la synthèse de ptérostilbène. L'expression de ces deux gènes est induite suite à une infection par P. viticola au niveau des feuilles de vigne. Deux autres gènes de la famille, VvROMT12 et VvROMT13, sont exprimés constitutivement au niveau des racines, et ne semblent pas répondre au stress. Des analyses métabolomiques sur des plants de Nicotiana benthamiana transgéniques exprimant ces deux ROMT ainsi que des tests enzymatiques in vitro ont été réalisés afin de déterminer la fonction des gènes ROMT12 et 13. L'ensemble de ces résultats fait apparaître une amplification remarquable des gènes impliqués dans la synthèse des stilbènes chez la vigne et ouvrent la voie à l'étude détaillée de la régulation de cette voie importante du métabolisme de défense de la vigne.

Thesis (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Plant-pathogen interactions have been intensively investigated in the last decade. This major drive towards understanding the fundamental aspects involved in plant disease resistance is propelled by the obvious agricultural and economical benefits that are intrinsically linked to disease and stress resistant plants. It is, therefore, not surprising that fundamental research in this area is not just restricted to model organisms, such as Arabidopsis and tobacco, but also extends to more traditional crop plants, such as maize, bean, soybean, apples, grapevine etc. In grapevine for instance, several genes involved in disease resistance have been isolated. One of these genes, encoding for a polygalacturonase inhibiting protein (PGIP), has been studied extensively. PGIPs are cell wall bound, contain leucine rich repeats (LRR) and are found in all dicotyledonous plants so far examined. In most cases, pgip genes occur in small multigene families and expression is often tissue specific and developmentally regulated. Up-regulation of PGIP-encoding genes typically occurs upon pathogen infection, treatment with elicitors, salicylic acid (SA), jasmonic acid (JA), cold treatment and wounding. Differential regulation and specificity have been shown to occur between members of the same multigene family. Differential regulation even extends to the utilization of separate pathways to induce pgip genes from the same family in response to a single stress stimulus. PGIPs interact with cell wall macerating polygalacturonases (PGs) that are secreted by pathogenic fungi during the infection process. The antifungal action of PGIPs is thought to depend on a dual action. The physical interaction of PGIP with PGs has an inhibitionary effect, resulting in (i) a slower fungal infection rate and (ii) the prolonged existence of long chain oligogalacturonides (OGs). These oligosaccharides are able to elicit a general plant defense response, enabling the plant to further retard or curb the spread of infection. The main objective of this study was to investigate the regulatory aspects underlying PGIP expression in grapevine. Unlike most characterized PGIP encoding genes from other dicotyledonous plant species, no evidence to support the existence of a V. vinifera PGIP multigene family could be found from either genetic or biochemical analyses. Recently, a genomic DNA fragment from Vitis vinifera cv Pinotage was pathogen interactions with regards to the fundamental processes underlying defense gene regulation.
AFRIKAANSE OPSOMMING: Die ooglopende voordele wat, vanuit 'n landboukundige én ekonomiese oogpunt, uit siekte- en stresbestande plante spruit, het gedurende die laaste dekade aanleiding gegee tot die ontwikkeling van plantpatogeen-interaksies as "n baie belangrike studieveld. Dit was dus ook te verwagte dat fundamentele navorsing in hierdie area nie net beperk gebly het tot modelorganismes soos Arabidopsis en tabak (ook natuurlik van landboukundige belang) nie, maar ook na meer tradisionele landbougewasse soos mielies, boontjies, sojaboontjies, appels, druiwe, ens. oorgevloei het. Verskeie siekteweerstands-verwante gene is byvoorbeeld al vanuit wingerd geïsoleer. Een só "n geen wat vir "n poligalakturonase-inhiberende proteïen (PGIP) kodeer, vorm deel van hierdie groep gene. Die funksie en regulering van PGIP's is baie goed bestudeer. Hierdie proteïene word normaalweg in die selwande van die meeste dikotiele plante aangetref. Leusienryke herhalings is algemeen in PGIP's en hierdie tipe van herhalings is kenmerkend van proteïene betrokke by proteïen-proteïen-interaksies. Verder word pgip-gene gewoonlik in klein multigeenfamilies aangetref, waar in die meeste gevalle die uitdrukking weefselspesifiek en die regulering spesifiek ten opsigte van die ontwikkelingsfase is. Verskeie faktore kan tot die induksie van pgip-gene lei, soos onder andere patogeen-infeksie, elisitoor-, salisiensuur-, jasmoonsuur- en kouebehandeling, asook verwonding. Differensiële regulering word in baie gevalle tussen lede van dieselfde multigeenfamilie aangetref. Hierdie differensiële regulering kan selfs bemiddel word deur onafhanklike reguleringsweë in reaksie op dieselfde induksiestimulus. PGIP's is in staat om te reageer met poligalakturonases (PGs), wat selwande afbreek en wat gedurende die infeksieproses deur swamme of fungi afgeskei word. Die effek van hierdie interaksie is tweeledig: (i) Die fisiese interaksie tussen PGIP en PG moduleer die aktiwiteit van die PG deur die ensiemaksie te inhibeer, en (ii) PGinhibisie lei tot die verhoogde stabiliteit van langketting-oligogalakturonades, molekules wat daartoe in staat is om die weerstandsrespons van plante te ontlok. Die inhibisie van die patogeen-PG's, tesame met die geïnduseerde weerstandrespons, stel die plant dan in staat om verdere infeksie te vertraag of te verhoed. Die doel van hierdie studie was om die onderliggende aspekte van PGIPregulering in wingerd te bestudeer. In teenstelling met die meeste plantspesies waar pgip-gene in klein multigeenfamilies aangetref word, is daar nie 'n pgip-multigeenfamilie in wingerd nie. Veelvuldige kopieë van In enkele pgip-geen word egter in die wingerdgenoom aangetref. Daar is onlangs in ons laboratorium In genoom-DNAfragment vanaf Vitis vinifera cv Pinotage geïsoleer wat die oopleesraam en 5'-stroomopsekwense van In PGIP-enkoderende geen (Vvpgip1) bevat. In hierdie studie is die uitdrukkingspatroon van Vvpgip1 ten opsigte van weefselspesifisiteit, korrelontwikkelingsfase, asook die effek van verskeie omgewings en patogeenverwante stres-stimuli ontleed. Die regulatoriese meganismes van Vvpgip1 bevat spesifieke in planta-ontwikkelingsfaseseine wat verder deur spesifieke faktore, insluitende omgewings- en patogeenstres, gereguleer word. In lyn hiermee is mRNS-transkripte van Vvpgip1 tot wortel- en korrelweefsels beperk, terwyl die mRNS-vlakke ook tussen verskillende korrelontwikkelingsfases wissel. Kumulatiewe uitdrukking kon waargeneem word in veráison-korrels in reaksie op verwonding en osmotiese stres. Die weefselspesifieke uitdrukkingspatroon tipies van wingerd-PGIP is in blare opgehef in reaksie op Botrytis cinerea-infeksie, verwonding, osmotiese stres, ouksien (indoolasynsuur) en salisiensuur. PGIP-uitdrukking word ook onderdruk deur In staurosporien-sensitiewe proteïenkinase, wat In goeie aanduiding is van die betrokkenheid van proteïenfosforilasie in die seintransduksiekaskade wat tot PGIPuitdrukking aanleiding gee. Die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare kan ook nageboots word in tabak wat met die Vvpgip1-geen en -promotor getransformeer is. PG-inhibisie-eksperimente met membraan-geassosieerde proteïenekstrakte van geïnduseerde wingerdblare het ook dieselfde profiel getoon as dié van PGIP wat deur die Vvpgip1-geen geënkodeer is. Die uitdrukkingsprofiel van PGIP in die transgeniese tabakplante het ook bewys dat die promotor van die Vvpgip1-geen vir die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare verantwoordelik is. In silica-analise van die promotorarea dui op die teenwoordigheid van verskeie cis-werkende elemente. Die kern promotor en transkripsie-aanvangsgedeelte is gevolglik eksperimenteel bepaal. Verder het uitdrukkingseksperimente met promotorfragmente verskeie dele van die promotor geïdentifiseer wat by stimulis-geassosieerde uitdrukking betrokke is. Posisioneel is hierdie fragmente in goeie konteks met die voorspelde cis-werkende elemente en kan dus die basis vorm vir verdere studies oor Vvpgip-regulering. Met hierdie studie word die eerste data verskaf waar die regulering van PGIP deur omgewingsverwante faktore verbind kan word met onwikkelingspesifieke toestande in die plant. Verder verskaf die resultate verdere bewyse vir die rol van PGIP in plant-patogeen-interaksies en lewer spesifieke bydraes tot die onderliggende prosesse wat by die regulering van siekteweerstandverwante gene betrokke is.

Effectiveness of a capture and return system for the partial retention of fermentation volatiles, as a means of improving white wine quality, was evaluated. Twenty-three aroma-active volatiles including ethyl esters, acetate esters, fusel alcohols, and fatty acids, were quantified using head-space solid phase microextraction with GC/MS. Volatile analysis of fermentations maintained at 15ºC demonstrated a trend of increased concentrations of ethanol, esters and ethyl esters of fatty acids and decreased concentrations of fusel alcohol acetates, fatty acids and higher alcohols in treatment wines. When fermentation temperature was maintained at 15ºC there was increased concentration and retention of fusel alcohols, fatty acids and higher alcohols compared to 15ºC. Sensory analysis of wines fermented at 15°C, using triangle difference testing, indicated variable differences in aroma among treatments.
Master of Science

L’ANS recombinante de Vitis vinifera (VvANS) a été exprimée chez E. coli, et purifiée par chromatographie d’affinité sur colonne Nickel. La production et purification de l'holoenzyme chargée en fer a été mise en point, afin d’éviter les réactions d'oxydation non-enzymatique incontrôlée en présence de sel de Fe(II). Un complexe VvANS-Fe(II) stable est formé en présence d'α- cétoglutarate et d'ascorbate, complexe qui est catalytiquement actif à PO2 ambiante en l'absence de sel de Fe(II). La transformation de la (+)-catéchine par VvANS a été étudiée avec ou sans ascorbate, en utilisant le complexe VvANSFe( II) ou l’holoenzyme co-incubée en présence de sulfate ferreux, afin d’étudier le rôle de l’ascorbate. Aucune activité enzymatique n’a été observée en l'absence d’ascorbate, ce qui indique qu'il s'agit d'un cofacteur indispensable de VvANS. Un adduit covalent ascorbate-cyanidine est produit in vitro, mais seulement en l'absence d'un autre réducteur nucléophile majeur, le glutathion GSH. Les deux stéréoisomères de la leucocyanidine (flavan-diols 3,4-cis et 3,4-trans), substrats potentiels de VvANS, mais non commerciaux, ont été synthétisés par réduction de la dihydroquercétine par NaBH4, puis identifiés par RMN du proton. L'analyse des deux stéréoisomères par spectrométrie de masse en tandem (MS/MS) montre que leurs voies de fragmentation MS/MS sont distinctes et peuvent être utilisées pour les distinguer lors de leur production. Les deux stéréoisomères sont stables en milieu aqueux congelé à -20°C. Douze flavonoïdes de quatre familles distinctes (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) ont été testés comme substrats potentiels. Tous les produits enzymatiques ont été purifiés par HPLC en phase inverse, puis identifiés par MS/MS, avec les résultats suivants: 1) Seuls les dihydroflavonols de configuration (2R,3R) sont acceptés comme substrats par VvANS dont l'activité diminue avec le nombre de groupements hydroxyles du cycle B. 2) Seuls les flavan-3-ols ou flavan-3,4-diols de configuration (2R,3S) ayant un catéchol ou trois OH phénoliques vicinaux sur le cycle B sont acceptés comme substrats. 3) La naringénine n'est pas substrat de VvANS, sans doute en raison de l'absence de groupement hydroxyle en C3. […] Le glutathion GSH est un puissant nucléophile, réducteur et piégeur de radicaux libres, qui est abondant dans la baie de raisin. Nous avons donc étudié son effet sur l'activité de VvANS avec tous les substrats identifiés. GSH n’a pas d'effet sur la transformation des dihydroflavonols et des flavan-3,4-diols, mais il modifie considérablement le mode de transformation de la (+)-catéchine et de la (+)-gallocatéchine. En présence de (+)-catéchine et de GSH, on observe deux produits majeurs, la cyanidine et un adduit thioéther cyanidine-glutathion, et le rendement de production est beaucoup plus élevé qu'en l'absence de GSH. De plus, l’adduit covalent ascorbate-cyanidine et le dimère issu de la (+)-catéchine obtenus lors de la réaction réalisée en l'absence de GSH ont disparu. Nos données suggèrent que l'adduit covalent cyanidine-glutathion est un thioéther en C4 qui fait l'objet d'un équilibre de tautomérisation céto-énolique en C3, et se décompose en cyanidine et GSH. En présence de (+)-gallocatéchine, un adduit thioéther similaire delphinidine-glutathion est aussi observé. Pour tester l'éventuelle spécificité de GSH, trois autres mercaptans (thiomalate, cystéine et cystéamine) ont été testés et aucun adduit similaire n’a été observé, ce qui suggère que GSH est un ligand spécifique, et pourrait être un coenzyme de VvANS. Nos résultats suggèrent que les anthocyanidines pourraient être produites in vivo à partir d'un substrat flavan-3-ol (catéchine ou gallocatéchine) via un intermédiaire thioéther de glutathion, alors que le stéréoisomère naturel (3,4-cis) de la leucocyanidine n'est pas transformé en cyanidine
Recombinant anthocyanidin synthase from Vitis vinifera (VvANS) has been expressed in E. coli, and purified by nickel affinity chromatography. The production and purification of the iron-loaded enzyme has been developed in order to avoid uncontrolled nonenzymatic oxidation reactions in the presence of Fe(II) salt. A stable VvANS-Fe(II) complex is formed in the presence of 2-oxoglutarate and ascorbate, and this complex is catalytically active at ambient PO2 in the absence of Fe(II) salt. The transformation of (+)-catechin by VvANS has been studied with and without ascorbate, by using either the VvANSFe( II) complex or the holoenzyme co-incubated with ferrous sulfate, to investigate the role of ascorbate. No enzyme activity has been observed in the absence of ascorbate, which means that it is an essential enzyme cofactor. A covalent adduct ascorbate-cyanidin is produced in vitro, but only in the absence of glutathione (GSH), another major nucleophilic and reducing agent. The two stereoisomers of leucocyanidin (3,4-cis et 3,4-trans flavan-diols) which were expected to behave as substrates of VvANS, are not commercial and were synthesized by reduction of dihydroquercetin by NaBH4, and characterized by proton NMR. The analysis of the two stereoisomers by means of tandem mass spectrometry (MS/MS) shows that their fragmentation pathways are distinct and may be used to distinguish them during their production. The two stereoisomers are stable in frozen aqueous medium at -20°C. Twelve flavonoids of four distinct families (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) were tested as potential substrates of VvANS. All enzymatic products were purified by means of reverse-phase HPLC and characterized by MS/MS, with the following results: 1) Only dihydroflavonols of (2R,3R) configuration are accepted as substrates by VvANS, the activity decreasing with the number of hydroxyl groups of ring B. 2) Only flavan-3-ols or flavan-3,4-diols of (2R,3S) configuration having either a catechol or three vicinal phenolic OH on ring B are accepted as substrates. 3) Naringenin is not substrate of VvANS, most likely because a C3 hydroxyl group is missing. […] Glutathione GSH is a powerful nucleophilic and reducing agent as well as a free radical scavenger, which is abundant in grape berries. We therefore studied its effect on VvANS activity with all identified substrates. GSH has no effect on the transformation of dihydroflavonols and flavan-3,4-diols, but it considerably modifies the transformation pattern of (+)- catechin and (+)-gallocatechin. In the presence of (+)-catechin and GSH, we observe two major products, cyanidin and a cyanidin-glutathione thioether, with production yields which are much higher than in the absence of GSH. Moreover, the ascorbate-cyanidin covalent adduct and the (+)-catechin dimer that had been obtained in the absence of GSH have disappeared. Our data suggest that the cyanidin-glutathione adduct is a C4-thioether which is in equilibrium between the two keto-enolic tautomeric forms at C3, and decomposes into cyanidin and GSH. In the presence of (+)-gallocatechin, a similar delphinidin-glutathione thioether adduct is also observed. In order to test the possible specificity of GSH as a cofactor, three other mercaptans (thiomalate, cysteine and cysteamine) were tested, and no similar product was observed, which suggests that GSH is a specific ligand, and might be a coenzyme of VvANS. Our results suggest that anthocyanidins could be produced in vivo from a flavan-3-ol substrate (catechin or gallocatechin) via a glutathione thioether intermediate, whereas the natural 3,4-cis stereoisomer of leucocyanidin is not transformed into cyanidin by VvANS

This diploma thesis deals with application of pressurized liquid extraction (PFE) on grape skins of Vitis vinifera cultivars Alibernet and Svatovavřinecké to gain extraction of anthocyanin pigments in the form of 3-monoglucosides. This thesis deals with comparison efficiency pressurized liquid extraction (PFE) for the range temperatures 40–120 °C and the Soxhlet extraction with four extraction solvents. The identification of the anthocyanin pigments by high-performance liquid chromatography with diode array detection (HPLC/DAD) based on Synergi C12 column separation (Phenomenex) was performed. The anthocyanin pigments were identified at wavelength 520 nm. The complete amount of anthocyanin pigments grape skins was depended on the extraction solvent and temperature in range 0,38–244,06 mg/g for cultivar Alibernet and 0,28–105,01 mg/g for cultivar Svatovavřinecké. The contents of anthocyanin pigments was determined in the samples of wine both cultivars in the range 3,83–2836,84 mg/l too. PFE followed by HPLC/DAD employing Synergi C12 column provides relatively fast, quantitative and reproducible determination of anthocyanins in grape skins and wines.