This major thesis expands the issue of the bachelor thesis "The issue of raising laying hens considering on introduction of enriched cages" and adds new knowledge relating to the topic feed rations and their influence on the final production. The theoretical part of the thesis analyses the basic theoretical concepts cohering with this topic, focused on creating a comprehensive overview of the structure and quality of the eggs, the basic ingredients in the feed rations, qualitative parameters of grapes as the modified additive provided by us and last but not least there is processed the recent overview of the current state of large-scale farmers in the Czech republic. The practical part of the thesis aims to test the effect of the proposed feed with an additive in the form of the crushed grape seeds, on the strength of the eggshell, by homogeneous samples of the breeds through from us proposed measuring instrument. Subsequently, a statistical evaluation of measured results is realized. In conclusion, the consideration is extended to the economic evaluation of costs and profits in case of the implementation of the suggested feed ration into practice in order to quantify the difference compared to the commonly used feed ration.

Orientador: João Domingos Rodrigues / Banca: Elizabeth Orika Ono / Banca: Manuel Abílio de Queiróz / Banca: Teresinha Costa Silveira de Albuquerque / Banca: José Carlos Fachinello / Resumo: O presente trabalho foi conduzido na Fazenda Cooperyama - Juazeiro- BA, com o objetivo de estudar a eficiência de reguladores vegetais na brotação e características dos cachos da uva 'Itália'. Para avaliação da brotação das gemas foram efetuadas observações em dias alternados determinando-se o número de brotos. Os tratamentos considerados foram: T1: Testemunha; T2: 2,45 g L-1 de cianamida hidrogenada; T3: 0,045 mg L-1 de cinetina, 0,025mg L-1 de ácido giberélico e 0,025 mg L-1 de ácido indolilbutírico; T4: 0,09 mg L-1 de cinetina, 0,05 mg L-1 de ácido giberélico e 0,05 mg L-1 de ácido indolilbutírico; T5: 0,045 mg L-1 de cinetina, 0,025mg L-1 de ácido giberélico e 0,025 mg L-1 de ácido indolilbutírico + 10 kg de nitrogênio e 9 kg de Ca ha-1; T6: 0,09 mg L-1 de cinetina, 0,05mg L-1 de ácido giberélico e 0,05 mg L-1 de ácido indolilbutírico + 10 kg de nitrogênio e 9 kg de cálcio ha-1; T7: 0,1 mg L-1 de cinetina + 10 kg de nitrogênio e 9 kg de cálcio ha-1; T8: 0,2 mg L-1 de cinetina + 10 kg de nitrogênio e 9 kg de cálcio ha-1 e T9: 0,045 mg L-1 de cinetina, 0,025mg L-1 de ácido giberélico e 0,025 mg L-1 de ácido indolilbutírico + 2,45 g L-1 de cianamida hidrogenada + 10 kg de nitrogênio e 9 kg de cálcio ha-1. Para caracterização dos cachos foram avaliados a massa, comprimento e largura média dos cachos; massa, comprimento e diâmetro médio das bagas; massa seca dos engaços; número médio de bagas/cacho; número médio de cachos/planta; produtividade média/planta; sólidos solúveis, acidez titulável e ratio. Os tratamentos considerados foram 2,5; 20 e 20 mg L-1 de ácido giberélico em pó; 0,019; 0,25; 0,038; 0,5; 0,076; 1,0 mL L-1 de ácido giberélico líquido; 0,01; 0,05, 0,075; 0,1, 0,5% de cinetina, ácido giberélico e ácido indolilbutírico, respectivamente e 1,5; 3,0; 6,0; 20,0; 40,0; 80,0 mL L-1 de ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The trial was carried out in the farm Cooperyama - Juazeiro-BA, with the objective to study the efficiency of plant growth regulators on sprouting and on the cluster characteristics of 'Itália' grapes. To evaluate the bud sprouting, observations were done on alternate days and the number of sprouts were registered. The treatments considered were: T1: Control; T2: 2,45 g L-1of the hydrogen cyanamide ; T3: 0,045 mg L-1of kinetin, 0,025 mg L-1 of gibberellic acid the 0,025 mg L-1of indolilbutiric acid; T4: 0,09 mg L-1 of kinetin, 0,05 mg L-1 of gibberellic acid the 0,05 mg L-1 of indolilbutiric acid; T5: 0,045 mg L-1 of kinetin, 0,025 mg L-1 of gibberellic acid the 0,025 mg L-1 of indolilbutiric acid 0,045 mg L-1 of kinetin, 0,025 mg L-1 of gibberellic acid the 0,025 mg L-1 of indolilbutiric acid + 10 kg the nitrogen e 9 kg the calcium ha-1; T6: 0,09 mg L-1 of kinetin, 0,05 mg L-1 of gibberellic acid the 0,05 mg L-1 of indolilbutiric acid + 10 kg the nitrogen e 9 kg the calcium ha-1; T7: 0,1 mg L-1 of kinetin + 10 kg the nitrogen e 9 kg the calcium ha-1; T8: 0,2 mg L-1 of kinetin + 10 kg the nitrogen e 9 kg the calcium ha-1 e T9: 0,045 mg L-1of kinetin, 0,025 mg L-1 of gibberellic acid the 0,025 mg L-1 of indolbutiric acid + 2,45 g L-1of the hydrogen cyanamide + 10 kg the nitrogen e 9 kg the calcium ha-1. For cluster characterization, weight, length and average width of clusters; weight, length and average diameter of berries; dry weight of rachis; average number of berries/cluster; average number of clusters/plant; average productivity/plant; soluble solids content; acidity and ratio were evaluated. The treatments considered were 2.5; 20 and 20 mg L -1 of gibberellic acid; 0.019; 0.25; 0.038; 0.5; 0.076; 1.0 mL L-1 of gibberellic acid liquid of 0.01; 0.05, 0.075; 0.1, 0.5 % of kinetin, gibberellic acid the indolilbutiric acid, respectivemant and 1.5; 3.0; 6.0; 20.0; ...(Complete abstract click electronic access below) / Doutor

A ferrugem da videira, causada pelo fungo Phakopsora euvitis, é uma doença importante para a viticultura brasileira por causar desfolha precoce e, consequentemente, prejudicar a maturação dos frutos e comprometer as safras seguintes. Por ser um patógeno biotrófico, a preservação de urediniósporos para estudos com o patógeno, se faz em folhas do hospedeiro vivo. Em outras espécies de ferrugens é possível conservar os esporos em condições controladas por longos períodos de armazenamento. Outro ponto importante, para auxiliar na compreensão da epidemiologia do patossistema, está relacionado com as condições ambientais favoráveis na germinação de urediniósporos e na patogenicidade do fungo em folhas de videira. Com o objetivo de preservar o patógeno e determinar as condições ambientais favoráveis para a doença, foram avaliados: (1) O efeito da temperatura e desidratação na manutenção da viabilidade dos esporos; (2) O efeito da temperatura e do período de molhamento na germinação de urediniósporos e na patogenicidade em mudas de \"Niágara Rosada\" inoculadas. Os resultados obtidos mostraram que a desidratação dos esporos proporcionou maior viabilidade destes ao longo do tempo, para todas as condições de armazenamento testadas. A desidratação seguida pelo armazenamento a -80°C conseguiu manter os esporos viáveis, com uma alta porcentagem de germinação, por até 150 dias de armazenamento. Os urediniósporos armazenados no ambiente, independente do processo de desidratação, não conseguiram manter sua viabilidade por um período superior a 15 dias. A faixa de temperatura para germinação de urediniósporos foi ampla, entre 10 e 30°C, com um ótimo em 20°C. A expressão de sintomas em plantas inoculadas foi maior nas temperaturas de 25 e 30°C. O período de molhamento mínimo estimado pelo modelo monomolecular foi de 7 horas para as plantas mantidas a 15 °C e 5 horas para as plantas mantidas a 20, 25 e 30 °C. Para períodos acima de 6 horas de molhamento, a maior severidade da doença ocorreu na temperatura de 30 °C. Não ocorreu sintomas em plantas inoculadas e mantidas a 35°C em nenhum dos períodos de molhamento testados. O período de latência da ferrugem foi de 7 dias para plantas inoculadas e mantidas a 25 e 30°C, estendendo-se para 13 dias quando as plantas foram mantidas a 15°C. / Grapevine rust, caused by Phakopsora euvitis, is an important disease in brazilian viticulture, causing early defoliation and jeopardizing the maturation of fruits and the following crop season. P. euvitis is a biotrophic pathogen, and for research purpose, uredospores are kept through inoculation in grapevine plants. In other species of rust, it is possible to preserve spore under controlled conditions for long periods of storage. Understand the favorable environmental conditions for uredospores germination and the pathogenicity of the fungus on grapevine leaves are important for the understanding of the pathosystem epidemiology. In order to preserve the pathogen and determine the favorable conditions for the disease were evaluated: (1) The effect of temperature and dehydration in maintaining the viability of the spores; (2) The effect of temperature and wetness period on uredospores germination and pathogenicity in \'Niágara Rosada\' inoculated plants. The results showed that spore dehydration maintained high viability, for all the tested storage conditions. Dehydration followed by storage at -80°C kept viable spores, with a high percentage of germination, for 150 days of storage. The uredospores stored in the environment, independent of the dehydration process, could not maintain their viability for a period exceeding 15 days. The temperature range for uredospores germination varied from 10 to 30°C, with an optimal at 20°C. The expression of symptoms in inoculated plants was higher at 25°C and 30°C temperatures. The minimum wetness period estimated by the monomolecular model was 7 hours for plants kept at 15 °C and 5 hours for plants kept at 20, 25 and 30 ºC. For wetness periods up to 6 hours, the highest disease severity occurred at 30 °C. There were no symptoms on inoculated plants maintained at 35°C in any of the tested wetness periods. The rust latency period was 7 days for inoculated plants kept at 25 and 30°C extending for 13 days when the plants were kept at 15°C.

O presente trabalho foi subdividido em quatro capítulos com os seguintes objetivos: (i) elaborar uma minuciosa revisão de literatura abordando os principais aspectos da interação viróide-hospedeiro e as relações evolutivas dos viróides e virusóides; (ii) identificar e caracterizar viróides associados a videiras, no Brasil; (iii) purificar, clonar e caracterizar, um RNA circular de seqüência totalmente desconhecida; (iv) estudar alguns aspectos relacionados à interação viróidehospedeiro. Inicialmente, foram identificadas e caracterizadas duas espécies de viróides (o Citrus exocortis viroid CEVd e o Hop stunt viroid, HSVd) isolados de videiras no Brasil. Para tal, promoveu-se extração de RNAs totais de folhas de Vitis vinifera ‘Cabernet Sauvignon’ e V. labrusca ‘Niagara Rosada’, seguida de RT-PCR com oligonucleotídeos específicos. Os fragmentos de DNA amplificados foram clonados e seqüenciados. Os resultados revelaram que as videiras estavam duplamente infectadas com o CEVd e HSVd. As análises filogenéticas mostraram que os clones de HSVd de videira agruparam-se com outros variantes de videira, formando um grupo separado de um segundo formado por variantes de citros. Já os clones de CEVd de videira agruparam-se com isolados de citros e videira. No capítulo 3, empregou-se um método para a clonagem e caracterização de um pequeno RNA circular (com aproximadamente 300 nucleotídeos) de seqüência totalmente desconhecida. Este RNA, quando submetido à eletroforese dupla em géis de poliacrilamida desnaturantes, apresentou um retardamento na migração, similar aos viróides. Após a clonagem de fragmentos do RNA, amplificados via RTPCR com oligonucleotídeos aleatórios (apresentando seis nucleotídeos degenerados no terminal 3&#145),os clones obtidos foram seqüenciados. A partir desses dados, dois oligonucleotídeos adjacentes de polaridades opostas foram desenhados e empregados para amplificar via RT-PCR a seqüência completa do RNA circular. A análise das seqüências revelou a presença da CCR (central conserved region) do Apple scar skin viroid (ASSVd), espécie tipo do gênero Apscaviroid, e compartilha similaridade com outros membros deste gênero, o que sugere fortemente que o RNA circular é um viróide recombinante. Finalmente, no capítulo 4, foram realizados experimentos que comprovaram a existência do motivo loop E (presente na CCR de algumas espécies dos Pospiviroidae) in vivo no PSTVd. Demonstrou-se também, utilizando ensaios de união in vitro (análise de retardo em gel, EMSA e entrecruzamento com luz ultravioleta), que as proteínas L5 e TFIIIA de Arabidopsis thaliana se unem especificamente ao PSTVd com a mesma afinidade que elas se unem ao seu substrato natural, o rRNA 5S, enquanto que a afinidade por um viróide cloroplástico (Avocado sunblotch viroid, ASBVd) foi significativamente menor. Estas duas proteínas devem participar na síntese e movimento intracelular do PSTVd in vivo. / The present work has been divided into four chapters to: (i) review the main points in viroid-host interactions and present different aspects in the evolutionary relationship of the viroids and virusoids; (ii) identify and characterize viroids infecting grapevine in Brazil; (iii) purify, clone and sequence what appears to be a novel citrus viroid; (iv) study some aspects related to the viroid-host protein interactions. Firstly, two viroid species (Citrus exocortis viroid, CEVd and Hop stunt viroid, HSVd) were identified and characterized from grapevine in Brazil. Total RNAs, extracted from leaves of Vitis vinifera ‘Cabernet Sauvignon’ and V. labrusca ‘Niagara Rosada’, were RT-PCR amplified with specific primers for the five viroids described infecting grapevines. The resulting products were separated by agarose gel electrophoresis and the DNA fragments of the expected full-size were eluted, cloned and sequenced. The grapevines analyzed were doublyinfected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants. On the other hand, a method for cloning small circular RNAs of unknown sequence has been applied to an RNA of this kind from citrus (with ca. 300 nucleotides). This RNA, when analyzed by PAGE in denaturing conditions, showed the slow mobility typical of viroid RNAs. After denaturation, the purified RNA was RT-PCR amplified using a primer with six randomized positions at its 3? terminus, with the resulting products being then cloned and sequenced. From these data, two adjacent primers of opposite polarities were designed and used to RT-PCR amplify the complete sequence. Analysis of the sequences revealed the presence of the CCR (central conserved region) of the Apple scar skin viroid (ASSVd), the type member of the genus Apscaviroid, and scattered similarities with other members of this genus, suggesting that the circular RNA is a viroid recombinant. Finally, UV irradiation of infected tissue has revealed the existence in vivo of an RNA motif (loop E) in Potato spindle tuber viroid (PSTVd), the type member of the family Pospiviroidae (nuclear viroids), and RNA-protein binding followed by eletrophoretic mobility shift (EMSA) and UV cross-linking label transfer assays have shown that transcription factor IIIA (TFIIIA) and L5 ribosomal protein from Arabidopsis thaliana bind this RNA in vitro with the same affinity as they bind 5S rRNA, whereas the affinity for a chloroplastic viroid (Avocado sunblotch viroid, ASBVd) is significantly lower. These two proteins may participate in synthesis and delivery of PSTVd in vivo.

Au vignoble, des nuits froides peuvent se produire pendant la floraison et en particulier au moment de la méiose ovulaire dans les fleurs. La vigne est particulièrement vulnérable puisque la méiose ovulaire se produit lors de la transition du métabolisme de la plante d'un état hétérotrophe à un état autotrophe. Bien que la feuille soit l'organe photosynthétique principal, la jeune inflorescence de vigne possède un statut particulier et participe activement à l'effort reproducteur en exportant la majorité du carbone qu'elle assimile, permettant ainsi le développement des feuilles. Par conséquent, le métabolisme carboné de l'inflorescence lors du développement reproducteur peut être impliqué dans le futur rendement. Chez la vigne, le phénomène de coulure, propre à chaque cépage, peut engendrer des pertes de rendement importantes lorsque la plante est exposée à un stress. Le Pinot noir (PN) est considéré comme un cépage relativement « résistant » à la coulure alors que le taux d'abscission chez le Gewurztraminer (GW) augmente considérablement lors de conditions climatiques défavorables. Chez le PN, l'inflorescence effectue une photosynthèse inférieure et une respiration de nuit supérieure à celle de la feuille. L'activité et la régulation des deux photosystèmes sont très différentes entre ces deux organes lors des premiers stades de développement et les activités des deux photosystèmes sont supérieures au niveau de l'inflorescence. Néanmoins, la protection de la chaîne photosynthétique contre l'excès d'énergie est plus efficace dans la feuille. Contrairement à la feuille, l'activité photosynthétique de l'inflorescence évolue au cours de son développement. En effet, l'activité de la chaîne photosynthétique ainsi que la photosynthèse nette diminuent progressivement au cours de la floraison et le flux cyclique des électrons se met en place pour être finalement supérieur à celui de la feuille. L'activation de ce flux peut alors permettre une synthèse accrue d'ATP, une protection de la chaîne photosynthétique contre les dommages provoqués par un excès d'énergie ou encore la réparation des photosystèmes. Lorsque la nuit froide survient lors de la méiose ovulaire, le métabolisme carboné de l'inflorescence de PN est différemment modifié selon l'intensité du stress. Ainsi, après une nuit à 4°C, la modification de l'activité photosynthétique de l'inflorescence est due à des limitations de nature non-stomatique alors qu'après une nuit à 0°C, cette modification est due à des limitations de nature stomatique. Une nuit à -3°C altère profondément l'activité photosynthétique de l'inflorescence. Ces nuits froides induisent également une accumulation de glucides. Lors du développement floral en conditions optimales, le PN et le GW présentent une activité photosynthétique et un métabolisme carboné différents. La régulation des flux linéaire et cyclique des électrons est également différente. Ce dernier semble avoir une fonction différente chez ces deux cépages avec notamment une possible implication dans la réparation du PSII et/ou dans une synthèse d'ATP accrue à la fin du processus de floraison chez le PN. La chaîne photosynthétique du GW semble mieux protégée ce qui peut expliquer le rendement supérieur de ce cépage en conditions optimales. Néanmoins, l'exposition à une nuit froide entraine des modifications différentes de l'activité de l'inflorescence chez ces cépages avec une perturbation plus importante chez le GW. En effet, chez ce cépage, seule la photosynthèse nette est perturbée suite à la nuit froide alors que chez le PN, les processus de photosynthèse et de respiration sont modifiés. L'activité de la chaîne photosynthétique ainsi que l'activité métabolique de l'inflorescence de GW est également davantage affectée. De manière intéressante, nos résultats suggèrent que ces différentes perturbations de l'activité de l'inflorescence sont dues à des régulations différentes. / In the vineyard, cold night can occur during flowering and particularly at time of female meiosis in flowers. In grapevine, stress vulnerability is enhanced because female meiosis occurs when the whole plant physiology is switching its carbon nutrition from mobilization of wood reserves to photosynthesis in the leaves. Nevertheless, although leaf is the major photosynthetic organ, in grapevine, the young inflorescence has a particular status and takes part in the reproductive effort by exporting the majority of assimilated carbon, allowing in particular the leaves development. Consequently, the inflorescence metabolism during this phase can ultimately determines yield. In grapevine, coulure phenomenon, differing according to the cultivar, can generate important yield losses when a stress occurs. Pinot noir (PN) is considered as a cultivar relatively “resistant” to coulure phenomenon whereas Gewurztraminer (GW) abscission rate considerably increases under environmental stress.In PN, inflorescence performs a lower photosynthesis and a higher dark respiration than leaves. Functioning and regulation of PSI and PSII are very different between inflorescence and leaf during the first developmental stages and activities of these photosystems are higher in the inflorescence. Nevertheless, the photosynthetic chain against excess energy is more efficient in the leaf. Contrary to the leaf, the inflorescence photosynthetic activity evolves during the floral development. Indeed, photosynthetic chain activity and net photosynthesis progressively decrease and the cyclic electron flow appears and is higher than in leaf. This activation could provide ATP, protection against photodamage or repair of the photosystems.When cold night occurs at the female meiosis stage, carbohydrate metabolism of the PN inflorescence is differently modified according to the intensity of the cold stress. At 4°C, photosynthesis in the inflorescence is impaired through non-stomatal limitations, whereas at 0°C it is affected through stomatal limitations. A freezing night (-3°C) severely deregulates photosynthesis in the inflorescence. Cold nights also induce accumulation of sugars.Comparing PN and GW, different photosynthetic activity and carbohydrate metabolism have been showed during the floral development under optimal conditions. Regulations of the linear and cyclic electron flow are also different and the cyclic electron flow seems to have a different aim with particularly an implication in the recovery of PSII and ATP synthesis at the end of the flowering process in PN. GW could have higher protection of the photosynthetic chain and consequently gets a higher yield under optimal conditions. Nevertheless, chilling night impacts differently the activity of the inflorescences of both cultivars with higher modification in GW inflorescence. Indeed, in this cultivar, only the net photosynthesis is altered whereas in PN, both net photosynthesis and respiration processes are modified. The photosynthetic chain activity and metabolical activity of the inflorescence are also more affected by the cold night in GW. Interestingly, our results suggest that the different fluctuation of the inflorescence activities as response to the chilled night is due to different regulations.

Le réchauffement climatique planétaire annoncé ne sera pas sans conséquence sur le métabolisme de la baie et en particulier sur la teneur en composés phénoliques. Les objectifs de cette thèse visent à mieux comprendre les processus de régulation associant le microclimat des baies et la synthèse des composés phénoliques. Par des approches moléculaires et biochimiques, ce travail a permis de mieux décrire les réponses spécifiques des baies de raisin (cultivar Cabernet-Sauvignon) aux facteurs rayonnement et température.L'analyse du transcriptome des baies exposées soit à un stress thermique soit à un stress lumineux a mis en lumière deux processus, une réponse rapide ayant lieu dès les premières heures de traitement et une réponse apparaissant au bout de plusieurs jours d’exposition aux stress. Cette étude a également permis de valider le système expérimental de découplage des effets du rayonnement et de la température.L'analyse des profils d’expression d’une vingtaine de gènes intervenant dans la voie de biosynthèse des flavonoïdes révèle des différences dans la réponse transcriptionnelle des gènes en fonction du stress et du stade développement auquel il a été appliqué. Néanmoins cette réponse n'est pas ou peu corrélée avec les variations des teneurs en anthocyanes et flavonols observées. Les teneurs en anthocyanes sont fortement réduites par l’action de la chaleur alors que les teneurs de certains flavonols augmentent sous l’influence du rayonnement. Au niveau des acides présents dans la pulpe des baies, l’acide malique voit sa teneur réduite sous l’effet de la chaleur ainsi que d’une forte intensité lumineuse. Les analyses montrent un impact important du stress thermique sur la teneur de la phénylalanine, de la tyrosine et de la lysine dans la pellicule.Parallèlement, l'initiation d’une étude du métabolome des pellicules de baies de raisin a été entreprise par UPLC-ESI-LTQ-Orbitrap™ et a permis d'acquérir des bases solides pour optimiser le protocole utilisé en vue d'analyses futures. Cette étude permet de dresser une liste préliminaire de métabolites d’intérêts.Enfin, le gène VvGOLS1 (Galactinol synthase 1) voit son expression stimulée dans les baies exposées au stress thermique, ce qui se traduit par une accumulation de galactinol, précurseur de la voie des RFOs. Un rôle de molécule «signal» est envisagé pour le galactinol. Un régulateur de la transcription de VvGOLS1 a également été identifié par des expériences d'expression transitoire en protoplastes. Il s'agit du facteur de transcription VvHsfA2, dont l'expression est également stimulée par le stress thermique. Dans ce contexte, la caractérisation des gènes de la famille des Hsfs (Heat Stress Factors) a été initée. / Global warming will affect berry metabolism, and especially phenylpropanoïd contents. This PhD work aimed to acquire a better understanding on the cellular processus linking the microclimate and the phenolic synthesis. By molecular and biochemical approaches, we extended this study to detail specific responses taking place in berries under heat and light stress.Transcriptomic analysis of heat-stressed and light-stressed berries showed the existence of two processes that occur in exposed berries. The first one triggers a rapid and transient expression of genes within the first hours of treatment. The second one mobilizes a set of genes showing increase in their expression after several days of stress exposure. Furthermore, this study validated the experimental set used to discriminate the effects of light and temperature, respectively.Expression analysis of 20 genes involved in the flavonoid biosynthetic pathway revealed strong differences among the transcriptional responses, depending on the nature of stress and the developmental stage of the berry. However, expression patterns of genes involved in the biosynthesis of flavonoid could not fully explain the changes in anthocyanin and flavonol contents. This suggests that additional regulation processes such as post-traductional modifications of enzymes or metabolite degradation might take place in berries under abiotic stress. Anthocyanin content decreases under heat stress whereas flavonol content increases under high light. Malic acid increases in berry exposed to heat stress and high light. Moreover, heat-stressed berries showed an accumulation of phenylalanine, tyrosine and lysine in skin but not in pulp.In parallel, a metabolomic analysis was initiated on stress exposed berry skins by using UPLC-ESI-LTQ-Orbitrap™ technology. The first experiments revealed contrasted metabolite contents in berries according to the stress applied, and highlighted several metabolites of interest. The preliminary assays will help optimize this powerful tool for futures analysis.Finally, expression of VvGOLS1 (Galactinol synthase 1) was strongly induced in grape berries exposed to heat stress, in good agreement with the observed galactinol accumulation. Role of galactinol as a signaling molecule is discussed. Transient expression experiments revealed that VvGOLS1 expression is regulated at the transcriptional level through VvHsfA2 action. VvHsfA2 expression is also stimulated under heat stress. In this context, characterization of the grapevine heat stress factors was initiated.

O presente trabalho foi subdividido em quatro capítulos com os seguintes objetivos: (i) elaborar uma minuciosa revisão de literatura abordando os principais aspectos da interação viróide-hospedeiro e as relações evolutivas dos viróides e virusóides; (ii) identificar e caracterizar viróides associados a videiras, no Brasil; (iii) purificar, clonar e caracterizar, um RNA circular de seqüência totalmente desconhecida; (iv) estudar alguns aspectos relacionados à interação viróidehospedeiro. Inicialmente, foram identificadas e caracterizadas duas espécies de viróides (o Citrus exocortis viroid CEVd e o Hop stunt viroid, HSVd) isolados de videiras no Brasil. Para tal, promoveu-se extração de RNAs totais de folhas de Vitis vinifera ‘Cabernet Sauvignon’ e V. labrusca ‘Niagara Rosada’, seguida de RT-PCR com oligonucleotídeos específicos. Os fragmentos de DNA amplificados foram clonados e seqüenciados. Os resultados revelaram que as videiras estavam duplamente infectadas com o CEVd e HSVd. As análises filogenéticas mostraram que os clones de HSVd de videira agruparam-se com outros variantes de videira, formando um grupo separado de um segundo formado por variantes de citros. Já os clones de CEVd de videira agruparam-se com isolados de citros e videira. No capítulo 3, empregou-se um método para a clonagem e caracterização de um pequeno RNA circular (com aproximadamente 300 nucleotídeos) de seqüência totalmente desconhecida. Este RNA, quando submetido à eletroforese dupla em géis de poliacrilamida desnaturantes, apresentou um retardamento na migração, similar aos viróides. Após a clonagem de fragmentos do RNA, amplificados via RTPCR com oligonucleotídeos aleatórios (apresentando seis nucleotídeos degenerados no terminal 3&#145),os clones obtidos foram seqüenciados. A partir desses dados, dois oligonucleotídeos adjacentes de polaridades opostas foram desenhados e empregados para amplificar via RT-PCR a seqüência completa do RNA circular. A análise das seqüências revelou a presença da CCR (central conserved region) do Apple scar skin viroid (ASSVd), espécie tipo do gênero Apscaviroid, e compartilha similaridade com outros membros deste gênero, o que sugere fortemente que o RNA circular é um viróide recombinante. Finalmente, no capítulo 4, foram realizados experimentos que comprovaram a existência do motivo loop E (presente na CCR de algumas espécies dos Pospiviroidae) in vivo no PSTVd. Demonstrou-se também, utilizando ensaios de união in vitro (análise de retardo em gel, EMSA e entrecruzamento com luz ultravioleta), que as proteínas L5 e TFIIIA de Arabidopsis thaliana se unem especificamente ao PSTVd com a mesma afinidade que elas se unem ao seu substrato natural, o rRNA 5S, enquanto que a afinidade por um viróide cloroplástico (Avocado sunblotch viroid, ASBVd) foi significativamente menor. Estas duas proteínas devem participar na síntese e movimento intracelular do PSTVd in vivo. / The present work has been divided into four chapters to: (i) review the main points in viroid-host interactions and present different aspects in the evolutionary relationship of the viroids and virusoids; (ii) identify and characterize viroids infecting grapevine in Brazil; (iii) purify, clone and sequence what appears to be a novel citrus viroid; (iv) study some aspects related to the viroid-host protein interactions. Firstly, two viroid species (Citrus exocortis viroid, CEVd and Hop stunt viroid, HSVd) were identified and characterized from grapevine in Brazil. Total RNAs, extracted from leaves of Vitis vinifera ‘Cabernet Sauvignon’ and V. labrusca ‘Niagara Rosada’, were RT-PCR amplified with specific primers for the five viroids described infecting grapevines. The resulting products were separated by agarose gel electrophoresis and the DNA fragments of the expected full-size were eluted, cloned and sequenced. The grapevines analyzed were doublyinfected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants. On the other hand, a method for cloning small circular RNAs of unknown sequence has been applied to an RNA of this kind from citrus (with ca. 300 nucleotides). This RNA, when analyzed by PAGE in denaturing conditions, showed the slow mobility typical of viroid RNAs. After denaturation, the purified RNA was RT-PCR amplified using a primer with six randomized positions at its 3? terminus, with the resulting products being then cloned and sequenced. From these data, two adjacent primers of opposite polarities were designed and used to RT-PCR amplify the complete sequence. Analysis of the sequences revealed the presence of the CCR (central conserved region) of the Apple scar skin viroid (ASSVd), the type member of the genus Apscaviroid, and scattered similarities with other members of this genus, suggesting that the circular RNA is a viroid recombinant. Finally, UV irradiation of infected tissue has revealed the existence in vivo of an RNA motif (loop E) in Potato spindle tuber viroid (PSTVd), the type member of the family Pospiviroidae (nuclear viroids), and RNA-protein binding followed by eletrophoretic mobility shift (EMSA) and UV cross-linking label transfer assays have shown that transcription factor IIIA (TFIIIA) and L5 ribosomal protein from Arabidopsis thaliana bind this RNA in vitro with the same affinity as they bind 5S rRNA, whereas the affinity for a chloroplastic viroid (Avocado sunblotch viroid, ASBVd) is significantly lower. These two proteins may participate in synthesis and delivery of PSTVd in vivo.

Ce travail de thèse porte sur l’effet du rayonnement UV-B sur la physiologie foliaire et la maturation des baies chez la Vigne (Vitis vinifera L. cv Tempranillo), dans le contexte du changement climatique en cours. Dans ce but, des expériences ont été menées en conditions contrôlées en serre, sur des boutures fructifères. Les plantes ont été exposées à trois doses de rayonnement UV-B biologiquement actifs (0, 5,98 et 9,66 kJ.m-2.jour-1), soit à partir de la nouaison, soit à partir de la véraison, et ce jusqu’à la pleine maturité. Le rayonnement UV-B a été appliqué seul ou en combinaison avec d’autres stress abiotiques (déficit hydrique, taux de CO2 et température élevés). L’impact de ces stress sur l’activité photosynthétique, les contenus en pigments et en composés photoprotecteurs, ainsi que sur les activités des enzymes produisant des composés antioxydants, ont été étudiés. La phénologie, les profils d’accumulation des flavonols et des anthocyanes, ainsi que le contenu en acides aminés des baies ont également été analysés, de même que l’expression des principaux gènes régulateurs et structuraux de la voie de biosynthèse des polyphénols. Les résultats obtenus montrent que les effets des UV-B sur la physiologie foliaire peuvent être découpés en deux phases : une première phase transitoire de diminution de l’activité photosynthétique, suivie d’une phase d’acclimatation due à la production de composés photoprotecteurs (flavonoïdes essentiellement) et à l’activité d’enzyme de détoxification des forme actives de l’oxygène (superoxyde dimutase, catalase, ascorbate peroxidase). Le déficit hydrique, le stress thermique et un taux de CO2 élevé (700ppm, +4ºC) ne modifient pas le processus d’acclimatation aux UV-B ; en revanche on note une interaction positive entre les UV-B d’une part et la tolérance aux températures et au taux de CO2 élevé d’autre part, atténuant les dommages oxydatifs dus à l’induction de sénescence par ces deux derniers facteurs. La maturité des baies est retardée par les UV-B et le déficit hydrique, et ce phénomène et amplifiés lorsque ces deux stress sont appliqués en combinaison ; alors que les hautes températures et un taux de CO2 élevé ont l’effet inverse. Dans ce dernier cas, le UV-B rayonnement atténue les effets du couple température/CO2 élevés. Ces effets sur la phénologie de la Vigne ont pu être reliés à des modifications de l’activité photosynthétique des feuilles. En ce qui concerne la composition des baies, l’augmentation du rayonnement UV-B et le déficit hydrique ont augmenté les concentrations en anthocyanes et en potassium des moûts et diminué leur acidité, ce qui peut s’expliquer en partie par une augmentation du ratio de masse pellicule/pulpe. L’augmentation des teneurs en anthocyanes et flavonols des pellicules observée en réponse aux UV-B a pu être reliée à l’induction de gènes structuraux (CHS, F3’H, FLS, UFGT and GST) et régulateurs (MYBF1 et MYBA1) de la voie de biosynthèse des flavonoïdes. Ces changements quantitatifs étaient toujours associés à des changements qualitatifs, avec une augmentation de la part relative des flavonols mono- et disubstitués, en raison d’une compétition de la flavonol synthase avec les F3’ et F3’5’ hydroxylases pour les mêmes substrats. Une interaction notable a été observée entre le rayonnement UV-B et le déficit hydrique, sur les profils d’hydroxylation des flavonols, ce qui s’explique par le fait que ces deux facteurs agissent sur deux étapes distinctes de la voie de biosynthèse. / The aim of the thesis was to assess the effect of UV-B radiation on grapevine Vitis viniferacv. Tempranillo leaf physiology and grape berry composition, framed within the climatechange scenario. Experiments were conducted under glasshouse controlled conditions withfruit-bearing cuttings. Plants were exposed to three UV-B biologically effective doses (0,5.98, 9.66 kJ m-2 d-1) either from fruit set or veraison to maturity. The combined effects of UVand water deficit, as well as, UV-B and elevated CO2-temperature (700ppm, +4ºC), appliedfrom fruit set to maturity were also tested. Gas exchange, Chlorophyll a fluorescence, lipidperoxidation, antioxidant enzyme activity, UV-B absorbing compound levels and chlorophylland carotenoid concentration were determined in leaves. Berry development was assessedquantitatively (e.g. elapsed time to reach phenological stages). Amino acid, anthocyanin andflavonol concentrations and profiles were analyzed in berries, as well as, transcript profilingof regulatory and structural genes involved in flavonoid biosynthesis.The results show that initial down-regulation of photosynthesis was followed by anacclimation, mediated by the accumulation of UV-B absorbing compounds and antioxidantresponse elicitation (flavonoids and antioxidant enzymes). Water deficit and elevated CO2-temperature did not alter UV-B acclimation process, however, UV-B did led to certain degreeof cross-tolerance to elevated CO2-temperature, avoiding the senescence-induced oxidativedamage. Berry technological maturity (ca. 22ºBrix) was delayed by UV-B exposure and waterdeficit, especially when combined, whereas it was hastened by elevated CO2-temperature. Inthe last case, UV-B attenuated the effect of elevated CO2 and temperature. Changes in berryripening rates were associated with changes in photosynthetic performance.UV-B radiation and water deficit induced lower grape must acidity, mediated by increases inrelative skin mass or potassium levels rather than a decrease in organic acid concentration.In addition this increase in relative skin mass may have contributed to higher anthocyaninconcentration in the must. Grape berry skin flavonol and anthocyanin concentration wasincreased by UV-B, mainly due to the up-regulation of the structural (CHS, F3’H, FLS, UFGTand GST) and regulatory genes (MYBF1 and MYBA1) committed to their synthesis.Quantitative changes in flavonol concentration induced by UV-B were always associated withqualitative changes in flavonol profile (i.e. increased relative abundance of mono- anddisubstituted flavonols), as a result of the competition of FLS with flavonoid hydroxylases(F3’H and F3’5’H) for the same substrates. The independent up-regulation of FLS and F3’5’Hby UV-B radiation and water deficit, respectively, resulted in an intaractive effect on theflavonol B ring hydroxylation pattern. Under elevated CO2-temperature anthocyanin-sugaraccumulation was decoupled. However, UV-B partially alleviated this uncoupling by upregulatinganthocyanin biosynthesis and modulating berry ripening rates.UV-B radiation greatly influenced grapevine leaf physiology and berry composition. Theseresponses to UV-B were modulated, to a greater or lesser extent, by other factors linked toclimate change (water availability, atmospheric CO2 levels and temperature).

Les recherches sur les interactions porte-greffe/greffon chez la vigne en relation avec l’environnement perdurent depuis plusieurs décennies, mais les mécanismes physiologiques sous-jacents de la vigueur conférée sont toujours incompris. Ce manque de connaissance constitue un frein dans le développement des porte-greffes existants pour contrôler la vigueur et la productivité, ou dans la recherche de nouveaux génotypes de porte-greffe mieux adaptés aux conditions futures de production. L’objectif de ce travail est de comprendre par une approche de biologie intégrative couplant expérimentation et modélisation comment le porte-greffe interagit spécifiquement avec son greffon (et vice versa) pour modifier dès les premières étapes du greffage, les caractéristiques physiologiques de la plante entière afin de coordonner le développement et la croissance des parties aériennes avec celle des parties racinaires. L’azote étant considéré comme un élément-clef de contrôle de la croissance et de l’allocation de la biomasse au sein d’une plante, un accent particulier est porté sur le rôle de la nutrition azotée dans le contrôle trophique de la croissance du couple porte-greffe/greffon. Un travail expérimental en serre a été mené pour caractériser par marquage isotopique les flux d’azote (15N) et de carbone à l’échelle de la plante entière au sein de deux combinaisons de porte-greffe/greffon au stade végétatif : l’une conférant une forte vigueur (CS/1103P), l’autre une faible vigueur (CS/RGM), en réponse à une variation de la disponibilité externe en nitrate. Cette étude sur le couplage entre fonctions d’acquisition et d’utilisation des ressources azotées et carbonées a été complétée par un phénotypage dynamique de la croissance aérienne, de la répartition de biomasse entre les organes et de la composition biochimique et minérale des principaux organes de la plante. Nous avons ainsi pu appréhender les signaux de communication entre la partie aérienne et la partie racinaire de la vigne greffée, ce qui a abouti à l’élaboration d’un modèle conceptuel simplifié du fonctionnement de la vigne greffée. Une première version d’un modèle mécaniste basé sur un formalisme source-puits prenant en compte l’acquisition et l’allocation de C et N au sein de deux compartiments aérien et racinaire, ainsi que leur plasticité vis-à-vis de la disponibilité exogène et endogène en ressources a été élaborée. A terme, le modèle devrait permettre d’identifier des paramètres génétiques clef au niveau racinaire explicitant les différences de croissance et vigueur conférée observées selon les combinaisons porte-greffe/greffon / Research on rootstock/scion interactions in grapevine in relation to the environment persisted for several decades, but the physiological mechanisms determining the rootstock effect on scion vigour are still misunderstood. This lack of knowledge hampers the development of existing rootstocks to control the vigor and productivity, or research new rootstock genotypes better adapted to future conditions of production. The objective of this work is to understand by an integrative biology approach coupling experimentation and modeling how the rootstock interacts specifically with the scion (and vice versa) to change in the early stages of grafting , the physiological characteristics of the whole plant to coordinate the development and growth of the aerial parts with the root parties. Nitrogen is considered a key element in the control of the growth and the biomass allocation within a plant, and a particular emphasis is placed on the role of nitrogen nutrition in the nutritional control of the grafted grapevine growth. Experimental work was conducted in a greenhouse to characterize by isotopic labeling nitrogen (15N) and carbon flow within the whole plant for two rootstock/scion combinations at vegetative stage : one giving a strong vigour (CS/1103P), the other a low vigour (CS / RGM), in response to a change in the external nitrate availability. This study on the coupling between acquisition functions and use of nitrogenous and carbonaceous resources was completed by a dynamic phenotyping aerial growth, the distribution of biomass between the organs and the biochemical and mineral composition of the principal organs of plant. We were able to understand the communication signals between the aerial part and the root part of grafted vines, which led to the development of a simplified conceptual model of the functioning of the grafted vines. A first version of a mechanistic model based on a source-sink formalism taking into account the acquisition and allocation of C and N in both aerial and root compartments and their plasticity to the availability of exogenous and endogenous resource was developed.

L'industrie viti-vinicole est affectée par la présence d’Ochratoxine A (OTA) dans ses produits en raison de la contamination des raisins par des souches fongiques d'Aspergillus section Nigri, notamment A. carbonarius. Le vin et le jus de raisin sont considérés comme les deuxièmes contributeurs en Europe à l'ingestion par les consommateurs de cette mycotoxine ayant des effets néphrotoxiques, neurotoxiques et tératogènes. L'objectif principal de ce travail est de proposer des méthodes alternatives aux traitements phytosanitaires chimiques pour prévenir la contamination en OTA dans les raisins et le vin, dans le respect de l'environnement et de la santé des producteurs et consommateurs. Différents traitements ont été comparés en station expérimentale après contamination artificielle du cépage Mourvèdre par une souche d’A. carbonarius fortement productrice d’OTA (CP-OTA) isolée de raisin : un fongicide chimique (Scala®) servant de témoin conventionnel et 3 traitements alternatifs, un extrait de plante agissant comme éliciteur (Stifénia®), et Saccharomyces cerevisiae et Trichoderma atroviride utilisés comme agents biologiques antagonistes. Deux parcelles non traitées ont servi de témoins non traités, l'une a été artificiellement contaminée. Une réduction significative de la teneur en OTA dans les jus (de 38 à 42 %) a été observée pour les traitements avec le fongicide chimique, la levure en tant qu’agent biologique et l’éliciteur Stifenia permettant une amélioration notable de la qualité sanitaire des jus et une réduction en dessus de la norme à 2 g/Kg. Les dénombrements microbiologiques et la Q-PCR utilisant des amorces spécifiques d’A. carbonarius ont montré les plus fortes réductions de son occurrence dans les jus de raisin et les rafles après traitement avec l’éliciteur. L’analyse des résultats de PCR-DGGE a permis de bien de discriminer les différents traitements par rapport à des modifications d’écosystèmes. Des espèces des genres Penicillium et Fusarium non productrices d’OTA ont été isolée des baies traitées par le Stifénia® alors qu’elles n’ont pas été mises en évidence sur les autres parcelles. Les deux traitements biologiques et le traitement par éliciteur ont considérablement augmenté l'épaisseur des peaux de baies (quantités de cire et de cuticule et épaisseur de la peau de la baie). Ceci pourrait être due à des mécanismes de résistance des baies de raisin à certains agents pathogènes et pourraient expliquer simultanément la réduction d’OTA ainsi que l'amélioration de la qualité globale du jus de raisin, qui présentent notamment des teneurs en polyphénols augmentées. Le trans-6-nonenal et trans-2-octenal, rencontrés à plus haute concentration dans les feuilles traitées au Stifénia® lors de l’analyse des profils des composés organiques volatils (COV) des feuilles ont montré une activité antifongique sur la croissance et toxinogenèse du CP-OTA alors qu’aucune activité antifongique de la poudre Stifénia® n’a pu être mesurée. Cela pourrait expliquer en partie le mode d'action de défense de la plante suite au traitement éliciteur par production au niveau des feuilles des COV induisant des modifications métaboliques sur le CP-OTA et engendrant les teneurs d’OTA réduites dans les raisins. Suite aux modifications d’écosystème obtenus, l’autre possibilité envisagée de défense induite par le Stifénia serait la promotion de souches microbiennes antagonistes des souches fongiques mycotoxinogènes. Lors de test d’antagonisme, certaines souches des genres Penicillium et Fusarium, isolées sur parcelles traitées au Stifenia, ont eu un effet positif de réduction de la croissance et toxinogenèse du CP-OTA. C'est notamment le cas pour Penicillium adametzioides qui présente la meilleure réduction de toxinogenèse du CP-OTA. Le traitement par éliciteur présente donc de très bonnes alternatives aux traitements phytosanitaires chimiques pour lutter contre les champignons toxinogènes ainsi que des potentialités de nouveaux agents biologiques. / The grape and wine industry is affected by the presence of Ochratoxin A (OTA) in its products because of contamination of grapes by strains of Aspergillus section Nigri. Grapes and wine are considered as the second contributors in Europe to the ingestion of this mycotoxin with nephrotoxic, neurotoxic and teratogenic effects. The main objective of this work is to provide non-chemical alternative methods to control OTA contamination in grapes and wine, in respect with environment and health of producers and consumers. Different treatments were compared in experimental vineyard PECH-ROUGE of INRA and IFV, Narbonne, France on near parcels after artificial contamination of the Mourvèdre grape cultivar by A. carbonarius: (OTA producing fungus; OTA-PF) a chemical fungicide (Scala®); Saccharomyces cerevisiae and Trichoderma atroviride as antagonists; and a plant extract as elicitor (Stifénia®). Two untreated parcels served as controls, one was artificially contaminated. A significant reduction (38 - 42%) was observed in the OTA juice content by the chemical, yeast bioagent and elicitor treatments with juice safety improvement under the standard at 2 g/Kg. The microbiological enumeration and Q-PCR using universal and specific primers for A. carbonarius had shown the highest reduction of its occurrence on grapes and stems from elictor treatment. The DGGE gave an overview on their effect on the fungal ecosystem, that showed higher similarity between the non-contaminated and elicitor treatment (76%) followed by yeast one and the lowest treatment was the contaminated one. The results obtained from traditional methods of isolation showed that the elicitor treatment had a higher proportion of fungal species from Penicillium and Fusarium genera not isolated in the other treatments. The two biological treatments and one elicitor treatment significantly increased the thickness of the berry skins in general (wax, cuticle layers and skin thickness), which could be related to the enhancement of the disease resistance of the grape berries to certain pathogens and could also simultaneously explain the OTA reduction and the grape juice quality improvement whith particular increasment of polyphenol contents. Further study was conducted in order to understand the Stifénia® mode of action because the reduction effect of the black aspergilli incidence and the OTA contamination while the isolated strains still have the high ability of producing OTA. Trans-6-nonenal and trans-2-octenal, which recognized in Stifénia® treatment leaves with the highest significant concentration regarding to their concentration with the chemical treatment during volatile organic compound (VOC) analyses, have antifungal activity against the OTA-PF growth and OTA production with low concentrations. No antifungal activity of the Stifénia® powder against the OTA-PF mycelial growth or toxigenesis were measured. That may partially explain the mode of action of plant defence by producing leaf VOCs that induce positive changes on the OTA-PF and its OTA contents in grapes. Due to ecosystem changes observed, an other potential effect of the induced defense with Stifénia treatment could be to promote fungal strains with antagonistic effect on A. carbonarius. In vitro antagonistic test was performed with Stifénia® non-Aspergillus isolates from Penicillium and Fusarium genera. Certain strains had a positive effect on mycelial growth reduction on OTA-PF and have also an effect on OTA production of OTA-PF. Penicillium adametzioides showed the highest reduction of toxigenesis of OPT-PF. This could be accomplished by applying as the elicitor one of the tested fungi with an antagonistic effect on OTA production, such as P. adametzioides. The Elicitor treatment therefore offers very good alternatives to chemical treatments to fight against toxigenic fungi directly or by giving new potential biological agents.