Thèse ou mémoire avec insertion d'articles. / Le chancre bactérien de la tomate causé par Clavibacter michiganensis (Cm) est l'une des maladies bactériennes les plus dévastatrices affectant l'industrie de la tomate dans le monde entier. Actuellement, aucun cultivar résistant n'est commercialement disponible et les contrôles chimiques et biologiques offerts sont inefficaces. Afin d'augmenter nos outils disponibles pour lutter contre cet agent pathogène, cette étude a permis de développer une méthode d'inoculation artificielle pour induire le chancre bactérien sur les plants de tomate en serre afin d'uniformiser les résultats des différentes études sur Cm. Nous avons comparé deux méthodes d'inoculation, le scalpel et la seringue, avec deux niveaux de fertilisation faible et élevé. Après avoir évalué le pourcentage de feuilles nécrosées et la hauteur des plants, les résultats ont montré que l'inoculation à la seringue avec une faible fertilisation était la méthode d'inoculation la plus efficace, permettant le développement d'une échelle à plusieurs niveaux qui peut être utilisée pour étudier l'interaction entre les plants de tomate et les isolats de Cm. Dans cette étude, nous avons également développé un multiplexe TaqMan qPCR spécifique et sensible pour détecter Cm. Une détection précise est une étape cruciale pour confirmer les foyers de la maladie et permettre le développement de stratégies de gestion. Les résultats faussement positifs et négatifs constituent un problème majeur des méthodes de détection existantes. Ainsi, deux gènes chromosomiques liés à la virulence de Cm, rhuM et tomA, ont été utilisés comme cibles spécifiques. Le gène, α-tubuline, a été inclus dans chacun des multiplexes comme contrôle interne afin d'améliorer la fiabilité du test. La spécificité a été évaluée avec 12 souches de Cm et 15 souches bactériennes, y compris d'autres Clavibacter spp. et des bactéries pathogènes de la tomate provenant de différents lieux géographiques. Notre test a détecté spécifiquement toutes les souches de Cm et jusqu'à 10 copies d'ADN plasmidique pour toutes les paires d'amorces et les sondes. Le test a été validé sur des échantillons de tissus et de semences infectés artificiellement. Il a détecté avec précision la présence de Cm dans tous les échantillons infectés analysés. Le test de diagnostic développé est hautement spécifique, sensible et fiable pour la détection de Cm et peut être adapté à des fins multiples telles que les programmes de certification des semences, la surveillance, la biosécurité, les études épidémiologiques et l'évaluation de nouvelles méthodes de lutte.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-26T14:00:38Z No. of bitstreams: 1 2011- C?ntia Let?cia da Silva Rosa.pdf: 3366742 bytes, checksum: 280ece7f9ee6afde0d927d4a7dd0c3ef (MD5) / Made available in DSpace on 2016-08-26T14:00:38Z (GMT). No. of bitstreams: 1 2011- C?ntia Let?cia da Silva Rosa.pdf: 3366742 bytes, checksum: 280ece7f9ee6afde0d927d4a7dd0c3ef (MD5) Previous issue date: 2011-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / This study aimed to evaluate accessions of tomato 'heirloom' whose cultivation is still little known in Brazil, grown organically, and its physical-chemical, nutritional and sensory characteristics after processing to obtain concentrated pulp. We used the non-hybrid tomatoes (Lycopersicum esculentum Mill) San Marzano, Chico Grande, Amish Paste and called EUA 05. The fruits were produced in the period from May to September 2010, the sector of Horticulture Department of Plant Science, of UFRRJ. In fresh fruit were analyzed total soluble solids (TSS), acidity (TTA), pH, quantification and identification of carotenoids, color of skin and pulp, texture instrumental and analysis of pesticides. Access San Marzano was not processed because of bacterial contamination in the field. The process was composed of the stages of pre-washing, washing / screening, milling, pulping, concentration, filling, exhaustion, closing, heat treatment / cooling and storage. It were added in the formulation of 1% salt, 0.4% sugar and citric acid for pH correction for microbial control. It was performed viscosity and pre-commercial sterility analyses in addition to those cited in the fresh tomato, both in the pulps in this study as six commercial brands on the market. For the sensorial analysis of the product manufactured was performed the Quantitative Descriptive Analysis (QDA) with trained judges and acceptance test with consumers. With respect to the TSS in tomatoes fresh, the Chico Grand and San Marzano showed the highest values (5.2? Brix). Access Chico Grande showed greater balance between acidity and sugar content in fruit, established by the TSS / TA (? Brix /%) of 19.2. Regarding the content of lycopene, access San Marzano had a higher average level of 6029 ?g/100g and more red coloration observed in the amount of a *= 29.68. No pesticide residues were found in the samples for organochlorine and organophosphorus pesticides. Results for the SST of pulps showed that accessions Amish Paste and EUA 05 stood out with values of 10.1 ? Brix. Regarding ATT access Amish Paste showed higher citric acid (1.445 g/100g) and lower pH value (3.66), which can lead to an impairment of sensory quality. The efficiency of the processing of pulp was 73% for access Chico Grande, Amish Paste 72% and 81% for EUA 05. After analyzing the lycopene content of the samples, we observed that the sample Amish Paste (3346.0 ?g/100g) and EUA 05 (3375.0 ?g/100g) showed the highest values with no statistical difference between them, corroborating the red color represented by lower values of hue angle. With respect to viscosity, the samples Chico Grande (246cP) and EUA 05 (235cP) stood out by the highest maximum viscosity. The results found in ADQ characterized the pulps by the attributes of astringency, taste salty, sour and acid flavor, and the commercialpulps by the attributes sweet aroma and sweet taste, flavor and aroma of tomato pulp. With respect to acceptance, the lowest scores were attributed to pulp in this study. However, samples Chico Grande (4.41) and the EUA 05 (4.48) had the highest score for overall acceptability with statistically significant differences with regard to the sample Amish Paste (3.06). It can be concluded with respect to fresh tomatoes that tomato 'heirloom' evaluated showed good quality in relation to balance acidity/sugar, and lycopene content. In pulps prepared from the three accessions 'heirloom' studied, concentrated pulp obtained from tomato Amish Paste and USA 05 showed the best characteristics for the food industry, with respect to soluble solids, lycopene content and red color. However, access Chico Grande showed higher viscosity when compared to other accessions. After the sensory analysis concluded that there was a rejection by the majority of consumers with regard to all the pulps obtained from tomatoes 'heirloom' due to significant changes in aroma and taste sour, salty taste as well as during the preparation / Este trabalho teve como objetivo geral avaliar acessos de tomate ?heirloom? cujo cultivo ainda ? pouco conhecido no Brasil, cultivados no sistema org?nico, e suas caracter?sticas f?sicoqu?micas, nutricionais e sensoriais ap?s o processamento para obten??o de polpa concentrada. Foram utilizados os tomates n?o h?bridos (Lycopersicum esculentum Mill) San Marzano, Chico Grande, Amish Paste e o denominado EUA 05. Os frutos foram produzidos no per?odo de maio a setembro de 2010, no setor de Horticultura do Departamento de Fitotecnia, da UFRRJ. Os frutos in natura foram caracterizados pelas an?lises de s?lidos sol?veis totais (SST), acidez total titul?vel (ATT), pH, quantifica??o e identifica??o dos caroten?ides, cor da casca e da polpa, textura instrumental e an?lise de pesticidas. O processamento e as an?lises foram realizados na Embrapa Agroind?stria de Alimentos, Rio de Janeiro-RJ. O processamento foi composto pelas etapas de pr?-lavagem, lavagem/sele??o, tritura??o, despolpamento, concentra??o, envase, exaust?o, fechamento, tratamento t?rmico/resfriamento e armazenamento. O acesso San Marzano n?o foi utilizado devido ? contamina??o bacteriana no campo. Foram adicionados na formula??o 1% de sal, 0,4% de a??car e ?cido c?trico para corre??o do pH para controle microbiol?gico. As mesmas an?lises realizadas no tomate in natura e ainda as an?lises de viscosidade, coliformes a 45?C, salmonelas, pr?-esterilidade comercial e avalia??o sensorial foram realizadas nas polpas concentradas e em seis marcas comerciais do mercado. Para realiza??o da an?lise sensorial utilizou-se a An?lise Descritiva Quantitativa (ADQ) com equipe treinada e Teste de Aceita??o com consumidores. Com rela??o ao teor de SST nos tomates in natura os acessos Chico Grande e San Marzano apresentaram os maiores valores (5,2 ?Brix). O acesso Chico Grande apresentou maior balan?o entre acidez e o teor de a??cares no fruto, estabelecido pela rela??o SST/ATT (?Brix/%) de 19,2. Com rela??o ao teor de licopeno o acesso San Marzano apresentou maior teor m?dio de 6029 ?g/100g, bem como colora??o mais vermelha observada pelo par?metro instrumental a*=29,68. N?o foram encontrados res?duos de agrot?xicos organofosforados e organoclorados em nenhuma das amostras. Nas polpas concentradas os resultados encontrados para a an?lise SST demonstraram que os acessos Amish Paste e EUA 05 se destacaram com valores de 10,1?Brix. O acesso Amish Paste apresentou maior valor de acidez (1,445g/100g), bem como menor valor de pH (3,66), o que levou a um comprometimento da sua aceita??o sensorial. O rendimento ap?s despolpamento dos tomates foi de 73% para o acesso Chico Grande, 72% para Amish Paste e de 81% para EUA 05. Ap?s a an?lise do teor de licopeno das amostras, observou-se que a amostra Amish Paste (3346,0?g/100g) e EUA 05 (3375,0?g/100g) apresentaram os maiores valores sem diferen?a estat?stica entre si, corroborando com a colora??o vermelha representada pelos menores valores do ?ngulo hue (?h). As polpas Chico Grande (246cP) e EUA 05 (235cP) se destacaram pelos maiores valores de viscosidade m?xima. A avalia??o sensorial (ADQ) caracterizou as polpas obtidas pelos atributos adstring?ncia, gosto salgado, gosto ?cido e aroma ?cido; e as polpas comerciais pelos atributos aroma doce, gosto doce, sabor e aroma caracter?stico de polpa de tomate. Com rela??o ? aceita??o, as menores notas foram atribu?das ?s polpas deste estudo. No entanto, as amostras Chico Grande (4,41) e EUA 05 (4,48) apresentaram a maior nota de aceita??o global com diferen?a estat?stica significativa com rela??o ? amostra Amish Paste (3,06). Pode-se concluir com rela??o ao tomate in natura que os acessos de tomate ?heirloom? avaliados apresentaram boa qualidade com rela??o ao equil?brio acidez/a??cares e teores de licopeno. Nas polpas elaboradas dos tr?s acessos ?heirloom? estudados, a polpa concentrada obtida do tomate EUA 05 e Amish Paste apresentaram melhores caracter?sticas para a ind?stria de alimentos, com rela??o ao teor de s?lidos sol?veis, teor de licopeno e colora??o vermelha. No entanto, o acesso Chico Grande apresentou valores maiores de viscosidade quando comparados aos outros acessos. Ap?s an?lise sensorial concluise que houve uma rejei??o pela maioria dos provadores com rela??o a todas as polpas obtidas dos tomates ?heirloom? devido a altera??es significativas no aroma e sabor ?cido, bem como no gosto salgado, decorrentes de sua formula??o.

Made available in DSpace on 2015-03-26T13:42:32Z (GMT). No. of bitstreams: 1 texto completo.pdf: 806747 bytes, checksum: 15d877e808731540d6a5b0cb3ec099c7 (MD5) Previous issue date: 2007-03-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The plants resist to the infection caused by pathogens using constitutive and induced defenses. The induced defense is activated by the plants when they recognize general elicitors and effector proteins. This recognition leads to the rapid activation of the defense responses, including the synthesis of Pathogenesis-related proteins - PR proteins. There are 17 known PR-protein families (PR-1 to PR-17) that are expressed in response to pathogens and/or chemical inductors. The genes that codes for PR-1 proteins share a high sequence identity and are used as markers of the systemic acquired resistance (SAR). However the biochemical function of these proteins is still unknown. Antimicrobial activities were reported only for basic PR-1 proteins. PR-1 genes are members of gene families in many plant species. Based on sequence analysis of sequences deposited in public database the following possible PR-1 gene family members from tomato plant were identified: PR-1aP4 (accession numbers AJ011520 and M69247); P1p14 (Y08804, M69248 and 68738); PR1A1 (X71592); PR1A2 (Y08844); PR1D (AJ001627) in the NCBI database and two unigenes sequences from the Solanaceae Genomics Network database, SGN-U213451 and SGN- U220473. The genes PR-1aP4 and P1p14 and the unigenes SGN-U213451 and SGN-U220473 encode basic PR-1 proteins and the genes PR1A1, PR1A2 and PR1D encode acid proteins. The gene represented by the unigene SGN-U213451 seems encode the P14c protein, that has a reported antimicrobial activity and until this moment is not in the databases. In this work were developed essays to detect the expression of PR-1 family members by real-time PCR. The analyzed genes can be distinguished by their quantitative and qualitative expression pattern. PR-1aP4, P1p14, PR1A1 and the SGN-U213451 had a higher level of expression in younger leaves in response to A. solani; while, the gene PR1A2 was more induced in the older leaves of these plants, where severity of the disease is greater. The gene represented by the SGN-U220473 did not show induction by A. solani. The P1p14 and PR1A2 expression was induced by benzothiadiazole (commercial product Bion) and jasmonic acid, respectively. Lower gene expression levels were obtained with chemical induction showing that more refined kinetic induction essays of PR-1 genes of the tomato plant in response to chemical inductors are needed in order to establish which specific signaling molecule is able to trigger the expression of each family member. / As plantas resistem à infecção por patógenos utilizando defesas constitutivas e induzidas. A defesa induzida é ativada pelo reconhecimento pelas plantas de elicitores gerais e proteínas de avirulência do patógeno. Este reconhecimento leva à rápida ativação de respostas de defesa, que incluem a síntese de proteínas relacionadas à patogênese (proteínas PR). São conhecidas 17 famílias de proteínas PR (PR-1 a PR-17) que são expressas em resposta a patógenos e ou a indutores químicos. Os genes que codificam proteínas PR-1 constituem famílias gênicas em diversas espécies vegetais. Com base na análise de seqüências depositadas em bancos de dados foram identificados sete possíveis membros da família gênica PR-1 do tomateiro, os genes: PR-1a P4 (números de acessos: AJ011520 e M69247); P1p14 (Y08804, M69248 e 68738); PR1A1 (X71592); PR1A2 (Y08844); PR1D (AJ001627) depositados no NCBI e as seqüências de dois unigenes depositadas no SGN, o SGN-U213451 e SGN-U220473. Os genes PR-1a P4 e P1p14 e os representados pelos unigenes SGN- U213451 e SGN-U220473 codificam proteínas PR-1 básicas, enquanto os genes PR1A1, PR1A2 e PR1D codificam proteínas ácidas. O gene representado pelo unigene SGN-U213451 parece codificar a proteína P14c que apresenta atividade antimicrobiana e até o momento não está anotada nos bancos de dados. Neste trabalho, foram desenvolvidos ensaios para a detecção da expressão desses genes por PCR em tempo real em resposta à infecção por Alternaria solani e a aplicação de indutores químicos. Apenas para o gene PR1D não foi possível obter oligonucleotídeos adequados para efetuar análises específicas de expressão por PCR em tempo real. A análise da cinética de expressão demonstrou que os genes analisados podem ser diferenciados pelo seu padrão qualitativo e quantitativo de expressão. Os genes PR-1a P4, P1p14, PR1A1 e o SGN-U213451 tiveram maior indução da expressão no terço superior de plantas inoculadas com A. solani; enquanto o gene PR1A2 foi mais induzido no terço inferior destas plantas, onde a severidade da doença é maior. Já o gene representado pelo SGN- U220473 não apresentou indução após a inoculação com A. solani. A expressão dos genes P1p14 e PR1A2 foi induzida pela aplicação de benzotiadiazole (produto comercial Bion) e ácido jasmônico, respectivamente. Menores níveis de expressão dos genes foram detectados nos ensaios com indutores químicos comparativamente à indução biótica indicando que ensaios mais refinados de cinética de indução de genes PR-1 do tomateiro em resposta a indutores químicos são necessários para estabelecer quais sinais moleculares induzem a expressão de cada membro desta família.

La tomate (Solanum lycopersicum) est l’espèce modèle pour l’étude des fruits charnus. Le développement du fruit de tomate a été décrit comme une succession de phases de développement distinctes, dans lesquelles les régulations hormonales jouent un rôle crucial. Afin de mieux comprendre la régulation du développement du fruit de tomate, des approches ciblées sur des catégories particulières de protéines régulatrices (F-Box) ainsi que des approches non ciblées de génomique fonctionnelle ont été menées dans le laboratoire. L'objectif de cette thèse est de contribuer à la validation fonctionnelle de gènes candidats mis en évidence dans ces travaux: trois protéines à F-Box (SlFB2, SlFB11 et SlFB24) et deux facteurs de transcription (SlHAT22 et SlTGA2.1). Au cours de cette thèse, la caractérisation de lignées RNAi précédemment générés pour les protéines SlFB2, SlFB11 et SlFB24 a été poursuivie. Ce travail n’a pas permis pas de conclure sur le rôle de ces protéines à F-Box dans le développement du fruit de tomate. Mais elle a permis d'isoler un mutant d'insertion dans une lignée RNAi SlFB2, spécifiquement affecté au niveau des fruits. Ce mutant est caractérisé par l'absence de développement de tissu loculaire, ce qui entraine une altération de la forme des graines, ainsi qu'une augmentation de la fermeté du fruit. Le site d'insertion de l'ADN-T dans ce mutant n'est pas encore identifié. En outre, la caractérisation des lignées RNAi SlFB11, a permis de proposer l'implication de cette protéine dans la régulation des méristèmes apicaux et floraux. La caractérisation fonctionnelle de SlHAT22 et SlTGA2.1 a été initiée par la génération de lignées transgéniques présentant une altération du niveau d'expression de ces facteurs de transcription (sur-expression et RNAi) ou une altération de la fonction du facteur de transcription par utilisation de la technologie CRES-T. La caractérisation phénotypique des lignées transgéniques, ainsi que des analyses métaboliques préliminaires ont révélé que SlTGA2.1 et SlHAT22 sont impliqués dans des processus de régulation au cours du développement précoce du fruit de tomate. En outre, ils ont également suggéré que SlTGA2.1 est impliqué dans la régulation du murissement du fruit de tomate. / Tomato (Solanum lycopersicum) is a model species for the study of fleshy fruits. Tomato fruit development has been described as a sequence of distinct developmental phases where different hormones play crucial regulatory roles. To further gain insight into the regulation of tomato fruit development, targeted approaches focused on particular classes of regulatory proteins (F-Box) as well as global functional genomics approaches were undertaken in the laboratory. The aim of this thesis was to contribute to the functional validation of candidate genes isolated from such approaches: three F-Box proteins (SlFB2, SlFB11 and SlFB24) and two transcription factors (SlHAT22 and SlTGA2.1).During this PhD thesis, the characterization of SlFB2, SlFB11 and SlFB24 RNAi lines previously generated was pursued. This work does not allow to conclude about the role of these F-Box in tomato fruit development. But it allowed to isolate an insertion mutant in a SlFB2 RNAi line, specifically affected at the fruit level. This mutant is characterized by the absence of locular tissue development and subsequent alteration of seed shape, as well as by an increase in fruit firmness. The insertion site of the T-DNA in this mutant is not yet identified. In addition, characterization of SlFB11 RNAi lines, allowed to propose the implication of this protein in the regulation of the shoot apical and floral meristems.Functional characterization of SlHAT22 and SlTGA2.1 was initiated by the generation of transgenic lines carrying an alteration of the transcription factor (TF) expression level (OE and RNAi lines) or with an alteration of TFs function using the CRES-T technology. Phenotypical characterization of the transgenic lines, together with preliminary metabolic analyses revealed that SlTGA2.1 and SlHAT22 are implicated in regulatory processes during tomato early fruit development. In addition, they also suggested that SlTGA2.1 is involved in the regulation of tomato fruit ripening.

La réduction de l’expression de deux gènes codant pour la L-galactono-lactone-1,4-déshydrogénase (GalLDH) et de la GDP-D-mannose-3’,5’-épimérase (GME), enzymes de la voie de biosynthèse de l’ascorbate, a permis de mieux comprendre le rôle physiologique de ces enzymes chez la tomate. D’une part, l’étude de la GalLDH a mis en évidence la régulation complexe du métabolisme de l’ascorbate et la fonction essentielle de cette protéine au sein de la chaîne de transport des électrons au niveau mitochondrial. D’autre part, ce travail a révélé le rôle central de la GME à la fois pour la biosynthèse de l’ascorbate et la biosynthèse des polysaccharides pariétaux, notamment les mannanes et le rhamnogalacturonane II. Chez les plantes sous-exprimant la GME, nous avons pu noter l’incidence de perturbations de la structure pariétale sur les propriétés mécaniques des tiges et des fruits ainsi que sur la fécondation. Ces modifications ont notamment engendré une fragilité accrue des tiges et une stérilité partielle. La GME est donc déterminante pour la qualité nutritionnelle et organoleptique du fruit de tomate. Enfin, dans le cadre d’une approche de biologie intégrative, nos résultats associés aux données issues de plantes sous-exprimant des gènes codant pour des enzymes de la voie de recyclage de l’ascorbate chez la tomate ouvrent des perspectives originales pour l’approfondissement des connaissances sur la régulation et sur l’intégration du métabolisme de l’ascorbate dans le fonctionnement de la cellule. / Down-regulation of two genes encoding the L-galactono-1,4-lactone dehydrogenase (GalLDH) and the GDP-D-mannose-3',5'-epimerase (GME), enzymes of ascorbate biosynthesis pathway, led to a better understanding of the physiological role of these enzymes in tomato plants. On one hand, the study of GalLDH highlighted the complex regulation of ascorbate metabolism and the essential function of this protein in mitochondrial electron transport chain. Moreover, this work revealed the central role of the GME for both the ascorbate biosynthesis and the biosynthesis of cell wall polysaccharides, including mannans and rhamnogalacturonan II. In the GME-silenced plants, we found that modifications of the cell wall structure change the mechanical properties of stems and fruit as well as the fertilization. These changes led to an increase of stem fragility and to an increase of sterility. Therefore, GME plays a crucial role regarding the nutritional and organoleptic quality of tomato fruit. Finally, within the context of a systems biology approach, our results associated to datas obtained with plants silenced for recycling pathway related genes lead to the prospect to unravel the knowledges on the regulation and the integration of ascorbate metabolism in cell functions.

La méthylation de l'ADN génomique est l'un des principaux mécanismes épigénétiques qui conduisent à des changements stables et héréditaires de l'expression des gènes sans que cela s’accompagne de la modification de la séquence d'ADN sous-jacente. Elle fait référence à l'addition d'un groupement méthyl sur le carbone 5 des cytosines (5meC). Ces dernières années, l’étude des mécanismes régulant la mise en place et le maintien de de cette méthylation est devenu un thème de recherche importante, en raison de son rôle essentiel dans la régulation du fonctionnement du génome des plantes et des mammifères. La distribution des 5meC sur l’ensemble du génome d’un organisme, encore appelé méthylome, peut être déterminée par différentes méthodes dont le séquençage de l’ADN génomique après traitement au bisulfite de sodium (WGBS ou méthyl C séq). Chez les végétaux, la méthylation de l’ADN peut se produire dans tous les contextes de séquence incluant les motifs symétriques CG et CHG et le contexte dissymétrique CHH (H pouvant être A, T ou C). En fonction du contexte de séquence, la méthylation des cytosines est mise en place et maintenue par trois types différents d'ADN méthyltransférase. [ ] Chez la plante-modèle Arabidopsis, la déméthylation active de l'ADN joue un rôle essentiel dans l'empreinte maternelle et la déméthylation l’ADN génomique lors du développement de l’albumen, mais elles ne semblent pas jouer de rôle essentiel pendant le développement de la plante chez cette espèce. La méthylation de l’ADN génomique peut aussi être perdue après la réplication de l’ADN, lorsque les mécanismes devant assurer son maintien ne sont pas actifs. On parle alors de déméthylation passive de l’ADN génomique. [ ] En conclusion, les observations présentées dans ce travail fournissent un cadre de travail permettant d’analyser les mécanismes moléculaires responsables de la déméthylation de l'ADN se produisant pendant la maturation des fruits de tomate. Ici, nous présentons une analyse complète des conséquences d’une réduction de l’expression du gène de SlDML2 sur le trancriptome et le métabolome des fruits, tout au long de leur développement. La corrélation entre les profils d’expression de gènes réalisées lors de ce travail ( variété WVA106) et les changements de la distribution de la méthylation de l’ADN telles que décrites chez la variété Ailsa craig montre qu’en plus d'un rôle général dans la régulation des gènes directement impliqués dans plusieurs voies métaboliques, plusieurs gènes codant pour des facteurs de transcription ainsi que des régulateurs épigénétiques sont également susceptibles d'être directement contrôlés par la méthylation de leur région promotrice. Cependant, nous ne pouvions pas établir une relation stricte entre la diminution de la méthylation de l'ADN et l'induction de l'expression des gènes, car de nombreux gènes présentant une diminution du niveau de méthylation de l'ADN dans leur région promotrice pendant la maturation des fruits sauvages correspondent à des gènes normalement réprimés. Ceci suggère que la méthylation active de l'ADN serait nécessaire à leur répression pendant le processus de maturation. Ainsi la relation entre la déméthylation de l'ADN et l'expression des gènes pourrait être plus complexe et ne se limiterait pas à la simple hypothèse de départ de ce travail: la déméthylation de l'ADN est nécessaire à l'expression de gènes induits au cours de la maturation. La déméthylation active de l'ADN pourrait également être nécessaire à la répression de gènes exprimés uniquement lors des phases précoces du développement des fruits et réprimés lors du murissement. / DNA methylation is one of the epigenetic mechanisms that lead to stable and heritable changes in gene expression without alteration on DNA sequence. DNA methylation refers to the addition of a methyl group to the fifth position of the cytosine ring. In recent years, DNA methylation is becoming more and more widely studied, because of its importance in mammals and plants. Methylated cytosines distribution can be determined across the genome at single-nucleotide resolution, that is methylome, using whole genome bisulfite-sequencing (BS-seq) approaches. [ ] Solanum lycopersicum (tomato) is an important agronomic crop and the main model to study the development and ripening process of climacteric fleshy fruit. Recent studies have now shown that the development and ripening of fleshy fruits relies on the establishment and maintenance of differential transcription patterns and complex regulatory pathways that involve both genetic and hormonal controls are operating at these developmental phases. However, it appears that a full understanding of fruit development and ripening will not be achieved based only on genetic models as suggested by recent studies, which showing an important decrease in global methylation level and demethylation at specific promoters during fruit ripening. [ ] In conclusion, the observations presented in this work provide a framework for analysis of the molecular mechanism of DNA demethylation during fruit ripening of tomato. Here, we provide a comprehensive analysis of the knock down SlDML2 on the trancriptome, metaoblom and DNA methylation in the promoter analysis. The large transcriptional reprogramming that occured in mutant during fruit ripeing was correlated alterations in DNA methylation. Here we highlight the central role of active DNA demethylation during tomato fruit ripening. In addition to a general role in the regulation of genes directly involved in several metabolic pathways, we also found that several transcription factors as well as epigenetic regulators are also likely under direct methylation control. However, we could not establish a district relationship between DNA reduction of DNA methylation and induction of gene expression, as not all DEGs containing a type-a DMRs (decreased DNA methylation during fruit ripening) do not correspond to genes normally induced in WT and repressed in transgenic plants. Some were corresponding to an opposite situation and in a few cases more complex methylation pattern (several DMRs) were also found. Indeed these conclusions are based on methylation analysis obtained in another variety. They might however reflect the situation of WVA106 fruits, although some variations are expectable when the methylome of DML RNAi fruits will be analyzed. Hence the relationship between DNA demethylation and gene expression might be more complex than expected, and not limited to the starting hypothesis of this work: DNA demethylation is an absolute requirement for the expression of critical ripening induced genes. This is indeed clearly in this study, but the analysis presented here also suggest that DNA demethylation might also be necessary for the repression of several genes as well. In addition, from the rencent study in Arabidopsis, ROS1 were found preferentially targets transposable elements (TEs) which are closer to protein coding genes and intergenic regions, which suggesting that ROS1 may prevent DNA methylation spreading from TEs to nearby genes. While in tomato, as our analysis, we found the methylation level of promoter of a number of genes was altered during fruit ripening, therefore, through methylome analysis, we will also get the preference of DNA methylation on TE, this analysis will give us idea that demethylation in fleshy fruit may has other distinct function as it is in Arabidopsis.

A preocupação com os danos provocados à saúde do trabalhador rural e ao meio ambiente devido ao uso indiscriminado de agrotóxicos tem aumentado nos últimos anos. O uso generalizado e intensivo destas substâncias tem gerado diversos problemas relacionados à saúde pública e ao desequilíbrio ambiental, incluindo: intoxicações de agricultores, contaminação de alimentos, água e solos. O tomate é hoje, uma hortaliça bastante conhecida e de elevado consumo no mundo. Sua cultura é bastante afetada por quebras de rendimento e depreciação da qualidade de matéria- prima, em razão da ocorrência de doenças, pragas e estresses abióticos. Apesar da preocupação com o monitoramento dos resíduos de agrotóxicos nos alimentos, poucas metodologias analíticas podem alcançar resultados de alta qualidade simultaneamente para uma gama extensiva de agrotóxicos. No presente estudo, foi desenvolvida e validada a metodologia de análise de multirresíduos QuEChERS (quick, easy, cheap, effective, rugged, and safe) para quantificação de resíduos de agrotóxicos em tomate. Este método provê resultados de forma rápida, fácil, com custo acessível e com alta qualidade. Foram analisados agrotóxicos utilizados principalmente no controle de insetos, sendo: buprofezina, carbofurano, \'alfa\'-endossulfam, \'beta\'-endossulfam, sulfato de endossulfam e monocrotofós. Para isso, realizou-se a fortificação do tomate, previamente homogeneizado, com soluções contendo os agrotóxicos em 3 níveis de fortificação (0,0625; 0,25 e 1,00 mg/Kg). A purificação dos extratos foi realizada através de clean-up dispersivo, e em seguida os extratos foram analisados por CG-EM modo SIM. Neste estudo avaliaram-se os seguintes parâmetros de validação do método: linearidade da curva analítica, sensibilidade, limite de detecção (LD), limite de quantificação (LQ), o efeito matriz, bem como a precisão e a exatidão (em termos de percentual de recuperação). A faixa linear de concentração das curvas analíticas situou-se entre 0,25 a 4,0 ng.\'mü\'L-1 com valores de r2 maiores que 0,99 (na matriz). A técnica CG-EM modo SIM promoveu a quantificação (critérios de recuperação entre 70 e 120% e valores de CV% menores que 20%) de todos os agrotóxicos estudados. Portanto, conclui-se que o método mostrou-se adequado às análises multirresíduos dos agrotóxicos em tomate, apresentando sensibilidade e seletividade adequadas, e todos os parâmetros de validação encontram-se de acordo com os limites sugeridos para validação de métodos cromatográficos. Foram coletadas amostras (n=33) em varejões na cidade de Piracicaba e os níveis residuais de agrotóxicos apresentaram-se abaixo dos limites de detecção para os produtos analisados. Os produtos acefato, deltametrina, difenoconazole e fenpropatrina foram avaliados no sistema CG-EM e não apresentaram sensibilidade e seletividade nas condições cromatográficas aplicadas. A implementação de novos métodos cromatográficos para análise de resíduos de agrotóxicos em alimentos devem ser fomentados para contribuir com o monitoramento eficiente, visando avaliar a qualidade e segurança dos alimentos consumidos pela população, caracterizar as fontes de contaminação e proporcionar uma avaliação quanto ao uso inadequado e não autorizado dos agrotóxicos / The concernment about the damage caused to the health of rural workers and the environment due to indiscriminate use of pesticides has increased in recent years. The commonly and intensive use of these substances has created several problems related to public health and environmental disequilibrium, including poisoning of farmers, contamination of food, water and soil. Nowadays, the tomato is a well known vegetables and high consumption in the world. Its culture is highly affected by loss of yield and depreciation of the quality of raw material, due to the occurrence of diseases, pests and abiotic stresses. Considering the concernment about the monitoring of pesticide residues in food, few analytical methods can achieve high quality results for both a wide range of pesticides. In this study, was developed and validated the methodology of multiresidue analysis QuEChERS (quick, easy, cheap, effective, rugged, and safe) for quantification of residues of pesticides in tomatoes. This method provides results quick, easy, inexpensive and high quality. Were analyzed mainly pesticides used to control insects, are: buprofezina, carbofuran, \'alfa\'-endossulfam, \'beta\'- endossulfam, sulphate endossulfam and monocrotophos. Then, performed a spiking of tomatoes homogenized previously, with solutions containing pesticides, in 3 of fortification levels (0.0625, 0.25 and 1.00 mg/kg). The purification of the extracts was performed by dispersive clean-up, and then the extracts were analyzed by GC-MS using SIM mode. This study evaluated the following parameters for validation of the method: linearity, sensitivity, limit of detection (LOD), limit of quantification (LOQ), matrix effect, precision and accuracy (in terms of percentage of recovery). The linear range of concentration of the analytical curves was between 0.25 to 4.0 ng.\'mü\'L-1 with r2 values of greater than 0.99 (in the matrix). The technique GC-MS SIM mode promoted quantification (criteria of recovery between 70 and 120% and RSD% values of less than 20%) of all pesticides studied. Therefore, concluded that the method proved to be appropriate for multiresidue analysis of pesticides in tomatoes, showing sensitivity and selectivity adequate, and all parameters of validation are according with the limits suggested for validation of chromatographic methods. Samples were collected (n = 33) in the Piracicaba city in local market and the levels of pesticide residues were below the limits of detection for the products analyzed. The products acephate, deltamethrin, difenoconazole and fenpropathrin were evaluated in the GC-MS and showed no sensitivity and selectivity in the chromatographic conditions applied. The implementation of new chromatographic methods for analysis of pesticide residues in food should be encouraged to contribute to efficient monitoring to evaluate the quality and safety of food consumed by the population, identify the sources of contamination and provide an assessment as to the misuse and use unauthorized pesticides

Cereais como a cevada e o arroz e leguminosas como o feijão, são importantes fontes de alimento no mundo. Alcançar maiores índices de produtividade não é apenas um fator econômico, mas social. Os reguladores vegetais têm o papel de estimular os diversos processos do desenvolvimento vegetal. Defensivos agrícolas passaram a serem estudados também como estimulantes vegetais, ao ser verificado que certas substâncias promoviam efeitos positivos na produtividade de cultivos. A fim de estudar esses efeitos em parâmetros de produtividade, quanto ao uso de substância específica e se ocorre diferença quando se modifica a fonte de reguladores vegetais numa formulação, foi realizada pulverização foliar com doses fixas dos produtos: (GA+NAA+BA), (GA+IBA+CN), nano TiO2 (nano dióxido de titânio) e tiametoxam em plantas de feijoeiro, cevada, arroz e em tomateiro cultivar Micro-Tom. Além disso, foi instalado um experimento com feijão via tratamento de sementes, com doses fixas dos produtos já citados, em rizotron e em vasos para avaliar o crescimento radicular. Os experimentos foram conduzidos em casa de vegetação no Departamento de Ciências Biológicas da ESALQ/USP. Os resultados obtidos foram submetidos à análise de variância e ao teste de Duncan através do software estatístico SAS. Em feijoeiro (aplicação via foliar) e tomateiro não foram encontrados efeitos significativos dos produtos na produtividade das plantas, contudo, alguns parâmetros foram influenciados por determinados tratamentos. Foi observada possível fitotoxidez no cultivo de tomate com o uso de tiametoxam ao se avaliar sua produtividade em relação ao controle. Em cevada, a aplicação de nano TiO2 aumentou a altura das plantas, o índice SPAD e o índice de colheita. Em arroz o tratamento GA+NAA+BA aumentou a altura das plantas e o tratamento tiametoxam promoveu maior número de perfilhos. Na análise do tratamento de sementes em vasos os resultados não foram significativos. Entretanto, o uso de nano TiO2 influenciou na área radicular. Em rizotron foi observado o maior CRV (crescimento radicular vertical) no tratamento com GA+NAA+BA. Ao final dos experimentos pode-se constatar também que o tratamento com GA+NAA+BA teve desempenho semelhante ao tratamento GA+IBA+CN. / Cereals like barley, rice and legumes like beans are important sources of food in the world. To achieve higher rates of productivity is not just an economic but a social factor. Plant regulators have the role of stimulating the various processes of plant development. Agricultural defensives were also studied as plant stimulants, as it was verified that certain substances promoted positive effects on crop productivity. In order to study these effects in productivity parameters, regarding the use of specific substance and if difference occurs when the source of plant regulators in a formulation is modified, a fixed dose of foliar spray was carried out: (GA+NAA+BA), (GA+IBA+CK), nano TiO2 (nano titanium dioxide) and thiamethoxam in common bean, barley, rice and tomato (Micro-Tom cultivar) plants. In addition, a bean experiment was installed through seed treatment with fixed doses of the mentioned products, in rhizotron and in pots to evaluate root growth. The experiments were conducted in a greenhouse at the Biological Sciences Department of ESALQ/USP. The results were submitted to analysis of variance and the Duncan test through SAS statistical software. In bean (foliar application) and tomato, no significant effects of the products were found on plant productivity; however, some parameters were influenced by certain treatments. Possible phytotoxicity was observed in tomato leaves with the use of thiamethoxam when evaluating its productivity in relation to the control. In barley the application of nano TiO2 increased the plant height, SPAD chlorophyll and the harvest indexes. In rice, the treatment GA+NAA+BA increased the plant height and the treatment with thiamethoxam increased the number of tillers. In the seed treatment in pots, analyses of the results were not significant. However, the use of nano TiO2 influenced the root area. In rhizotron, the highest CRV (vertical root growth) was observed in the GA+NAA+BA treatment. At the end of the experiments, it could be seen that the GA+NAA+BA treatment performed similarly to GA+IBA+CK treatment.

Preocupados com a segurança dos alimentos, consumidores em todo o mundo estão pagando prêmios de preço por \"alimentos naturais\", supostamente livres de qualquer tipo de contaminador. Nesse cenário, desenvolve-se a chamada agricultura orgânica. Esta pesquisa compreende a análise do sistema agroindustrial do tomate salada orgânico no Estado de São Paulo, sob dois prismas: o comportamento do consumidor, ou seja, o que o consumidor espera desse produto, e como se dá a coordenação desse sistema agroindustrial para atender o consumidor. A primeira parte da pesquisa compreende a análise dos atributos valorizados pelo consumidor de tomate orgânico, com vistas a comprovar ou não a hipótese que este paga um adicional de preço visando ao consumo de um produto seguro. Para isso, foi realizada uma pesquisa empírica com consumidores, com base no método da Conjoint analysis. A segunda parte da pesquisa consiste na descrição e análise da estrutura de governança desse sistema, com o objetivo de identificar os mecanismos de coordenação adotados para garantir a autenticidade e inocuidade dos produtos, benefícios intrínsecos não facilmente observáveis, mas que são exigidos pelos consumidores de tomate orgânico. A base empírica para a análise foi constituída de entrevistas com representantes de vários segmentos que compõem o Sistema Agroindustrial do tomate orgânico. O trabalho revela que o consumidor de tomate orgânico é diferenciado e busca um alimento seguro. No entanto, as estruturas de governança que coordenam esse sistema de produção são frágeis para garantir o que o consumidor deseja. Preservar a reputação e o prêmio de preço em um fraco ambiente regulatório é um desafio real, especialmente para produtos frescos. Esta pesquisa investiga como os agentes gerenciam esse sistema, visando a garantir sua vantagem competitiva. / Concerned with food safety, consumers all over the world are paying price premiums for \"natural foods\", supposedly free of any type of contaminant. In this scenario, the so-called organic agriculture is developed. This research analyzes the agribusiness of the organic salad tomato in the State of São Paulo, through two prisms: consumer behavior, that is, what the consumer expects from this product, and how this agribusiness is coordinated in order to satisfy the consumer. The first part of the research involves the analysis of the attributes valued by the consumer of organic tomato, with a view to showing whether or not the hypothesis that he or she pays a higher price in order to consume a safe product is correct. To this end, an empirical survey was conducted on consumers, based on the method of Conjoint analysis. The second part of the research consists of a description and analysis of the governance structure of this system, with the objective of identifying coordination mechanisms adopted to guarantee the authenticity and innocuity of the produce, intrinsic benefits not easily observable, but that are demanded by the consumers of organic tomato. The empirical basis for the analysis was made up of interviews with representatives of various segments that compose the Agroindustrial System of organic tomato. The work reveals that the organic tomato consumer is differentiated and seeks a safe food. Nevertheless, the governance structures coordinating this production system are too fragile to guarantee what the consumer desires. To preserve reputation and the price premium in a weak regulatory environment is a real challenge, especially for fresh produce. This research investigates how the agents manage this system, aiming at guaranteeing their competitive advantage.

As plantas se defendem do ataque de patógenos através de um sistema imune composto por duas fases. A primeira delas é mediada por receptores localizados na membrana celular ou intracelularmente, os quais são conhecidos como receptores de reconhecimento padrão (do inglês, pattern recognition receptors - PRR). Esses receptores reconhecem moléculas derivadas de microrganismos, as quais são conservadas evolutivamente e são chamadas de padrões moleculares associados a patógenos (do inglês, pathogen-associated molecular patterns - PAMPs). Esse reconhecimento dispara uma resposta de defesa conhecida como PTI (do inglês, PAMP-triggerd immunity - PTI). Alguns patógenos foram aptos a sintetizar moléculas capazes de suprimir a PTI e essas moléculas são denominadas de efetores. A resposta que ocorre devido à ação dos efetores é chamada de susceptibilidade disparada por efetores (do inglês, effector-triggered susceptibility - ETS). Entretanto, plantas resistentes podem reconhecer os efetores através de proteínas de resistência localizadas intracelularmente, ativando a imunidade disparada por efetores (do inglês, effector-triggeredimmunity - ETI). De modo geral, as respostas advindas da PTI e da ETI são similares, mas a segunda é ativada mais rapidamente e é mediada por um único gene de resistência R. Por essa razão, a ETI é conhecida como uma resposta à doença qualitativa e as plantas não desenvolvem sintomas, caracterizando a interação incompatível. Por outro lado, a PTI é mediada por diversos genes e as respostas de defesa são tardias, possibilitando a disseminação do patógeno pelas células da planta e a ocorrência da doença, o que caracteriza a interação compatível. Nas respostas de defesa, moléculas como o óxido nítrico, as poliaminas e o ácido salicílico participam do processo de sinalização. O sistema antioxidante da planta é ativado de modo a mitigar os efeitos das espécies reativas de oxigênio e o metabolismo da planta é alterado. Dessa maneira, o estudo das respostas de defesa contra patógenos, pode ser uma ferramenta útil para estabelecer controles efetivos para as doenças de plantas / Plants defend themselves from pathogen attack through an active immunity system composed by two phases. The first is mediated by cell surface and intracellular pattern recognition receptors (PRR), which recognizes conserved molecules derived from microbes known as pathogen-associated molecular patterns (PAMPs). This recognition triggers a defense response called PAMP-triggered immunity (PTI). Throughout evolution, pathogens were able to synthesize molecules capable of suppressing PTI. These molecules are named effectors and they are responsible for effector-triggered susceptibility (ETS). However, resistant plants can recognize effectors by intracellular resistance (R) proteins, initiating effector-triggered immunity (ETI). In general, responses derived from PTI and ETI are the same, but the latter is activated faster and is mediated by a single R gene. For this reason, ETI-response is also known as qualitative disease response (QDR) and plants do not develop disease symptoms, characterizing the incompatible interaction. On the other hand, PTI is mediated by several genes and the defense response is delayed, enabling the pathogen to spread out and to cause disease. This interaction is known as compatible. In defense responses, molecules like nitric oxide, polyamines and salicylic acid can participate in signaling process. The antioxidant system can be activated to quench the ROS effects and the plant metabolism is altered. In this sense, studying defense responses against pathogens can help to develop tools to establish effective control methods for plant disease