O desenvolvimento dos órgãos vegetativos e florais das angiospermas depende da ação combinada e finamente regulada de eventos de proliferação e expansão celular. Estudar os genes envolvidos com a regulação destes processos permite ampliar nossa compreensão sobre o desenvolvimento da flor, de seus diferentes órgãos, do processo reprodutivo como um todo, além de permitir produzir modificações de interesse econômico. Um gene codificando uma proteína da família F-box foi identificado na biblioteca TOBEST de cDNAs de estigma/estilete de N. tabacum (Quiapim et al., 2009; Abbad, 2012). A maioria das proteínas F-box pertence ao complexo SCF (formado principalmente pelas proteínas SKP1, CUL1 e F-box), participando da marcação de proteínas alvo para a degradação pela via ubiquitina-proteassomo. O gene identificado no TOBEST demonstrou expressão preferencial nos órgãos florais e foi denominado FEF1 (Flower Expressed F-box 1). Plantas de silenciamento e superexpressão deste gene indicaram alterações no tamanho dos órgãos florais, incluindo o pistilo, foco principal de estudo em nosso laboratório (Abbad, 2012). O screening de duplo-híbrido de uma biblioteca de cDNAs de estigma/estilete de N. tabacum identificou a interação com uma SKP1, indicando que a FEF1 poderia atuar junto ao complexo SCF (Abbad, 2012). No presente trabalho foram realizadas análises macroscópicas e microscópicas em pistilos de plantas transgênicas da geração T1, que permitiram: 1) verificar a estabilidade dos transgenes e das alterações fenotípicas na descendência; 2) quantificar e analisar estatisticamente as alterações de tamanho do pistilo; e 3) verificar as alterações em nível celular, que resultaram nas alterações do tamanho do pistilo, ocorridas nas plantas transgênicas. As plantas de silenciamento apresentaram redução estatisticamente significativa do comprimento de pistilos e da largura dos ovários. As análises histológicas permitiram verificar que ocorreu a redução da proliferação celular na zona secretória do estigma e no parênquima do ovário destas plantas. Por outro lado, as plantas de superexpressão demonstraram aumento estatisticamente significativo do comprimento dos pistilos e da largura de estigmas e ovários. Nestas plantas, foi verificado o aumento do número de células na zona secretória do estigma e parênquima do ovário. A interação entre FEF1 e a SKP1 foi confirmada em experimento de BiFC (Bimolecular Fluorescence Complementation), corroborando a participação dessa F-box no complexo SCF. A interação entre estas proteínas ocorre no citoplasma das células vegetais, indicando que este é o local de atuação de FEF1. A participação no complexo SCF confere a essa F-box o papel de seleção dos alvos a serem poliubiquitinados pelo complexo. A análise de candidatos do screening revelou três novos parceiros de interação de FEF1, todos fatores de transcrição da classe I da família TCP, relacionados com a regulação da proliferação celular. Estas proteínas são candidatas à degradação no proteassomo, sinalizada pela marcação promovida pelo complexo SCFFEF1. Deste modo, propomos que a FEF1 desempenhe uma função na regulação do desenvolvimento e do tamanho final dos órgãos florais, mais especificamente do pistilo, através da regulação dos níveis de fatores de transcrição, como as TCPs aqui encontradas, envolvidas com o controle da proliferação celular. / Angiosperms vegetative and flowering organs development depends on a combined influence of finely regulated events of cell proliferation and expansion. The study of genes involved with the regulation of this processes allows the expansion of our knowledge about the flower and its organs development, of the reproductive process and allows the production of modifications of economic interest. One gene coding for an F-box family protein was identified in the TOBEST stigma/style cDNA library of N. tabacum (Quiapim et al., 2009; Abbad, 2012). The majority of F-box proteins belong to the SCF (mainly composed of the SKP1, CUL1 and F-box proteins) complex, participating in the signalization of target proteins for degradation through the ubiquitin-proteasome pathway. The gene identified on TOBEST presented preferential expression on the floral organs and was named FEF1 (Flower Expressed F-box 1). Transgenic plants silencing and overexpressing this gene indicated alteration of the floral organs, including the pistil, the main focus of study in our laboratory (Abbad, 2012). A yeast two-hybrid screening of a N. tabacum stigma/style cDNA library revealed the interaction with a SKP1 protein, indicating that FEF1 possibly functions with the SCF complex (Abbad, 2012). In the present work macroscopic and microscopic analysis of the pistils of T1 generation of transgenic plants were performed, which allowed us to: 1) Confirm the transgene and phenotypic stability through generations; 2) Quantify and statistically analyze the size alterations on the pistils; 3) Analyze the cellular modifications that produced the pistils size alterations observed in the transgenic plants. The plants silencing FEF1 presented a statistically significant reduction of the pistil length and ovary width. Histological analysis allowed the observation that a reduction in cell proliferation occurred in the secretory zone of the stigma and in the ovary parenchyma. On the other hand, overexpression plants presented statistically significant enlargement of pistil length and of stigma and ovary width. In these plants it was observed an increase in cell number in the stigma secretory zone and ovary parenchyma. The interaction between FEF1 and SKP1 was confirmed on a BiFC (Bimolecular Fluorescence Complementation), reinforcing the participation of this F-box protein in the SCF complex. The interaction between these proteins was observed to occur in the cytoplasm of plant cells, indicating that this is the cellular compartment of FEF1 action. The participation in the SCF complex confers this F-box the role of selecting targets for polyubiquitination by the complex. The analysis of candidates of the screening revealed three new interaction partners of FEF1, all of them transcription factors of the class I TCP family, related to the regulation of cell proliferation. These proteins are candidates for degradation by the proteasome, signalized by the polyubiquitination promoted by the SCFFEF1 complex. We propose that FEF1 has a role in the regulation of the development and final size of the floral organs, particularly the pistil, by regulation the levels of transcription factors like the TCPs here revealed, involved with the control of cell proliferation.

Made available in DSpace on 2014-08-20T13:44:29Z (GMT). No. of bitstreams: 1 dissertacao_alexandro_de_paula.pdf: 1347086 bytes, checksum: 5f7aa723fd96d4d6ee7beb8f49b68039 (MD5) Previous issue date: 2012-11-01 / The main goal of this study was to determine the best harvesting time of seed capsule so that the tobacco seed (Nicotiana tabacum L.) presents the best germination percentage and vigor, seeking a combination of maturity, vigor and germination. The study was conducted in the 2010/2011 crop season, at the Agronomic Center of Universal Leaf Tobacco Company, in Rio Pardo, RS, using the hybrid seed of Virginia type ULT 163,. Five sampling periods were established as treatment in a field of commercial production of tobacco seeds (T1 - 30% dry capsules and green sepals, T2 - 90% dry capsules and green sepals; T3 - dried capsules and dried sepals; T4 - 50 % dry stalk; T5 - 100% dry stalk) with eight replicates each treatment. There were germination and vigor, first germination and accelerated aging. It was found that the tobacco harvest seeds from hybrid cultivar ULT 163 may be performed without significant changes in germination and vigor among treatments T3 to T5 consisting of sepals and capsule brown in color until the total drying peduncle. / O objetivo deste trabalho foi verificar qual o melhor momento de colheita da cápsula de sementes de modo que a semente de tabaco (Nicotiana tabacum L.) apresente o maior vigor e a maior porcentagem de germinação, buscando uma combinação entre maturidade, vigor e germinação. O trabalho foi desenvolvido no Centro Agronômico da empresa Universal Leaf Tabacos LTDA., em Rio Pardo, RS, utilizando o híbrido de semente tipo Virgínia ULT 163, na safra 2010/2011. Estabeleceram-se cinco momentos de colheita como tratamentos em uma lavoura para produção comercial de sementes de tabaco (T1 30% das capsulas secas e sépalas verdes; T2 90% das capsulas secas e sépalas verdes; T3 capsulas secas e sépalas secas; T4 50% pedúnculo seco; T5 100% pedúnculo seco), com oito repetições cada. Realizaram-se testes de germinação e de vigor - primeira contagem de germinação e envelhecimento acelerado. Verificou-se que a colheita das sementes de tabaco da cultivar híbrida ULT 163 pode ser realizada sem alterações significativas na germinação e no vigor entre os tratamentos T3 a T5 que consiste da cápsula e sépalas na cor marrom até a secagem total do pedúnculo.

Made available in DSpace on 2014-08-20T13:44:37Z (GMT). No. of bitstreams: 1 dissertacao_gizele_gadotti.pdf: 196227 bytes, checksum: 149ecb1f928b4cb8a0872d291ffc90fd (MD5) Previous issue date: 2006-08-11 / The disposable information about small seeds, include tobacco, especially about the perform densimetric table, it haven t been contemplate to literature. The present work was carried through with the objective to verify the perform tobacco seeds process in densimetric table. The seeds had initially been clean in air machine. The terminal axle of discharge of the densimetric table of width 50 cm was dividend in five parts: low with 9 cm, low and high intermediate each with 13 cm, high with 15 cm and discharge going out stone with 3 cm. The treatments had constituted of the fraction gotten in the feeding deposit and of the five parts discharged each part of the discharge. This work comprised two studies, the first involved seven combinations of the densimetric table regulations and the second seven tobacco cultivars. Tobacco seeds discharged on the high fraction in densimetric table discharge zone presents superior physiological quality as that seeds in the low fraction. The densimetric table, properly regulated, presents efficiency in the improvement of the physiological quality in the tobacco seeds lots to remove of the fraction discharge in low part zone of discharge. / A disponibilidade de informações sobre sementes pequenas, inclusive de tabaco, especialmente sobre a influência da utilização da mesa densimétrica na performance das sementes, não vem sendo contemplada pela pesquisa. O presente trabalho foi desenvolvido com o objetivo de avaliar o desempenho de sementes de tabaco beneficiadas em mesa densimétrica. As sementes inicialmente limpas em máquina a ar foram beneficiadas em mesa densimétrica. O eixo terminal de descarga de largura de 50 cm foi dividido em cinco partes: baixa com 9 cm, intermediária baixa e alta cada uma com 13 cm, alta com 15 cm e mais uma bica de saída de pedras com 3 cm mais longitudinalmente ao eixo terminal de descarga. Os tratamentos constituíram na fração coletada no depósito de alimentação e nas cinco frações descarregadas em cada uma das partes da descarga. O trabalho compreendeu dois estudos, o primeiro envolvendo sete combinações de regulagens da mesa densimétrica e o segundo sete cultivares de tabaco. Sementes de tabaco descarregadas na parte alta da zona de descarga da mesa densimétrica apresentam qualidade fisiológica significativamente superior as sementes descarregadas na parte baixa. A mesa densimétrica regulada conforme as características do lote, apresenta eficiência no aprimoramento da qualidade fisiológica de lotes de sementes de tabaco pela remoção da fração descarregada na parte baixa da zona de descarga.

Made available in DSpace on 2014-08-20T13:44:41Z (GMT). No. of bitstreams: 1 tese_everton_maksud_medeiros.pdf: 4288165 bytes, checksum: 749373d882062c9a1c5eaf350df60fb4 (MD5) Previous issue date: 2008-06-02 / The objective of this first work was to evaluate the efficience of the accelerated aging test to the evaluation of the vigor in tobacco seeds (Nicotiana tabacum L.). Three hybrids of Virginia type seeds were used: CSC 405, K 326 and CSC 459 with two quality levels (high and medium). The seeds were evaluated according to their standard germination and vigor: first counting, traditional accelerated aging, aging with salt saturated solution, and salt non saturated solution in the following condition: (temperature of 38°C and exposition time of 18 hours). (41°C with period of 12, 24, 36, 48, 60 and 72 hours) and (temperature of 45°C with period of 72 hours). It allowed to conclude that: a) the vigor of the portion of the tobacco seeds of hybrids CSC 405, K 326 and CSC 459 can be evaluated through the accelerated aging test with salt saturated solution using temperature of 45 ± 2°C and exposition time of 72 hours; b) the vigor of the portion of the tobacco seeds of hybrids CSC 405 and CSC 459 can be evaluated: - through the traditional accelerated aging test using temperature of 38 ± 2°C and exposition time of 18 hours or temperature of 41 ± 2°C and period of 60 hours; - through accelerated aging test with salt non saturated solution using temperature of 38 ± 2°C and period of 18 hours or temperature of 41 ± 2°C and period of 48 hours; - through accelerated aging test salt saturated solution using temperature of 41± 2°C and period of 48, 60 and 72 hours c) the vigor of tobacco seeds is not evaluated correctly in the first counting of the germination test. The aim of this work was to determine the harvest time of the seeds aiming at the attainment of seeds with a better physiological maturation possible. Seeds of five tobacco hybrids of Virginia Type were used: CSC 416, CSC 439, CSC 444, CSC 458 and CSC 459. The seeds were sowed in a foam tray and conducted in the Float system, producing changes with good quality, achieving the ideal size to the ix transplant, the spacing between lines was 2,0m and 0,5m between plants and there were about 10.000 plants.ha-1. It was 500Kg.ha-1 of NPK in the formulation 10-16-10 of basis fertilization and two saltpetre applications with 200Kg.ha-1 of covering fertilization. According to the planned management it was executed the elimination of the main stem, leaving two to three secondary stems. The plants were pollinated in different days, marking the pollinated flowers. The physiological quality was evaluated by the germination tests and accelerated aging test according to the established methodology in the first work. To determine the coloration of the fruits in the right moment of the crop, they were photographed in all days of the crop in order to obtain a standard of colours, aiming to facilitate further seeds crop. The obtained results allowed to conclude that: a) maturation point of tobacco seeds changes from the twenty-first to twenty-eighth day after anthesis; b) the pollination time does not interfere in physiological maturity point of tobacco; c) it is not possible to determine the physiological maturity point based on the fruits colour. / O objetivo do trabalho primeiro trabalho foi avaliar a eficiência do teste de envelhecimento acelerado para a avaliação do vigor em sementes de fumo (Nicotiana tabacum L.). Utilizaram-se três híbridos de sementes do tipo Virgínia, a CSC 405, K 326 e CSC 459, com dois níveis de qualidade, alta e média. As sementes foram avaliadas quanto ao poder germinativo e vigor: primeira contagem, envelhecimento acelerado tradicional, envelhecimento com solução salina saturada e solução salina não saturada nas seguintes condições: (temperatura de 38ºC e período de exposição de 18 horas), (41ºC com período de 12, 24, 36, 48, 60 e 72 horas) e (temperatura de 45ºC com período de 72 horas). Concluiu-se que: a) o vigor de lotes de sementes de fumo, dos híbridos CSC 405, K 326 e CSC 459 podem ser avaliados através do teste de envelhecimento acelerado com solução salina saturada utilizando temperatura de 45±2ºC e período exposição de 72 horas. b) o vigor de lotes de sementes de fumo dos híbridos CSC 405 e CSC 459 podem ser avaliados: - através do teste de envelhecimento acelerado tradicional utilizando temperatura de 38±2ºC e período de exposição de 18 horas ou temperatura de 41±2ºC e período de 60 horas; - através do teste de envelhecimento acelerado com solução salina não saturada utilizando temperatura de 38±2ºC e período de 18 horas ou temperatura de 41±2ºC e período de 48 horas; - através do teste de envelhecimento acelerado com solução salina saturada utilizando temperatura de 41±2ºC e período de 48, 60 e 72 horas c) o vigor de sementes de fumo não é avaliado corretamente na primeira contagem do teste de germinação. O objetivo do segundo trabalho foi determinar a época de colheita das sementes, visando a obtenção de sementes com a maior qualidade fisiológica possível. Foram utilizadas sementes de cinco híbridos de fumo do tipo Virgínia - CSC 416, CSC 439, CSC 444, vii CSC 458 e CSC 459. As sementes foram semeadas em bandejas de isopor e conduzidas no sistema Float produzindo mudas de qualidade, atingindo o tamanho ideal para o transplante, o espaçamento entre linhas foi de 2,0m e 0,5m entre plantas, contendo cerca de 10.000 plantas.ha-1. Foram utilizados 500kg.ha-1 de NPK na formulação 10-16-10 de adubação de base e duas aplicações de salitre com 200kg.ha-1 de adubação de cobertura. De acordo com o manejo planejado realizouse a eliminação da haste principal, deixando duas a três hastes secundárias. As plantas foram polinizadas em diferentes dias, marcando as flores polinizadas. A qualidade fisiológica foi avaliada pelos testes de germinação e de envelhecimento acelerado conforme metodologia determinada no primeiro trabalho. Para determinar a coloração dos frutos no momento ideal de colheita, os frutos foram fotografados em todos os dias de coleta, para obter um padrão de cores visando facilitar futuras colheitas de sementes. Os resultados obtidos permitem concluir que: a. O ponto de maturidade das sementes de fumo varia do vigésimo primeiro ao vigésimo oitavo dia após a antese. b. A época de polinização não interfere no ponto de maturidade fisiológica. c. Não é possível determinar o ponto de maturidade fisiológica baseado na cor do capulho.

Dans un contexte de réduction des intrants chimiques, l’utilisation des phytosanitaires naturels stimulant l’immunité végétale ouvre la porte vers une nouvelle approche de protection des plantes. Ces composés éliciteurs regroupés sous le terme de «Stimulateurs des Défenses naturelles des Plantes» (SDP) activent le système défensif de la plante la rendant plus résistante aux bio-agresseurs. Les SDP, de nature diverse, se présentent sous forme de composés uniques ou en mélange dans les extraits végétaux. Au cours de ma thèse, nous avons démontré l’activité SDP des extraits de marc de raisin. Les extraits issus de sous-produits de la vigne, marc de raisin rouge, marc de raisin blanc et pépins de raisin induisent diverses réactions de défense au sein de plantes modèles. Nous avons focalisé notre étude sur l’extrait de marc de raisin rouge (EMR) stimulant l’immunité chez le tabac. Infiltré sur feuilles, l’EMR induit la réponse de type HR caractérisée par l’apparition de lésions chlorotiques et accumulation de composés autofluorescents dans les tissus infiltrés. Ces réactions de défense locales ont été observées également chez l’arabette et la tomate. L’EMR déclenche les réponses LAR et SAR avec l’accumulation des transcrits des gènes de défense dans les feuilles de tabac et ce quelque soit son mode d’application (infiltration ou pulvérisation). Le mode d’action de l’EMR a été abordé sur cultures cellulaires de tabac BY-2. L’EMR induit une forte alcalinisation du milieu extracellulaire avec une mobilisation du calcium (Ca2+), l’expression des gènes de défense et la mort cellulaire. Une étude pharmacologique de la mort cellulaire suggère la mise en place de mort cellulaire programmée (PCD) dans les cellules de tabac. La caractérisation de la voie de signalisation activée par l’EMR a été étudiée avec le mutant NahG de tabac incapable d’accumuler l’acide salicylique (SA). Les réponses de défense (HR, LAR et SAR) sont faiblement induites par l’EMR chez le mutant nahG. L’EMR provoque une réponse de type HR fortement réduite avec une faible accumulation des composés autofluorescents et une diminution drastique de l’accumulation des transcrits des gènes PR suggérant l’intervention du SA dans l’induction des réactions de défense. Le degré de protection induit par l’EMR a été déterminé sur le pathosystème tabac/Phytophthora parasitica. Pulvérisé sur feuilles, l’EMR réduit de 45% les zones infectées par l’oomycète. Ce degré de protection semble être le résultat de l’activité antimicrobienne de l’EMR combinée à l’activité SDP. Aucune protection n’a été observée chez le mutant nahG confirmant l’implication de SA dans la résistance induite par l’EMR. Le fractionnement de l’EMR a permis de simplifier la formule active des extraits de raisin et d’identifier un mélange de molécules potentiellement capables d’induire l’activité SDP. Les composés actifs sont de nature polyphénolique et contiennent de la procyanidine B2 capable à elle seule d’induire la réponse de type HR et l’expression de l’antimicrobien PR1. Cependant, il semble que cette molécule agisse en association avec d’autres composés polyphénoliques pour stimuler le système défensif de la plante. / In order to reduce chemical inputs, the use of natural phytosanitary products stimulating plant immunity are emerging approaches in phytoprotection. These elicitor compounds known as "Plant Defense Inducers" (PDI) activate the plant defense system and improve their resistance to pests attack. PDI are single molecule or mixture of compounds extracted from plant. In my thesis, we demonstrated the PDI activity of different grape marc extracts. The winery byproducts, red grape marc extract, white grape marc extract and grape seed extract all induced various defense reactions in several plant models. We focused our study on the red grape marc extract (GME) which stimulates the immunity system in tobacco plants. When infiltrated into tobacco leaves, GME induced HR-like response characterized by the appearance of chlorotic lesions and accumulation of autofluorescent compounds in infiltrated tissues. Similar local defense reactions have been observed in Arabidopsis thaliana and tomato. GME also triggered LAR and SAR responses and induced defense gene transcript accumulation in tobacco leaves after infiltration or spraying. The GME mode of action was studied using the suspension-cultured cells of tobacco BY-2. GME induced rapid alkalinization of extracellular medium with calcium mobilization, expression of defense genes and cell death. A pharmacological approach of this defensive phenomenon suggests the establishment of programmed cell death (PCD) in tobacco cells. The characterization of the signaling pathway activated by GME was studied using tobacco nahG mutant unable to accumulate salicylic acid (SA). Defense responses (HR, LAR and SAR) induced by GME were impaired in the nahG mutant. GME drastically reduced HR-like response symptoms and PR transcript accumulation. These data suggest the implication of SA in the GME-induced plant defense reactions. The GME-induced protection was evaluated in the model pathosystem of compatible interaction between Nicotiana tabacum and Phytophthora parasitica var. nicotianae (Ppn). GME could reduce by 45% the infected areas induced by the oomycete on tobacco leaves. This level of protection was the result of the combined antimicrobial and PDI actions of GME. GME had no protecting effect against Ppn on NahG leaves evidencing the involvement of SA in the GME-induced resistance. GME fractionation led to identification of a bioactive molecule mixture capable of inducing the PDI activity. The active compounds are polyphenolics and involve procyanidin B2 which is by itself able to induce the HR-like response and PR1 transcript accumulation. This compound should act in combination with other polyphenolic molecules to stimulate the full plant defense reactions.

Les bactéries du sol produisant des antifongiques comme le 2,4-diacétylphloroglucinol(DAPG) protègent les racines des plantes vis-à-vis des champignons phytopathogènes. Néanmoins, les conditions de fonctionnement de ces populations bactériennes dans le sol restent très mal connues. Dans certains sols, dits résistants aux maladies, ces bactéries phytoprotectrices sont présentes à des effectifs importants et leur activité est suffisante pour protéger la plante malgré la présence du pathogène. L'objectif de cette thèse a été de comprendre la relation entre la résistance des sols à la maladie de la pourriture noire des racines de tabac, et la fonction de synthèse du DAPG chez les bactéries du genre Pseudomonas. Dans la situation de référence de Morens (Suisse), les sols résistants diffèrent des sols sensibles par la présence de vermiculite, argile capable de relarguer du fer. On sait que la présence de vermiculite améliore la phytoprotection assurée par les Pseudomonas producteurs de DAPG, mais les mécanismes moléculaires sous-jacents restent inconnus. Dans un premier temps, la quantification de ces bactéries par une nouvelle méthode de PCR quantitative développée ici, a confirmé que leurs effectifs sont élevés dans les sols résistants, mais aussi dans les sols sensibles, suggérant que la résistance puise plutôt dépendre d'une plus forte expression de la fonction de synthèse du DAPG. Dans un second temps, l'étude de l'expression des gènes de synthèse du DAPG en système de sol artificiel, à l'aide de la souche rapportrice P. protegens phlA-gfp, a montré que la présence de vermiculite dans le sol se traduit par une plus forte biodisponibilité du fer pour les Pseudomonas, induisant une plus forte expression des gènes de synthèse du DAPG et la protection du tabac. En conclusion, la résistance des sols de Morens à la maladie de la pourriture noire des racines est conditionnée par plusieurs facteurs abiotiques et biotiques, dont la biodisponibilité du fer qui régule l'expression des gènes de synthèse du DAPG chez Pseudomonas / Soil bacteria producing antimicrobial compounds like 2,4-diacetylphloroglucinol (DAPG) protect plants from soil-borne phytopathogens. Nevertheless, the functioning of these bacterial populations in the soil is largely unknown. In certain soils, termed disease- suppressive soils, these bacteria are present at high numbers and their activity is sufficient to assure effective plant protection in the presence of the pathogen. The aim of this thesis was to understand the relation between soil suppressiveness towards black root rot of tobacco, and the 2,4-diacetylphloroglucinol synthesis ability of certain Pseudomonas. In Morens region (Switzerland), suppressive soils differ from conducive soil by the presence of vermiculite, an iron-releasing clay. It is known that DAPG-producing Pseudomonas provide better plant protection in the presence of vermiculite, but the molecular basis of this interaction is still unknown. First, the quantification of these bacteria, through a new real-time PCR method developed here, confirmed that high numbers of DAPG-producing Pseudomonas occur in suppressive soils, as well as in conducive ones, raising the possibility that suppressiveness depends rather on a higher expression of DAPG synthetic genes. Second, expression studies of DAPG synthetic genes using a P. protegens ph/A- gfp reporter strain and artificial soil systems, confirmed that the presence of vermiculite in the soil can translate into higher iron bioavailability for Pseudomonas, triggering higher expression of DAPG synthetic genes and effective plant protection. In conclusion, black root rot suppressiveness of Morens soils is determined by several abiotic and biotic factors, among which iron bioavailability regulating the expression of DAPG synthetic genes in plant-protecting Pseudomonas

Nous avons défini le complexe tabagique selon quatre champs applicables : les pipes à fumer, le tabac, le rôle du tabac dans les rapports Autochtones-Européens et dans les échanges. Le but de ce mémoire est de mieux saisir ce que les pipes à fumer de la période historique nous dévoilent dans le contexte de l’archipel montréalais. Le dépouillement des rapports de fouille a permis de retirer d’innombrables fragments de pipes à fumer que nous retrouvons dans les contextes archéologiques de l’archipel montréalais pour la période 1642−1760. Les récits d’époque nous informent sur les habitudes reliées à la consommation du tabac ainsi que les rôles du tabac et des pipes à fumer dans les relations entre Autochtones et Européens et des processus d’échange. Des recherches sur le tabac sont venues ajouter des éléments cruciaux à ces deux sources d’information. Cette étude a été limitée à l’archipel montréalais, plus une aire de 10 km autour de celui-ci. La période étudiée est de 1642 à 1760. Nous avons constaté que les fragments des pipes à fumer se retrouvent majoritairement dans les zones de contact démontrant ainsi l’importance de ces objets dans les échanges. Ces zones de contact sont les endroits où le métissage prend place. Le tabac, que nous ne pouvons dissocier des pipes à fumer, joue aussi un rôle majeur dans les relations amérindiennes-européennes. Des dons de Nicotiana tabacum ont permis de solidifier des liens de confiance primordiaux entre les deux groupes dans les processus d’échange. Le tabac et les pipes à fumer, ont aussi subit le processus de transfert culturel, mais, le degré varie selon le type de pipes à fumer. / We defined the smoking complex according to four applicable fields: smoking pipes, tobacco, Indigenous -European relations and the role of tobacco in trade. The purpose of this research is to better understand what smoking pipes reveal to us in the context of the Montreal archipelago. The review of the excavation reports allowed us to retrieve information regarding the innumerable fragments of smoking pipes that we find in the archaeological contexts of the Montreal archipelago for the period 1642−1760. The ethnohistorical publications inform us about the habits and customs related to the use of tobacco as well as the roles that tobacco and smoking pipes played in Indigenous-European relations and exchanges. Tobacco studies have also added crucial elements to these two sources of information. This study was limited to the Montreal archipelago, plus an area of 10 km around it. The study period is from 1642 to 1760. We found that the fragments of smoking pipes are mostly found in contact zones demonstrating the cultural importance of these objects. Nicotiana tabacum strengthened bonds of trust between the two groups. These contact zones are the places where métissage takes place. Inseparable from smoking pipes, tobacco also played a major role in Amerindian-European relations. Gifts of Nicotiana tabacum favoured consolidation between the two groups and built the primordial trust necessary in exchange processes. Tobacco and smoking pipes are also part of the process of cultural transfer, but to a variable degree according to the type of smoking pipes.

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style. / Dissertation (MSc)--University of Pretoria, 2003. / Genetics / Unrestricted

Dans son environnement la plante est confrontée à une variété de microorganismes bénéfiques, neutres et pathogènes, qui sont fortement dépendants des ressources carbonées qu’elle libère dans le sol. Le transport de sucres, processus clé de la physiologie de la plante, est essentiel pour les interactions plantes-microorganismes et leur devenir. Au cours de l'évolution, les plantes ont acquis des mécanismes leur permettant de percevoir les signaux microbiens du milieu extérieur, et aboutissant à la transduction d’un signal spécifique puis à des réponses biologiques adaptées (défense versus mutualisme) à la stratégie du microorganisme. Ces réponses assurent la survie et le développement des plantes. Mes travaux de thèse, menés avec un système « d’interaction » simplifié, contribuent à une meilleure compréhension des mécanismes sous-jacents au déterminisme des interactions plantes-microorganismes. Ce système a permis d’étudier, sur des suspensions cellulaires de N. tabacum, les réponses cellulaires précoces déclenchées suite à la perception de molécules microbiennes provenant de microorganismes à stratégie pathogène avirulent ou à stratégie mutualiste. Nous avons mesuré des évènements de signalisation et des flux de sucres induits en réponse à ces molécules microbiennes. Nos résultats ont mis en évidence que les chitotétrasaccharides (CO4), sécrétés par les champignons mycorhiziens à arbuscules dans les stades pré-symbiotiques de l’interaction, mobilisent les mêmes événements de signalisation précoce (H2O2 dépendant de la protéine rbohD, Ca2+ cytosolique, activation de MAPK) que la cryptogéine, un éliciteur des réactions de défense ; mais avec des réponses différentes en terme d’intensité et de cinétique. Les CO4 et la cryptogéine ont par ailleurs montré des impacts distincts sur les flux de sucres et l’expression de transporteurs impliqués. En complément nous avons montré un effet de la modification de la dynamique membranaire associée à la clathrine sur des évènements de signalisation déclenchés par la cryptogéine, ainsi que dans les flux entrants de sucres et l’expression de gènes de transporteurs de sucres. Enfin, l’analyse in silico de l’interactome de transporteurs de sucres chez la plante modèle A. thaliana, nous a permis d’apporter des connaissances supplémentaires quant aux évènements de régulations des transporteurs de sucres et l’identification de protéines régulatrices putatives en interaction avec ces derniers. L’ensemble de ces travaux ouvrent la voie à de nouvelles recherches visant à élucider les mécanismes cellulaires et moléculaires impliqués dans la mise en place des interactions entre plantes et microorganismes. / In their natural environment plants are in close interaction with beneficial, neutral, or pathogenic microbes, which are highly dependent on carbon resources exuded by plant roots. Sugar transport, which is a key process of plant physiology, is essential to support the fate of plant-microbe interactions. During evolution, plants have acquired the ability to perceive microbial molecules, initiating specific signal transduction cascades and leading to adapted response for microbe lifestyles (avirulent, virulent, or benefic). Plant survival will depend on the nature of the induced mechanisms. My PhD work, carried out on a simplified experimental system, contributes to the understanding of mechanisms underlying the determinism of plant-microbe interactions. We used Nicotiana tabacum cells in suspension exposed to microbial molecules derived from mutualistic or avirulent microbes. Using such a simplified system, we analyzed elements of the early signaling cascade and sugar fluxes. We have shown that CO4, which is originating from AMF, initiate early signaling components (rbohD-dependent H2O2, cytosolic Ca2+, MAPK activation) as cryptogein, a defense elicitor, but with distinct profile and amplitude. Those two molecules (CO4 and cryptogein) are responsible of different effects on sugar fluxes and the expression of the underlying sugar transporter genes. In addition, we presented an impact of the alteration of clathrin-mediated process on early signaling events triggered by cryptogein, as well as inward sugar fluxes and expression of sugar transporter genes. Finally, in silico analyses of sugar transporter interactome in Arabidopsis thaliana has provided some possible regulation mechanisms through the identification of new candidate proteins involved in sugar transporter regulation. These information open new perspectives towards a better understanding of the cellular and molecular mechanisms involved in plant-microbe interactions.

The aim of this study was to verify the possibility of ibuprofen degradation by selected plant cultures and determination of activities of antioxidant enzymes (peroxidase, catalase, ascorbate peroxidase and glutathione-S-transferase) as markers of oxidative stress caused by ibuprofen. Nicotiana tabaccum (cv. La Burley 21, cv. SR 1 and their GMOs) and Nicotiana glauca were used as experimental plants. The rate of removal of ibuprofen tested by tobacco was decreasing in the following order: N. tabaccum SR1 > N. tabaccum Zm-P60-1-T4 > N. tabaccum TRI 2T2 > N. glauca > N. tabaccum TRI 2T1 > N. tabaccum cv. La Burley > N. tabaccum Zm-P60-1-T5. As the most suitable tobacco for the removal of ibuprofen seemed untransformed N. tabaccum SR1. The long-term experiment showed that plant stress is being manifested even after longtime. N. tabaccum cv. La Burley 21 seemed to be the most tolerant to ibuprofen in compare with the total enzyme activities in cultures with the presence of ibuprofen and controls. N.glauca was the least tolerant cultivar. Keywords: phytoremediation, ibuprofen, Nicotiana tabaccum, Nicotiana glauca, HPLC, peroxidase, catalase, ascorbate peroxidase, glutathion-S-transferase