A soja é uma das culturas mais antigas e praticadas no mundo, tendo sua importância pelo elevado teor de proteínas, sendo utilizada na alimentação humana e animal, além de ser o principal produto de exportação brasileira. Apesar da alta produção nacional, a cultura possui perdas de produtividade pelos ataques de insetos-pragas, dentre eles, a mosca-branca Bemisia tabaci Biótipo B (Hemiptera: Aleyrodidae). Seu controle tradicional é realizado com inseticidas, mas atualmente métodos alternativos estão sendo avaliados, considerando a não contaminação do ambiente e a saúde humana. O presente trabalho teve como objetivo avaliar o potencial de indutores de resistência no processo de defesa vegetal contra a mosca-branca em plantas de soja. Foram realizados dois experimentos na Universidade Tecnológica Federal do Paraná, Campus Dois Vizinhos-PR, no ano de 2017. Sementes de soja da cultivar BRS 284 foram semeadas em vasos de polietileno com capacidade para 10 litros, contendo solo proveniente de lavoura. O cultivo ocorreu em casa de vegetação, sendo que os vasos ficaram dispostos nas bancadas até a fase fenológica V6, onde foram aplicados os indutores conforme os tratamentos. Os tratamentos foram aplicados por microasperção sendo: ASM (0,005%), AS (2Mm), fertilizante foliar composto por fosfito de potássio (0,004%); quitosana (1%) , silício (0,25%) e testemunha (água destilada). O primeiro experimento buscou avaliar o potencial dos indutores quanto a capacidade de ativar mecanismos de defesa vegetal, considerando a presença e a ausência da mosca branca. Para tanto, aplicou- se os indutores, sendo que para a condição de ausência de insetos, os vasos permaneceram em gaiola individual com tela anti-afídica, para evitar o contato com o inseto praga. Então, realizou-se a coleta do material vegetal em intervalos de 0, 24, 48, 96 e 168 horas após a aplicação dos indutores de resistência. Avaliaram-se proteínas totais, açúcares totais e redutores, compostos fenólicos, taninos e a atividade das enzimas peroxidases, fenilalanina amônia-liase (FAL) e quitinase. O segundo experimento buscou avaliar a preferência de oviposição em função da aplicação dos indutores. Após 24 horas da aplicação dos indutores, 500 adultos coletados não sexados de mosca-branca foram liberados no centro dos vasos sobre a bancada, tendo chance de escolha entre os tratamentos. Após 48 horas da infestação inicial, coletaram-se dois folíolos do terço mediano das plantas, de cada tratamento. Os folíolos foram avaliados em microscópio estereoscópio para a quantificação do número de ovos. A área foliar total dos folíolos também foi calculada, utilizando-se o software Image J. Os indutores de resistência possuem capacidade de ativar o metabolismo primário através da síntese de proteínas totais, bem como demonstram potencial na ativação de mecanismos de defesa entre eles, a rota dos fenilpropanóides com a ativação da enzima FAL e a formação de compostos fenólicos. Ainda demonstram ativar enzimas relacionadas a patogenicidade como as peroxidases e quitinase, tais ativações possuem especificidade quanto ao indutor e o tempo de ativação. O uso dos indutores quando desafiados com insetos demonstraram maior efetividade de ativação da enzima FAL, peroxidade e quitinase, enzimas estas relacionadas ao processo de defesa vegetal a insetos. Os indutores ASM, silício e quitosana possuem potencial de redução da oviposição da mosca-branca, o que pode estar relacionado a ativação de mecanismos de defesa vegetal. / Soybean is one of the oldest and most practiced crops in the world,and its importance is due to its high protein content,its use in food for humans and animals, as well as being the main Brazilian export product. Despite the high national production, the crop has productivity losses due to attacks of insect pests, among them, the whitefly Bemisia tabaci Botype B (Hemiptera: Aleyrodidae). Its traditional control is carried out with insecticides, but currently alternative methods are being evaluated considering the noncontamination of the environment and human health. The present work had as objective to evaluate the potential of resistance elicitors in the process of vegetal defense against the whitefly in soybean plants. Two experiments were carried out at the Federal University of Technology - Paraná, Campus Dois Vizinhos-PR, in 2017. Soybean seeds of cultivar BRS 284 were sown in polyethylene pots with capacity of 10 liters, containing soil gathered from crops at UTFPR. Cultivation occurred in a greenhouse, and the vessels were placed on benches until the V6 phenological phase, when the elicitors were applied by microaspersion according to the treatments: ASM (0.005%), SA (2 Mm), foliar fertilizer composed of potassium phosphite (0.004%); chitosan (1%), silicon (0.25%) and control (distilled water). The first experiment aimed to evaluate the potential of the elicitors to activate plant defense mechanisms, considering the presence and absence of the whitefly. For this, the elicitors were applied, and for the condition of absence of insects, the vases remained in individual cages with anti-aphid screen, to avoid contact with the insect pest. Then, the plant material was collected at intervals of 0, 24, 48, 96 and 168 hours after the application of resistance elicitors. Total proteins, total and reducing sugars, phenolic compounds, tannins and the activity of the enzymes peroxidases, phenylalanine ammonia-lyase (FAL) and chitinase were evaluated. The second experiment sought to evaluate the oviposition preference due to the application of the elicitors. After 24 hours of application, 500 not sexed whitefly adults were released in the center of the vases on the bench, having a choice among treatments. After 48 hours of the initial infestation, two leaflets of the median third of the plants were collected from each treatment. The leaflets were evaluated under stereomicroscope for the quantification of the number of eggs. The total leaf area of the leaflets was also calculated using Image J. The resistance elicitors have the capacity to activate the primary metabolism through the synthesis of total proteins, as well as demonstrate the potential in the activation of defense mechanisms among them, the route of the phenylpropanoids with the activation of the enzyme FAL and the formation of phenolic compounds. They also activated pathogenic enzymes such as peroxidases and chitinase, such activations have specificity for the elicitor and the activation time. The use of elicitors when challenged with insects demonstrated greater effectiveness of activation of the enzyme FAL, peroxidase and chitinase, these enzymes related to the process of plant defense against insects. ASM, silicon and chitosan elicitors have the potential to reduce oviposition of the whitefly, which may be related to the activation of plant defense mechanisms.

Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq) / Os terpenos constituem a classe de produtos naturais com maior diversidade química e estrutural, estando associados ao metabolismo das plantas e às interações destas com outros organismos. Estes compostos, as enzimas que os sintetizam e as plantas que os produzem são amplamente estudados em diferentes aspectos. Para melhor compreensão da expressão de terpenos em plantas sob estresse biótico, as espécies Varronia curassavica e Pistacia palaestina foram estudadas neste trabalho. Folhas de V. curassavica contém óleo essencial rico em sesquiterpenos com propriedades anti-inflamatórias, especialmente α-humuleno and β-cariofileno. O objetivo deste estudo foi avaliar as respostas do seu metabolismo em função da aplicação de dois eliciadores naturais no conteúdo de sesquiterpenos de V. curassavica. Para isso, as plantas receberam a aplicação de acibenzolar-S-metil (500 mg L-1), 1,6 β-D-glucano obtido a partir de Lasiodiplodia theobromae (50 mg L-1) e água destilada como controle, sendo realizada as avaliações de trocas gasosas, atividade das enzimas fenilalanina amônia-liase, superóxido dismutase, peroxidase e catalase, bem como a análise química do óleo essencial. Acibenzolar-S-metílico reduziu significativamente a taxa líquida de assimilação de carbono e a concentração intercelular de CO2, enquanto que 1,6 β-D-glucano reduziu significativamente apenas a concentração intercelular de CO2. O maior rendimento de óleo essencial (0.819%) foi obtido em plantas eliciadas por 1,6 β-D-glucano. As proporções relativas e a quantidade de α-humuleno e β-cariofileno não diferiram entre os tratamentos, porém os eliciadores aumentaram significativamente a atividade da enzima guaiacol peroxidase. Os terpenos estão presentes nas folhas e galhas de Pistacia palaestina. Apesar do mecanismo de desenvolvimento das galhas ainda não ter sido completamente elucidado, sabe-se que espécies de afídeos como Baizongia pistaciae L. manipulam anatomicamente, fisiologicamente e quimicamente as plantas hospedeiras em seu benefício. Por este motivo, o isolamento e a caracterização funcional dos genes que codificam terpeno sintases em galhas induzidas por B. pistaciae, bem como sua expressão relativa por RT-qPCR em folhas e galhas de P. palaestina foram os objetivos deste trabalho. A expressão heteróloga foi realizada em E. coli pLYS-BL21, sendo as reações enzimáticas feitas com proteínas purificadas e usando geranil difosfato (GPP) ou farnesil difosfato (FPP) para testar a atividade das enzimas como mono- e sesquiterpeno sintases, respectivamente. Para o experimento de RT-qPCR, foi selecionado um gene referência entre actina, ciclofilina, fosfoglicerato quinase, RNA polimerase II, α-tubulina e ubiquitina. Em seguida, realizaram-se reações com as terpeno sintases para avaliar as diferenças nos níveis de expressão em folhas não colonizadas e galhas. Foram isoladas e caracterizadas duas monoterpeno sintases (PpTPS281 e PpTPS809) e uma sesquiterpeno sintase (PpTPS232) em P. palaestina. PpTPS281 produziu exclusivamente D-limoneno a partir de GPP, enquanto PpTPS809 produziu vários monoterpenos a partir de GPP e PpTPS232 catalisou a formação de diferentes sesquiterpenos a partir de FPP. Os resultados da RT-qPCR mostraram que o gene actina é o mais apropriado para ser usado na comparação de expressão de genes de folhas não colonizadas e galhas induzidas por B. pistaciae. Os níveis de expressão dos três genes foram significativamente aumentados em galhas (de 2,21- a 96.5-vezes), quando comparados com folhas. / Terpenes are a large and diverse class of natural products, being associated with plant metabolism and interactions with other organisms. Nowadays compounds and enzymes of the terpenes pathway in plants are widely studied in different aspects. Varronia curassavica and Pistacia palaestina were the selected species to study the biotic stress effects on terpenoids expressions. Leaves of V. curassavica are the commercial source α-humulene and β-caryophyllene, sesquiterpenes with anti-inflammatory properties. The objective of this study was to evaluate the effect of two natural elicitors on the sesquiterpene content of V. curassavica. Thus, field grown plants received the application of acibenzolar-S-methyl (500 mg L-1), 1,6 -D-glucan obtained from Lasiodiplodia theobromae (50 mg L-1) and distilled water as a control. Gas exchange rate, terpene enzymes such as phenylalanine ammonia-lyase, superoxide dismutase, guaiacol peroxidase and catalase activity and essential oil content in leaves were measured. Acibenzolar-S-methyl reduced significantly the net carbon assimilation rate and the intercellular CO2 concentration, while1,6 -D-glucan reduced significantly only the intercellular CO2 concentration. The highest essential oil yield (0.819%) was obtained in plants elicited with 1,6 -D-glucan. The content of α-humulene and β-caryophyllene did not differ among treatments however the elicitors provided a significant increase in guaiacol peroxidase activity. Terpenes are present in Pistacia palaestina in leaves and in horn-shaped galls. The mechanism of gall development remains unknown, however it is clear Baizongia pistaciae L., an aphid species, manipulates their hosts anatomy, physiology, and chemistry for their own benefit. To isolate and functional characterize terpene synthase genes from galls induced by B. pistaciae as well as their gene relative expression by RT-qPCR in leaves and galls of P. palestina were the aims of this study. The heterologous expression was performed in E. coli pLYS-BL21 cells, being enzymatic assay reactions made with the purified proteins using geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) to test for possible mono- and sesquiterpene synthase activity, respectively. For relative real-time PCR, it was selected an appropriate reference gene between actin, cyclophilin, phosphoglycerate kinase, RNA polymerase II, α-tubulin and ubiquitin. After selection, it was performed reactions with terpene synthases genes to evaluate differences in expression levels in P. palaestina non-colonized leaves and galls. Two monoterpene synthases (PpTPS281 and PpTPS809) and one sesquiterpene synthase (PpTPS232) were isolated and characterized in P. palaestina. PpTPS281 produced exclusively D-limonene from GPP, while PpTPS809 produced several monoterpenes from GPP and PpTPS232 catalyzed the formation of different sesquiterpenes from FPP. Real-time PCR results showed that actin is the most proper gene to be used for genes expression studies between non-colonized P. palaestina leaves and galls induced by B. pistaciae. The levels of expression of the genes were significantly upregulated in galls (from 2,21- to 96.5-fold) when compared to leaves.

Le cuivre (Cu) est un nutriment essentiel à la vie des organismes mais aussi, lorsqu’il est présent en excès, un constituant toxique de la cellule. Pour faire face à des concentrations élevées en Cu dans l’environnement, les plantes ont développé des mécanismes complexes d’homéostasie et de tolérance au Cu. L’objectif de ce travail est d’apporter une contribution à la compréhension de ces mécanismes en utilisant la plante modèle Arabidopsis thaliana. Dans un premier temps, nous avons montré que l’excès de Cu2+ entraînait une réorganisation de l’architecture du système racinaire caractérisée par une inhibition de la croissance de la racine primaire et l’augmentation de la densité de racines latérales. Nous avons mis en évidence plusieurs processus métaboliques qui pourraient être impliqués dans cette réorganisation, tels que des changements dans l’homéostasie minérale, le statut hormonal, et la production de lignine. Une approche de génétique classique a ensuite été entreprise afin de mieux comprendre les composants moléculaires impliqués dans cette réponse. Un criblage de mutants a été effectué sur excès de Cu2+ dans le but d’isoler des mutants présentant un phénotype racinaire altéré par le Cu2+. Un mutant sensible au Cu2+ (appelé cop29) a été sélectionné en raison de sa forte inhibition de croissance racinaire sur excès de Cu2+. Le clonage positionnel du mutant, combiné avec des approches génomiques, a permis d’identifier le gène At3g14190 comme étant le gène muté responsable du phénotype de cop29. Ce gène encode une protéine de fonction encore inconnue. La caractérisation phénotypique de cop29 a révélé que le mutant était également sensible à l’excès de Zn2+, Mn2+ et Na+. De plus, nous avons montré qu’en présence d’un excès de Cu2+ ou de NaCl le mutant présentait des concentrations en K+ significativement plus faibles que le type sauvage. Etant donné que le maintien de l’homéostasie du K+ joue un rôle essentiel dans la tolérance des plantes à l’excès de Cu ou de NaCl, nous avons émis l’hypothèse que ces faibles concentrations en K+ pourraient être la cause du phénotype de cop29. Par ailleurs, l’utilisation des banques de données génomiques et protéomiques a mis en avant que la protéine COP29 pourrait également jouer un rôle dans la régulation du cycle cellulaire. Les perspectives consisteront à découvrir la fonction de la protéine COP29 et contribueront ainsi à mettre en évidence un nouveau composant moléculaire impliqué dans la tolérance des plantes aux stress. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

This research explored fate of organic contaminants in aquatic plant systems through (i) experimental development of relationships to describe sorption, uptake and enzymatic processing of contaminants by plants and inhibition of aquatic plants by contaminants and (ii) incorporation of experimental relationships into a conceptual model which describes contaminant fate in aquatic plant systems. This study focused on interactions of aquatic plants L. minor and M. aquaticum with halogenated phenols. 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4-dichlorophenol (2,4-DCP) are precursors for the highly toxic and heavily applied herbicides 2,4,5-T and 2,4-D and were examined in detail. Chlorophenols are generally resistant to microbial degradation, a property which may limit microbial remediation options as effective alternatives for clean up of contaminated sites. Relationships for fundamental interactions between plants and contaminants that dictate uptake, enzymatic processing and sequestration of contaminants by aquatic plants were established. An assay which quantified production of oxygen by plants was developed to quantify plant metabolic activity and inhibition. Uptake of chlorinated phenols depended on plant activity and aqueous phase concentration of contaminant in the protonated form. Therefore, plant activity, contaminant pKa and media pH were established as critical parameters controlling rate of contaminant uptake. A conceptual model was developed which incorporated plant activity and inhibition into a mathematical description of uptake of organic contaminants by aquatic plants. The conceptual model was parameterized using experimental data delineating effect of plant activity, inhibition and speciation on contaminant uptake and the model was verified using independently gathered data. Experimentation with radio-labeled chlorinated phenols established that contaminants were sequestered internal to plants by plant enzymatic processing. 19F NMR was established as a technique to quantify transformation and conjugation products internal to plants and contaminant assimilation by plants and demonstrated that multiple metabolites containing the parent compound were present and quantifiable internal to plants. Finally, fate of plant-sequestered contaminants in an anaerobic bioassay was examined using Desulfitobacterium sp. strain Viet1. The results of this study address the role of aquatic plants in sequestration of contaminants in surface waters that indicate the potential and limitations of use of aquatic plants in natural and engineered treatment systems.

Durant cette thèse nous nous sommes attachés au développement d'une méthode globale de création de réseaux métaboliques chez des espèces biologiques non classiques pour lesquelles nous possédons peu d'informations. Classiquement cette reconstruction s'articule en trois points : la création d'une ébauche métabolique à partir d'un génome, la complétion du réseau et la vérification du résultat obtenu. Nous nous sommes particulièrement intéressés au problème d'optimisation combinatoire difficile que représente l'étape de complétion du réseau, en utilisant un paradigme de programmation par contraintes pour le résoudre : la programmation par ensemble réponse (ou ASP). Les modifications apportées à une méthode préexistante nous ont permis d'améliorer à la fois le temps de calcul pour résoudre ce problème combinatoire et la qualité de la modélisation. L'ensemble de ce processus de reconstruction de réseau métabolique a été appliqué au modèle des algues brunes, Ectocarpus siliculosus, nous permettant ainsi de reconstruire le premier réseau métabolique chez une macro-algue brune. La reconstruction de ce réseau nous a permis d'améliorer notre compréhension du métabolisme de cette espèce et d'améliorer l'annotation de son génome. / In this thesis we focused on the development of a comprehensive approach to reconstruct metabolic networks applied to unconventional biological species for which we have little information. Traditionally, this reconstruction is based on three points : the creation of a metabolic draft from a genome, the completion of this draft and the verification of the results. We have been particularly interested in the hard combinatorial optimization problem represented by the gap-filling step. We used Answer Set Programming (or ASP) to solve this combinatorial problem. Changes to an existing method allowed us to improve both the computational time and the quality of modeling. This entire process of metabolic network reconstruction was applied to the model of brown algae, Ectocarpus siliculosus, allowing us to reconstruct the first metabolic network of a brown macro-algae. The reconstruction of this network allowed us to improve our understanding of the metabolism of this species and to improve annotation of its genome.