Hemicelluloses are synthesized by proteins encoded by genes from the cellulose synthasegene superfamily. One subgroup of this gene family is the cellulose synthase-like B, which islargely uncharacterized and unexplored. The common model organism Nicotianabenthamiana has one such gene in its genome, NbCslB, encoding a membrane protein. Theexpression of this gene has previously been studied in vivo, but in order to study the protein invitro a viable solubilization and purification protocol is required. This study evaluated the useof the detergent n-Dodecyl β-D-maltoside (DDM) for solubilization, followed by purificationusing immobilized metal ion affinity chromatography (IMAC), and thereafter reconstitutionof the protein into proteoliposomes. SDS-PAGE as well as Western blot analyses showed thatthe purification was successful and provided a pure sample of protein. Throughout theanalyses performed, an anti-FLAG antibody was discovered to bind well to the protein, andthereby be especially useful for analysis. An activity assay was performed on the purifiedprotein, to characterize its function and evaluate whether the protein had maintained itsactivity and conformation after the steps of purification and reconstitution. No activity couldbe detected in the enzymatic assay, which indicated that the purification protocol may havebeen too rough on the protein, that the reconstitution was not successful, or that the assayconditions were not optimal. These results can be used as a base for future research, where theprotocols for solubilization, purification, and reconstitution should be further refined in orderto obtain an end result where the purified protein is active. When an active and pure proteinsample is achieved, it will be possible to perform further attempts at characterizing thefunction of the protein using enzymatic activity assays. Additionally, the results showed thatthe choice of antibody can be crucial for proper analysis of this protein. / Hemicellulosa syntetiseras av proteiner vars gener återfinns i genfamiljen cellulosasyntas. Enundergrupp till denna genfamilj är cellulosasyntasliknande B, en grupp som till stor del ärokarakteriserad och outforskad. Den vanliga modellorganismen Nicotiana benthamiana haren sådan gen i sitt genom, NbCslB, som kodar för ett membranprotein. Hur denna genuttrycks har tidigare studerats in vivo, men for att kunna studera proteinet in vitro krävs etthållbart protokoll för solubilisering och rening. Denna studie utvärderade användningen avlösningsmedlet n-Dodecyl β-D-maltoside (DDM) för solubilisering, följt av rening medimmobiliserad metalljon-affinitetskromatografi (IMAC), och efter det rekonstitution avproteinet till proteoliposomer. SDS-PAGE och Western blot analyser visade att reningen varlyckad, och att ett rent proteinprov erhållits. När analyserna genomfördes upptäcktes att enanti-FLAG antikropp band särskilt väl till proteinet, och därmed var mycket användbar vidanalys. En aktivitetsanalys genomfördes med det renade proteinet för att karakterisera dessfunktion och utvärdera huruvida proteinet hade bevarat sin aktivitet och konformation efterrening och rekonstitution. Ingen aktivitet kunde detekteras i den enzymatiskaaktivitetsanalysen, vilket indikerade att reningen eventuellt var för hård mot proteinet,alternativt att rekonstitutionen inte var lyckad, eller att förhållandena för analysen inte varoptimala. Dessa resultat kan användas som en bas för framtida forskning om proteinet, därprotokollen för solubilisering, rening och rekonstitution bör vidareutvecklas för att uppnå ettslutresultat där det renade proteinet är aktivt. När ett aktivt och rent proteinprov uppnåtts ärdet möjligt att genomföra ytterligare försök att karakterisera proteinets funktion medenzymatiska aktivitetsanalyser. Resultaten visade också att valet av antikropp kan varaavgörande för att ordentligt kunna analysera detta protein.

Объектом исследования были растения табака (Nicotiana tabacum L.), выращенные в трех разных условиях: контроль (р-р Кнопа), р-р Кнопа с добавлением 100 µM CuSO4 и 300 µM CuSO4. Цель работы – изучить анатомо-морфологическое строение, активность антиоксидантных ферментов растений табака в период последействия ионов меди, оценить способность табака к восстановлению после стресса. Исследовали всхожесть семян, биомассу и анатомо-морфологические характеристики корня, стебля и листа. Определяли биохимические параметры (содержание пероксида водорода, продуктов ПОЛ, фенольных соединений, активность ферментов – КАТ, АПО, СОД, цитозольных и апопластных ГПО и БПО, изоформы пероксидаз). Проведенное исследование дает представление о том, как изменяются прорастание, анатомо-морфологические и биохимические параметры растений N. tabacum при последействии ионов меди. Выявлено, что воздействие 100 µM привело к увеличению биомассы корня, при этом длина корней, высота стеблей, толщина корней и стеблей, а также площадь листьев снизились. Действие 300 µM привело к более сильному снижению данных параметров, а также уменьшению сухой массы стеблей и листьев. Содержание пероксида водорода и уровень ПОЛ увеличился при действии 100 и 300 µM. Содержание фенольных соединений снизилось в корнях при 100 µM и увеличилось в корнях и листьях и 300 µM и 100 µM соответственно. Влияние ионов меди активировало работу ферментов антиоксидантной защиты – АПО и КАТ во всем растении, цитозольной ГПО и апопластных ГПО и БПО в корнях, в то же время активность СОД снизилась в обоих вариантах опыта. Белковый электрофоре показал, снижение активности общих для тканей корня, стебля и листьев изоформ пероксидаз. Отмечено снижение ферментативной активности изоформ, найденных в стеблях, в случае предобработки растений 300 µM, при этом активность специфичных для корня изоформ оставалась высокой во всех вариантах опыта. / The object of the study were tobacco plants (Nicotiana tabacum L.) grown under three different conditions: control (Knop's solution), Knop's solution with 100 µM CuSO4 and 300 µM CuSO4. The purpose of the work was to study the anatomical and morphological structure, the activity of antioxidant enzymes in tobacco plants after prolonged exposure to copper ions and their after-action, to assess the ability of tobacco to recover from stress. Seed germination, biomass and anatomical and morphological characteristics of the root, stem and leaf were studied. Biochemical parameters were determined (the content of hydrogen peroxide and phenolic compounds, the activity of enzymes - CAT, APX, SOD, cytosolic and apoplastic GPX and BPX). The study gives an idea of how the germination, anatomical, morphological and biochemical parameters of N. tabacum plants change under the influence of copper ions. It was revealed that exposure to 100 μm led to an increase in root biomass, whereas the length of roots, height of stems, thickness of roots and stems, as well as leaf area were decreased. The effect of 300 μM led to even lower decrease in these parameters, as well as a decrease in dry weight of stems and leaves. The content of hydrogen peroxide and the level of lipid peroxidation increased upon exposure to 100 and 300 µM. The content of phenolic compounds decreased in roots at 100 µM and increased in roots and leaves at 300 µM and 100 µM, respectively. The influence of copper ions activated the work of antioxidant defense enzymes - APO and CAT in the whole plant, cytosolic GPO and apoplastic GPO and BPO in the roots, at the same time, SOD activity decreased in both variants of the experiment. Protein electrophoresis showed a decrease in the activity of peroxidase isoforms common for the root, stem and leaves. A decrease in the enzymatic activity of the peroxidase isoforms found in the stems was noted in plants pretreated with 300 μM, while activity of the root specific isoforms remained high in all variants of the experiment.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2015-2016 / Cette étude visait à caractériser la croissance, la capacité photosynthétique, la concentration en azote et protéines totales solubles, la production de protéines recombinantes (HA) ainsi que la quantité de lumière interceptée à différents stades de développement de plants de Nicotiana benthamiana afin d’optimiser la production de vaccins. L’évolution des réponses physiologiques étudiées fut similaire chez toutes les feuilles primaires, suggérant que le processus de sénescence s’initie et progresse de façon semblable indépendamment de leur ordre d’initiation. Toutefois, la superposition des patrons temporels de sénescence et de croissance foliaire a mené à un rendement HA maximal se situant invariablement dans la partie médiane du plant lorsqu’exprimé sur une base foliaire. À l’échelle du plant entier, nos résultats suggèrent qu’il est possible d’augmenter la production de vaccins en récoltant les plants à un stade de développement plus tardif, ou en augmentant la densité de culture et en récoltant ces plants plus tôt. / Nicotiana benthamiana is a wild relative of tobacco increasingly used as a plant protein expression platform to produce recombinant vaccine antigens against the influenza virus. Investigation on the physiological determinants of this production is essential to optimize and regulate vaccines production following a new flu outbreak. We examined the photosynthetic photon flux density, growth, light-saturated photosynthesis, total soluble protein, nitrogen content and recombinant protein production at different phenological stages. The similar evolution of the studied physiological responses suggested that the senescence process is initiated and progresses in a similar way in all primary leaves, regardless of the order of initiation. In contrast, the superposition of the time pattern of senescence with that of leaf growth shows that maximal HA yield expressed on a leaf basis is invariably located in the middle part of the plant. At the whole plant scale, our results suggest that it is possible to increase the production of antigens by harvesting plants at a later developmental stage, or by increasing plant density and harvesting these plants earlier.

Essa pesquisa objetivou avaliar métodos para estimar o vigor das sementes de fumo (Nicotiana tabacum L.) variedade Virgínia, cultivar CSC 439, nuas e peletizadas representadas por cinco lotes de sementes. Essas sementes foram submetidas aos seguintes testes de vigor: condutividade elétrica (0,5; 0,8; e 1,0 g e 2,5; 4,0 e 5,0 g de sementes nuas e peletizadas, respectivamente, hidratadas por 2, 4, 6, 8 e 24h em 25 mL de água destilada à 25 °C), envelhecimento acelerado (41 °C e 43 °C por 12 e 24h) com água (100% UR) e com solução salina de NaCl saturada (76% UR) e deterioração controlada (graus de umidade de 20% e 24% para sementes nuas e 8% e 12% para peletizadas, a 40 °C e 43 °C por 24 e 48h). As avaliações foram realizadas aos 7, aos 10 e aos 16 DAS (dias após a semeadura). Adicionalmente foi determinado o grau de umidade e realizados os testes de germinação, de primeira contagem de germinação e, a emergência da plântula e a velocidade de emergência da plântula. O delineamento experimental foi o inteiramente casualizado com quatro repetições. Os dados foram submetidos separadamente à análise de variância e a comparação das médias pelo teste de Tukey a 5% de probabilidade. Todas as análises foram repetidas uma vez. Conclui-se que o teste de condutividade elétrica não é eficiente para ordenar os lotes de semente de fumo, nuas e peletizadas, em diferentes níveis de vigor. Para o teste de envelhecimento acelerado as condições mais adequadas são 41 ºC por 12 horas de exposição com avaliação aos 7 dias após a semeadura, utilizando água (100% UR) para as sementes nuas e solução salina de NaCl (76% UR) para as sementes peletizadas. Para o teste de deterioração controlada, as combinações mais adequadas para as sementes nuas são 24% de água, exposição a 43 °C por 24h e avaliação aos 7 dias após a semeadura e para as sementes peletizadas 8 % de água a 43 °C por 48h e avaliação aos 16 dias. / The objective of this research was to evaluate methods for estimating the physiological quality of tobacco seeds (Nicotiana tabacum L.) \'Virginia variety, CSC 439\' cultivar. For this, five original seeds lots and five coated seed lots were used. The seed vigor were evaluated by electrical conductivity test (0.5, 0.8, and 1.0 g and 2.5, 4.0 and 5.0 g of original and coated seeds, respectively, hydrated for 2, 4, 6, 8 and 24 hours in 25 mL of distilled water at 25 ° C), accelerated aging test (41 ° C and 43 ° C for 12 and 24 hours) with water and saturated salt solution (NaCl) and controlled deterioration test (moisture content 20% and 24% for original seeds and 8% and 12% for coated seeds at 40 ° C and 43 ° C for 24 and 48). The evaluations were performed at 7, 10 and 16 DAS (days after sowing). Additionally, it was determined the seed the moisture content, germination test, first counting, seedling emergence and speed of seedling emergence. The experimental design was a completely randomized and the means were compared by Tukey test (5%). In conclusion, the electrical conductivity test is not efficient to sort lots of original and coated tobacco seeds in different levels of vigor. On the accelerated aging test the most adequate conditions are observed at 41 ºC for 12 hours of exposition and evaluations performed at 7 days after sowing, by using water (100% HR) for the original seeds and NaCl saturated salt solution (76% HR) for coated seeds. On the controlled deterioration test for the tobacco seeds the most adequated conditions are observed with the combinations of 24% of moisture content for the original seeds at 43 °C for 24 hours on evaluations performed at 7 days after sowing and 8% moisture content at 43 °C for 48 hours of exposition for coated seeds.

Plant secondary metabolites are essential for normal growth and development in plants ultimately affecting crop yield. They play roles ranging from appearance of the plants to defending against pathogen attack and herbivory. They have been used by humans for medicinal and recreational purposes amongst others. Glycosyltransferases catalyze the transfer of sugars from donor substrates to acceptors. Glucosyltransferases are a specific type of glycosyltransferases known to transfer glucose molecules from a glucose donor to a glucose acceptor (aglycone) producing the corresponding glucose secondary metabolite or glycone, in this case glucosides. It was hypothesized that SBIP68, a tobacco putative glucosyltransferase-like protein glucosylated salicylic acid. Salicylic acid is an essential plant defense secondary metabolite. SBIP68 was cloned and heterologously expressed in both prokaryotic and eukaryotic systems. Results from activity screening suggest that SBIP68 is a UDP-glucose flavonoid glucosyltransferase with broad substrate specificity. Further studies are required to fully characterize SBIP68.

Programa de Doctorado: Desarrollo sostenible: manejos forestal y turístico.

SUMMARY PtaRHE1 is a poplar (Populus tremula x P. alba) gene encoding a REALLY INTERESTING NEW GENE (RING) domain-containing protein. RING proteins are largely represented in plants and play important roles in the regulation of many developmental processes as well as in plant-environment interactions. In this thesis, we present a functional characterization of PtaRHE1. To gain further insight into the role of this gene, molecular and genetic alteration approaches were used. The results of in vitro ubiquitination assays indicate that PtaRHE1 protein is a functional E3 ligase and this activity was shown to be specific with the human UbCH5a, among the tested ubiquitin-conjugating enzymes. Histochemical GUS stainings showed that the PtaRHE1 promoter is induced by plant pathogens and by elicitors such as salicylic acid and cellulase and is also developmentally regulated. In silico predictions and the transient expression of PtaRHE1-GFP fusion protein in N. tabacum epidermal cells revealed that PtaRHE1 is localized both in the plasma membrane and in the nucleus. The localization of expression of PtaRHE1 in poplar stem by in situ hybridization indicated that PtaRHE1 transcripts are localized within the cambial zone mainly in ray cells, suggesting a role of this gene in vascular tissue development and/or functioning. The overexpression of PtaRHE1 in tobacco resulted in a pleiotropic phenotype characterized by a curling of leaves, the formation of necrotic lesions on leaf blades, growth retardation as well as a delay in flower transition. Plant genes expression responses to PtaRHE1 overexpression provided evidence for the up-regulation of defence and/or programmed cell death (PCD) related genes. Moreover, genes coding for WRKY transcription factors as well as for MAPK, such as WIPK, were also found to be induced in the transgenic lines as compared to the wild type (WT). Taken together, our results suggest that the E3 ligase PtaRHE1 plays a role in the signal transduction pathways leading to defence responses against biotic and abiotic stresses. Identification of PtaRHE1 target(s) is required in order to fully assess the role of this E3 ligase in the ubiquitination-mediated regulation of defence response./ RÉSUMÉ PtaRHE1 est un gène qui code pour une protéine possédant un domaine RING (REALLY INTERESTING NEW GENE) chez le peuplier (Populus tremula x P. alba). Les protéines de type RING sont très répandues chez les végétaux où elles jouent de rôles importants dans la régulation de plusieurs processus de développement et également dans les interactions plantes-environnement. Dans le cadre de ce travail, nous avons procédé à la caractérisation fonctionnelle du gène PtaRHE1. Dans le but de découvrir la fonction de ce gène, nous avons adopté une stratégie faisant usage d’approches moléculaires ainsi que de l’altération de l’expression génique. Les résultats obtenus montrent que la protéine PtaRHE1 est une E3 ligase et que cette activité enzymatique est spécifique à l’Ubiquitin-Conjugating enzym humaine UbCH5a. Les résultats du test histochimique GUS ont montré que le promoteur du gène PtaRHE1 est induit par des pathogènes et aussi par l’acide salicylique et la cellulase. Par ailleurs, ce promoteur est aussi régulé au cours du développement végétal. Les prédictions in silico et l’expression transitoire d’une fusion traductionnelle GFP-PtaRHE1, au niveau de l’épiderme des feuilles du tabac N. tabacum, ont révélé que la protéine PtaRHE1 se situe tant au niveau de la membrane cytoplasmique qu’au niveau du noyau. La localisation de l’expression du gène PtaRHE1, par les techniques d’hybridation in situ, montre que les transcrits de ce gène se retrouvent principalement au niveau des cellules de rayon, dans la zone cambiale, suggérant que ce gène pourrait jouer un rôle dans le développement ou la formation du tissu vasculaire. La surexpression du gène PtaRHE1 chez le tabac a conduit à l’obtention d’un phénotype pléiotropique caractérisé par un recroquevillement (incurvation) des feuilles, la formation des lésions nécrotiques sur le limbe, un retard de croissance ainsi qu’un retard dans la transition florale. L’analyse de la réponse de l’expression de différents gènes à la surexpression de PtaRHE1 a mis en évidence l’induction des gènes liés à la défense et ou à la mort cellulaire programmée. En outre, l’expression des gènes codant pour des facteurs de transcription WRKY et aussi des MAPKs, tel que WIPK, était aussi plus élevée chez les plantes transgéniques comparées au type sauvage. Les résultats de ce travail suggèrent que PtaRHE1, comme E3 ligase, pourrait jouer un rôle dans la transduction des signaux cellulaires conduisant aux réactions de défense contre les stress biotiques et abiotiques. L’identification de la (des) cible(s) de PtaRHE1 est indispensable pour la compréhension du rôle de cette protéine dans la régulation des réponses de défense par l’intermédiaire de l’ubiquitination.

Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.

Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.

Ce projet avait pour but la production de protéines recombinantes pour des procédures vaccinales dans un système végétal. Les protéines choisies ont été d'une part, deux protéines virales d'un Arterivirus, le virus du syndrome reproducteur et respiratoire porcin (PRRSV), les protéines GP5 et M, et d'autre part une protéine du flagelle, la flagelline ou FljB de Salmonella typhimurium. Le PRRSV est responsable de pertes économiques majeures dans l'industrie porcine mondiale et l'efficacité des vaccins actuels contre ce virus est limitée. La protéine M a été choisie pour sa propriété à former un hétérodimère avec la protéine GP5 qui augmente la réponse immunitaire. La glycoprotéine GP5, quant à elle, possède deux épitopes dans sa région N-terminale dont un joue un rôle majeur puisqu' il est capable de stimuler la production d'anticorps neutralisants nécessaires à la clairance du virus. Enfin FljB, qui est une protéine bien conservée chez les bactéries Gram négatives, a été retenue pour ses propriétés adjuvantes intéressantes de par sa capacité à se lier au récepteur 5 de type toll (TLR5) et à induire des réponses immunitaires systémiques et mucosales. Les hypothèses pour cette étude étaient que la production d'une protéine recombinante M::GP5 intégrant trois mutations (M::GP5mut) à des sites stratégiques de celle-ci induirait une meilleure réponse immunitaire que la protéine GP5 sauvage ; que l'administration par voie orale de plante produisant la protéine M::GP5mut induirait une immunité protectrice contre le PRRSV ; que les propriétés adjuvantes de FljB produite chez Nicotiana benthamiana devraient être comparables à celles de FljB recombinante produite chez E. coli. Pour répondre à ces questions, un système transitoire d'expression chez N. benthamiana a été choisi. Il a l'avantage de pouvoir produire des protéines rapidement et à des niveaux élevés. L'immunogénicité de la protéine M::GP5mut, et les propriétés adjuvantes de la flagelline ont été testées dans un système murin. Dans le cas de la flagelline, l'ovalbumine (OVA) a été choisie comme immunogène. Peu de résultats ont été obtenus dans le cadre du projet des protéines du PRRSV du fait de l'absence de production de celles-ci à un niveau détectable bien que la présence des ARNm ait été confirmée. En revanche, pour FljB, il a été montré que l'administration par voie orale de celle-ci induisait une réponse immunitaire contre l'OVA d'une intensité égale à celle produite par l'administration du mélange OVA avec de la FljB recombinante produite par E. coli et supérieure à celle de l'OVA administrée seule. De plus, FljB recombinante produite dans N. benthamiana a permis d'obtenir une réponse plus précoce qu'avec l'utilisation de FljB recombinante produite chez E. coli. En conclusion, il a pu être montré grâce à FljB que le système de production utilisé était efficace et que FljB est un bon adjuvant qui conserve ses propriétés quand elle est produite chez N. benthamiana. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : PRRSV, FljB, expression transitoire, adjuvant, réponse immunitaire, Nicotiana benthamiana