Le fer est un élément essentiel pour presque tous les êtres vivants cependant, il est peu biodisponible et est toxique dans sa forme libre car il engendre des formes réactives de l'oxygène via la réaction de Fenton. Pour se procurer le fer, les microorganismes sécrètent de petites molécules nommées sidérophores ayant une très forte affinité pour Fe3+. Les sidérophores sont requis pour la pathogénie de plusieurs agents pathogènes sur hôtes animaux ou végétaux, mais ce sont également des éliciteurs de défense. Des travaux antérieurs ont montré que les sidérophores activent les défenses et les gènes de réponse à la carence en fer chez Arabidopsis thaliana. L’activation de réponses de défense par le sidérophore requiert un niveau physiologique de fer dans la plante indiquant que le fer participe à la mise en place de ce processus. Au cours de ma thèse, les réponses globales de la plante A. thaliana au sidérophore deferrioxamine (DFO) ont été étudiées par une approche transcriptome. Les résultats obtenus montrent que le principal processus activé est l’immunité. En utilisant des chélateurs de fer différents, j’ai montré que l’effet chélation du fer est responsable de l’activation de l’immunité. Le traitement sidérophore provoque également une perturbation de l’homéostasie du fer et d’autres métaux dans la plante. Dans un mutant irt1 affecté dans le transport de plusieurs métaux lourds dont le fer, l’activation des défenses par la DFO est compromise. Par ailleurs, j’ai étudié l’effet du statut en fer de la plante sur sa sensibilité à la bactérie pathogène Dickeya dadantii et sur l’expression des défenses. Il apparait que le fer est requis pour la mise en place de plusieurs processus de défense en réponse à D. dadantii. Les plantes carencées en fer sont plus résistantes à l’infection. Une quantité de fer physiologique dans la plante est requise pour la multiplication bactérienne et pour l’expression des facteurs de pathogénie, les pectate lyases. Le marquage du fer par la méthode Perls’-DAB-H2O2 montre que celui–ci est très peu abondant dans les tissues végétaux contenant les bactéries qui, elles, sont chargées de fer. Dans l’ensemble, nos résultats montrent que le fer est requis dans l’arsenal défensif de la plante mais qu’il est également un facteur limitant pour le cycle infectieux de D. dadantii. / Iron is an essential element for almost all living organisms, however, it is not bioavailable and is toxic in its free form as it generates reactive oxygen species via the Fenton reaction. To obtain iron, microorganisms secrete small molecules named siderophores with very high affinity for Fe3 +. Siderophores are required for the pathogenesis of several pathogens on animals or plants, but they also are elicitors of defenses. Previous work has shown that siderophores activate defenses and iron deficiency response genes in Arabidopsis thaliana. The activation of defense responses by the siderophore requires a physiological level of iron in the plant indicating that iron is involved in the activation of this process. In my thesis, the global response of the plant A. thaliana to the siderophore deferrioxamine (DFO) has been studied by a transcriptomic approach. The results obtained show that the main process being activated is immunity. By using different iron chelating agents, I have shown that the iron chelation effect is responsible for the activation of immunity. The siderophore treatment also causes disturbance in the homeostasis of iron and other metals in the plant. In an irt1 mutant affected in the transport of heavy metals including iron, activation of defenses by the DFO is compromised. In addition, I studied the effect of iron status of the plant on its susceptibility to the pathogenic bacteria Dickeya dadantii and on the expression of defenses. It appears that iron is required for the establishment of several defense processes in response to D. dadantii. The iron deficient plants are more resistant to infection. A physiological amount of iron in the plant is required for bacterial growth and for expression of the virulence factors, pectate lyases. Iron staining by the Perls' -DAB - H2O2 method shows that low abundance of this metal in plant tissue coincides with the presence of bacteria, which contain high amounts of iron. Overall, our results show that iron is required in the defense arsenal of the plant but it is also a limiting factor for the infectious cycle of D. dadantii.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Phenolic compounds in plants have attracted considerable attention during the last decade due to their contribution to human health in reduce the risk of chronic diseases. The grape is one of the largest sources of these substances and like other plants, the synthesis of these compounds is usually associated with defense responses against stress factors, also called elicitors. More recently, ultrasound has been studied as possible abiotic elicitors of secondary metabolites due to mechanical stress and microstreaming induced by acoustic cavitation. Thus, this work aimed evaluate the application of different power densities (53 and 113 W cm-2) and exposure times (1, 5 and 10 minutes) to ultrasound in Isabel and Cabernet Sauvignon grapes about the effects on total polyphenols of juices made from this two cultivars. The samples were also subjected to the determination of total anthocyanins, tannins, total soluble solids, total acidity, pH and sensory evaluation. The Isabel cultivar showed more significant results in response to ultrasound, an increase of up to 83% in the levels of total polyphenols of juice, as well as significant increase in the content of total anthocyanins and tannins. These results were also accompanied by an increase in total soluble solids and total acidity of the juices. The application of ultrasound emphasized color, odor and taste of the Isabel grape juice, and the sonicated treatments were preferred by tasters. About Cabernet Sauvignon cultivar, it only showed a little variation in the most parameters. Power density and exposure time to ultrasound influenced the most of parameters evaluated in juices regarding to degree of response to sonication. Treatment with 5 and 10 min of exposure to ultrasound and power density of 113 W cm-2, is the combination that proved more effective for the elicitation of phenolic compounds by activation of plant secondary metabolism, improving the quality of the Isabel grape juice. / Compostos fenólicos em plantas têm atraído grande atenção durante a última década devido à sua contribuição para a saúde humana, podendo reduzir os riscos de doenças crônicas. A uva é uma das maiores fontes dessas substâncias e assim como os demais vegetais, tem a síntese desses compostos geralmente associada a respostas de defesa contra fatores de estresse, também chamados elicitores. Mais recentemente, o ultrassom vem sendo estudado como possível elicitor abiótico de metabólitos secundários devido a estresse mecânico e microagitação induzidos por cavitação acústica. Dessa forma, este trabalho foi conduzido com o intuito de avaliar a aplicação de diferentes densidades de potência (53 e 113 W cm-2) e tempos de exposição (1, 5 e 10 minutos) ao ultrassom em uvas Isabel e Cabernet Sauvignon quanto aos efeitos sobre os polifenóis totais dos sucos elaborados com as duas cultivares. As amostras também foram submetidas à determinação de antocianinas totais, taninos, sólidos solúveis totais, acidez total, pH e análise sensorial. A cultivar Isabel mostrou resultados mais significativos em resposta ao ultrassom, com elevação de até 83% nos níveis de polifenóis totais do suco, bem como, importante aumento no conteúdo de antocianinas totais e taninos. Estes resultados também foram acompanhados por um aumento nos sólidos solúveis totais e acidez total dos sucos. A aplicação do ultrassom acentuou cor, odor e sabor dos sucos de uvas Isabel, sendo os tratamentos sonicados os preferidos pelos provadores. Quanto a cultivar Cabernet Sauvignon, esta somente apresentou pequena variação na maioria dos parâmetros analisados. A densidade de potência e tempo de exposição ao ultrassom influenciaram a maioria dos parâmetros avaliados nos sucos quanto ao grau de resposta à sonicação. Tratamentos com 5 e 10 min de exposição ao ultrassom, com densidade de potência de 113 W cm-2 mostraram ser a combinação mais efetiva para a elicitação de compostos fenólicos mediante ativação do metabolismo secundário da planta, melhorando a qualidade dos sucos de uva Isabel.

Der pilzliche Erreger der Schwarzen Sigatokakrankheit, Mycosphaerella fijiensis (Mf), befällt fast alle Sorten (cv) der angebauten Banane Musa sp. Dabei sind weder die Reaktionen der Wirtspflanzen gegenüber dem Pathogen noch dessen Pathogenität hinreichend charakterisiert. Eine besondere Bedeutung wurde für pilzliche Sekundärmetaboliten des Pentaketid-Biosyntheseweges als spezifische Toxine innerhalb des Pathosystems postuliert. Die vorliegende Arbeit hat die experimentelle Untersuchung der Wirt-Pathogeninteraktionen zur Zielsetzung und wendet Methoden der Gewebekultur und Chromatographie zur bio-chemischen und physiologischen Charakterisierung des pflanzlichen und pilzlichen Stoff-wechsels an. Bezüglich der pflanzlichen Abwehrmechanismen von resistenten Musa cv konnte eine hypersensitive Reaktion (HR), die Aktivierung des Enzyms PAL und die Anreicherung von post-infektionellen Substanzen zur Hemmung des Pilzwachstums nachgewiesen werden. Im Er-regermetabolismus wurden unter in vitro-Bedingungen die Pentaketide Flaviolin, 2-Hydroxy-juglon, Juglon und 2,4,8-Trihydroxytetralon (-THT) bestimmt. Die Konzentration an 2,4,8-THT konnte sowohl durch die Anwendung des synthetischen Wirkstoffes Tricyclazol ® sowie durch natürliche Aktivatoren aus interzellulärem Blattgewebe resistenter Musa cv stark gesteigert werden. Mf-Rohextrakte und ausgewählte pilzliche Substanzen wurden auf ihre Dosis-Wirkungsbeziehung gegenüber verschiedenen Musa cv untersucht. Unter in vivo-Bedingungen Mf-inokulierter Bananenpflanzen führte die Anwendung des Wirkstoffes zu einer als "zerstörerische HR" bezeichneten Nekrotisierung des Blattes in anfälligen und resistenten Sorten. Diese Ergebnisse belegen die Bedeutung des 2,4,8-THT für die Ausprägung nekrotischer Blatt-symptome, die in Abhängigkeit von der Konzentration zum jeweiligen Zeitpunkt Musa cv-spezifische Wirkungen hervorruft: In der resistenten Sorte führte die Steigerung von 2,4,8-THT zur HR und der Elizitierung postinfektioneller Abwehrreaktionen, die zur Inkompatibilität zwischen Pathogen und Wirtspflanze führen; in der anfälligen Sorte erreicht 2,4,8-THT erst nach Ausbildung einer kompatiblen Interaktion mit biotropher Ernährung toxische Dosen und wirkt als Virulenzfaktor in der nekrotrophen Phase der Pathogenese. / The fungal pathogen causing black Sigatoka disease, Mycosphaerella fijiensis (Mf), attacks almost all varieties (cv) of cultivated bananas and plantains Musa sp. However, neither the reactions of host plant in relation to the pathogen nor its pathogenicity has been characterized in detail. A special significance has been attributed to fungal secondary metabolites of pentaketid pathway as host-specific toxins within the pathosystem. The present study refers to the experimental investigation of host-pathogen interactions using tissue culture as well as chromatographic methods in order to characterize biochemical and physiological metabolisms of plant and fungus. With reference to plant defense mechanisms of resistant Musa cv, hypersensitive reaction (HR), activation of phenylalanine-ammonia lyase and accumulation of postinfectional substances which blocked fungal growth have been demonstrated. Using in vitro conditions, the pentaketide metabolites flaviolin, 2-hydroxyjuglone, juglone and 2,4,8-trihydroxytetralone (2,4,8-tht) had been determined. 2,4,8-tht concentration was significantly increased through the application of the synthetic compound tricyclazole ® and through natural activators extracted from resistant Musa cv intercellular space of leaf tissue. Dose-effect relationship between crude extracts and selected fungal substances applied to different Musa cv were investigated. Using in vivo conditions, the application of tricyclazole ® to host plants inoculated with Mf resulted in extensive necrosis of susceptible and resistant Musa cv leaves, characterized as "devastating HR". These results proof the importance of 2,4,8-tht for the development of necrotic leaf symptoms that causes host-specific reactions depending on their concentration at different moments of pathogenesis: The resistant Musa variety increases 2,4,8-tht causing HR and elicitation of postinfectional defense reactions that lead to incompatibility between pathogen and host plant; in the susceptible variety, 2,4,8-tht attains toxic doses only after establishment of a compatible interaction including biotrophic nutrition of the pathogen, acting as a virulence factor during the necrotrophic phase of pathogenesis.

O nim (Azadirachta indica) é conhecido na Ásia devido a várias propriedades biológicas conhecidas desde a antigüidade. Os estudos referentes à ação inseticida dessa planta restringem-se a análise de seus mecanismos de ação sobre insetos e também de seus efeitos sobre trabalhadores rurais que fazem uso de produtos a base de nim; não havendo, na literatura pesquisada, trabalhos relativos aos impactos causados sobre o sistema vegetal. As plantas, assim como outros organismos, possuem a capacidade de se defenderem contra ataque de patógenos. Uma das respostas desencadeadas pelo reconhecimento do patógeno pelas células vegetais é a reação de hipersensibilidade (RH), que envolve a morte imediata das células do sítio primário de infecção, oferecendo resistência ao crescimento do patógeno. A RH é caracterizada pela necrose dos tecidos onde primeiro se manifestou a infecção, e este processo de morte celular programada envolve uma série de sinais que ainda não estão completamente elucidados. Neste trabalho, foram estabelecidas as condições do meio de cultura de células de Rubus fruticosus para os estudos com extrato de sementes de nim, avaliado o efeito elicitor deste sobre a cultura. Foram obtidos extratos hidroalcoólicos E1 e E2 e suas respectivas frações lioflizadas, L1 e L2. Estes extratos apresentaram maior teor de açúcares e lipídeos em sua composição e revelaram potencial antioxidante. Detectou-se a presença de AZA-A em L1 e L2, por meio de CLAE, cujos teores foram de 5,03 e 1,1 mg/mL, respectivamente, com tempo de retenção em torno de 9,5 minutos, confirmado por meio de análises via espectrometria de massas. O extrato L2 foi fracionado nas frações L2 inicial e AZA2. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e destas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e suas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. As células de Rubus fruticosus (1,8g) foram incubadas em tampão citrato de sódio contendo o extrato L2 e as frações L2 inicial e AZA2, separadamente, até a concentração de 5 mg/mL, por 1h, em temperatura ambiente. Após este período, os compostos fenólicos, proteínas e açúcares redutores foram determinados no meio extracelular e intracelular por métodos colorimétricos. O efeito destas frações e do extrato L2, na produção de EROs em células intactas de Rubus fruticosus, foi analisado usando a sonda diacetato 2,7-diclorofluoresceína (LEE et al., 1999; MURATA et al., 2001). Os resultados obtidos indicam que AZA isolada não teve efeito sobre respostas de defesa. A fração L2 inicial teve aumento de fenólicos intracelulares, de açúcares redutores extracelulares e diminuição de EROs, com o aumento da concentração do elicitor, indicando potencial antioxidante e mecanismo de defesa. O extrato L2 também demonstrou potencial antioxidante e protetor das células com o aumento da concentração do elicitor, além de possuir ação inseticida. / The neem (Azadirachta indica) is known in Asia due to their several biological properties. The studies on the insecticide action of neem extracts have only been restrict to the insect mechanisms and their effects on rural workers; studies on the impact in the vegetable system are not available. The plants, like the other organisms, have the ability to self-defend against attack of patogens. The hypersensitive response (RH), a type of programmed cell death (PCD) in plants, is triggered by plant cells when they recognize the patogen, and is characterized by necrosis of tissues in the local region surrounding the infection; the signals involved are still not completely elucidated. The present study evaluated the effects of neem extracts in Rubus fruticosus cell. The powdered seeds were submitted to two consecutive extractions with ethanol:water (1:1, v/v) at room temperature for 10 minutes, yielding E1 and E2 fractions. The solvent was evaporated and the aqueous extracts were concentrated and lyophilized, resulting in two samples, L1 and L2. They are used for analyses by high performance liquid chromatograph (HPLC) in C-18 column (4.6 x 250 mm), with acetonitrile-water (4:6 v/v) as mobile phase, flow rate 1 mL/min, monitored at 214 nm. The principal compound of this fraction was azadirachtin (5.03 and 1.1 mg/mL, respectively), the retention time was 9.5 min; it was confirmed using mass spectrophotometry. The L2 extract was partially fractionated by high performance liquid chromatograph (HPLC) in semi preparative C-18 column. The main fractions, analyzed by colorimetric methods, ESI-MS, were L2 initial and AZA2. The Rubus fruticosus cells (18-21 days; 1.8 g) were incubated in sodium citrate buffer containing L2, L2 initial ans AZA2 at concentrations up to 5 mg/mL, for 1h, at room temperature. After this period, the phenolic compounds, proteins and reducing sugar were determined in the extracellular and intracellular medium by colorimetric methods. Also, the effects of these fractions over the production of reactive oxygen species (ROS) in intact cell of Rubus fruticosus, was analyzed using 2,7-dichloro-fluorescein diacetate. AZA2 had no effect on the defense response. The initial L2 fraction increased the phenolic compounds in the intracellular medium and the reducing sugars in the extracellular medium. The same fraction showed an inhibitory effect on ROS and also increased the concentration of the elicitor. These results indicate the antioxidant potential and protector effect of the L2 initial. The L2 extract also demonstrated antioxidant and protective potential of cells with the increase of the elicitor concentration. Therefore, in parallel with its insecticide action, the neem extract contributes to the self-defense ability of the plants.

O nim (Azadirachta indica) é conhecido na Ásia devido a várias propriedades biológicas conhecidas desde a antigüidade. Os estudos referentes à ação inseticida dessa planta restringem-se a análise de seus mecanismos de ação sobre insetos e também de seus efeitos sobre trabalhadores rurais que fazem uso de produtos a base de nim; não havendo, na literatura pesquisada, trabalhos relativos aos impactos causados sobre o sistema vegetal. As plantas, assim como outros organismos, possuem a capacidade de se defenderem contra ataque de patógenos. Uma das respostas desencadeadas pelo reconhecimento do patógeno pelas células vegetais é a reação de hipersensibilidade (RH), que envolve a morte imediata das células do sítio primário de infecção, oferecendo resistência ao crescimento do patógeno. A RH é caracterizada pela necrose dos tecidos onde primeiro se manifestou a infecção, e este processo de morte celular programada envolve uma série de sinais que ainda não estão completamente elucidados. Neste trabalho, foram estabelecidas as condições do meio de cultura de células de Rubus fruticosus para os estudos com extrato de sementes de nim, avaliado o efeito elicitor deste sobre a cultura. Foram obtidos extratos hidroalcoólicos E1 e E2 e suas respectivas frações lioflizadas, L1 e L2. Estes extratos apresentaram maior teor de açúcares e lipídeos em sua composição e revelaram potencial antioxidante. Detectou-se a presença de AZA-A em L1 e L2, por meio de CLAE, cujos teores foram de 5,03 e 1,1 mg/mL, respectivamente, com tempo de retenção em torno de 9,5 minutos, confirmado por meio de análises via espectrometria de massas. O extrato L2 foi fracionado nas frações L2 inicial e AZA2. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e destas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e suas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. As células de Rubus fruticosus (1,8g) foram incubadas em tampão citrato de sódio contendo o extrato L2 e as frações L2 inicial e AZA2, separadamente, até a concentração de 5 mg/mL, por 1h, em temperatura ambiente. Após este período, os compostos fenólicos, proteínas e açúcares redutores foram determinados no meio extracelular e intracelular por métodos colorimétricos. O efeito destas frações e do extrato L2, na produção de EROs em células intactas de Rubus fruticosus, foi analisado usando a sonda diacetato 2,7-diclorofluoresceína (LEE et al., 1999; MURATA et al., 2001). Os resultados obtidos indicam que AZA isolada não teve efeito sobre respostas de defesa. A fração L2 inicial teve aumento de fenólicos intracelulares, de açúcares redutores extracelulares e diminuição de EROs, com o aumento da concentração do elicitor, indicando potencial antioxidante e mecanismo de defesa. O extrato L2 também demonstrou potencial antioxidante e protetor das células com o aumento da concentração do elicitor, além de possuir ação inseticida. / The neem (Azadirachta indica) is known in Asia due to their several biological properties. The studies on the insecticide action of neem extracts have only been restrict to the insect mechanisms and their effects on rural workers; studies on the impact in the vegetable system are not available. The plants, like the other organisms, have the ability to self-defend against attack of patogens. The hypersensitive response (RH), a type of programmed cell death (PCD) in plants, is triggered by plant cells when they recognize the patogen, and is characterized by necrosis of tissues in the local region surrounding the infection; the signals involved are still not completely elucidated. The present study evaluated the effects of neem extracts in Rubus fruticosus cell. The powdered seeds were submitted to two consecutive extractions with ethanol:water (1:1, v/v) at room temperature for 10 minutes, yielding E1 and E2 fractions. The solvent was evaporated and the aqueous extracts were concentrated and lyophilized, resulting in two samples, L1 and L2. They are used for analyses by high performance liquid chromatograph (HPLC) in C-18 column (4.6 x 250 mm), with acetonitrile-water (4:6 v/v) as mobile phase, flow rate 1 mL/min, monitored at 214 nm. The principal compound of this fraction was azadirachtin (5.03 and 1.1 mg/mL, respectively), the retention time was 9.5 min; it was confirmed using mass spectrophotometry. The L2 extract was partially fractionated by high performance liquid chromatograph (HPLC) in semi preparative C-18 column. The main fractions, analyzed by colorimetric methods, ESI-MS, were L2 initial and AZA2. The Rubus fruticosus cells (18-21 days; 1.8 g) were incubated in sodium citrate buffer containing L2, L2 initial ans AZA2 at concentrations up to 5 mg/mL, for 1h, at room temperature. After this period, the phenolic compounds, proteins and reducing sugar were determined in the extracellular and intracellular medium by colorimetric methods. Also, the effects of these fractions over the production of reactive oxygen species (ROS) in intact cell of Rubus fruticosus, was analyzed using 2,7-dichloro-fluorescein diacetate. AZA2 had no effect on the defense response. The initial L2 fraction increased the phenolic compounds in the intracellular medium and the reducing sugars in the extracellular medium. The same fraction showed an inhibitory effect on ROS and also increased the concentration of the elicitor. These results indicate the antioxidant potential and protector effect of the L2 initial. The L2 extract also demonstrated antioxidant and protective potential of cells with the increase of the elicitor concentration. Therefore, in parallel with its insecticide action, the neem extract contributes to the self-defense ability of the plants.

L’objectif de ce travail est la valorisation des molécules bioactives présentes initialement dans Gelidium sesquipedale. Les rhodophycées agarophytes dont Gelidium sesquipedale sont exploitées à l’échelle industrielle pour l’agar, un phycocolloïde aux propriétés gélifiantes, qu’elles contiennent en abondance. Une multitude de coproduits sont générés lors de l’extraction de l’agar. Ces derniers, peu étudiés, ne sont pas valorisés alors qu’ils constituent une source potentielle de molécules d’intérêts. En premier lieu, le process industriel d’extraction de l’agar a été adapté à l’échelle du laboratoire afin de récupérer ces co-produits dont l’analyse a montré la richesse en glucides. Ils ont par la suite été fractionnés pour isoler les oligosaccharides dont certains sont connus comme éliciteurs chez les plantes. Ainsi, plusieurs fractions oligosaccharidiques ont été obtenues avec un rendement estimé à 15,7% de Gelidium sesquipedale sec. Les fractions sélectionnées ont été caractérisées par CPG, ESI-MS, RMN et perméthylation ce qui a permis d’élucider les structures des oligosaccharides qu’elles contiennent et de révéler notamment la présence de dérivés de floridoside dont le Gal2glycérol, le Gal3glycérol et le Gal4glycérol qui sont des molécules originales chez Gelidium sesquipedale non décrites à ce jour chez les algues rouges. Une dernière partie a consisté en la mesure de l’activité élicitrice de ces fractions qui a pu être vérifiée sur des plantes de tomate à travers des mesures de marqueurs biochimiques relatifs à l’expression des réactions de défense chez la plante. En conclusion, les coproduits issus de l’extraction de l’agar représentent une source de pSDNs (phyto stimulateur des défenses naturelles chez la plante) ; ils offrent une nouvelle perspective de développement à l’industrie de l’agar. / This work aims at promoting the bioactive molecules initially present in Gelidium sesquipedale. The rhodophycea agarophytes, including Gelidium sesquipedale, are used for industrial extraction of agar, a phycocolloid with gelling properties, which they contain in abundance. A multitude of co-products are generated during the extraction of the agar. These co-products have only been studied a little, hence not valued, while they constitute a significant source of molecules of interest. Firstly, the industrial agar extraction process was adapted on a laboratory scale, in order to recover these coproducts, which were subsequently subjected to an analysis, which revealed the presence of carbohydrates as major components. They were submitted to a fractionation process to obtain oligosaccharidic fractions, with a potential of elicitor activity, and a yield estimated at 15.7% of dry Gelidium sesquipedale. Also, a follow-up of co-products from batches of Gelidium sesquipedale harvested in different years from 2014 to 2016, enabled the comparison of the composition of the various co-products, depending on the year of the harvest, and thus to evaluate the variability of the initial resource. In addition, the impact of an extraction factor, being the sodium concentration, and the comparison with an industrial co-product produced by this process, were carried out. The retained fractions were characterized by GPC, ESI-MS, NMR and permethylation that allowed the elucidation of the structures of the oligosaccharides they contain, and revealed in particular the presence of floridoside derivatives including Gal2glycerol, Gal3glycerol and Gal4glycerol, which are original molecules in Gelidium sesquipedale, not described to date in red algae. A final part consisted in measuring the activity of these fractions as elicitor that could be estimated on tomato plants through measurements of biochemical markers relating to the expression of defense reactions in the plant. In conclusion, the co-products from agar extraction represent a source of pSDNs (phyto stimulator of natural defense in the plant) and give a new perspective to the agar industry.

Les extensines sont des glycoprotéines pariétales appartenant à la famille des HRGPs (Hydroxyprolin-rich glycoproteins) impliquées dans plusieurs fonctions telles que la croissance, le développement et la défense des plantes contre les pathogènes. Toutefois, leur mode d’action dans la réponse immunitaire végétale n’est pas encore bien connu et reste à élucider. Les extensines interviennent dans le renforcement de la paroi, un des premiers remparts cellulaires contre les pathogènes, en se liant entre elles de manière intra- et intermoléculaire. Ce « cross-linking » est catalysé par des enzymes peroxydases spécifiques et nécessite une correcte conformation des extensines, laquelle est conférée par leur partie glycosylée. Dans ce projet de thèse, nous avons donc entrepris d’étudier l’impact de la glycosylation des extensines sur la défense racinaire et tenté de caractériser, de manière préliminaire, des peroxydases potentiellement impliquées dans le « cross-linking » chez Arabidopsis thaliana. Des techniques d’immunocytochimie réalisées sur une sélection de mutants affectés dans la glycosylation des extensines ont révélé une modulation de la distribution des extensines dans la racine d’A. thaliana en réponse à une élicitation avec un peptide bactérien, la flagelline 22. L’un des résultats majeurs de cette étude a été de montrer l’importance de l’arabinosylation des extensines dans la colonisation de la racine par l’oomycète pathogène Phytophthora parasitica. Ainsi, l’ensemble de ces résultats nous apermis d’élaborer un modèle proposant d’illustrer l’importance de l’arabinosylation des extensines dans l’organisation et l’architecture de la paroi, modulant ainsi l’adhésion du pathogène sur les cellules de la racine et influençant in fine la colonisation de cette dernière. / Extensins are cell wall glycoproteins involved in various biological processes including plantprotection. However, their mode of action in plant immunity response is not clearly established and remains to be elucidated. Extensins are able to strengthen the cell wall, one of the first cellular barriers against pathogens, through intra- and intermolecular cross-links. This cross-linking is catalysed by specific peroxidase enzymes and requires a correct conformation of extensins conferred by their glycan moiety. This PhD project aimed to investigate the impact of extensin glycosylation in root defence and to characterize, as a preliminary study, the peroxidases potentially involved in the extensin crosslinking in Arabidopsis thaliana. Through immunocytochemistry techniques on mutants impaired withextensin glycosylation, we have revealed that a modulation of extensin distribution occurs in A. thaliana root in response to elicitation with the bacterial peptide, flagellin 22. We have also showed that extensin arabinosylation plays a major role, although probably indirect, in the root colonization by the pathogen oomycete Phytophthora parasitica. We have therefore elaborated a model proposing to illustrate the importance of extensin arabinosylation in the cell wall organization and architecture,modulating pathogen adhesion on root cells and influencing in fine root colonization.

La vigne est une culture pérenne sensible à de nombreux bioagresseurs et sur laquelle il est nécessaire de réaliser de nombreux traitements pesticides, susceptibles de causer des problèmes environnementaux, de santé humaine et d’apparition de résistance au sein des populations de bioagresseurs. Aujourd’hui, il est indispensable de développer des stratégies nouvelles de lutte contre les bioagresseurs, plus raisonnées mais permettant de conserver une viticulture compétitive. L’utilisation de produits de biocontrôle semble, en ce sens, être une approche prometteuse permettant d’allier agriculture durable et intensive.Deux écoproduits généralistes à fort potentiel ont été identifiés, comme possédant des actions intéressantes sur les principales maladies cryptogamiques de la vigne. Le premier est un extrait naturel de plante, sans action fongicide directe mais capable de stimuler efficacement et de façon systémique les défenses de la plante. Le second, quant à lui, est un microorganisme qui possède une forte action antagoniste fongicide, mais aussi la capacité à stimuler les défenses de plante. Dans un premier temps, des études en conditions contrôlées ont mis en évidence l’efficacité des deux actifs pour inhiber le développement de diverses souches d’Erysiphe necator, Plasmopara viticola, Botrytis cinerea et Botryosphaeriaceae. En parallèle, des expérimentations au vignoble, ont confirmé le fort potentiel de ces produits de biocontrôle, avec des bonnes efficacités, particulièrement stable avec l’extrait naturel. Ces différentes études nous ont permis d’identifier et d’élaborer des stratégies d’utilisation pour ces deux produits de biocontrôle. / Grapevine is a perennial crop sensitive to many fungal pathogens that require numerous pesticide treatments. However, its uses lead to environmental, human health and fungicide resistance problems. Developing sustainable pest management strategies while keeping a good wine quality is of major importance. In this sense, the use of bio-pesticides products seems to be a promising approach to combine sustainable and intensive agriculture.Two generalist bio-pesticides of great potential have been preliminary identified, forits actions on major fungal diseases of grapevine. The first one is a natural plant extract, with no direct fungicide action but able to systemically stimulate plant defenses. The second one is a microorganism showing strong antagonist fungicide actions, and important ability to stimulate plant defenses. First, the studies conducted in controlled conditions have demonstrated the effectiveness of both products in the suppression of various isolates of Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Botryosphaeriaceae. In parallel,the good efficiencies of these products have been confirmed during vineyard assays. This was especially well demonstrated for the natural extract. As a whole, these studies confirm thepotential of these two products as promising bio-pesticides, of which the strategy of application have been further defined.