Young male rats fed a choline-deficient diet for three days accumulated triacylglycerol (TG) in the liver, and had reduced very low density lipoprotein (VLDL), but not high density lipoprotein (HDL), levels in the plasma. Cultured hepatocytes obtained from these rats were used as a model system to investigate how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis and lipoprotein secretion. When the cells were cultured in a medium free of choline and methionine, the secretion of TG and phosphatidylcholine (PC) was impaired. Supplementation of choline, methionine or lysophosphatidylcholine (lysoPC) to the culture medium increased the secretion of these lipids to normal levels, and stimulated PC biosynthesis. Fractionation of the secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of TG and PC from choline-deficient cells was mainly due to impaired secretion of VLDL. The secretion of HDL and lipid-free proteins (for example albumin), however, was not affected by choline and methionine deficiency. Supplementation of betaine and homocysteine also stimulated PC biosynthesis and enhanced hepatic VLDL secretion. However, supplementation of ethanolamine, N-monomethylethanol-amine or N, N-dimethylethanolamine did not correct the impaired VLDL secretion from the hepatocytes, although an active synthesis of phosphatidylmonomethyl-ethanolamine (PMME) and phosphatidyldimethylethanolamine was observed. Choline deficiency had no effect on the rate of incorporation of [³H]leucine into cellular apolipoprotein B, E and C or on the rate of disappearance of radioactivity from the labeled apolipoproteins. These results suggest that biosynthesis of PC is specifically required for hepatic VLDL (but not HDL) secretion, and any one of the three synthetic pathways, the CDP-choline pathway, methylation of phospha-tidylethanolamine (PE) or reacylation of lysoPC, is sufficient to provide the required PC. The total activity of cytidylyltransferase in liver was unchanged in choline deficiency. However, choline deficiency caused an abnormal distribution of cytidylyltransferase activity between rat liver cytosol and microsomes (mainly endoplasmic reticulum), a decrease in the cytosolic enzyme activity and an increase in the microsomal enzyme activity. In cultured hepatocytes from the choline-deficient rat, the abnormally distributed cytidylyltransferase activity could be rapidly reversed by the addition of choline, but not lysoPC, to the culture medium. The stimulated microsomal activity of cytidylyltransferase during choline deficiency might be a mechanism whereby the cells could more effectively utilize phosphocholine to maintain a normal CDP-choline level in the choline-deficient liver. Rat liver PE N-methyltransferase catalyzes all three transmethylation reactions in the conversion of PE to PC. The in vitro activity of PE N-methyltransferase was increased in choline-deficient livers using endogenous PE as the methyl group acceptor. However, no significant changes were observed in the enzyme activity when exogenous PMME was used as the methyl group acceptor. Addition of methionine to the cultured hepatocytes obtained from choline-deficient rats resulted in a concomitant reduction in cellular PE levels and the specific activity of PE-dependent methyltransferase. However, the specific activity of PMME-dependent methyltransferase was not significantly altered upon the addition of methionine. No change in PE N-methyltransferase activity was observed in the cultured hepatocytes supplemented with choline. Immunoblotting of PE N-methyltransferase, in crude liver microsomes and in membrane fractions of cultured hepatocytes, revealed that the enzyme mass was not altered by choline and methionine deficiency. Thus, hepatic PE N-methyltransferase is preserved in choline deficiency, and its activity is probably dependent on the availability of metabolic substrates (i.e. methionine and PE). / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Les phlorotannins, polymères du phloroglucinol, sont des composés phénoliques (CP) uniquement retrouvés chez les algues brunes (Phéophycées). Ces métabolites présentant des activités anti-oxydantes, interviendraient dans la formation de la paroi, mais à ce jour leur voie de biosynthèse reste non élucidée. L'annotation du génome de l'algue brune Ectocarpus, a permis d'identifier des gènes homologues codant pour des enzymes de la biosynthèse des CP chez les plantes terrestres. Une polyketide synthase de type III (PKSIII), a été caractérisée: elle synthétise le phloroglucinol. La recherche d'autres cibles a été poursuivie sur des gènes codant pour des chalcone-isomérases-like (CHIL), ainsi que pour des phénol-sulfotransférases homologues d'enzymes de sulfatation des flavonoïdes. Les CHIL se sont révélées être des fatty acid binding protein (FAP) impliquées dans le métabolisme des acides gras. L'intérêt pour cette nouvelle famille a justifié leur caractérisation biochimique puis fonctionnelle par complémentation de mutants FAP d'Arabidopsis thaliana. L'élucidation progressive des voies de biosynthèse des phlorotannins a servi de base pour étudier les mécanismes de régulation de ce métabolisme chez les Phaeophycées. En combinant des approches intégrées d'expression de gènes cibles, de dosages et de profilages de phlorotannins solubles, nous avons pu montrer que ces composés assurent une protection constitutive chez Fucus vesiculosus en réponse aux UV-B, et que leur métabolisme serait induit très précocement au cours de l'herbivorie. Le développement d'outils moléculaires spécifiques de ces voies métaboliques, ouvre de nouvelles perspectives en écophysiologie et en écologie. / Phlorotannins are polymers of phloroglucinol that are specific phenolic compounds of brown algae (Phaeophyceae). These metabolites present antioxidant activities and are potentially involved in the formation of cell-walls but their biosynthetic pathway is currently uncharacterized. The genome annotation of the brown algae Ectocarpus provided some information about conserved genes which are implicated in the synthesis of phenolics in terrestrial plants. One polyketide synthase of type III (PKSIII) has been successfully characterized: it produces phloroglucinol. The search for other targets has been pursued in brown algae focusing mainly on chalcone isomerase-like (CHI-like) genes, as well as on phenol-sulfotransferases, which are implicated in the sulfation of flavonoids. The characterization of CHIL has revealed their implication in fatty acid binding (FAP). However, the level of interest for this new family has led to their biochemical characterization and to functional studies by complementation of gene in the Arabidopsis thaliana FAP mutant. The progressive elucidation of the phlorotannin biosynthesis pathway has been used in order to discover mechanisms which regulate this metabolism in brown algae. By combining integrated approaches of gene expression profiling with the quantification and profiling of soluble phlorotannins, we have shown that these metabolites ensure the constitutive protection in Fucus vesiculosus against UV-B radiation and could also be induced as a very early response to grazing. The development of specific molecular tools for this metabolic pathway opens some news perspectives in ecophysiological and ecological studies.

Penicillium expansum est un contaminant commun des pomaceae (pommes et poires) causant la pourriture bleue. Ce champignon est le principal responsable de la présence de patuline dans les pommes et ses produits dérivés. Actuellement, la voie de biosynthèse de la patuline n’est que partiellement élucidée et le cluster de gènes correspondant n’est décrit que chez Aspergillus clavatus, champignon tellurique incapable de se développer dans les pommes. La caractérisation moléculaire de la voie de biosynthèse de la patuline est la condition sine qua none à toute étude visant à comprendre la régulation de la biosynthèse de la patuline, mais également à toute action permettant de limiter sa synthèse. C’est pourquoi le premier objectif de cette thèse a été de caractériser le cluster de gènes spécifique de la voie de biosynthèse chez Penicillium expansum. Celui-ci est caractérisé par une taille de 40 kb et contient les 15 mêmes gènes qu’Aspergillus clavatus, les seules différences résidant dans l’organisation et l’orientation des gènes. La caractérisation de la seconde étape de la voie de biosynthèse de la patuline a été ensuite entreprise chez Aspergillus clavatus, organisme modèle. Le gène patG code pour l’acide 6-méthylsalicylique décarboxylase responsable de la conversion de l’acide 6-méthylsalicylique en m-crésol. Pour faire suite au premier objectif, la régulation de la voie de biosynthèse de la patuline a été étudiée. Pour cela, une souche mutante pour le facteur de régulation spécifique à la patuline patL a été généré puis la production de patuline ainsi que l’expression des gènes du cluster analysés. Les résultats de cette étude ont montré que le gène patL joue le rôle d’interrupteur au sein du cluster. L’absence de patL conduit à une extinction totale de l’expression des gènes du cluster et à une abscence de production de patuline par Penicillium expansum. Dans cette même étude, des tests de pathogénicité ont été entrepris sur des pommes de différentes variétés démontrant ainsi que la patuline peut être un facteur de virulence facilitant l’infection de certaines variétés de pommes telles que la Golden Delicious ou la Pink Lady. Enfin, l’influence de la lumière a été évaluée en analysant l’impact de différentes longueurs d’ondes sur la croissance et la production de patuline de Penicillium expansum. Que ce soit in-vitro ou in-vivo, la croissance et la production de patuline sont très affectés par les lumières blanche, bleue et rouge. Favoriser le stockage des pommes sous les lumières blanche, bleue ou rouge plutôt qu’à l’obscurité pourrait devenir un moyen de prévention contre la contamination par Penicillium expansum. En conclusion, cette thèse présente un aspect fondamental avec la caractérisation du cluster de gènes chez Penicillium expansum et la caractérisation de la seconde étape de la voie de biosynthèse de la patuline ; mais aussi un aspect appliqué avec l’utilisation des lumières de différentes couleurs comme méthode de prévention contre Penicillium expansum durant le stockage des pommes. / Penicillium expansum is the common contaminant of apples and the causal agent of blue mold rot. This fungus is the main patulin producer in apple based products. Actually, the patulin biosynthesis is partially elucidates and the gene cluster has been elucidated in Aspergillus clavatus, a telluric fungi unable to grow on apples. The molecular characterization of the patulin biosynthetic pathway is the key step for a better understanding of the mechanisms leading to patulin production and will help to define strategies to reduce its presence in apple products. The first objective of this thesis was the characterization of the patulin gene cluster in Penicillium expansum. The latter includes the same 15 genes as in Aspergillus clavatus but in a different order and orientation. Then, the second step of this biosynthetic pathway has been characterized and the patG gene encode for the 6-methylsalicylic decarboxylase involved in the 6- methylsalicylic acid conversion into m-cresol. The second objective consists of the study of the patulin regulation. For that, a patL mutated strain was generated and the patulin production and the patulin gene cluster expression were assessed. The mutation of this gene results in a down-regulation of the rest of the genes in the cluster associated with a lack of patulin production. Pathogenicity tests on apples revealed that patulin could act as a virulence factor in some apple varieties, like Golden Delicious or Pink Lady. In the last part of this thesis, the influence of different wavelength lights on the growth and the patulin production by Penicillium expansum were assessed in vitro and in vivo. In both cases, growth and patulin production were significantly affected under white, blue and red lights. Consequently, the apple storage under these lights could be a good alternative to the storage in the dark. In conclusion, this thesis presents a fundamental aspect that consist in the characterization of the patulin gene cluster in Penicillium expansum and the characterization of the second step of this pathway. An applied aspect is also provided by the use of the different wavelength lights to prevent the Penicillium expansum contamination during apple storage.

No tubo digestivo das larvas de Anastrepha fraterculus e Anastrepha pickeli há β-glicosidases capazes de clivar dissacarideos, β-glicosídeos tóxicos produzidos por plantas e substratos sintéticos. As β-glicosidases de A. fraterculus são pouco ativas e as de A. pickeli são bastante ativas sobre alguns compostos, entre eles linamarina, um glicosídeo cianogênico. Esse composto está presente, em altas concentrações, no fruto da mandioca do qual a larva se alimenta. A. fraterculus alimenta-se do fruto da goiaba e aparentemente consegue o carboidrato que necessita por ação de α-glicosidases, que são bem mais ativas do que as β. O fruto da mandioca não é tão nutritivo e A. pickeli deve aproveitar a glicose da linamarina para obter energia e consegue desintoxicar-se do aglicone tóxico. Rhynchosciara americana apresenta quatro β-glicosidases nas membranas microvilares intestinais, sendo três delas β-galactosidases. Dessas, duas são ativadas por Triton X-100 sendo que a glicosidase, de maior mobilidade eletroforética é ativada por este composto, com uma Ka de 4µM, um α de 0,5 e um β de 2. β-tioglicosidases foram demonstradas em afideos. Nós verificamos que ocorre a clivagem do tioglicosídeo sinigrina após separação das β-glicosidases digestivas do Lepidoptera Diatraea saccharalis por cromatografia hidrofóbica. Nesse inseto, a mesma enzima é capaz de clivar O- e S-glicosídeos com atividades semelhantes. Enzimas com essas características nunca foram descritas anteriormente. Esses experimentos ilustram a viabilidade das adaptações dos insetos na utilização de compostos formados for ligações β-glicosídicas, viabilizando a exploração de nutrientes normalmente inacessíveis a outros animais. / Anastrepha fraterculus and Anastrepha pickeli have in their midguts 13-glycosidases able to hydrolase dissaccharides, synthetic substrate and plant toxic β-glucosides. β-glycosidases from A. fraterculus have low activity and the enzymes from A. pickeli may be highly active depending on the substrate used. Linamarin, a cyanogenic β-glucoside present in A. pickeli food (Manihot fruit) is easly hydrolysed by A. pickeli β-glycosidases (A. fraterculus eats on guava fruits and may obtain carbohydrate through the action of α-glycosidases, that are much more active them the β-glycosidases). A. pickeli probably uses glucose derived from linamarinan avoiding the effects of the toxic aglycon. Rhynchosciara americana has 4 β-glycosidases (3 galactosidases and I glucosidase) in their intestinal microvilar membranes. Two of these enzymes are activated by Triton X-100. In β glucosidase the activation has Ka= 4µM, α=0,5 e β=2. β-thioglycosidases occur in Aphids. One digestive β-glucosidase from Diatraea saccharalis resolved by hydrofobic chrornatography hydrolyses sinigrin. The same enzyme may hydrolyse O- and S-glucosides with the same efficienly. Enzymes with this specificity have never been described before. In this study we shown some adaptations of insects to use substrates with β-glycosidic bonds, allowing these organisrns to explore nutrients usualy avoided by other animals.

O gene hrcA é encontrado em quase todos os ramos da árvore filogenética das eubactérias, e seu produto, a proteína HrcA, funciona como repressor da expressão dos operons de choque térmico groESL e dnaKJ, ligando-se à seqüência repetida invertida denominada CIRCE (controlling inverted repeat of chaperonin expression) presente na região regulatória destes operons. O sistema HrcA-CIRCE está, portanto, amplamente representado nas eubactérias. Particularmente, em Caulobacter crescentus, uma α-proteobactéria, este sistema está envolvido no controle da expressão do operon groESL durante o ciclo celular da bactéria. Conhecer a estrutura e as interações de HrcA é importante para entender este processo. Neste trabalho são apresentadas as análises de seqüência das HrcA\'s de C. crescentus e de Xylella fastidiosa, uma proteobactéria do grupo γ, as quais são muito similares. Este estudo levou à proposta de um modelo estrutural com a delimitação dos domínios da proteína, os dobramentos de cada domínio, com base nas interações da HrcA de C. crescentus com o elemento CIRCE e ATP, que estão sendo caracterizadas em nosso laboratório, assim como a atribuição de aminoácidos e motivos conservados funcionais. Adicionalmente, embora a expressão da HrcA recombinante de X. fastidiosa não tenha tido sucesso, a HrcA recombinante de C. crescentus purificada tem se prestado aos ensaios espectroscópicos, ainda que tenha sido detectada uma microagregação que está sendo enfrentada com um protocolo de purificação baseado no uso de α ciclodextrina. Os estudos espectroscópicos preliminares da HrcA C. crescentus dão suporte ao modelo estrutural proposto. / The hrcA gene is found in almost all branches of the filogenetic tree of eubacteria, and its product, the protein HrcA, functions as a repressor regulating the expression of the heat shock operons groESL and dnaKJ, by binding to the inverted repeat sequence called CIRCE (controlling inverted repeat of chaperonin expression). The system HrcA-CIRCE, therefore, is widely represented in eubacteria. Specifically in Caulobacter crescentus, an α-proteobacterium, this system is involved in the cell-cycle control of groESL expression (Baldini et al, 1998). Knowledge of the structure of HrcA and its interactions is important to understand this process. This work presents the analysis of the sequences of HrcA from C. crescentus and Xylella fastidiosa, a proteobacterium of the γ group, which are very similar. A structural model has been proposed, with protein domain delimitation, specific domain folding, based on known interactions of C. crescentus HrcA with the CIRCE element and ATP, obtained in our laboratory, as well as assignment of functional residues and conserved motifs. Additionally, even though no sucess was obtained the expression of recombinant HrcA from X. fastidiosa, purified recombinant HrcA from C. crescentus has been shown to be suitable for spectroscopic studies, in spite of microagregation observed, which is being faced with a purification protocol based on the use of α cyclodextrin. The preliminary spectroscopic studies of HrcA from C. crescentus support the proposed structural model.

Os polihidroxialcanoatos constituem um grupo de poliésteres acumulados por inúmeras bactérias na forma de grânulos intracelulares, que podem representar até 80% da massa seca celular. No presente trabalho, foi avaliado o comportamento de diferentes linhagens quanto a capacidade de metabolizarem ácidos graxos livres e óleo de soja para a produção de biopolímeros e, posteriormente, em biorreator, algumas linhagens selvagens e recombinantes foram testadas na presença ou não de um inibidor da b-oxidação. Experimentos em frascos agitados, mostraram valores de teor de PHA em até cerca de 70% na biomassa total com cerca de 5% de monômeros de cadeia média permitindo selecionar as linhagens Cupriavidus necator e Burkholderia cepacia para os ensaios seguintes. Experimentos em biorreator mostraram que, as variáveis manipuláveis quanto ao fluxo de óleo de soja, de co-substratos e o inibidor de b-oxidação não influenciaram, significativamente, no rendimento de P3HB/3HAmcl formado quando utilizado Burkholderia cepacia IPT-048, entretanto, contribuíram na síntese de 3HV. Durante os ensaios, o aumento do número de cópias do gene phaB em Cupriavidus necator, aparentemente, não contribuíram para o aumento do rendimento de 3HHx, porém, outras variáveis manipuláveis deverão ser propostas para a confirmação. / The poly-3-hydroxyalkanoates (PHA) are a group of polyesters accumulated for several bacteria in the intracellular granule form, that can represent up to 80% of the dry mass cellular. The main advantage of the biopolymers on the synthetic materials is its degradation in the environment. Recycable raw materials can be used as carbon sources for the production biodegradable polymer. Some polymers have appeared in literature having mechanical characteristic considered appropriate in such a way for use in packings (flexible and covering films), how base for controlled release of asset to be applied in the pharmacological-medical and foods area. The P3HB-co-3HAmcl are 3-hydroxybutyrate copolymers and 3-hydroxyacyl of 6 or more carbon atoms which has aroused interest for to present intermediate properties between HB and HAmcl, having taken care of the requirements for diverse applications. Some strains are distinguished, however, assays in bottles agitated with the Cupriavidus sp. and Burkholderia sp. strains, using as substratum greasy free acid and soy oil, had led the promising results. In the present work, first, was evaluated the behavior of different strains (Cupriavidus necator DSM545 IPT-026, C. necator H16 IPT-027, Pseudomonas sp. IPT-066, Burkholderia cepacia IPT-048 and B. sacchari IPT-189) how much to metabolizer capacity of free fatty acids and soybean oil for the production of biopolymers and, later, in tank bioreactor some wild strains and recombinant had been tested together or not of an inhibitor of the boxidation (acid acrylic). Experiments in bottles with agitation, had shown values of PHA in about 70% of the total biomass with about 5% monomers chain average allowing to select the Burkholderia cepacia IPT-048 and Cupriavidus necator DSM545 strains for the following assays. Experiments in tank bioreactor had shown that, the interactions between the substratum, co-substratum and the inhibitor of boxidation had influenced in the amount of formed P3HB/3HAmcl when used the Burkholderia cepacia IPT-048 strains, contributing for the synthesis of 3HV and 3HHx. When used only the soybean oil (5g/L) was gotten an amount of HHx with about 6 mol% with Burkholderia cepacia strains. In this same condition was verified that after some determined period also proved monomer HV in an amount of 0.20 mol%. Adding the acid acrylic (0.18 g/L) were gotten 1.50 mol% of HHx, however, differently of the previous condition the amount of HV varied of 0.91 - 2.77 mol%. A conclusion for the condition soybean oil and acrylic acid is that, the strains is using the acrylic fatty acid as carbon source, or either, the formation of acrylil-CoA with consequent formation of chains with 5 carbons. Similar results had been gotten when analyzed extracted polymer, or either, after the stage of extraction with chloroform was verified that the amount varied of 1.60 - 2.14 mol%. When added to a cosubstratum in the phase of accumulation (caproic fatty acid) and absence of the acrylic acid, it did not have the increase of HHx in relation to the previous variable, as much that, the amount of monomer reached about 0.50 mol%. In this condition, the presence of the acrylic fatty acid also did not contribute for the HV synthesis, not having been observed monomers during the assays. In the following phase, in which we work with Cupriavidus necator and a recombinant (Cupriavidus necator::phaB) strains, was verified that the gotten amount of HHx had been similar, about 0.30 mol%. Our hypothesis, we believe that the increase of the number of copies of the gene phaB could contribute for the increase of the carbon flow in the direction to raise the amount of 3HB how much of other monomers. As in the assays in bottles with agitation substrat caproic and caprylic fatty acids they had been distinguished also when availabled 2g/L and the soybean oil of in a concentration of 5g/L, we analyze the strains recombinant with soybean oil and caprylic fatty acid with 5 g/L. The insistence in the use of the caprylic fatty acid was based on the fact of the possibility of strains to oxidate part of fatty acids for the production of cells and energy and, at the moment that had the nitrogen limitation, to begin the accumulation phase dividing two carbons for the production of acetyl-CoA and the remain of the chain was stored in the form of 3HHx. The caprylic fatty acid disponibility it did not have the monomer attainment, therefore, Cupriavidus necator and Cupriavidus necator::phaB had its replyed inhibited capacity by the substratum. In the added soybean oil bottles with and without the acrylic fatty acid the behavior it was the opposite to the observed with caprylic fatty acid. During the incubation period, for both the strains, the amount of PHA, and consequently, the percentage of 3HB in the dry mass cellular was of 22.95% (100 mol%) and 25.90% (100 mol%) in 28ª and 24ª hours, respectively. In this condition, in the recombinant strains, also was observed the presence of HHx between 32ª and 49ª hour with a maxim amount of 1.10 mol%. From the results it can be concluded that the Burkholderia cepacia is considered a promising strains for the attainment of HHx and HV allowing that soybean oil with and without the acrylic fatty acid, respectively. The increase in the number of copies of the gene phaB in Cupriavidus necator apparently did not contribute with the increase of 3HHx efficiency however, other handling variables will be proposed for confirmation.

Poucos trabalhos foram publicados envolvendo a biossíntese do AA em plantas desde sua descoberta em 1928. O mecanismo de biossíntese era um mistério até 1998 quando Wheeler, Jones e Smirnoff demonstraram que a L-galactose é um precursor chave desta importante vitamina. Utilizando-se açúcares marcados e frios pudemos confirmar o mecanismo Smirnoff-Wheeler de biossíntese do AA. Neste trabalho nós apresentamos os resultados alcançados usando alguns supostos precursores e alguns frutos como o morango, a goiaba e o mamão papaya, e alguns legumes como o brócolis, alguns deles ricos em AA. As técnicas de HPLC e espectrofometria UVNIS foram utilizadas na determinação do AA. Os vegetais foram mantidos em soluções dos precursores frios por 24 horas e então analisados quanto ao teor de AA. Os resultados do uso de compostos marcados foi analisado utilizando-se a cintilografia líquida (LSC). Os açúcares extraídos do brócolis, do mamão papaya e da goiaba que foram infiltrados com D-[U-14C] manose, L-[1-14C] galactose e D-[U-14C] glucose-1-P mostraram diferentes padrões de distribuição entre os açúcares envolvidos no mecanismo Smirnoff-Wheeler de biossíntese do AA. Em folhas de goiabeira encontramos altos teores de ácido desidroascórbico e pequena quantidade de AA, diferentemente do conteúdo de AA nos frutos. Nossos resultados confirmaram que a L-galactono-1 ,4-lactona é um precursor bastante eficiente do AA em frutos e proveram algumas evidências do envolvimento da 0- manose no mecanismo de biossíntese do AA em plantas. / Since the first isolation of the Ascorbic Acid (AA) in 1928, a few papers have been published leading with the determination the AA biosynthetic pathway in plants. This pathway was a mystery until recently when in 1998 Wheeler, Jones and Smirnoff demonstrated that L-galactose is a key precursor of this important vitamin. Using radiolabeled and non-radiolabeled sugars we were capable of giving support to the Smirnoff-Wheeler pathway of AA biosynthesis. In this work we present the results reached using some putative precursors and some fruits and vegetables as strawberry, guava, papaya and broccoli some of them very rich in AA. The techniques used for the AA analysis included UVNIS spectrophotometry and HPLC methods. The fruits and vegetables were maintained in the solution of the non-radiolabeled precursors for 24 hours and then analyzed for the AA content. The results of the use of radiolabeled precursors were analyzed using liquid scintillation (LSC). The sugars extracted from broccoli , papaya and guava, that were supplied with D-[U-14C] mannose, L-[1-14C] galactose and D-[U-14C] glucose-1-P dipotassium salt were analyzed using HPLC amperometric method and LSC showed different pattern of distribution between the sugars involved in the Smirnoff-Wheeler AA biosynthesis pathway. In guava leaves we found a high content of dehydroascorbic acid and low amount of AA, unlikely of the content of AA in the fruit. Our results confirmed that L -galactono-1 ,4-lactone is a very effective precursor of AA in fruits and provided some evidence of the involvement of D-mannose in AA biosynthesis in plants.

Le Dimethylsulfoniopropionate (DMSP) est une molécule à fort impact écologique couramment produite par le phytoplancton marin, mais très rarement chez les plantes à fleurs: seulement chez quelques genres (dont Spartina chez les Poacées). Bien que les étapes enzymatiques impliquées dans la voie de biosynthèse du DMSP soient connues chez les spartines, son origine ainsi que les gènes impliqués restent encore à découvrir chez les plantes. Cette étude s’est fixée pour objectif de contribuer à élucider les mécanismes à l’origine de cette fonction chez les spartines. Cette question a été appréhendée à travers différentes approches : biochimique, métabolomique, transcriptomique, génomique comparative et phylogénétique. Les résultats ont montré que la capacité à synthétiser le DMSP a une origine unique au sein du genre Spartina et se serait mise en place il y a 3-10 millions d’années. Cette capacité est intervenue chez l’ancêtre d’un des deux principaux clades (hexaploïde) de spartines, puis a été héritée chez toutes les espèces dérivant de ce clade (hexaploïdes à dodécaploïdes). Les espèces de l’autre clade (tétraploïde) et leurs descendants (quel que soit leur niveau de ploïdie) n’accumulent pas de DMSP. En utilisant les génomes séquencés des espèces de Poacées ainsi que les ressources génomiques et transcriptomiques disponibles chez les spartines, les gènes candidats intervenant dans les 4 étapes de la voie de biosynthèse proposée dans la littérature ont été explorés. L’identification des gènes intervenant dans les deux étapes intermédiaires, supposées spécifiques de la capacité de synthèse du DMSP représente un véritable défi dans la mesure où seules des activités enzymatiques putatives ont été proposées à ce jour (sans connaissance des enzymes spécifiques ni de leur séquence protéique). Nous avons pu identifier une série de gènes candidats pour chacune des deux fonctions concernées (décarboxylase et amine oxydase), comparer leur niveau de transcription entre les espèces DMSP+ et DMSP-, et prédire leur localisation cellulaire. De plus, des analyses d’activités enzymatiques ont permis de formuler de nouvelles hypothèses et pistes de recherches sur l’émergence de cette nouvelle voie de biosynthèse chez les spartines. / Dimethylsulfoniopropionate (DMSP) is an ecologically important molecule produced by most marine phytoplankton species, but very rarely by flowering plants: only in a few genera (including Spartina in Poaceae). Despite the different enzymatic steps involved in DMSP biosynthesis are well known, the origin of the function and the genes encoding the different enzymes are yet to be discovered. To explore the evolutionary mechanisms involved in the DMSP accumulation in Spartina, we used various approaches, including biochemical analyses, metabolomics, transcriptomics, comparative genomics and phylogenetics. Notably, we demonstrate that the ability to synthesize DMSP evolved once in the Spartina genus, sometimes 3-10 million years ago. This functional innovation occurred following the emergence of the hexaploid clade, and was inherited by all Spartina species deriving from this hexaploid ancestor. Spartina species belonging to the tetraploid clade and their deriving species do not accumulate DMSP (whatever their ploidy level). Using Poaceae sequenced genomes as well as Spartina genomic and transcriptomic resources obtained in our laboratory, candidate genes involved in the four different enzymatic steps of the DMSP biosynthesis pathway were searched. Identifying genes involved in the intermediate (2nd and 3rd) steps that are specific to this pathway was particularly challenging as only putative enzymatic activities have been proposed so far (corresponding protein sequences and genes are unknown). A set of candidate genes potentially involved in these two steps (with decarboxylase and amine oxydase activities) were identified and their transcription levels were compared among DMSP producing (DMSP+) and non-producing (DMSP-) Spartina species. Their putative cellular localization was also predicted. Moreover, enzymatic activity assays open new hypotheses and research perspectives regarding this enigmatic biosynthesis pathway in Spartina.

Salt stress is a severe environmental challenge in agriculture, limiting the quality and productivity of the crops around the globe. Plant growth promoting rhizobacteria (PGPR) is proposed as a friendly solution to overcome those challenges. The desert plant endophytic bacterium, Enterobacter sp. SA187 has shown plant growth promotion and salt stress tolerance beneficial effect on the model plant Arabidopsis thaliana in vitro as well as under the field conditions on different crops. SA187 has a distinguished morphology of yellow colonies (SA187Y) that could be due to carotenoid biosynthesis. However, the bacteria tend to lose the yellow color upon incubation with the plants and the colonies turn to white (SA187W). In comparison to SA187Y, SA187W shows 50% reduction on the beneficial impact on A. thaliana fresh and dry weight of root and shoot system. By counting the CFU/plant, we showed that SA187Y and SA187W both have similar colonization rate in both shoots and roots. Under non-salt conditions, optimal bacterial colonization was observed on day 8 after inocubation, however, under the salt stress condition, the optimal colonization was observed at day 4. Moreover, during the time period of the incubation of the SA187Y with the plants, there was a consistent noticeable loss of the yellow color of the colonies. This change in color is only observed eight days after transfer and the number of white colonies increases with the increase of the incubation time. In addition, SA187W was GFP-tagged by Tn7 transposon system and visualized by confocal laser scanning microscopy. The SA187W-GFP colonies have shown a similar colonization pattern as SA187Y-GFP, bacteria were colonizing the differentiation zone and cell elongation zone in the roots. Finally, the gene expression of the carotenoid biosynthesis pathways genes in SA187Y showed an overall higher gene expression compared to SA187W. In conclusion, the color loss seems to affect the beneficial impact of the bacteria on plants. However, the reduced beneficial impact is not due to the colonization efficiency of bacteria on the plant roots but could be due to a regulation of gene expression of carotenoid biosynthesis.

No description available.