Metabolic characterization of green pods from Vanilla planifolia accessions grown in La Reunion.

Large phenotypic variation has been observed between the cultivated vanillas since a single genetic source of Vanilla planifolia was spread to the Indian Ocean and the Indonesia in the 19th century. In order to differentiate the cultivated vanilla plants, genetic studies have been conducted in the past on the plants grown in various regions such as the French island, La Réunion. However, the genetic difference was not big enough to differentiate diverse accessions of V. planifolia. In this study, metabolomics, in which genetic variation could be amplified, was employed to delve into the variation between the cultivated vanilla plants. To obtain a broad view of the metabolome, nuclear magnetic resonance (NMR) spectroscopy was applied to the analysis of V. planifolia green pods. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) of the data showed that the accessions could be differentiated according to their glucovanillin and glucosides A and B contents. Furthermore, a correlation between the glucovanillin content and the pod length, number of flower and growth capacity of the accessions has been observed from the multivariate data analysis

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage

<p>Abstract</p> <p>Background</p> <p><it>Vanilla planifolia </it>is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and <it>in vitro </it>mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of <it>V. planifolia </it>through indirect shoot differentiation from callus culture is rare and may be a successful use of in <it>vitro </it>techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.</p> <p>Results</p> <p>For embryogenic callus induction, seeds obtained from 7-month-old green pods of <it>V. planifolia </it>were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l^-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l^-1) and TDZ (0.5 mg l^-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l^-1) and TDZ (0.3 mg l^-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l^-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (<it>P </it>< 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.</p> <p>Conclusion</p> <p>The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.</p

Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling

Background: The respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas. Methods: In this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico. Results: Even though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species. Conclusions: We were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species

Metabolic Investigation of the Mycoplasmas from the Swine Respiratory Tract

International audienceBackgroundThe respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas.MethodsIn this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico.ResultsEven though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species.ConclusionsWe were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species

Caractérisation métabolomique par RMN de Vanilla planifolia

Vanilla planifolia, orchidée épiphite florifère, est la principale source naturelle de l'arôme de vanille. Largement utilisé dans les produits laitiers, les boissons, les pâtisseries et les parfums, cet arôme est le résultat d'un processus complexe : de huit à neuf mois après la fécondation des fleurs, les gousses matures sont récoltées et traitées pendant environ un an afin de libérer leur bouquet aromatique. Aujourd'hui, plus de la moitié de la production mondiale de vanille provient de Madagascar. Pour faire face à cette concurrence, les producteurs de la Réunion se tournent vers la production de vanille "haut de gamme". L'exploitation des vanilliers les plus intéressants du point de vue aromatique est donc favorisée. Toutefois, les programmes d'amélioration se heurtent au manque de connaissances sur la physiologie de la plante. Il devient alors essentiel de mieux comprendre les mécanismes physiologiques et biochimiques impliqués dans la production aromatique des gousses de V. planifolia. Dans ce travail de thèse, une analyse des métabolites présents dans les gousses vertes et les feuilles de vanille a été effectuée par Résonance Magnétique Nucléaire. Cette technique permet l'évaluation qualitative et quantitative des métabolites primaires (sucres, acides animés et organiques...) et secondaires (composés phénoliques...) présents dans la plante dans diverses conditions physiologiques : au cours du développement de la gousse, lors d'une infection virale, selon les saisons ou encore sur différentes accessions.Vanilla planifolia, a flowering epiphitic orchid, is the major natural source of vanilla flavour. Largely used in dairy products, beverages, bakeries and perfume, vanilla flavour is obtained after a long process: from eight to nine months after flower pollinisation, mature pods are harvested and then prepered during about one year in order to release the characteristic vanilla aroma. Nowadays, more than half vanilla pods world production comes from Madagascar. To face the concurrence, a solution could be develop higher quality pods. Selection of the most aromatic vanilla plant is then preferred. Nevertheless, amelioration program are facing up to a lack of knowledge in vanilla plant physiology. It is now essential to understand more the physiological and biochemical mechanisms implied in the aromatic production of V. planifolia pods. In this thesis, a metabolomic analysis of vanilla green pods and leaves has been performed by nuclear magnetic resonance. This technique has allowed the qualitative and quantitative analysis of primary (sugar, amino and organic acids...) and secondary metabolites (phenolic compounds...) present in vanilla plant according to various physiological conditions: developing pods, viral infection, inter-accession or seasonal variation

Metabolic changes in different developmental stages of vanilla planifolia pods.

The metabolomic analysis of developing Vanilla planifolia green pods (between 3 and 8 months after pollination) was carried out by nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Multivariate data analysis of the 1H NMR spectra, such as principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA), showed a trend of separation of those samples based on the metabolites present in the methanol/water (1:1) extract. Older pods had a higher content of glucovanillin, vanillin, p-hydroxybenzaldehyde glucoside, p-hydroxybenzaldehyde, and sucrose, while younger pods had more bis[4-(β-d-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A), bis[4-(β-d-glucopyranosyloxy)-benzyl]-2-(2-butyl)tartrate (glucoside B), glucose, malic acid, and homocitric acid. A liquid chromatography−mass spectrometry (LC−MS) analysis targeted at phenolic compound content was also performed on the developing pods and confirmed the NMR results. Ratios of aglycones/glucosides were estimated and thus allowed for detection of more minor metabolites in the green vanilla pods. Quantification of compounds based on both LC−MS and NMR analyses showed that free vanillin can reach 24% of the total vanillin content after 8 months of development in the vanilla green pods

Modélisation du réseau métabolique des cellules de Leucémies Aiguës Myéloïdes pour comprendre les différences métaboliques liées à la mutation sur IDH1

International audienceLes leucémies aigues myéloïdes (LAM) sont des maladies hématologiques liées à la transformation maligne de progéniteurs hématopoïétiques dans la moelle osseuse aboutissant à la destruction du tissu sanguin sain. Des mutations récurrentes ont été observées chez les patients atteints de LAM dont l'une sur des enzymes clés du métabolisme : les isocitrate déshydrogénases 1 et 2 (IDH1, IDH2). La mutation IDH1-R132H conférerait aux cellules LAM une flexibilité métabolique induisant des dépendances métaboliques et énergétiques spécifiques, qui seraient responsables de la chimiorésistance de ces cellules. Dans cette étude, nous avons utilisé une approche globale de modélisation in silico du réseau métabolique des cellules LAM, basée sur des données de transcriptomique et d'exométabolomique, afin d'identifier l'ensemble des voies métaboliques activées par la mutation IDH1-R132H dans ces cellules. À partir du réseau métabolique humain global (Recon2; Thiele et al., 2013) et des données d'expression de gènes obtenues sur des cellules LAM avec ou sans la mutation IDH1-R132H, nous avons utilisés des algorithmes d'optimisation permettant de générer des modèles de réseaux métaboliques représentant spécifiquement le réseau métabolique fonctionnel de cellules LAM avec et sans mutation, c'est-à-dire les réactions du réseau global qui sont spécifiquement actives dans les cellules étudiées. Plusieurs algorithmes publiés visant à l'identification de réseaux tissu-ou cellule-spécifiques à partir de données de transcriptomique ont été testés et adaptés. Les données d'exométabolomique ont été utilisées, via des méthodes de modélisation sous contraintes, pour contextualiser les modèles cellules-spécifiques générés et rendre compte de la production ou consommation des métabolites mesurés. La comparaison des réseaux cellules-spécifiques générés a permis notamment de mettre en évidence que les voies du métabolisme des acides gras étaient différemment utilisées chez les cellules mutées ou non mutées. Les modèles générés pourront être utilisés pour identifier des dépendances métaboliques présentes spécifiquement dans les cellules LAM mutées

Biological variation of Vanilla planifolia leaf metabolome

International audienc

Procédé de discrimination des bactéries Escherichia Coli et Shigella par spectrométrie RMN

Priority Data: 1555269 - 09.06.2015 FRThe invention relates to a method for the proton NMR spectrometry-based differentiation of Escherichia colibacteria and Shigella bacteria, said method comprising the steps of: obtaining at least one supernatant of at least one suspension of bacteria that could be Escherichia coli or Shigella; preparing at least one sample suitable for proton NMR spectrometry analysis, by mixing at least one fraction of at least one supernatant with a deuterated buffer; obtaining at least one NMR spectrum of the at least one sample; analysing the peaks of the at least one spectrum corresponding to the following metabolites: acetate, alanine, aspartate, ethanol, lactose, lysine, Na-acetyllysine, propionate, serine, succinate, threonine and valine; determining if it is Escherichia coli or Shigella bacteria.La présente invention concerne un procédé de différenciation par spectrométrie RMN u proton entre une bactérie Escherichia coli et une bactérie Shigella, ledit procédé omportant les étapes consistant à : - Obtenir au moins un surnageant d'au moins une suspension de bactéries susceptibles d'être Escherichia coli ou Shigella; - Préparer au moins un échantillon adapté à une analyse en spectrométrie RMN du proton, par mélange d'au moins une fraction du au moins un surnageant avec un tampon deutéré; - Obtenir au moins un spectre RMN du moins un échantillon, - Analyser les pics dudit au moins un spectre correspondant aux métabolites suivant : acétate, alanine, aspartate, éthanol, lactose, lysine, Na-acétyllysine, propionate, sérine, succinate, thréonine et valine; - Déterminer s'il s'agit d'une bactérie Escherichia coli ou Shigella