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

Exploring the Phytobeneficial and Biocontrol Capacities of Endophytic Bacteria Isolated from Hybrid Vanilla Pods.

peer reviewedIn this study, 58 endophytic bacterial strains were isolated from pods of two hybrid vanilla plants from Madagascar, Manitra ampotony and Tsy taitra. They were genetically characterized and divided into four distinct phylotypes. Three were associated to genus Bacillus species, and the fourth to the genus Curtobacterium. A selection of twelve strains corresponding to the identified genetic diversity were tested in vitro for four phytobeneficial capacities: phosphate solubilisation, free nitrogen fixation, and phytohormone and siderophore production. They were also evaluated in vitro for their ability to biocontrol the growth of the vanilla pathogenic fungi, Fusarium oxysporum f. sp. radicis vanillae and Cholletotrichum orchidophilum. Three bacteria of phylotype 4, m62a, m64 and m65, showed a high nitrogen fixation capacity in vitro, similar to the Pseudomonas florescens F113 bacterium used as a control (phospate solubilizing efficiency respectively 0.50 ± 0.07, 0.43 ± 0.07 and 0.40 ± 0.06 against 0.48 ± 0.03). Strain t2 related to B. subtilis showed a higher siderophore production than F113 (respectively 1.40 ± 0.1 AU and 1.2 ± 0.1 AU). The strain m72, associated with phylotype 2, showed the highest rate of production of Indole-3-acetic acid (IAA) in vitro. Bacteria belonging to the pylotype 4 showed the best capacity to inhibit fungal growth, especially the strains m62b m64 and t24, which also induced a significant zone of inhibition, suggesting that they may be good candidates for controlling fungal diseases of vanilla. This competence was highlighted with spectral imaging showing the production of lipopeptides (Iturin A2 and A3, C16 and C15-Fengycin A and C14 and C15-Surfactin) by the bacterial strains m65 confronted with the pathogenic fungi of vanilla.FEDER BIOMED HUB Technology Suppor

Étude des mécanismes de défense mis en jeu lors de l'interaction entre Ralstonia solanacearum et des Solanacées

Solanum torvum, résistant au flétrissement bactérien, est une espèce sauvage apparentée à S.melongena. La connaissance des bases biologiques des mécanismes de résistance de cette espèce sauvage face à r. solanacearum devrait contribuer à faire progresser les connaissances dans l'interaction solanacées/ r.solanacearum. L'objectif de ce travail a été d'appréhender certains mécanismes induits lors de l'infection par r. solanacearum. Linfection de S. torvum par R.solanacearum a permis de montrer la mise en place très précoce de tyloses limitant la pénétration de la souche 1609 au sein du systèmes racinaire. Par contre, d'autres souches telle que JT516, colonisent aisément la plante, induisant des réponses d'ordre biochimique et physiologique. De plus, l'évaluation du pouvoir pathogène de différentes souches de phylotype II génétiquement proche après inoculation de 8 cultivars de Solanacées a aussi permis d'observer une grande variabilité de pathogénicité face à un même hôte végétal.Solanum torvum, resistant to bacterial wilt, is a wild species closely related to Solanum.melongena. knowledge of the biological bases of the mechanisms of resistance of this wild species against R. solanacearum should contribute to improve the comprehension of the interaction Solanaceae/R. solanacearum. The major aim of this work was to study some mechanisms induced during R.solanacearum infection. The infection of S. torvum with R.solanacearum showed a very rapid induction of thylles. This structure was able to limit the penetration of strain 1609 in roots. . On the contrary, other strains such as JT516, colonize easilyn the host, inducing biochemical and physiological responses. Moreover, the evaluation of pathogenicity of different genetically related strains belonging to the phylotype II after inoculation of 8 Solanaceae cultivars revealed a high variability of pathogenicity after infection of the same host plant.SAINT DENIS/REUNION-Droit Lettre (974112101) / SudocSudocFranceReunionFRR

Medicinal plants, malaria and biotechnology

The first part of the talk will be dedicated to the investigation of medicinal plants with the objective to identify new antimalarial treatments. According to the last World Malaria Report [1], there were 584 000 deaths for 198 millions malaria cases worldwide in 2013. Particularly, the disease caused an estimated 437 000 African children died before their fifth birthday, still in 2013. Malaria is caused by a parasite, Plasmodium sp. and transmitted by Anopheles mosquitoes. The problem of parasite resistance towards common available medicines such as chloroquine, mefloquine, quinine, is increasing. In this context, the vegetal kingdom remains the main source of pharmacologically active compounds against this parasitic infection as attested by the famous quinine, isolated from Cinchona sp., artemisinin extracted from Artemisia annua and also atovaquone derived from lapachol found in several Bignoniaceae. All these substances are related to plants with traditional use against fever and malaria. Beside these well-known examples, various new antiplasmodial compounds are frequently discovered from Nature, particularly following an ethnopharmacological approach, as reviewed by several authors in recent years [2-6]. Then, the pharmacological and phytochemical study of plants from traditional pharmacopoeias can be of first interest not only to discover new antimalarial “lead compounds”, but also to valorize local vegetal species whose efficacy and safety would have been demonstrated in laboratory and by clinical investigations [7,8]. Some results obtained with Dicoma tomentosa from Burkina-Faso [9] and Terminalia mollis from Rwanda [10] will be presented. In the second part of the talk, two applications of biotechnology for the production of artemisinin and paclitaxel and then some works developed at the ‘Université de la Réunion’ will be presented. In the framework of this collaboration, Psiadia arguta, an endemic plant from Reunion Island, which is known to have cytotoxic, anti-plasmodial and anti-inflammatory properties, was subjected to micropropagation. The objective of the work was to compare the biological properties and the phytochemical composition of callus, vitroplants and acclimatized plants of Psiadia arguta [11]. 1. WHO, World Malaria Report 2014, December 2014, Geneva (http://www.who.int/malaria/publications/world_malaria_report_2014/en/). 2. Batista R, Silva Ade J Jr, de Oliveira AB: Plant-derived antimalarial agents: new leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules 2009, 14:3037-72. 3. Bero J, Frédérich M, Quetin-Leclercq J : Antimalarial compounds isolated from plants used in traditional medicine. Journal of Pharmacy and Pharmacology 2009, 61:1401–1433. 4. Bero J and Quetin-Leclercq J: Natural products published in 2009 from plants traditionally used to treat malaria. Planta Medica 2011, 77:631-40. 5. Kaur K, Jain M, Kaur T, Jain R: Antimalarials from nature. Bioorganic & Medicinal Chemistry 2009, 17:3229–3256. 6. Nogueira CR and Lopes LMX: Antiplasmodial Natural Products. Molecules 2011, 16:2146-2190 7. Ginsburg H and Deharo E: A call for using natural compounds in the development of new antimalarial treatments – an introduction. Malaria Journal 2011, 10 (suppl. 1):S1 8. Willcox M, Graz B, Falquet J, Diakite C, Giani S, Diallo D: A “reverse pharmacology” approach for developing an antimalarial phytomedicine. Malaria journal 2011, 10(suppl1):S8 9. Jansen, O., Tits, M., Angenot, L., Nicolas, J.-P., De Mol, P., Nikiema, J.-B., & Frédérich, M : Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15-O-acetate as the main active compound. Malaria Journal 2012, 11, 289. 10. Muganga, R., Angenot, L., Tits, M., & Frédérich, M : In vitro and in vivo antiplasmodial activity of three Rwandan medicinal plants and identification of their active compounds. Planta Medica 2013, 80(6), 482-489. 11. Mahy Justine, Comparative study of biological activities and analysis of volatile compounds of Psiadia arguta in various cultures: vitroplants and acclimatized plants. Mémoire de M2, 2013, Université de Liège/Université de la Réunion

Etude des composés volatiles de la gousse noire de la vanille par la méthode headspace statique couplée à la GC-MS/MS : mise en évidence d’une relation terroir/mode de culture/bouquet aromatique

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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