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Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.
A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level : from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation ; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.
Maritime pine somatic embryos require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome (2D-SDS-PAGE with MS identification) of immature somatic embryos, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous ABA (GC-MS), soluble sugars (HPLC), starch, and confocal laser microscope observations. This multi-scale, integrated analysis was used to unravel early molecular and physiological events involved in somatic embryo development. Under unfavourable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation which may be antagonistic to somatic embryo development. Under favourable conditions (9G), somatic embryos adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin like protein and ubiquitin-protein ligase could be used as predictive markers of somatic embryo development whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in development of pine somatic embryos following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.
The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. In order to further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.
The chiral separation of a new antianginal agent has been investigated on a chiral cellulose column with UV and circular dichroism (CD) detection. This benzoxathiepin derivative under development has two stereogenic centers whose (R,S) stereoisomer shows an interesting, antianginal activity. After optimisation of the mobile phase composition, a baseline-resolved separation of the four stereoisomers was achieved on a Chiralcel OJ-H chiral column by using methanol-ethanol-diethylamine (25:75:0.1, v/v/v) as mobile phase. The CD detection system allowed quantitation and a linear response was observed within a 10-200 mu g mL(-1) concentration range (r(2) = 0.9966) and limit of quantification down to 2 jig mL(-1) was achieved. (c) 2005 Elsevier B.V. All rights reserved.
Reported here is an analytical method enabling the stereochemical resolution of a new antianginal compound possessing two stereogenic centers, leading to four stereoisomers. Only one of these isomers is currently under development as a novel antianginal agent and consequently, the other three isomers are considered as unwanted chiral impurities. Therefore, an enantioselective method is required in order to check its enantiomeric purity. This paper presents a method exploiting the high efficiency of capillary electrophoresis and the complexing properties of cyclodextrins to achieve the separation of the four stereoisomers of this weakly basic compound (pK(a) = 7.4).
Studies of the perturbing effect of chiral solvating agents (CSAs) 5a and mostly of 5c upon the NMR spectra of chiral Delta(2)-oxazoline 1 demonstrated the ability of these fluoroalcohols to afford diastereomeric solvates from these solutes. Thus, for all tested Delta(2)-oxazolines 1Aa-d, 1Ba, and 1e there is at least one possibility to proceed to their enantiomeric discrimination either by H-1 or F-19 NMR using these CSAs (see Fig. 1). NMR results are discussed from substrate and CSA structure standpoints and a solvation model is proposed on the basis of the inequivalence senses generally observed. Then the method was applied to extracts of incubated locust tissues obtained by solid phase extraction (SPE) after a partial unmasking of the substrate 1.
The 300 MHz H-1 NMR spectra of 2-ethyl Delta(2) -oxazoline 1m have been studied in CCl4, CD3CN and C6D6 solutions, in the presence of the achiral lanthanide shift reagent (LSR), tris (6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato)-europium III, 4 known as Eu(fod)(3), (see Sch. 1). All the protons of 1 were deshielded at various extent, and the sequence observed for their Deltadelta suggested a major complexation at the basic N(3) center of the heterocycle. Then the chiral monosubstituted oxazoline 1e and the disubstituted oxazolines 1Aa-d and 1Ba, were studied in the presence of chiral LSR, tris-[D, D dicampholylmethanato] europium III Eu(dcM)(3) 5 and tris-[3-(heptafluoropropylhydroxy-methylene)-d-camphorato] praseodym III Pr(hfC)(3) 6. H-1 NMR, and eventually F-19[H-1] NMR spectra were mostly recorded in C6D6 solution.
Modern thin-layer chromatography (TLC) was used for the evaluation of Delta (2) -oxazolines-1,3 I and N-acylaziridine VII structures, as potential proinsecticides of carboxylic acids III. Thus the unmasking(2) of the active principles III from Delta (2) -oxazolines-1,3 Ia-c and N-acylaziridine VIIc was monitored by spotting aliquots directly onto RP-18 TLC plates, without any sample pretreatment during in vitro assays performed in concentrated locust tissues. To achieve a good separation of carboxylate IIIa from endogenous components of the tissues, a short preliminary development with methanol or ion-pairing was necessary. From UV-TLC chromatograms (densitograms) it appeared that in a phosphate buffer at pH 7.4, the oxazoline Ia with a C-2 substituent devoid of alpha -ramification or alpha,beta -in saturation hydrolysed slowly into the corresponding beta -hydroxylamide VIa and intermediate aminoester Va.
Treatment of 4,6-dinitrobetlzofuroxan (DNBF) with the imidazoline 1-NRf is found to afford a zwitterionic nitrogen-bonded complex (2-NRf) which, in the presence of base (Et3N), undergoes a slow but quantitative transformation to give 7-hydroxy-4,6-dinitrobmzofurazan (5) as the final product. Overall, an oxygen transfer has thus occurred from the N-oxide function to the carbocyclic moiety of DNBF. The key point in this transformation is shown to be a facile abstraction of the sp(3) hydrogen bonded at C-7 of 2-NRf, providing important new evidence that the parent DNBF structure is extremely electron-withdrawing (’super-electrophile’). The overall conversion is also an unusual case of a catalytic process in which the catalysts (both 1-NRf and Et3N) partake to form covalent reaction intermediates and thereby lower the activation energy, resulting in a facile reaction. (C) 2001 Elsevier Science Ltd. All rights reserved.
Maître de conférences , Spectrométrie de masse fonctionnelle et métastases