[show abstract][hide abstract] ABSTRACT: Flax (Linum usitatissimum) is grown for its oil and its fiber. This crop, cultivated in temperate regions, has seen a renewed interest due to the presence of abundant molecules of interest for many applications. Little information is available about the behavior of flax during osmotic stress; yet this is considered a major stress that causes significant yield losses in most crops. To control the presence of this stress better, flax behavior was investigated following the application of osmotic stress and the response was examined by applying increasing concentrations of PEG 8000. This resulted in the reorganization of 32 metabolites and 6 mineral ions in the leaves. The analysis of these two types of solute highlighted the contrasting behavior between a higher metabolite content (particularly fructose, glucose and proline) and a decrease in mineral ions (especially nitrate and potassium) following PEG treatment. However, this reorganization did not lead to a greater accumulation of solutes, with the total amount remaining unchanged in leaves during osmotic stress.
Journal of plant physiology 08/2013; · 2.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: The bacterium Enterobacter ludwigii Ez-185-17, member of the family Enterobacteriaceae, was isolated from the root nodules of plants harvested in the nuclear power region of Chernobyl. Under batch culture conditions, the bacteria produce a high-molecular-mass exopolysaccharide (EPS). After purification, the structure of this EPS was determined using a combinatory approach including monosaccharide composition (GC-FID, HPAEC-PAD) and branching structure determination (GC-MS), as well as 1D/2D NMR (H, C) and ESI-MS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged hexasaccharide with a repeating unit composed of d-galactose, d-glucose, l-fucose, d-glucuronic acid (2:1:2:1) and substituted with acyl and pyruvyl groups. The metal-binding properties of the exopolysaccharide were then investigated, and the results seem to indicate that the EPS decreased Cd sequestration in flax seeds.
[show abstract][hide abstract] ABSTRACT: Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1->6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.
PLoS ONE 01/2013; 8(7):e68373. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trehalose is a non-reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana.
After clearly highlighting a co-elution of trehalose in these two species by a trehalase assay and liquid chromatography-high resolution mass spectrometry analysis, gas chromatography-mass spectrometry (GC-MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC-PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC-MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (-0.30 MPa), the quantity (31.49 nmol g(-1) dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.
Physiologia Plantarum 08/2012; · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: From green tea leaves, two distinct pectin fractions were obtained based on their solubility in water. Polyphenols were detected only in the easily water soluble fraction (P1). The estimated uronic acids/neutral sugars ratio was 1.7 in the easily water soluble pectin fraction (P1), and 1.0 in the less water soluble fraction (P2). Homogalacturonan sequences (HGAs) corresponded to about 62% of the P1 pectin fraction but only 47% of the P2 fraction. After degradation of the two pectin fractions by pectin lyase, chemical studies revealed rhamnogalacturonan RG I and RG II regions present in the P1 pectin fraction, whereas only RG I sequences were detected in the P2 pectin fraction. The degree of substitution was lower for HGAs of the P1 pectin fraction than P2. Different acetylation patterns for the two fractions were observed. Polyphenols extracted simultaneously with pectins were present only in HGA fractions from P1.
[show abstract][hide abstract] ABSTRACT: In wheat, little is known about disease resistance inducers and, more specifically, about the biological activities from those derived from endogenous elicitors, such as oligogalacturonides (OGAs). Therefore, we tested the ability of two fractions of OGAs, with polymerization degrees (DPs) of 2-25, to induce resistance to Blumeria graminis f. sp. tritici and defense responses in wheat. One fraction was unacetylated (OGAs-Ac) whereas the second one was 30% chemically acetylated (OGAs+Ac). Infection level was reduced to 57 and 58% relative to controls when OGAs-Ac and OGAs+Ac, respectively, were sprayed 48 h before inoculation. Activities of various defense-related enzymes were then assayed in noninoculated wheat leaves infiltrated with OGAs. Oxalate oxidase, peroxidase, and lipoxygenase were responsive to both OGAs-Ac and OGAs+Ac, which suggests involvement of reactive oxygen species and oxilipins in OGAs-mediated responses in wheat. In inoculated leaves, both fractions induced a similar increase in H₂O₂ accumulation at the site of fungal penetration. However, only OGAs+Ac led to an increase in papilla-associated fluorescence and to a reduction of formed fungal haustoria. Our work provides the first evidence for elicitation and protection effects of preventive treatments with OGAs in wheat and for new properties of acetylated OGAs.
[show abstract][hide abstract] ABSTRACT: Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone, produces a non-referenced high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was determined using a combination approach including monosaccharide composition (GLC-FID, HPAEC-PAD), determination of glycosylation sites (GLC-EIMS) and 1D/2D NMR ((1)H, (13)C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a repeating unit composed of D-galactose, D-glucose, D-mannose and D-glucuronic acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose and glucose residues, respectively, as presented in the following structure:
Carbohydrate research 06/2010; 345(9):1163-73. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family (Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family (Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 x 10(6) and 3 x 10(6) Da. Five different compositions of EPS were characterized by physico-chemical analyses and (1)H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs.
Research in Microbiology 03/2010; 161(2):101-8. · 2.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Homogalacturonic and homoglucuronic acid sequences, with various molecular weights and corresponding monomer, were mixed with cholesterol or linolenic acid in controlled conditions (pH, temperature, concentration), then studied by 1H and 13C NMR. The variation of 13C NMR chemical shifts showed the formation of interactions between cholesterol or linolenic acid and carbohydrate sequences depending the molecular weight. Galacturonic acid sequences revealed to be more suitable than glucuronic acid sequences to entrap cholesterol or linolenic acid molecules probably due to the difference of conformation of the two molecules.
Comptes Rendus Chimie - C R CHIM. 01/2010; 13(4):443-448.
[show abstract][hide abstract] ABSTRACT: Flaxseed mucilage from Linum usitatissimum L. species was constituted by arabinoxylan (about 75%) and pectin (about 25%). A new procedure was developed to obtain only arabinoxylans which implicated treatment of the pectin fraction by enzymatic hydrolysis with pectinase. Then three processes of depolymerization were evaluated on arabinoxylans. First, a thermic hydrolysis in mild acid conditions was performed and an ultrafiltration process was used as purification method. Second, the potential of xylanases from different glycoside hydrolase families for arabinoxylan-oligosaccharides (AXOS) production was tested, and finally a radical depolymerization was conducted. Average molecular weights were determined by high pressure size exclusion chromatography coupled with multiple angle laser light scattering (MALLS), and carbohydrate compositions were determined by high pH anion exchange chromatography pulse amperometric detector (HPAEC-PAD). Both chemical and enzymatic treatments were inefficient to convert arabinoxylans from flaxseed mucilage into AXOS. Only radical depolymerization process was allowed to obtain arabinoxylan-oligosaccharides presenting different molecular weights (11.9 x 10(3) to 1.9 x 10(3) g mol(-1)) with satisfactory yields (75% to 35%).
Journal of Agricultural and Food Chemistry 11/2009; 57(23):11308-13. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, we showed that oligogalacturonic acid (OGA) purified from flax pectin inhibit in vitro osteoclastic bone resorption in a dose-dependent manner. The OGA inhibitory effect was neither linked to an effect on osteoclast apoptosis, nor to an inhibition of cathepsin K activity. By means of an in vitro collagen degradation assay we demonstrated that OGA prevented triple-helical type I collagen cleavage by cathepsin K in a dose and chain length dependent manner. This inhibition was not restricted to cathepsin K, since collagenolytic activity of other lysosomal cysteine proteases, such as cathepsin B and cathepsin L, as well as matrixmetalloproteinases such as MMP-9 were also inhibited. Interestingly, using non-collagen substrates we demonstrated that OGA does not inhibit the proteolytic activity of cathepsin B and L, suggesting that OGA inhibits collagen degradation without affecting the lysosomal cysteine enzyme proteolytic activity. Finally, preliminary study using surface plasmon resonance (SPR) showed that OGA binds to type I collagen but not to albumin, consistent with a specific effect on collagen. These results suggest that the observed inhibition of collagen degradation by OGA may be due to its ability to bind to the collagen molecule. By masking the collagen surface, OGA may render the collagen cleavage site less accessible to enzymes and thus prevent its enzymatic degradation.
[show abstract][hide abstract] ABSTRACT: Since the past decades, oligosaccharides are considered for their potential biological activities. To exploit them, it was essential to obtain pure molecules in large amounts. Several strategies were developed to produce specific sugar sequences with specific substitution patterns from land plants and algae polysaccharides. Then, pure oligosaccharides were analyzed for their potential biological activities and relations between oligomers structure and function were tackled. First they can be health beneficial molecules when they are added to the diet to enhance the growth of probiotic bacteria, in that case, oligomers that resist to the digestive process are used as specific substrate for the growth of health beneficial bacteria. In other cases, oligomers have to interact with receptors on cells. In this instance, a specific conformation is needed to allow the sugar sequence to establish specific linkages with the receptor. So, to be adapted to the receptor, the oligosaccharides have to present specific groups to the receptor, there, the polymerization degree of oligosaccharides as well as the flexibility of the glycosidic linkages has to be considered.
Current opinion in microbiology 07/2009; 12(3):261-73. · 7.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biological activities, priming and protective effects of two oligogalacturonides fractions (OGAs) were assayed during a compatible wheat/Blumeria graminis f. sp. tritici interaction. These fractions were obtained from commercial polygalacturonic acid. They both consisted of oligogalacturonides with polymerisation degrees (DP) ranging from 2 to 25, and one of them was a 30% chemically acetylated fraction. A 5 g x L(-1) solution of each fraction was infiltrated in the first leave of ten-days-old plantlets, and activities of defence-related enzymes were measured 48H post-treatment. Among them, oxalate oxidase and peroxidase activities increased, suggesting an elicitation due to both fractions of oligogalacturonides. Some of the pre-treated plantlets were subsequently submitted to powdery mildew infection. As revealed by 3,3'-diaminobenzidine (DAB) staining, the accumulation of hydrogen peroxide (H2O2) at the penetration site of the fungus increased 21H after inoculation to the same extent in areas of plantlets infiltrated by both fractions. On the other hand, the intensity of fluorescence associated with papillae was higher when plantlets were pre-infiltrated with the acetylated fraction, whereas no difference was observed between control plantlets and those treated with the non-acetylated fraction. Moreover, microscopic assessment of the number of haustoria occurring 40H post-inoculation showed it was only reduced when acetylated fraction was used. Despite different modes of action of these molecules, a similar 45% protective effect occurred in both cases when the oligogalacturonides fractions were sprayed on ten-days-old plantlets.
Communications in agricultural and applied biological sciences 01/2009; 74(3):681-5.
[show abstract][hide abstract] ABSTRACT: A glucuronan lyase (EC 220.127.116.11) was immobilized on a monolithic Convective Interaction Media (CIM) disk. The immobilization yield was equal to 29% of the initial activity and 35% of the initial protein amount. Degradations of three glucuronans with various O-acetylation degrees were investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase was inhibited by the O-acetylation degree like the free enzyme. (1)H NMR analyses were used to study the O-acetylation degree of oligoglucuronans and demonstrated that the average degrees of polymerization were inclusive between 4 and 13 after 24h of degradation. This first immobilization of a glucuronan lyase constitutes a new tool to produce oligoglucuronans.
Carbohydrate Research 07/2008; 343(15):2687-91. · 2.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: The large production of acidic oligosaccharides was investigated by non-enzymatic depolymerization of polygalacturonic acid (PGA) using free hydroxyl radical hydrolysis process from H2O2/copper (II) system. A large amount of oligogalacturonides (OGAs) with degrees of polymerization up to 6 were fractionated, and characterized by ESI-Q/TOF-MASS spectrometry and 1H NMR spectroscopy. This efficient production of uronic oligosaccharides from PGA constitutes an original process to produce bioactive compounds in large scale up.
International Journal of Biological Macromolecules 07/2008; 43(3):257-61. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of Mg2+ on glucuronan O-acetylation and production by the Rhizobium meliloti M5N1 CS strain was studied. Magnesium ion induced the production of a more acetylated exopolysaccharide containing a greater molar ratio of 2,3-di-O-acetyl residues.
[show abstract][hide abstract] ABSTRACT: Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.
Journal of Chromatography B 04/2008; 863(2):266-72. · 2.49 Impact Factor