Search

International audience; Carbohydrates have a wide variety of structures whose complexity and heterogeneity challenge the field of analytical chemistry. Tandem mass spectrometry, with its remarkable sensitivity and high information content, provides key advantages to addressing the structural elucidation of polysaccharides. Yet, classical fragmentation by collision-activated dissociation (CAD) in many cases fails to reach a comprehensive structural determination, especially when isomers have to be differentiated. In this work, for the first time, vacuum ultra-violet (VUV) synchrotron radiation is used as the activation process in tandem mass spectrometry of large oligosaccharides. Compared to low energy CAD (LE-CAD), photon activated dissociation brought more straightforward and valuable structural information. The outstanding feature was that complete series of informative ions were produced, with only minor neutral losses. Moreover, systematic fragmentation rules could be drawn thus facilitating the definitive assignments of fragment identities. As a result, most of the structures present in a complex mixture of oligogalacturonans could be comprehensively resolved, including many isomers differing in the position of methyl groups along the galacturonic acid backbone.

International audience; Background and Aims Pectin is a complex macromolecule, the fine structure of which is influenced by many factors. It is used as a gelling, thickening and emulsifying agent in a wide range of applications, from food to pharmaceutical products. Current industrial pectin extraction processes are based on fruit peel, a waste product from the juicing industry, in which thousands of tons of citrus are processed worldwide every year. This study examines how pectin components vary in relation to the plant source (orange, lemon, lime, grapefruit) and considers the influence of extraction conditions on the chemical and macromolecular characteristics of pectin samples. Methods Citrus peel (orange, lemon, lime and grapefruit) from a commercial supplier was used as raw material. Pectin samples were obtained on a bulk plant scale (kilograms; harsh nitric acid, mild nitric acid and harsh oxalic acid extraction) and on a laboratory scale (grams; mild oxalic acid extraction). Pectin composition (acidic and neutral sugars) and physicochemical properties (molar mass and intrinsic viscosity) were determined. Key Results Oxalic acid extraction allowed the recovery of pectin samples of high molecular weight. Mild oxalic acid-extracted pectins were rich in long homogalacturonan stretches and contained rhamnogalacturonan I stretches with conserved side chains. Nitric acid-extracted pectins exhibited lower molecular weights and contained rhamnogalacturonan I stretches encompassing few and/or short side chains. Grapefruit pectin was found to have short side chains compared with orange, lime and lemon. Orange and grapefruit pectin samples were both particularly rich in rhamnogalacturonan I backbones. Conclusions Structural, and hence macromolecular, variations within the different citrus pectin samples were mainly related to their rhamnogalacturonan I contents and integrity, and, to a lesser extent, to the length of their homogalacturonan domains.

International audience; Background and Aims Rhamnogalacturonan II (RGII) is a structurally complex pectic sub-domain composed of more than 12 different sugars and 20 different linkages distributed in five side chains along a homogalacturonan backbone. Although RGII has long been described as highly conserved over plant evolution, recent studies have revealed variations in the structure of the polysaccharide. This study examines the fine structure variability of RGII in wine, focusing on the side chains A and B obtained after sequential mild acid hydrolysis. Specifically, this study aims to differentiate intrinsic structural variations in these RGII side chains from structural variations due to acid hydrolysis. Methods RGII from wine (Vitis vinifera Merlot) was sequentially hydrolysed with trifluoroacetic acid (TFA) and the hydrolysis products were separated by anion-exchange chromatography (AEC). AEC fractions or total hydrolysates were analysed by MALDI-TOF mass spectrometry. Key Results The optimal conditions to recover non-degraded side chain B, side chain A and RGII backbone were 0.1 M TFA at 40 degrees C for 16 h, 0.48 M TFA at 40 degrees C for 16 h (or 0.1 M TFA at 60 degrees C for 8 h) and 0.1 M TFA at 60 degrees C for 16 h, respectively. Side chain B was particularly prone to acid degradation. Side chain A and the RGII GalA backbone were partly degraded by 0.1 M TFA at 80 degrees C for 1-4 h. AEC allowed separation of side chain B, methyl-esterified side chain A and non-methyl-esterified side chain A. The structure of side chain A and the GalA backbone were highly variable. Conclusions Several modifications to the RGII structure of wine were identified. The observed dearabinosylation and deacetylation were primarily the consequence of acidic treatment, while variation in methyl-esterification, methyl-ether linkages and oxidation reflect natural diversity. The physiological significance of this variability, however, remains to be determined.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.

The foaming properties of undenaturated potato raw proteins and of isolated patatin and 16–25 kDa fraction are investigated over a pH range of 4–8 with and without added NaCl. The patatin fraction was shown to be a very promising foaming agent with a foamability and foam stability equivalent or even superior to that of egg-white powder.

The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). “Homogeneous” populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

Water-soluble potato proteins were extracted and an acidic and a basic fraction were isolated by ion-exchange chromatography. The acidic fraction contained mainly patatin (67%), the major soluble protein of potato tubers. The basic fraction contained the 20–25-kDa proteins family (64%) and a 16-kDa polypeptide (22%), probably corresponding to the protease inhibitor potato II. Functionality as pH–solubility curves, thermocoagulation sensitivity, kinetics of creaming and resistance to coalescence were determined. The patatin fraction showed a sharp solubility minimum at pH 4 and a marked sensitivity to thermocoagulation at pH 4 or in the presence of NaCl at both pH 4 and 7. The 16–25-kDa fraction was highly soluble whatever the pH and could be readily thermocoagulated only at 80°C at pH 7 or in the presence of NaCl at both pH 4 and 7. Both fractions could not be thermocoagulated at 1 mg/mL at pH values far from pI (pH 7 for the patatin fraction and pH 4 for the 16–25-kDa fraction) and without NaCl. The 16–25-kDa fraction led to stable emulsions towards creaming and coalescence whatever the pH and ionic strength. The patatin fraction exhibited poorer emulsification properties and the stability of the emulsions obtained was more pH- and ionic strength-dependent with the better results obtained near pI in the absence of NaCl. A thermal treatment (80°C, 30 min) of the emulsions had a marked positive effect on the resistance to coalescence of the patatin emulsions whatever the pH and the ionic strength, while it had a deleterious effect on the resistance to coalescence of emulsions from the 16–25-kDa fraction.

International audience; Plant cell walls are complex, multi-macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell-wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell-wall matrix glycan sub-populations and inter-connections. The method combines chromatographic separations with use of glycan-directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell-wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonanI sub-populations and their modulation in A.thaliana organs.

National audience; Pectin methylesterase (PME) catalyses the de-methylesterification of pectin in plant cell walls during cell elongation. While it is generally assumed that fungal PMEs have a random mode of action, plant PMEs are thought to act in a processive manner to generate long stretches of non-methylesterified residues. However, it is unlikely that all plant PMEs have a strictly similar mode of action. One A. thalianaPME gene (PME3) has been over-expressed in an heterologous system and its protein product purified by affinity chromatography. Homogalacturonans (HGs) from citrus pectin with varied degrees of methyl esterification (DM) have been generated and characterized. Kinetic properties of PME3 have been determined. PME activity from the purified PME3 was performed on the characterized HG substrates with respect to pH. PME3 shows a highest activity at a basic pH but displays different affinities towards specific methyl esterification patterns on the pectin substrates. Demethylesterified blocks assessed by measuring the degree of blockiness and absolute degree of blockiness will be presented. PME activities are also regulated by endogenous pectin methylesterase inhibitors (PMEIs), which control the DM of HG. PMEI7 inhibits PME3 enzyme activity. Docking analysis indicates that the inhibition of PME3 occurs via the PMEI7 interaction with a PME ligand-binding cleft structure.

International audience; Conditions for simple derivatization of reducing carbohydrates via adipic acid dihydrazide microwave-assisted condensation are described. We demonstrate with a diverse set of oligo- and polysaccharides how to improve a restrictive and labor intensive conventional conjugation protocol by using microwave-assisted chemistry. We show that 5 min of microwave heating in basic or acidic conditions are adequate to generate, in increased yields, intact and functional glycosylhydrazides, whereas hours to days and acidic conditions are generally required under conventional methods.

A casein kinase II purified from the lipolytic yeast Yarrowia lipolytica has been used to phosphorylate purified soy storage proteins and their isolated constitutive subunits. β-Conglycinin appeared to be a good substrate for the enzyme, glycinin being much poorer. Phosphate was incorporated specifically in the α and α‘ subunits of β-conglycinin and in the acidic subunits of glycinin. Phosphorylation stochiometries of 0.73 mol of P/mol for β-conglycinin and 0.20 mol of P/mol for glycinin were determined. To improve the accessibility of the phosphorylation sites of glycinin and β-conglycinin for the enzyme, these proteins were succinylated at different rates. Both glycinin and β-conglycinin became better substrates after extensive succinylation. Phosphate incorporation after succinylation was still occurring in the α and α‘ subunits of β-conglycinin but also in an unknown low molecular weight fraction. The acidic subunits of succinylated glycinin were still mainly phosphorylated, but the basic subunits bec...

Activity of an esterase from Pseudomonas fluorescens subsp. cellulosa (XYLD) on an insoluble feruloylated hemicellulose substrate (de-starched wheat bran) was dependent on the source of added endo-xylanase. The esterase exhibited high selectivity for the nature, position of linkage and size of the feruloylated oligosaccharides generated by hydrolysis of the hemicellulose. Increased affinity of XYLD with increasing size of the oligosaccharide substrate suggests that optimal activity is observed on substrates with at least 4 sugars.

International audience; Partially degraded sugar beet (Beta vulgaris) pectins were characterised in terms of galacturonic acid, neutral sugar and ferulic acids contents. It was shown that the total neutral sugar content is correlated with the ferulic acid content. One pectin (C) was further characterised by size exclusion chromatography coupled to refractive index and UV detectors (SEC-RI-UV). This gave the opportunity to estimate how the ferulic acid and neutral sugar contents changed with hydrodynamic radius. Pectin C was found to be heterogeneous in composition with neutral sugar-rich fractions of both high and low hydrodynamic radii. A neutral sugar-poor fraction was found at intermediate hydrodynamic radii.

International audience; A partially degraded sugar beet pectin (C) was found to be heterogeneous in composition with neutral sugar-rich fractions of both high and low hydrodynamic radii. A neutral sugar-poor fraction was found at intermediate hydrodynamic radii. In this paper we demonstrate using both conformation zoning and the global analysis method that fractions of sugar beet pectin rich in neutral sugar side chains (RG-I regions) are less flexible than those rich in galacturonic acid (HG regions). (C) 2012 Elsevier Ltd. All rights reserved.

Cell wall composition, pectin, and hemicellulose fine structure variation were assessed in peach and related genotypes with contrasted texture and fruit shape. Cell walls were prepared from four commercial peaches, eight genotypes from the Jalousia × Fantasia peach cross, and six genotypes from the Earlygold peach × Texas almond cross. Sugar composition was determined chemically while fine structure of homogalacturonan pectin and xyloglucan hemicellulose were assessed by coupling pectin lyase and glucanase degradation, respectively, with MALDI-TOF MS analysis of the degradation products. The results indicate clear compositional and structural differences between the parents and their related genotypes on the basis of pectin versus cellulose/hemicellulose content and on the fine structure of homogalacturonan and xyloglucan. A relation between methyl- and acetyl-esterification of pectin with fruit shape is revealed in the Fantasia × Jalousia peach genotypes., This work has been supported in part by funds from the program EU FP6 ISAFRUIT (Contract No. FP6-FOOD 016279-2).

International audience; Three series of model homogalacturonans (HGs) covering a large range of degree of methylesterification (DM) were prepared by chemical and/or enzymatic means. Randomly demethylesterified HGs, HGs containing a few long demethylesterified galacturonic acid stretches, and HGs with numerous but short demethylesterified blocks were recovered. The analysis of the degradation products generated by the action of a purified pectin lyase allowed the definition of two new parameters, the degree of blockiness, and the absolute degree of blockiness of the highly methylesterified stretches (DBMe and DBabsMe, respectively). By combining this information with that obtained by the more traditional endopolygalacturonase digestion, the total proportion of degradable zones for a given DM was measured and was shown to permit a clear differentiation of the three types of HG series over a large range of DM. This double enzymatic approach will be of interest to discriminate industrial pectin samples exhibiting different functionalities and to evaluate pectin fine structure dynamics in vivo in the plant cell wall, where pectin plays a key mechanical role.

International audience; Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

The activity of two forms of ferulic acid esterase (FAE) from Aspergillus niger on a synthetic feruloylated substrate (methyl ferulate) and on 11 different feruloylated oligosaccharides from sugar-beet pulp and wheat bran was determined. The enzymes exhibited different specificities for the various feruloylated substrates and were more active on certain substrates of cell-wall origin than on methyl ferulate. Both enzymes preferred the arabinose residue to which ferulic acid is attached in the furanose form. FAE-1 had no clear preference for the type of linkage involved between the ferulic acid units and the oligosaccharide chain. In contrast, FAE-III had a clear requirement for ferulic acid to be attached to O-5 of the Ara f ring while no catalysis was observed when ferulic acid was attached to O-2. Both enzymes showed maximum activity on feruloylated trisaccharides. An increase in the length of the oligosaccharide chain did not preclude catalysis, but feruloylated oligosaccharides of a dp > 3 were hydrolysed at a reduced rate. Our results support the hypothesis that different kinds of ferulic acid esterases exist with different specificities for the oligosaccharide chain of the feruloylated substrates.

The amount of water-soluble pectins was largely increased after extrusion-cooking of lemon fibres. These pectins showed the ability to form a gel in the presence of sucrose and at acidic pH. The gels obtained with the water-extracted pectins after extrusion-cooking and with pectins acid-extracted on a laboratory scale were softer than those prepared with commercial citrus pectins. The water-extracted pectins after extrusion-cooking and the pectins acid-extracted on a laboratory scale contained long neutral side-chains and required a higher sucrose concentration to gel than the commercial citrus pectins. The extruded lemon fibres showed the ability to form gels in the presence of sucrose and at acidic pH. The gels obtained with the extruded fibres containing some water-solube pectins of high molecular weight were stronger than those obtained with the extruded fibres containing higher amounts of more depolymerised water-soluble pectins. The extruded fibres containing 12.5–14.9% of water-soluble pectins of high molecular weight (intrinsic viscosity: 413–504 mL/g) were those showing the better gelation properties.

Enzymatically and chemically de-esterified lime pectins : physico-chemical characterisation, polyelectrolyte behaviour and calcium binding properties

International audience; Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.

Imbibed Arabidopsis (Arabidopsis thaliana) seeds are encapsulated by mucilage that is formed of hydrated polysaccharides released from seed coat epidermal cells. The mucilage is structured with water-soluble and adherent layers, with cellulose present uniquely in an inner domain of the latter. Using a reverse-genetic approach to identify the cellulose synthases (CESAs) that produce mucilage cellulose, cesa5 mutants were shown to be required for the correct formation of these layers. Expression of CESA5 in the seed coat was specific to epidermal cells and coincided with the accumulation of mucilage polysaccharides in their apoplast. Analysis of sugar composition showed that although total sugar composition or amounts were unchanged, their partition between layers was different in the mutant, with redistribution from adherent to water-soluble mucilage. The macromolecular characteristics of the water-soluble mucilage were also modified. In accordance with a role for CESA5 in mucilage cellulose synthesis, crystalline cellulose contents were reduced in mutant seeds and birefringent microfibrils were absent from adherent mucilage. Although the mucilage-modified5 mutant showed similar defects to cesa5 in the distribution of sugar components between water-soluble and adherent mucilage, labeling of residual adherent mucilage indicated that cesa5 contained less cellulose and less pectin methyl esterification. Together, the results demonstrate that CESA5 plays a major and essential role in cellulose production in seed mucilage, which is critical for the establishment of mucilage structured in layers and domains.

International audience; A very highly methylated homogalacturonan (HG) was produced and further used to generate model HG series. HG samples were characterized with respect to their macromolecular features, to their degree and pattern of methyl-esterification, and to their calcium-binding properties. All HG samples were of similar molar mass. Randomly demethylesterified HG samples (B-series) of DM >= 40, exhibiting low degree of blockiness (DB), absolute degree of blockiness (DBabs) and calcium binding properties, were classically obtained by alkali deesterification of the very highly methylated "mother" HG. HGs generated by the action of plant pectin-methylesterase (p-PME) on the very highly methylated "mother" HG (P-series) exhibited high DB, DBabs and calcium binding properties, whatever their DM, in consistency with the presence of few long deesterified blocks of GalA. HGs generated by the action of p-PME on "mother" HGs of DM similar to 80 arising from the B-series (BP-series) exhibited intermediate DB, DBabs and calcium binding properties, in agreement with the presence of numerous but short deesterified blocks of GalA. Manipulating the initial DM of randomly methyl-esterified "mother" HGs prior to enzymatic block-wise demethylesterification allowed the recovery of various HG types with respect to the length and number of demethylesterified GalA stretches. (C) 2011 Elsevier Ltd. All rights reserved.

The effects of extrusion-cooking on pea hulls were investigated. Two batches of pea hulls of different average particle size (80 μm and 500 μm) were extruded under various conditions with a twin screw extruder (Clextral BC 45). In all cases, the water-solubility of the product increased (3·6–15·3%) with the severity of the treatment, monitored by the specific mechanical energy (115–363 kWh/t). A redistribution from insoluble dietary fibre to soluble dietary fibre, indicating a partial solubilisation of fibre without extensive degradation of the polymeric structure, was observed. The solubilised material was composed of pectic substances and hemicelluloses. The hydration properties of pea hulls differed much within the particle size distribution of the products and were not markedly modified by extrusion-cooking treatment.

The soluble fraction (9%) obtained by aqueous extraction of an extruded pea hull fibre gave 3 peaks on DEAE-Sepharose-6B. The first population (25%) was not bound to the gel and contained xylose, arabinose and glucose (15:6:1), the second one (12%), eluted for an ionic strength of 0·08 m, contained xylose, arabinose and glucuronic acid (18:1:1), and the third one (63%), eluted for an ionic strength between 0·3 m and 0·8 m, contained galacturonic acid, arabinose, xylose, rhamnose and galactose (5·3:3:1·8:1·7:1). The structure of these fractions was studied through enzymic degradation, methylation, 1H-NMR and 13C-NMR. The first one was composed mainly of a heteroxylan having a (1 → 4)-β-d-xylan backbone (average d.p. 103) with only very few branching points at O-2, and of a slightly branched arabinan. The second one was an acidic xylan. Both heteroxylans were acetylated. The third one was pectic in nature with a main chain consisting of (1 → 4) linked galacturonic acid, partly substituted at the O-2 and O-3 positions, and (1 → 2) linked rhamnose residues which were mainly substituted at the O-4 position. This acidic fraction was appreciably hydrolyzed by an endopolygalacturonase, indicating the presence of homogalacturonan regions in the molecules. Arabinose, galactose and xylose were present as side-chains.

International audience; With the aim of mimicking the plant cell wall, a layer by layer approach was used to build a thin film consisting of successive adsorption of pectin and extensin. Elaboration of the thin film was monitored by surface plasmon resonance, quartz crystal microbalance, and ellipsometry. All data indicate that formation of the film was successful and that growth occurred according to a nonuniform growth. It is likely that diffusion of the polymers occurred within the multilayer structure and that the final structure is not constituted by layered individual pectin and extensin films. Polymer rearrangements were also supported by the atomic force microscopy images that show a smoother surface after extensin adsorption than after pectin deposition

International audience; Acid extracted sugar beet (Beta vutgaris) pectin was subjected to enzymatic hydrolysis using fungal pectin methyl esterase (f-PME) and two endopolygalacturonanases (PGs land II). From the hydrolysate, the RG-I fraction was separated and purified by chromatographic techniques. This RG-I fraction was shown to be of high weight average molar mass (188,000 g/mol), but low intrinsic viscosity (36 ml/g), which is consistent with a random coil conformation(L(p) = 1.4 nm). The HG fraction was prepared by mild acid hydrolysis of acid extracted pectin. The HG fraction was found to have a relatively low weight average molar mass (20,000 g/mol), but a rather high intrinsic viscosity (77 ml/g), which is consistent with the HG fraction being rigid in solution (L(p) = 9.8 nm). Lower molar mass pectins are richer in HG regions and pectins of higher molar mass are richer in RG-I regions. We conclude that the degradation of the HG region has an important impact on intrinsic viscosity, but less on molar mass and the inverse is true for the degradation of RG-I region. This has important consequences in terms of the functionality of sugar beet pectin molecules. (C) 2010 Elsevier Ltd. All rights reserved.

International audience; Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides-BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [-> 2)-alpha-l-rhamnosep-(1 -> 4)-alpha-d-galacturonic acid p-(1 ->](7) structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose-galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples

International audience; Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.

Applications of enzymes in the upgrading of fruit- and vegetable-based waste co-products are reviewed. Fruit and vegetable by-products are largely underexploited and their valorisation into functional compounds and their application in food is a promising field. Over the last years, the increasing amount of scientific research in this field shows the growing interest for renewable plant resource utilisation. Pectin-degrading enzymes as well as enzymes active towards cellulose and xyloglucans are described and their potential and current uses are explained and the uses of the final products are documented. Non-scientific aspects such as the economics, legislation, and the consumer point of view are also discussed.

Summary Ambarella and mango peels are good sources of pectins (15–20%), with high degree of methylation (60–78%) and high molar masses. Ambarella and mango (Amelioree and Mango varieties) peel pectins were extracted using HCl or oxalic acid/ammonium oxalate (OAAO). Purified pectins were analysed for their flow behaviour and phase diagrams were established at pH 3 as sucrose vs. pectin concentration. The gelation kinetics and mechanical spectra of these pectin gels were studied and compared to those of commercial citrus (lime) pectins. At a concentration of 1% (w/v), all pectic solutions had a shear thinning behaviour but at 0.6% (w/v), only OAAO-extracted pectins exhibited such behaviour. Phase diagrams showed that at pH 3, gelation of OAAO mango extracted pectins was possible at low polymer concentration (0.2%; w/w) for a sucrose concentration of 60% (w/w). OAAO-extracted pectins exhibited a higher gelling ability than HCl-extracted ones. Sucrose (45–50%) and pectin (0.2–0.6%) concentration had a deep impact on the gel strength. Our results enable to conclude that the OAAO extraction from mango and ambarella peels allowed the recovery of pectins that exhibit high gelling properties.

International audience

Mention d'édition : 1. ed.; International audience

International audience; Pectin are extremely complex biopolymers made up of different structural domains. Enzymatic degradation followed by purification and structural analysis of the degradation products proved to be efficienttools for the understanding of pectin fine structure, including covalent interactions between pectic structural domains or with other cell wall polysaccharides. Due to its high sensitivity, high throughput andcapacity to analyze mixtures, mass spectrometry has gained more and more importance as a tool for oligosaccharides structural characterization in the past 10 years. This review will focus on the combined useof mass spectrometry and enzymatic digestion for pectins structural characterization.

International audience; Pectins are a family of highly complex multifunctional cell wall polysaccharides. Little is known on the relation between pectin structure, hydrodynamic properties, and cellular function. In this study, we took advantage of the Arabidopsis pectin mutant quasimodo2 (qua2), which specifically lacks half of its homogalacturonan blocks, to study the relationship between the amount of homogalacturonan blocks and the hydrodynamic properties of pectins. It was first shown that, in qua2 pectins, homogalacturonans had maintained the same size as those in the wild type. The persistence lengths of isolated homogalacturonan and rhamnogalacturonan-I blocks were then measured and it was shown that homogalacturonan was over 4-fold more rigid than rhamnogalacturonan-I. WT and qua2 pectins were next compared and it appeared that the specific reduction of the number of homogalacturonan blocks leads to an increased flexibility of qua2 pectins. These results show for the first time how mutant pectins can be used to demonstrate the opposite influence of rhamnogalacturonan-I and homogalacturonan blocks on the hydrodynamic properties of pectins.

International audience; Commercial acid-extracted sugar beet pectin was extensively hydrolysed using an endo-polygalacturonase (AnPGI from Aspergillus niger or AnPGII from A. niger or FmPG from Fusarium moniliforme) in combination with Aspergillus aculeatus pectin methyl-esterase (AaPME). The homogalacturonan-derived oligogalacturonates released were quantified by high-performance anion-exchange chromatography and their structure determined by mass spectrometry. The different endo-polygalacturonases exhibited variable tolerance towards acetyl groups. AnPGI was the most active and FmPG the less. A hypothetical homogalacturonan was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account FmPG observed requirements and hydrolysis products. A blockwise repartition of the acetyl groups onto sugar beet pectin homogalacturonan is proposed.

Extraction and use of pectins from ambarella peels could add value to the waste products arising from processing of the fruit. Dried alcohol-insoluble residues (AIR) of ambarella peels were treated separately with HCl, deionised water and oxalic acid/ammonium oxalate solutions, and the resulting pectin extracts analysed for some biochemical and physicochemical parameters. The results show that pectin yield (9–30% dry AIR), uronic acid (557–727 mg/g dry weight), neutral sugars (125–158 mg/g), degree of methylation (50–58%) and acetylation (4–6%), molar mass (263,000–303,000 g/mol) and intrinsic viscosity (179–480 ml/g) varied significantly (p

International audience; The extraction of pectins from mango peels can offer a way to upgrade byproducts arising from the processing of these tropical fruits. In this work, several extraction conditions (HCl, or deionised water, or ammonium oxalate) were tested in order to isolate pectins from the peels of two mango varieties (Amelioree and Mango) harvested in Cameroon. After determining uronic acid and neutral sugar contents of mango peels and their alcohol insoluble residues (AIR), extracted pectins were analysed for their uronic acid and neutral sugar contents, their degree of methylation and acetylation, and their average molar mass and intrinsic viscosity. The extraction method was shown to significantly act upon yield (9-32% dry AIR), uronic acid (262-709 mg/g dry weight) and neutral sugar (160-480 mg/g) contents of pectin, their degree of methylation (52-86%) and acetylation (1-8%), their molar mass (245,000-432,000 g/mol), and their intrinsic viscosity (320-1346 mL/g). The physicochemical behaviour of pectins could be influenced thereof. With a good recovery yield, a high average molar mass and intrinsic viscosity and a high degree of methylation, ammonium oxalate-extracted mango pectins present good characteristics to be exploited industrially for their gelling properties.

Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.

International audience; The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.

Publisher Summary Fruit and vegetable processing (preservation or production of juice, wine, sugar, starch, oil, etc.) generates huge amounts of wastes. Vegetable wastes are also often spread on land or composted. These wastes have high water content and are consequently perishable and microbiologically instable. It can result in environmental issues such as noxious odors or microbiological hazards. Wastes arising from fruit and vegetable processing industries are rich in cell wall materials. Such plant-derived wastes are generally heterogeneous as they may contain entire vegetable, leaves, stems, roots, tubers, seeds, stones, etc., which makes their upgrading rather difficult. Therefore, this chapter discusses the upgrading of plant-derived food processing by-products, mainly vegetable trimmings and residues from extraction. After an inventory of the main origins of the by-products, the upgrading of these plant wastes as a whole, of the constituting polymers, and of the monomeric components, will be addressed. The major solid waste is obtained by pressing of fruits or vegetables and solid–liquid separation. This pomace can contain pulp, peels, seeds, and stones. The further utilization of pomace depends on the fruit or vegetable type. Pomace is generally used for the extraction of valuable compounds and for the production of foodstuffs.

International audience; Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic! domains responsible for binding with cellulose microfibrils.

International audience; A comprehensive analysis was carried out of the composition of seed coat mucilage from Arabidopsis thaliana using the Columbia- 0 accession. Pectinaceous mucilage is released from myxospermous seeds upon imbibition, and in Arabidopsis consists of a water- soluble, outer layer and an adherent, inner layer. Analysis of monosaccharide composition in conjunction with digestion with pectolytic enzymes conclusively demonstrated that the principal pectic domain of both layers was rhamnogalacturonan I, and that in the outer layer this was unbranched. The macromolecular characteristics of the water- soluble mucilage indicated that the rhamnogalacturonan molecules in the outer layer were in a slightly expanded random- coil conformation. The inner, adherent layer remained attached to the seed, even after extraction with acid and alkali, suggesting that its integrity was maintained by covalent bonds. Confocal microscopy and monosaccharide composition analyses showed that the inner layer! can be separated into two domains. The internal domain contained cellulose microfibrils, which could form a matrix with RGI and bind it to the seed. In effect, in the mum5- 1 mutant where most of the inner and outer mucilage layers were water soluble, cellulose remained attached to the seed coat. Immunolabeling with anti- pectin antibodies indicated the presence of galactan and arabinan in the inner layer, with the latter only present in the non- cellulose- containing external domain. In addition, JIM5 and JIM7 antibodies labeled different domains of the inner layer, suggesting the presence of stretches of homogalacturonan with different levels of methyl esterification.

The dietary fibre in the prunes was prepared using the Association of Official Analytical Chemists (AOAC) method for the soluble and insoluble dietary fibres. Prunes are very rich in dietary fibre (6-7% of the fresh fruit) with approximatively 57% of soluble dietary fibre and 43% of insoluble dietary fibre. The soluble dietary fibre is rich in pectins whereas the insoluble dietary fibre contains mainly cellulose, with significant amounts of pectins and hemicelluloses. 1 ­ ABBREVIATIONS AIS: alcohol insoluble solids DF: dietary fibre SDF: soluble dietary fibre IDF: insoluble dietary fibre TDF: total dietary fibre 2 ­ INTRODUCTION Many western countries have recommended increased consumption of foods rich in dietary fibre because of their potential health benefit such as protection against cardiovascular diseases, diabetes, colon cancer, as well as regulation of the transit time (Dreher, 2001; Gibney, 2001). Prunes are recognized as a healthy food (Stacewitz et al., 2001; Tinker et al., 1991). They are dried plums, the fruits of Prunus domestica cv d'Ente. After harvest, the fruits are dried for 16-24h at 75°C to reduce the moisture content down to 21-23% ; the prunes are then stored and rehydrated to 33-35% before marketing (Somogyi, 1987). They may be consumed directly in the diet or more rarely after processing to juices or purees for exemple (Stacewitz et al., 2001; Tinker et al., 1991). Prunes have long been used to treat intestinal disorders such as constipation. They are known to contain dietary fibre, sugars, sugar alcohols (sorbitol) and other substances having effects the intestinal function as laxatives (Somogyi, 1987; Stacewitz et al., 2001; Tinker et al., 1991). Prunes are also a good source of energy, phenolic acids, minerals or vitamins (Dikeman et al., 2004; Donavan et al., 1998; Kayano et al., 2003; Piga et al., 2003; Somogyi, 1987; Stacewitz et al., 2001; Tinker et al., 1991). The literature is very scarce on the constitutive dietary fibres and their polysaccharides. A value of ~11 g/100 g dry matter was found for total dietary fibre (Piga et al., 2003); this value is in the range (6-16g/100 g fresh matter) given by Stacewitz et al. (2001). Some food table compositions (Englyst et al., 1988; Fang et al., 2002) gave values of 8-9 g/100 g dry matter for total dietary fibre with 55-67% of soluble dietary fibre. This paper deals with the determination of dietary fibres in various prune samples and the analysis of their constitutive polysaccharides. We have measured in this work the dietary fibre by the conventional enzymatic-gravimetric method and characterised the solubleand insoluble-dietray fibre by their sugar composition. Dietary fibre in prunes 425 3 ­ MATERIALS AND METHODS 3.1 Plant material The prunes were sampled in region of Villeneuve-sur-Lot (F). The 11 samples used in this study varied by their storage conditions, their moisture contents and their sizes; some were stoned. 3.2 Preparation of alcohol insoluble solids (AIS) The prunes were first stoned and cut into small pieces (d ~ 5 mm). The samples were grinded in boiling ethanol 80% (12 mL ethanol/g of sample) : after boiling for 20 min, the samples were filtered on a Büchner (G3) funnel. The insoluble material was washed with ethanol 80% until a sugar-free extract was obtained (phenol-sulfuric acid test). The residue (AIS) was washed successively with ethanol 96%, and acetone, then air-dried overnight at 40°C and weighed. 3.3 Determination of dietary fibre (DF) The dietary fibre was measured gravimetrically after removing the non fibre constituents by enzymatic means as described by Prosky et al. (1988). Duplicates (2 x 1 g) in a 0.08 M phosphate buffer pH 6.0 were analysed for soluble (SDF) and insoluble (IDF) dietary fibres. Samples were treated with a thermostable -amylase from Aspergillus niger (Termamyl 120 L, Novozymes, Denmark) and then digested with a protease from Bacillus licheniformis (P5380, Sigma Aldrich, France) and an amyloglucosidase from Aspergillus niger (A-3042, Sigma). SDF and IDF were separated by filtration on a G4 funnel. SDF was recovered from the filtrate by precipitation with 4 volumes of ethanol 96%, then dried by solvent exchange and under vacuum overnight at 40°C. IDF was dried by solvent exchange and under vacuum overnight at 40°C. A blank was performed by the same procedure. IDF and SDF values were obtained after corrections from the blank, and residual ash and proteins. Total dietary fibre (TDF) was calculated as the sum of IDF and SDF. Preliminary assays (data not shown) evidenced that very similar values of DF were obtained directly from the prunes and from AIS. Therefore, for easiness reasons, the starting material for the determination of DF was the AIS and not the entire fruit 3.4 Determination of the uronic acids SDF were first deesterified with 0.05 M NaOH for 30 min and neutralised La with 0.1M HCl, whereas the AIS and IDF (~ 25 mg), after grinding in liquid nitrogen, were prehydrolysed with 13 M H2SO4 for 1h at 25°C (Saeman et al., 1954). The uronic acids (as galacturonic acid) were determined by the automatic metahydroxybiphenyl method; Les teneurs en fibres alimentaires des pruneaux ont été mesurées par la méthode de l'AOAC (Association of Official Analytical Chemists) qui permet de quantifier les fibres solubles, les fibres insolubles et les fibres totales (somme des solubles et des insolubles). Les pruneaux sont riches en fibres puisque leur teneur est de 6-7% par rapport à la matière fraîche ; les fibres solubles représentent environ 57% et les insolubles 43%. Les fibres solubles sont particulièrement riches en substances pectiques alors que les fibres insolubles contiennent principalement de la cellulose avec des quantités significatives de pectines et d'hémicelluloses.

International audience; Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1 -> 4)-linked galactose and (1 -> 5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.

International audience; The organisation of sugar beet and potato cell walls was studied using alkaline extractions following a response surface methodology, simultaneously with solid-state C-13 NMR spectroscopy. The influence of two extraction parameters: NaOH concentration (0.05, 0.275, 0.5 M) and temperature (40, 65, 90 degrees C) on the composition (neutral and acidic sugars) of the residues recovered was established. Treatments of increasing harshness progressively washed off non-cellulosic polysaccharides from the cell walls. Alkaline treatments applied to sugar beet cell wall material (SB-CWM) revealed the presence of diverse pectin populations. The existence of distinct pectin populations in potato cell wall material (P-CWM) was less outstanding. Solid-state C-13 NMR applied to SB-CWM and P-CWM and residues after treatment by 0.275 M NaOH at 65 degrees C revealed two fractions of pectic arabinan and galactan side chains. One fraction was highly mobile, whereas the other one displayed restricted mobility.

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after (18)O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (beta-1-->4-linked-dimer), Ara (alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.