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International audience; Fibre bundles are groups of elementary fibres glued together thanks to the middle lamella, and are the main fraction in plant fibre composites. In this study, relationship between the mechanical properties of flax fibre bundles, chemical composition and cellulose structure were investigated. To do so, a sequential biopolymer extraction was implemented. Fibre bundles were first depectinated by oxalate extraction, and then the hemicelluloses were extracted by LiCl/dimethyl sulfoxide (DMSO) and KOH. The oxalate extract consisted of homogalacturonans and type I rhamnogalacturonans, while the LiCl extract was composed mainly of glucomannans and the KOH extract of xyloglucans. The KOH stage resulted in the appearance of cellulose II in flax bundles. The extraction of pectin and hemicelluloses led to the disappearance of the middle lamella concomitant with a decrease in the tensile Young's modulus and maximum strength. Finally, the fibre bundle composition, ultrastructure and mechanical properties are discussed together in view of the thin middle lamella.

The instrumental analysis of complex mixtures of sugars often requires derivatization to enhance the method’s selectivity and sensitivity. 1-Phenyl-3-methyl-5-pyrazolone (PMP) is a common sugar derivatization agent used in high-performance liquid chromatography (HPLC). Although many C18 column applications for PMP–sugar derivative analysis have been developed, their transferability is not straightforward due to variations in column chemistry and preparation technology. The aim of this study was to develop and validate an application for Zorbax Extend C18 columns for the analysis of 8 neutral and 2 acidic sugars commonly found in plant polysaccharides. The method was further compared to well-established alditol acetates and m-hydroxydiphenyl methods and employed for sugar profiling of selected agro-industrial wastes. The most influential separation factors were the mobile-phase pH and acetonitrile content, optimized at 8.0 and a 12–17% gradient, respectively. The method showed excellent linearity, repeatability and intermediate precision. High sensitivity was achieved, especially for neutral sugars, with an accuracy error range of 5–10% relative standard deviation. The sugar profiling results were highly correlated to the reference method for neutral sugars. The HPLC method was highly applicable for the evaluation of polysaccharides in selected wastes and showed advantages in terms of simplicity, accuracy in acidic sugar determination and suitability for their simultaneous analysis with neutral sugars.

Rhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage (SM) of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of SM polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in Rhamnogalacturonan-I (RG-I) elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage (AM). Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains.

International audience; Cell-to-cell adhesion is essential for establishment of multicellularity. In plants, such adhesion is mediated through a middle lamella composed primarily of pectic polysaccharides. The molecular interactions that influence cell-to-cell adhesion are not fully understood. We have used Arabidopsis (Arabidopsis thaliana) seed coat mucilage as a model system to investigate interactions between cell wall carbohydrates. Using a forward-genetic approach, we have discovered a gene, RUBY PARTICLES IN MUCILAGE (RUBY), encoding a protein that is annotated as a member of the Auxiliary Activity 5 (AA5) family of Carbohydrate-Active Enzymes (Gal/glyoxal oxidases) and is secreted to the apoplast late in the differentiation of seed coat epidermal cells. We show that RUBY is required for the Gal oxidase activity of intact seeds; the oxidation of Gal in side-chains of rhamnogalacturonan-I (RG-I) present in mucilage-modified2 (mum2) mucilage, but not in wild-type mucilage; the retention of branched RG-I in the seed following extrusion; and the enhancement of cell-to-cell adhesion in the seed coat epidermis. These data support the hypothesis that RUBY is a Gal oxidase that strengthens pectin cohesion within the middle lamella, and possibly the mucilage of wild-type seed coat epidermal cells, through oxidation of RG-I Gal side-chains.

The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms., This work was supported in part by an Advanced Grant from the European Research Council (Grant No. 322820) awarded to H.J.G. and B.H. supporting A.S.L., D.N., A.C. and N.T., a Wellcome Trust Senior Investigator Award to H.J.G. (grant No. WT097907MA) that supported J.B. and E.C.L. a European Union Seventh Framework Initial Training Network Programme entitled the “WallTraC project” (Grant Agreement number 263916) awarded to M-C.R. and H.J.G, which supported X.Z. and J.S. The Biotechnology and Biological Research Council project ‘Ricefuel’ (grant numbers BB/K020358/1) awarded to H.J.G. supported A.L.

International audience

On imbibition, Arabidopsis (Arabidopsis thaliana) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis.

International audience; According to the high interest in agro-industrial waste reutilisation, underutilised lignocellulosic materials, such as walnut shell (WS) and pea pod (PP), come in focus. The aim of this paper was to evaluate WS and PP as sources for the production of xylooligosaccharides (XOS). Hemicelluloses from WS and PP were recovered by combining varying parameters of delignification and alkaline extraction. At optimal recovery conditions, the fractions were further hydrolysed to XOS using GH11 endo-xylanase, by varying time and enzyme concentration. Xylose was predominant in the monomeric composition of the obtained hemicelluloses, building low-branched (arabino) glucuronoxylan, in WS exclusively, while in PP some xyloglucan as well. Delignification was essential for high recovery of total xylose from the materials, up to at least 70 %. High xylan conversions were obtained for 24 h hydrolysis, resulting in xylobiose and xylotriose when using low enzyme concentration, while in xylose and xylobiose with high enzyme concentration.

International audience; Rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains were isolated from ginseng pectin by alkali saponification and endo-polygalacturonase hydrolysis, then purified by anion-exchange and size-exclusion chromatography. Monoclonal antibody detection indicated that ginseng RG-I contained -> 4)-alpha-GalpA-(1 -> 2)-alpha-Rhap-(1 -> repeating units as backbone, with arabinan, galactan and type II arabinogalactan (AG-II) as side chains. The use of galactose- and arabinose-releasing enzymes, mass spectrometry analysis of the oligosaccharides generated by rhamnogalacturonan hydrolase, and glycosidic linkage analyses provided evidence that ginseng RG-I contains both single galactose-branched subunits and highly branched subunits with arabinan and AG-II side chains. RG-II was analyzed by sequential acid hydrolysis followed by mass spectrometry. Ginseng RG-II contains 9 galacturonic acid units as backbone. Side chain A is an octasaccharide with 0 similar to 1 methyl ether group. Side chain B is a nonasaccharide with 0 similar to 1 acetyl group. These results provide useful information for further investigation of structure-activity relationship of ginseng pectin.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

Golden apple or ambarella ( Spondias cytherea ) is a tropical tree belonging to the family of Anacardiaceae. Before encouraging its development, it was at first considered as a backyard tree or forest plant. During its fruit growth, maturation and ripening, many morphological, physiological and biochemical modifications occur. The ripe fruit is characterized by a sweet and pleasant flavor. It is a good source of minerals and vitamin C. Ambarella fruit is mostly eaten fresh but its pulp is suitable for making jams, pickles, relishes, soups, stews, and juices. Ambarella fruit pulp can be used as food additive to improve sensorial and physicochemical properties of food product. Pectic polysaccharides from the pulp can be used in the healing process. The peel of the fruit is a good source of fiber and pectin.

1,5-arabinan is an abundant structural feature of side chains of pectic rhamnogalacturonan-I which is a matrix constituent of plant cell walls. The study of arabinan in cells and tissues is driven by putative roles for this polysaccharide in the generation of cell wall and organ mechanical properties. The biological function(s) of arabinan is still uncertain and high quality molecular tools are required to detect its occurrence and monitor its dynamics. Here we report a new rat monoclonal antibody, LM6-M, similar in specificity to the published rat monoclonal antibody LM6 (Willats et al. (1998) Carbohydrate Research 308: 149-152). LM6-M is of the IgM immunoglobulin class and has a higher avidity for α-1-5-L-arabinan than LM6. LM6-M displays high sensitivity in its detection of arabinan in in-vitro assays such as ELISA and epitope detection chromatography and in in-situ analyses.AbbreviationsAraArabinoseBSABovine Serum AlbuminGalGalactoseGalAGalacturonic acidEDCEpitope detection chromatographyELISAEnzyme-Linked Immunosorbent AssaymAbMonoclonal antibodyPBSPhosphate-buffered salineRhaRhamnoseRG-IRhamnogalacturonan-I

International audience; Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore,wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes ( plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.

International audience; Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.

International audience; Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG-I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG-I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG-I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.

The effects of a processive pectin-methylesterase (PME) treatment on two different pectins, both possessing a high degree of methylesterification (DM), were investigated. While the starting samples were purportedly very similar in fine structure, the intermolecular DM distributions arising from their PME treatments were strikingly different. Herein, a simulation that illuminates the origin of this phenomenon is described. It is concluded that: (1) very different low-DM samples (with the same average DM) can be generated using the same processive PME, simply by a judicious choice of the high DM starting material; (2) observing the intermolecular DM distribution of the products of processive-PME-processing is an extremely sensitive discriminator of the fine structure of high DM starting materials; and (3) for PMEs with unknown action patterns the processive nature of the enzyme is most simply revealed by studying the changes it induces in the intermolecular DM distribution of very-highly-methylesterified homogalacturonans.

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

International audience; The ratio of the two component sugar residues comprising the anionic polysaccharide homogalacturonan (HG;namely, methylesterified or unmethylesterifed galacturonic acid (GalA)) has been controlled chemically or enzymatically in order to produce samples comprised of varying degrees and distributions of methylesterification(DM). Capillary electrophoresis (CE) has been used to characterize the samples produced and, by mapping the measured electrophoretic mobilities to biopolymer charge density, intermolecular distributions of the DM have been extracted. For chemically modified samples with random intramolecular patterns of methylesterification, the experimentally extracted intermolecular DM distributions agree well with the predictions of calculations based on the binomial theorem, demonstrating that the random nature of the demethylesterification process and, hence, the intramolecular DM patterns themselves, are directly reflected in the intermolecular distribution. Furthermore, this principle has been demonstrated by extending the work to the study of substrates with highly nonrandom DM distributions generated using a processive plant-pectinmethylesterase (pPME). An ensemble of polymer chains, generated in silico by a simulation optimized to match the experimentally measured intermolecular DM distribution, contains all possible information regarding the substrate and can further be interrogated to obtain, for example, the full Gal-A blocklength distribution.

International audience; We report a study of xyloglucan (XG)-cellulose interactions made possible by the preparation of various well-defined cellulosic and xyloglucosidic substrates. Bacterial microcrystalline cellulose (BMCC) as well as cellulose whiskers (CellWhisk) were used as cellulosic substrates. Xyloglucosidic substrates were obtained from Rubus cells and Tamarindus indica seeds. Different primary structure characteristics of XGs such as the backbone length and the nature of the side chains, as well as their repartition, were considered in order to examine the influence of the primary structure on their interaction capacity. Two complementary approaches were carried out: first, the determination of adsorption isotherms and its associated models, and second, an enthalpic study using isothermal titration calorimetry (ITC). This study highlighted that an increase of XG interaction capacity occurred with increasing XG molecular weight. Furthermore, we determined that a minimum of 12 glucosyl residues on the backbone is required to observe significant interactions. Moreover, both the presence of trisaccharidic side chains with fucosyl residues and an increase of unsubstituted glucosyl residues enhanced XG cellulose interactions. The evolution of adsorption isotherms with temperature and ITC measurements showed that two different processes were occurring, one exothermic and one endothermic, respectively. Although the presence of an exothermic interaction mechanism has long been established, the presence of an endothermic interaction mechanism has never been reported

International audience; Capillary electrophoresis has been used to characterize samples of the copolymeric anionic polysaccharides homo and rhanino-galacturonan (HG and RGI, respectively). In the case of HG, the ratios of the two component sugar residues, galacturonic acid and its methylesterified analogue, have been controlled chemically to produce samples comprised of varying degrees of methylesterification (DM). The mapping of the measured electrophoretic mobilities to the biopolymer charge density has been considered in some detail and for HG substrates with random intramolecular patterns of methylesterification it is shown that the experimentally extracted intermolecular DM distribution agrees well with the predictions of calculations based on the binomial theorem. This demonstrates that the intermolecular distribution of the methylesterification of pectin samples contains information on the intramolecular pattern by virtue of the fact that they are both manifestations of the same statistical ! process.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Assemblies of pectin, xyloglucan and cellulose were studied in vitro using two ternary systems. In the first one, xyloglucan concentration varied, while pectin amount was kept constant. In the second one, pectin concentration varied, whereas xyloglucan amount was fixed. The use of ternary systems allowed to put forward the hypothesis that pectin/cellulose and xyloglucan/cellulose associations may exist together or separately, depending on the proportion of non-cellulosic polysaccharides in cell walls. It can be hypothesized that pectin plays a double role within primary cell walls: (i) pectin loosely bound to cellulose, in xyloglucan-rich cell walls, (ii) pectin associated with cellulose, in xyloglucan-poor cell walls.

International audience; Binding modelling of tamarind and pea xyloglucans, sugar beet and potato pectins, and pectic side chains (branched arabinan, debranclied arabinan, galactan) onto microcrystalline Avicel cellulose and primary cell wall (PCW) cellulose was performed. The most commonly used binding models, namely the Langmuir, the Freundlich and the Scatchard models, were applied to the data. It appeared that the Freundlich model was more appropriate to describe the binding of all the polysaccharides used in this study. The heterogeneity index calculated from the slope of Freundlich isotherms highlights an important heterogeneity of Avicel and PCW cellulose surfaces, in agreement with the Scatchard representation.

International audience; Pectins are anionic polysaccharides that are sensitive to cations, a property that is widely used in food science. The interactions of a cationic lipid film (dimethyldioctadecylammonium bromide, DODAB) with a set of pectins bearing the same charge, which was either distributed randomly or pseudorandomly or blockwise, are investigated. The combination of Brewster angle microscopy BAM and infrared reflection-absorption spectroscopy IRRAS at the air-water interface reveals that pectin strongly binds to the cationic lipid film in forming a stacked layer underneath the lipid film. The detailed vibrational study of this stable mixed film indicates furthermore that pectin induces a disorder in the internal structure of the cationic film. The strong binding induces a splitting of the carboxylate stretching mode of pectin that is pressure and charge distribution dependent. The occurrence of an intermediate plateau below the collapse of the mixed film originates probably from a change in conformation of the pectin structure underneath the film.

International audience; Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells produce large amounts of mucilage that is released upon imbibition. This mucilage is structured into two domains: an outer diffuse layer that can be easily removed by agitation and an inner layer that remains attached to the outer seed coat. Both layers are composed primarily of pectic rhamnogalacturonan I (RG-I), the inner layer also containing rays of cellulose that extend from the top of each columella. Perturbation in cellulosic ray formation has systematically been associated with a redistribution of pectic mucilage from the inner to the outer layer, in agreement with cellulose-pectin interactions, the nature of which remained unknown. Here, by analyzing the outer layer composition of a series of mutant alleles, a tight proportionality of xylose, galacturonic acid, and rhamnose was evidenced, except for mucilage modified5-1 (mum5-1; a mutant showing a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one), for which the rhamnose-xylose ratio was increased drastically. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. mum5-1 mucilage exhibited very weak adsorption to cellulose. MUM5 was identified as a putative xylosyl transferase recently characterized as MUCI21. Together, these findings suggest that the binding affinity of xylose ramifications on RG-I to a cellulose scaffold is one of the factors involved in the formation of the adherent mucilage layer.

Plant cell walls are the earth's most abundant source of terrestrial biomass. When potatoes are processed for starch production, a low-value, cell wall-rich byproduct known as potato pulp is produced. The huge amounts of pulp available have motivated the reemergence of research devoted to potato cell wall polysaccharide extraction and use.

International audience; Pectins of different structures were extracted from citrus peels with water, oxalate, hot dilute hydrochloric acid and cold dilute sodium hydroxide. Homogalacturonans (HGs) were isolated from the four pectins by mild acid hydrolysis after deesterification. The study of their macromolecular properties revealed that they have similar number- and weight-average degrees of polymerisation, in the range of 81–117 galacturonic acid units, irrespective of the initial pectin. It was inferred that citrus peel pectins consist of HG strectches of similar lengths whatever the mode of extraction. Rhamnogalacturonan I (RG I) and rhamnogalacturonan II (RG II) were isolated from the oxalate- and the acid-extracted pectins using an endo-1,4-α-polygalacturonase followed by anion-exchange and size-exclusion chromatographies. RG I was composed mainly of arabinose, galactose, galacturonic acid and rhamnose, suggesting the presence of arabinan and/or (arabino)galactan side chains. Debranched RG I (dRG I) was obtained by treating the RG I arising from acid-extracted pectin with endo-1,5-α-l-arabinanase, endo-1,4-β-d-galactanase, α-l-arabinofuranosidase and β-d-galactosidase in admixture. The molar ratios of galacturonic acid to rhamnose of RGs I and dRG I were very close to 1/1, indicating a strict repeating [GalA–Rha]n pattern in the backbone. It is concluded that citrus pectins consist predominantly of HG, with a few RG I and a minor RG II fraction.

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.

Main conclusion The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls. Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links. Electronic supplementary material The online version of this article (doi:10.1007/s00425-015-2375-4) contains supplementary material, which is available to authorised users.

Two series of lime pectins with different degrees and patterns of methyl-esterification were successively fractionated by size exclusion followed by anion exchange chromatography. ‘Homogeneous’ populations with respect to molar mass and charge density were thereby obtained and their physico-chemical properties were investigated. Recovered fractions were characterised by a high galacturonic acid and a low neutral sugars content. The homogeneous population obtained for each pectin sample was treated with side-chain releasing enzymes (rhamnogalacturonan hydrolase, endo-β-(1→4)- d -galactanase, endo-α-(1→5)- l -arabinanase, α- l -arabinofuranosidase and β- d -galactosidase) and combinations of these enzymes. The reaction products were fractionated by anion exchange chromatography and their constitutive homogalacturonans were recovered. Each homogalacturonan was found homogeneous with respect to charge density and to molar mass (polydispersity index close to 1).

Sugar beet pectin was extracted from fresh sugar beet pulp and further deesterified by base, plant or fungal pectin esterase or esterified in acid methanol. The modified sugar beet pectins were characterised by chemical analyses, macromolecular and rheological properties, capillary electrophoresis and enzymatic fingerprinting. The majority of the modified sugar beet pectins had the same molar mass and neutral sugar content except for loss of arabinose by prolonged acid hydrolysis. The ferulic acid content was almost stable at about 0.8%. It was found that the enzymatically modified sugar beet pectins were more inhomogeneous due to the presence of acetyl groups hindering the attack by pectin esterases.

Various feruloylated arabinose- and galactose-containing mono- and disaccharides with known linkage configurations (2-O-(trans-feruloyl)-L-arabinopyranose, 5-O-(trans-feruloyl)-L-arabinofuranose, O-[2-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->5)-L-arabinofuranose, and O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose) were analyzed by electrospray ionization mass spectrometry using an ion trap or a quadrupole time-of-flight (Q-TOF) mass analyzer. Collision-induced dissociation (CID) experiments using the two mass analyzers generated similar tandem mass spectrometric (MS/MS) fragmentation patterns. However, the ester-bond cleavage ions were more abundant using the Q-TOF mass analyzer. Compared with the positive ion mode, the negative ion mode produces simpler and more useful CID product-ion patterns. For arabinose-containing feruloylated compounds, results obtained with both analyzers show that it is possible to assign the location of the feruloyl group to the O-2 or O-5 of arabinosyl residues. In the characterization of the 2-O-feruloyl and 5-O-feruloyl linkages, the relative abundance of the cross-ring fragment ions at m/z 265 (-60 u or -62 u after 18O-labelling) and at m/z 217 (-108 u or -110 u after 18O-labelling) play a relevant role. For galactose-containing feruloylated compounds, losses of 60, 90 and 120 Da observed in MS3 experiment correspond to the production of 0,2A1, 0,3A1 and (0,2A1-60 Da) cross-ring cleavage ions, respectively, fixing the location of feruloyl group at the O-6 of the galactose residue.

After degradation of sugar beet cell walls with Driselase® and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1 → 5)-linked arabinose units esterified by a central 8-O-4′ ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.

Five series of pectins with different levels and patterns of methyl esterification and/or different levels of acetyl esterification were produced by chemical or enzymatic treatment of an acid-extracted sugar beet pectin. The relationship between pKa and the degree of dissociation and the free fractions of monovalent and calcium counterions were determined on dilute pectin salt-free solutions. Calcium sensitivity was determined in more concentrated solutions. The presence of acetyl groups hindered the formation of calcium-pectinate precipitates in different manners according to the demethoxylation mode and the pectin concentration. The base-deesterified pectins appeared to have the most homogeneous methyl ester distribution. Pectins demethoxylated by fungus-PME exhibited a peculiar distribution of free galacturonic acid residues. Blocks of contiguous free galacturonic acid were generated by treatment of sugar beet pectin with a plant-PME. Those blocks were not long enough to induce abnormal polyelectrolyte behaviour or to promote dimerisation of pectic molecules in dilute solution but allow the formation of calcium-pectinate precipitates in concentrated medium for high DM. © 2003 Elsevier Science B.V. All rights reserved.

Partially acetylated and methylated oligogalacturonides produced by enzymatic hydrolysis of sugar beet pectin were analysed by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS). The (18)O labelling of the oligomer reducing end allowed the precise assignment of the fragments resulting from glycosidic bond and cross-ring cleavages. The collisional-induced dissociation of the C(i) and Z(j) fragment ions through sequential MS(n) experiments always displayed (0, 2)A-type cross-ring cleavage ions which were related to C(2)H(4)O(2) losses. These (0, 2)A ions appeared to be highly diagnostic ions allowing the precise location of the acetyl groups to the O-2 and/or O-3 of the acetylated galacturonic acid residues.

This work was supported by a grant from the Conseil Regional de Picardie, France and the European Regional Development Fund (PECTINHIB project) through a studentship awarded to ML' and from the Agence National de la Recherche (GALAPAGOS project, ANR-12-BSV5-0001; International audience; Pectin methylesterases (PMEs) play a central role in pectin remodeling during plant development. They are also present in phytopathogens such as bacteria and fungi. We investigated the substrate specificity and pH dependence of plant and fungi PMEs using tailor-made pectic substrates. For this purpose, we used two plant PMEs (from orange peel: Citrus sinensis and from Arabidopsis thaliana) and one fungal PME (from Botrytis cinerea). We showed that plant and fungi PMEs differed in their substrate specificity and pH dependence, and that there were some differences between plant PMEs. We further investigated the inhibition of these enzyme activities using characterized polyphenols such as catechins and tannic acid. We showed that PMEs differed in their sensitivity to chemical compounds. In particular, fungal PME was not sensitive to inhibition. Finally, we screened for novel chemical inhibitors of PMEs using a chemical library of similar to 3600 compounds. We identified a hundred new inhibitors of plant PMEs, but none had an effect on the fungal enzyme. This study sheds new light on the specificity of pectin methylesterases and provides new tools to modulate their activity. (C) 2015 Elsevier B.V. All rights reserved.

An experimental design was used to study the influence of different parameters (pH, temperature, time, and type of acid) on extraction of pectins from fresh sugar beet. The extraction conditions have important effects on the features of extracted pectins. Their composition (neutral and acidic sugars, degrees of esterification, amounts of ferulic and diferulic acids) and some physicochemical properties (molar mass, intrinsic viscosity) were determined. The type of acid used (HCl or HNO3) had no effect on the characteristics of extracted pectins. Different kinds of pectins can be obtained with good yields at pH 1. Galacturonic acid amounts of the extracted pectins were nearly constant whatever the extraction conditions, whereas the degrees of methylation and acetylation showed large variations. At pH 1, the extracts were particularly rich in rhamnogalacturonan regions, the nature and the quantity of side chains differing according to extraction conditions. The molar masses of extracted pectins were higher than those obtained from sugar beet pulp; beside the sole impact of raw material, possible cross-linking of pectic molecules through diferulic bridges is discussed.

Two series of pectins with different levels and patterns of methyl esterification were produced by treatment of a very highly methylated lime pectin with a fungus- or plant-pectin methylesterase. The interchain distribution of free carboxyl groups was investigated by size exclusion and ion exchange chromatography. "Homogeneous" populations with respect to molar mass or charge density were thereby obtained, and their composition, molar mass, and calcium binding properties were investigated. The composition varies from one size exclusion chromatography fraction to another, the highest molar mass fraction being richer in rhamnogalacturonic sequences and exhibiting a slightly higher degree of methylation (DM). Separation of pectins by ion exchange chromatography revealed a narrow charge density distribution for pectins deesterified by fungus-pectin methylesterase, in agreement with a multichain mechanism. Conversely, pectins deesterified by plant-pectin methylesterase exhibited a very large charge density distribution suggesting a processive mechanism. The interchain polydispersity with regard to DM was however shown to have no impact on calcium binding properties of the different fractions. The progressive dimerization through calcium ions with decreasing DM of pectins deesterified by plant-pectin methylesterase seems to be the result of a peculiar intrachain pattern of methyl esterification that can be attributed to a multiple attack mechanism.

Nature 544, 65–70 (2017); doi:10.1038/nature21725 In this Article, the list of symbols used to depict monosaccharides in the figures is not derived from the Consortium of Functional Glycomics or included in ref. 25. Instead, it follows the current Symbol Nomenclature for Glycans (SNFG) system, whichis described in ref. 1. The original Article has not been corrected.

In certain plant species, including Arabidopsis, seed coat epidermal cells accumulate a large quantity of polysaccharides that are released as mucilage upon imbibition. Once released, Arabidopsis seed coat mucilage appears as two distinct layers, the outer non-adherent composed of unsubstitutedrhamnogalacturonan I (RG-I), and the inner adherent one mainly made of RG-I and cellulose. CELLULOSE SYNTHASE5 and MUCILAGE-MODIFIED (MUM)5 have both been shown to be required for anchoring the mucilage pectin to the seed coat surface [1-5]. The identity and molecular role of MUM5 remained to be determined. A comparison of sequentially extracted seed mucilage components from cesa5 and mum5 mutants was carried out. Both mutants showed a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one. cesa5 further showed a clear reduction in cellulose content, whereas mum5 had a wild-type cellulose content, but had reduced Xyl contents in the outer mucilage layer. Accordingly, MUM5 was identified as a putative xylosyltransferase. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. The importance of having both a correctly synthesized cellulose scaffold and pectin structure will be discussed with regard to mucilage partitioning into non-adherent and adherent layers.

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.