High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls

From the Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom, § Danisco Biotechnology, Langebrogade 1, DK 1001 Copenhagen K, Denmark, Danisco Cultor, Edwin Rahrs Vej 38, DK-8220 Brabrand, Denmark, the ** Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands, and the Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, United Kingdom Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls

From the Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom, § Danisco Biotechnology, Langebrogade 1, DK 1001 Copenhagen K, Denmark, Danisco Cultor, Edwin Rahrs Vej 38, DK-8220 Brabrand, Denmark, the ** Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands, and the Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, United Kingdom Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants

Profiling the main cell wall polysaccharides of tobacco leaves using high-throughput and fractionation techniques

Nicotiana species are used to study agriculturally and industrially relevant processes, but limited screening methods are available for this species. A tobacco leaf cell wall preparation was fractionated using both chemical and enzymatic methods; the fractions obtained were subsequently analysed using rapid high-throughput wall profiling tools. The results confirmed previous data showing that mature tobacco leaf cell walls are predominantly composed of pectic homogalacturonans with lesser amounts of hemicellulosic arabinoxyloglucan and glucuronoxylan polymers. This confirmation provided proof that the profiling methods could generate good-quality results and paves the way for high-throughput screening of tobacco mutants where a range of biological processes, where the cell wall profile is important, are studied. A novel enzymatic oligosaccharide fingerprinting method was optimized to rapidly analyse the structure of XXGG-rich arabinoxyloglucans characteristic of Solanaceae species such as tobacco. Digestion profiles using two available xyloglucanase-specific endoglucanases: Trichoderma reseei EGII and Paenibacillus sp. xyloglucanase were compared showing that the latter enzyme has a higher specificity toward tobacco arabinoxyloglucans, and is better-suited for screening. This methodology would be suitable for species, such as tomato (Solanum lycopersicum) or potato (Solanum tuberosum), with similar XXGG-type motifs in their xyloglucan structure. © 2012 Elsevier Ltd. All rights reserved

Contrasting Cd accumulation of Arabidopsis halleri populations: a role for (1→4)-β-galactan in pectin

Cadmium (Cd) accumulation is highly variable among Arabidopsis halleri populations. To identify cell wall (CW) components that contribute to the contrasting Cd accumulation between PL22-H (Cd-hyperaccumulator) and I16-E (Cd-excluder), Cd absorption capacity of CW polysaccharides, CW mono- and poly- saccharides contents and CW glycan profiles were compared between these two populations. PL22-H pectin contained 3-fold higher Cd concentration than I16-E pectin in roots, and (1→4)-β-galactan pectic epitope showed the biggest difference between PL22-H and I16-E. CW-related differentially expressed genes (DEGs) between PL22-H and I16-E were identified and corresponding A. thaliana mutants were phenotyped for Cd tolerance and accumulation. A higher Cd translocation was observed in GALACTAN SYNTHASE1 A. thaliana knockout and overexpressor mutants, which both showed a lengthening of the RG-I sidechains after Cd treatment, contrary to the wild-type. Overall, our results support an indirect role for (1→4)-β-galactan in Cd translocation, possibly by a joint effect of regulating the length of RG-I sidechains, the pectin structure and interactions between polysaccharides in the CW. The characterization of other CW-related DEGs between I16-E and PL22-H selected allowed to identify a possible role in Zn translocation for BIIDXI and LEUNIG-HOMOLOG genes, which are both involved in pectin modification.info:eu-repo/semantics/publishe

Adaptation of Arabidopsis halleri to extreme metal pollution through limited metal accumulation involves changes in cell wall composition and metal homeostasis

info:eu-repo/semantics/publishe

Resistant starch diet induces change in the swine microbiome and a predominance of beneficial bacterial populations

Background Dietary fibers contribute to health and physiology primarily via the fermentative actions of the host’s gut microbiome. Physicochemical properties such as solubility, fermentability, viscosity, and gel-forming ability differ among fiber types and are known to affect metabolism. However, few studies have focused on how they influence the gut microbiome and how these interactions influence host health. The aim of this study is to investigate how the gut microbiome of growing pigs responds to diets containing gel-forming alginate and fermentable resistant starch and to predict important interactions and functional changes within the microbiota. Results Nine growing pigs (3-month-old), divided into three groups, were fed with either a control, alginate-, or resistant starch-containing diet (CON, ALG, or RS), and fecal samples were collected over a 12-week period. SSU (small subunit) rDNA amplicon sequencing data was annotated to assess the gut microbiome, whereas comprehensive microarray polymer profiling (CoMPP) of digested material was employed to evaluate feed degradation. Gut microbiome structure variation was greatest in pigs fed with resistant starch, where notable changes included the decrease in alpha diversity and increase in relative abundance of Lachnospiraceae- and Ruminococcus-affiliated phylotypes. Imputed function was predicted to vary significantly in pigs fed with resistant starch and to a much lesser extent with alginate; however, the key pathways involving degradation of starch and other plant polysaccharides were predicted to be unaffected. The change in relative abundance levels of basal dietary components (plant cell wall polysaccharides and proteins) over time was also consistent irrespective of diet; however, correlations between the dietary components and phylotypes varied considerably in the different diets. Conclusions Resistant starch-containing diet exhibited the strongest structural variation compared to the alginate-containing diet. This variation gave rise to a microbiome that contains phylotypes affiliated with metabolically reputable taxonomic lineages. Despite the significant microbiome structural shifts that occurred from resistant starch-containing diet, functional redundancy is seemingly apparent with respect to the microbiome’s capacity to degrade starch and other dietary polysaccharides, one of the key stages in digestion