rhamnogalacturonan-i and polygalacturonic-acid

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up approximately 90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of alpha-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.

Topics: Cell Wall; Pectins; Plants; Polysaccharides

Topics: Cell Wall; Cellulose; Chemical Phenomena; Chemistry; Galactans; Glucans; Glycoproteins; Models, Molecular; Pectins; Phytoalexins; Plant Cells; Plant Extracts; Plant Growth Regulators; Plant Physiological Phenomena; Plant Proteins; Plants; Polysaccharides; Protease Inhibitors; Sesquiterpenes; Terpenes; Xylans

Platycodonis Radix is widely used as homology of medicine and food in China; polysaccharides are thought to be one of its functional constituents. In this study, a pectic polysaccharide, PGP-I-I, was obtained from the root of the traditional medicine plant Platycodon grandiflorus through ion exchange chromatography and gel filtration. This was characterized being mainly composed of 1,5-α-L-arabinan and both arabinogalactan type I (AG-I) and II chains linked to rhamnogalacturonan I (RG-I) backbone linked to longer galacturonan chains. In vitro bioactivity study showed that PGP-I-I could restore the intestinal cellular antioxidant defense under the condition of hydrogen peroxide (H

Topics: Animals; Antioxidants; Cell Line; Chromatography, Gel; Chromatography, Ion Exchange; Dietary Carbohydrates; Galactans; Hydrogen Peroxide; Pectins; Plant Extracts; Plant Roots; Platycodon; Polysaccharides; Swine

Two pectic polysaccharides (WRSP-A2b and WRSP-A3a) have been obtained from Radix Sophorae Tonkinensis and comparatively investigated in terms of their physical properties and antioxidant activities. Monosaccharide composition, FT-IR, NMR and enzymatic analyses indicate that both WRSP-A2b (13.6 kDa) and WRSP-A3a (44.6 kDa) consist of homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains, with mass ratios of 0.9:1.8:1 and 2.3:2.9:1, respectively. The RG-I domains were further purified and characterized. Results show that WRSP-A2b contains a highly branched RG-I domain, primarily substituted with α-(1→5)-linked arabinans, whereas WRSP-A3a contains a small branched RG-I domain mainly composed of β-(1→4)-linked galactan side chains. WRSP-A3a exhibits stronger antioxidant activity in scavenging different radicals than WRSP-A2b, a finding that may be due to its higher content of GalA residues and HG domains. Our results provide useful information for screening natural polysaccharide-based antioxidants from Radix Sophorae Tonkinensis.

Topics: Antioxidants; Fabaceae; Galactans; Humans; Magnetic Resonance Spectroscopy; Monosaccharides; Pectins; Polysaccharides; Spectroscopy, Fourier Transform Infrared

The cell wall is essential for plant survival. Determining the relationship between cell wall structure and function using mutant analysis or overexpressing cell wall-modifying enzymes has been challenging due to the complexity of the cell wall and the appearance of secondary, compensatory effects when individual polymers are modified. In addition, viability of the plants can be severely impacted by wall modification. A useful model system for studying structure-function relationships among extracellular matrix components is the seed coat epidermal cells of Arabidopsis thaliana. These cells synthesize relatively simple, easily accessible, pectin-rich mucilage that is not essential for plant viability. In this study, we expressed enzymes predicted to modify polysaccharide components of mucilage in the apoplast of seed coat epidermal cells and explored their impacts on mucilage. The seed coat epidermal-specific promoter TESTA ABUNDANT2 (TBA2) was used to drive expression of these enzymes to avoid adverse effects in other parts of the plant. Mature transgenic seeds expressing Rhamnogalacturonate lyase A (RglA) or Rhamnogalacturonate lyase B (RglB) that degrade the pectin rhamnogalacturonan-I (RG-I), a major component of mucilage, had greatly reduced mucilage capsules surrounding the seeds and concomitant decreases in the monosaccharides that comprise the RG-I backbone. Degradation of the minor mucilage component homogalacturonan (HG) using the HG-degrading enzymes Pectin lyase A (PLA) or ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) resulted in developing seed coat epidermal cells with disrupted cell-cell adhesion and signs of early cell death. These results demonstrate the feasibility of manipulating the seed coat epidermal cell extracellular matrix using a targeted genetic engineering approach.

Topics: Arabidopsis; Arabidopsis Proteins; Pectins; Plant Mucilage; Seeds

Plant cell walls contain cellulose embedded in matrix polysaccharides. Understanding carbohydrate structures and interactions is critical to the production of biofuel and biomaterials using these natural resources. Here we present a solid-state NMR study of cellulose and pectin in

Topics: Arabidopsis; Cell Wall; Cellulose; Magnetic Resonance Spectroscopy; Methyltransferases; Pectins; Polysaccharides

The microwave-assisted extraction of pectin from sweet lemon peel (SLP) was optimized using Box-Behnken design. The highest pectin yield (25.31%) was observed under optimal condition (microwave power of 700 W, irradiation time of 3 min and pH of 1.5). The physicochemical, structural and some bioactivity of the SLP pectin isolated at optimum condition was evaluated. The SLP pectin was rich in galacturonic acid and galactose (87.2 mol%), high in molecular weight (615.836 kDa) and low in degree of esterification (1.2-35.1%). Furthermore, the SLP pectin was composed of 55.7% linear region (homogalacturonan) and 42.2% hairy region (rhamnogalacturonan-I). Also, the FT-IR and H-NMR results confirm the major presence of low methylated galacturonic acid rich structure in the isolated samples. In addition, SLP pectin showed good emulsifying and antioxidant properties. A pseudoplastic flow behavior was observed for SLP pectin solution at higher concentrations (1% w/v <). These results represent an inexpensive source for pectin extraction with high pectin yield and good properties.

Topics: Antioxidants; Biphenyl Compounds; Citrus; Emulsions; Esterification; Fruit; Galactans; Galactose; Hexuronic Acids; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Microwaves; Molecular Weight; Pectins; Picrates; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

The chemical structure of pea pectin was delineated using pectin-degrading enzymes and biochemical methods. The molecular weight of the pea pectin preparation was 488,000, with 50 % arabinose content, and neutral sugar side chains attached to approximately 60 % of the rhamnose residues in rhamnogalacturonan-I (RG-I). Arabinan, an RG-I side chain, was highly branched, and the main chain was comprised of α-1,5-l-arabinan. Galactose and galactooligosaccharides were attached to approximately 35 % of the rhamnose residues in RG-I. Long chain β-1,4-galactan was also present. The xylose substitution rate in xylogalacturonan (XGA) was 63 %. The molar ratio of RG-I/homogalacturonan (HG)/XGA in the backbone of the pea pectin was approximately 3:3:4. When considering neutral sugar side chain content (arabinose, galactose, and xylose), the molar ratio of RG-I/HG/XGA regions in the pea pectin was 7:1:2. These data will help understand the properties of pea pectin.

Topics: Arabinose; Galactans; Galactose; Glycoside Hydrolases; Hexuronic Acids; Molecular Structure; Pectins; Pisum sativum; Polysaccharides; Rhamnose; Xylose

Differences in the composition and the structural organisation of the extracellular matrix correlate with the morphogenic competence of the callus tissue that originated from the isolated endosperm of kiwifruit. The chemical composition and structural organisation of the extracellular matrix, including the cell wall and the layer on its surface, may correspond with the morphogenic competence of a tissue. In the presented study, this relationship was found in the callus tissue that had been differentiated from the isolated endosperm of the kiwiberry, Actinidia arguta. The experimental system was based on callus samples of exactly the same age that had originated from an isolated endosperm but were cultured under controlled conditions promoting either an organogenic or a non-organogenic pathway. The analyses which were performed using bright field, fluorescence and scanning electron microscopy techniques showed significant differences between the two types of calli. The organogenic tissue was compact and the outer walls of the peripheral cells were covered with granular structures. The non-organogenic tissue was composed of loosely attached cells, which were connected via a net-like structure. The extracellular matrices from both the non- and organogenic tissues were abundant in pectic homogalacturonan and extensins (LM19, LM20, JIM11, JIM12 and JIM20 epitopes), but the epitopes that are characteristic for rhamnogalacturonan I (LM5 and LM6), hemicellulose (LM25) and the arabinogalactan protein (LM2) were detected only in the non-organogenic callus. Moreover, we report the epitopes, which presence is characteristic for the Actinidia endosperm (LM21 and LM25, heteromannan and xyloglucan) and for the endosperm-derived cells that undergo dedifferentiation (loss of LM21 and LM25; appearance or increase in the content of LM5, LM6, LM19, JIM11, JIM12, JIM20, JIM8 and JIM16 epitopes).

Topics: Actinidia; Antibodies, Monoclonal; Bony Callus; Cell Wall; Endosperm; Epitopes; Extracellular Matrix; Fruit; Glucans; Immunohistochemistry; Microscopy, Electron, Scanning; Mucoproteins; Pectins; Plant Proteins; Polysaccharides; Xylans

Panax notoginseng is a widely used traditional Chinese medicine and has extensive pharmacological effects. In this work, water-soluble polysaccharides from Panax notoginseng were isolated and fractionated. One starch-like polysaccharide (PNPN) and six pectin fractions (PNPA-1A, PNPA-1B, PNPA-2A, PNPA-2B, PNPA-3A and PNPA-3B) were obtained. Monosaccharide composition, enzymatic hydrolysis, nuclear magnetic resonance and methylation analysis were combined to characterize their structures. PNPA-1A and PNPA-2A mainly contained 1,4-β-D-galactans, 1,5-α-L-arabinan and arabinogalactan II (AG-II). PNPA-3A was a typical rhamnogalacturonan I (RG-I) type pectin with 1,4-β-D-galactan and 1,5/1,3,5-α-L-arabinan side chains. PNPA-1B, PNPA-2B and PNPA-3B consisted of homogalacturonan (HG) as major domains, together with different ratios of RG-I and rhamnogalacturonan II (RG-II) domains. These results will provide basis for further investigation of structure-activity relationships of Panax notoginseng polysaccharides and be useful for the application of Panax notoginseng.

Topics: Galactans; Hydrolysis; Magnetic Resonance Spectroscopy; Monosaccharides; Panax notoginseng; Pectins; Polysaccharides; Water

Topics: Blood Proteins; Cell Membrane; Cell Wall; Citrus; Fruit; Galactans; Galectins; Humans; Hydrolysis; MCF-7 Cells; Pectins; Plant Cells; Polysaccharides; Protein Binding; Solubility; Structure-Activity Relationship; Water

A draft genome of Paenibacillus amylolyticus 27C64 was assembled and a total of 314 putative CAZymes in 108 different families were identified. Comparison to well-studied polysaccharide-degrading organisms revealed that P. amylolyticus 27C64 has as many or more putative CAZymes than most of these organisms. Four different pectic substrates and xylan supported growth but cellulose was not utilized. Measurement of enzyme activities in culture supernatants revealed low levels of cellulase activity, high levels of xylanase activity, and pectinase activities that adapted to the specific polysaccharides provided. Relative expression levels of each putative pectinase in cells grown with and without three different pectic substrates were evaluated with RT-qPCR and distinct sets of genes upregulated in response to homogalacturonan, methylated homogalacturonan, and rhamnogalacturonan I were identified. It is also noted that this organism's pectinolytic system differs from other well-studied systems and contains enzymes which are of value for further study.

Topics: Bacterial Proteins; Cellulose; Culture Media; DNA, Bacterial; Molecular Sequence Annotation; Paenibacillus; Pectins; Polygalacturonase; Sequence Analysis, DNA

Brachypodium distachyon (Brachypodium) is now well considered as being a suitable plant model for studying temperate cereal crops. Its cell walls are phylogenetically intermediate between rice and poaceae, with a greater proximity to these latter. By microscopic and biochemical approaches, this work gives an overview of the temporal and spatial distribution of cell wall polysaccharides in the grain of Brachypodium from the end of the cellularization step to the maturation of grain. Variation in arabinoxylan chemical structure and distribution were demonstrated according to development and different grain tissues. In particular, the kinetic of arabinoxylan feruloylation was shown occuring later in the aleurone layers compared to storage endosperm. Mixed linked β-glucan was detected in whole the tissues of Brachypodium grain even at late stage of development. Cellulose was found in both the storage endosperm and the outer layers. Homogalacturonan and rhamnogalacturonan I epitopes were differentially distributed within the grain tissues. LM5 galactan epitope was restricted to the aleurone layers contrary to LM6 arabinan epitope which was detected in the whole endosperm. A massive deposition of highly methylated homogalacturonans in vesicular bodies was observed underneath the cell wall of the testa t2 layer at early stage of development. At maturity, low-methylated homogalacturonans totally fulfilled the lumen of the t2 cell layer, suggesting pectin remodeling during grain development. Xyloglucans were only detected in the cuticle above the testa early in the development of the grain while feruloylated arabinoxylans were preferentially deposited into the cell wall of t1 layer. Indeed, the circumscribed distribution of some of the cell wall polysaccharides raises questions about their role in grain development and physiology.

Topics: Brachypodium; Cell Wall; Edible Grain; Endosperm; Glucans; Organ Specificity; Pectins; Polysaccharides; Xylans

The pulp of gabiroba fruits was submitted to a hot water extraction, giving rise to a crude pectin named GW. GW was shown to be composed mainly of arabinose (54.5%), galacturonic acid (33.5%), galactose (7.6%), and rhamnose (1.6%). GW was characterized by chromatographic and spectroscopic methods indicating the presence of homogalacturonans (HG) with a degree of methyl-esterification (DM) of 60% and rhamnogalacturonans I (RG-I). HG domain represents 31.9% and RG-I domain 65.3%. Furthermore, GW was submitted to sequential fractionation methods, giving rise to GWP-TEP fraction, structurally characterized by the predominance of HG regions, and confirmed by NMR analysis. The rheological behavior of GW was analyzed at 1%, 3%, and 5% (w/v) concentration with 0.1 mol L

Topics: Fruit; Myrtaceae; Pectins; Rheology; Viscosity

In the present study, the various properties of pectin extracted using microwave-assisted extraction (MAE) from eggplant peel and eggplant calyx (as food processing wastes of eggplant) were compared with each other. The eggplant peel pectin (EPP) exhibited higher extraction yield (29.17%) than eggplant calyx pectin (ECP; 18.36%). Both of EPP and ECP were high in methoxyl and rich in galacturonic acid. HPLC analysis showed that EPP was high in HG (homogalacturonan) (58.6%), while ECP was high in RG-I (rhamnogalacturonan-I) (44.9%). Also, higher phenolic contents were observed for EPP in comparing with ECP. Approximately in all of the functionalities (WHC (water holding capacity) and OHC (oil holding capacity), emulsifying and foaming properties, and antioxidant activity), EPP showed higher value rather than ECP.

Topics: Chromatography, High Pressure Liquid; Food Handling; Hexuronic Acids; Magnetic Resonance Spectroscopy; Microwaves; Pectins; Phenols; Solanum melongena; Spectroscopy, Fourier Transform Infrared; Water

Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (

Topics: Cloning, Molecular; Escherichia coli; Hydrogen-Ion Concentration; Paenibacillus polymyxa; Pectins; Polysaccharide-Lyases; Substrate Specificity

Pectic polysaccharides from New Zealand (NZ) spinach (

Topics: Aizoaceae; Bacteroides; Fermentation; Fruit; Gastrointestinal Microbiome; Magnoliopsida; New Zealand; Pectins; Plant Leaves; Polysaccharides

The chemical and sensory profiles of wines prepared from Cabernet Sauvignon grapes at different ripening stages vary greatly. Here, the soluble cell wall carbohydrate (SCWC) and phenolic profiles of wines were analyzed in parallel with the sensory evaluation of their mouthfeel and taste characteristics. Both SCWCs and phenolic compounds correlated with wine mouthfeel. When analyses were extended to specific classes of cell wall carbohydrates, it was shown that rhamnogalacturonan I/II, arabinan, arabinogalactan types I and II and xyloglucan from grapes were the key determinants of overall mouthfeel descriptors, particularly viscosity, astringency and roughness, whereas heteromannan from grapes was associated with mouth coating and chalkiness. A perceived sour taste was notably associated with higher homogalacturonan contents. This finding provides insights into the contributions of non-phenolic compounds to wine mouthfeel. The data provide opportunities for the development of simple monosaccharide marker assays to monitor major mouthfeel characteristics in red wines.

Topics: Astringents; Carbohydrates; Cell Wall; Galactans; Humans; Molecular Weight; Mouth; Pectins; Phenols; Taste; Vitis; Wine

Craterostigma plantagineum belongs to the desiccation-tolerant angiosperm plants. Upon dehydration, leaves fold and the cells shrink which is reversed during rehydration. To understand this process changes in cell wall pectin composition, and the role of the apoplastic glycine-rich protein 1 (CpGRP1) were analysed. Cellular microstructural changes in hydrated, desiccated and rehydrated leaf sections were analysed using scanning electron microscopy. Pectin composition in different cell wall fractions was analysed with monoclonal antibodies against homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II and hemicellulose epitopes. Our data demonstrate changes in pectin composition during dehydration/rehydration which is suggested to affect cell wall properties. Homogalacturonan was less methylesterified upon desiccation and changes were also demonstrated in the detection of rhamnogalacturonan I, rhamnogalacturonan II and hemicelluloses. CpGRP1 seems to have a central role in cell adaptations to water deficit, as it interacts with pectin through a cluster of arginine residues and de-methylesterified pectin presents more binding sites for the protein-pectin interaction than to pectin from hydrated leaves. CpGRP1 can also bind phosphatidic acid (PA) and cardiolipin. The binding of CpGRP1 to pectin appears to be dependent on the pectin methylesterification status and it has a higher affinity to pectin than its binding partner CpWAK1. It is hypothesised that changes in pectin composition are sensed by the CpGRP1-CpWAK1 complex therefore leading to the activation of dehydration-related responses and leaf folding. PA might participate in the modulation of CpGRP1 activity.

Topics: Arginine; Cell Wall; Craterostigma; Dehydration; Pectins; Phosphatidic Acids; Plant Leaves; Plant Proteins

Our previous paper reported the structure of ginseng pectic polysaccharides related to the cell migration inhibitory effects, but the underlying mechanisms are poorly understood. In this manuscript, rhamnogalacturonan I (RGI)-rich pectins prepared from ginseng pectin were investigated for their effect on cell migration. The results indicated that the combination of homogalacturonan (HG) and RGI-rich pectins exerted stronger effects than either HG- or RGI-rich pectin alone. Further studies revealed that the effects of HG- and RGI-rich pectins were dependent on pretreatment, which caused alterations in cell morphologies such as cell size and shape, focal adhesion, and the organization of actin filaments, suggesting that HG and RGI pectins exert synergistic effects on cell migration, likely through different ways. Morphological data and quantitative cell adhesion and spreading assays showed that HG- and RGI-rich pectin treatment decreased cell adhesion and cell spreading on the substratum, suggesting that HG- and RGI-rich pectins may exert their effects on cell migration via decreasing cell adhesion and cell spreading. Additionally, we showed that L-929 cells expressed little galectin-3 (Gal-3) and that lactose, an inhibitor of Gal-3 did not block the activities of HG- and RGI-rich pectins, implicating that cell migration inhibited by pectin did not correlate to Gal-3.

Topics: Actin Cytoskeleton; Animals; Cell Adhesion; Cell Line; Cell Movement; Cell Shape; Cell Size; Drug Synergism; Fibroblasts; Focal Adhesions; Galectin 3; Gene Expression; Lactose; Mice; Panax; Pectins

The aim of this study was to investigate the chemical structure and biological activity of a pectic fraction isolated from the aerial parts of A. campestris L. subsp. maritima Arcangeli. The chemical and spectroscopic analyses of the pectic fraction (ACP-E10) demonstrated that ACP-E10 was composed of homogalacturonan (HG) (60%) and rhamnogalacturonan-I (RG-I) (29%) regions. Side chains of the RG-I included mainly branched arabinans and type II arabinogalactans (AG-II). The molar mass of ACP-E10 determined by HPSEC-MALLS was 16,600g/mol. ACP-E10 was evaluated for its gastroprotective effect against ethanol-induced gastric lesions in rats. Oral pretreatment of animals with ACP-E10 (0.3, 3 and 30mg/kg) significantly reduced gastric lesions by 77±7.9%, 55±11.1% and 65±11.8%. ACP-E10 also maintained mucus and glutathione (GSH) contents in the gastric mucosa. In addition, ACP-E10 demonstrated antioxidant activity in vitro by the DPPH assay. These results demonstrated that the pectin from A. campestris had significant gastroprotective effects in vivo, which were likely attributable to their capacity to increase the protective defenses of gastric mucosa.

Topics: Animals; Anti-Ulcer Agents; Artemisia; Ethanol; Gastric Mucosa; Humans; Mucoproteins; Pectins; Phytotherapy; Plant Leaves; Plant Proteins; Polysaccharides; Rats; Stomach Ulcer

The matrix polysaccharides of plant cell walls are diverse and variable sets of polymers influencing cell wall, tissue and organ properties. Focusing on the relatively simple parenchyma tissues of four fruits - tomato, aubergine, strawberry and apple - we have dissected cell wall matrix polysaccharide contents using sequential solubilisation and antibody-based approaches with a focus on pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). Epitope detection in association with anion-exchange chromatography analysis indicates that in all cases solubilized polymers include spectra of HG molecules with unesterified regions that are separable from methylesterified HG domains. In highly soluble fractions, RG-I domains exist in both HG-associated and non-HG-associated forms. Soluble xyloglucan and pectin-associated xyloglucan components were detected in all fruits. Aubergine glycans contain abundant heteroxylan epitopes, some of which are associated with both pectin and xyloglucan. These profiles of polysaccharide heterogeneity provide a basis for future studies of more complex cell and tissue systems.

Topics: Cell Wall; Fragaria; Fruit; Glucans; Malus; Pectins; Polysaccharides; Solanum lycopersicum; Solanum melongena; Xylans

Topics: Adhesins, Bacterial; Animals; Bacterial Adhesion; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Epithelial Cells; Gastrointestinal Microbiome; Hydrogen-Ion Concentration; Limosilactobacillus reuteri; Mice; Microbial Interactions; Molecular Dynamics Simulation; Pectins; Protein Folding; Repetitive Sequences, Amino Acid; Sequence Homology, Amino Acid; Serine

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

Topics: Acetylation; Acetyltransferases; Arabidopsis; Arabidopsis Proteins; Botrytis; Gene Expression Regulation, Plant; Glucans; Mannans; Pectins; Plant Growth Regulators; Plant Leaves; Polysaccharides; Pseudomonas syringae; Transcriptome; Xylans

Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains cell-cell adhesion, among other functions. As the most complex wall polysaccharide, pectin is composed of several covalently linked domains, such as homogalacturonan (HG) and rhamnogalacturonan I (RG I). Pectin has widespread uses in the food industry and has emerging biomedical applications, but its synthesis remains poorly understood. For instance, the enzymes that catalyze RG I elongation remain unknown. Recently, a coexpression- and sequence-based

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Glucuronosyltransferase; Glycosyltransferases; Hydrolases; Microscopy, Electron, Scanning; Pectins; Phylogeny; Plant Mucilage; Polysaccharides; Seeds

Sijunzi Decoction (SJZD) is a formula used for the treatment of spleen deficiency and gastrointestinal diseases in Traditional Chinese Medicine. Polysaccharides are reported to be the main components of SJZD responsible for its bio-functions. However, highly purified and clearly characterized polysaccharides from SJZD are not well described. Here we obtained a purified polysaccharide (SJZDP-II-I) from SJZD using ion exchange chromatography and gel filtration. Structure analysis by FT-IR and NMR identified SJZDP-II-I as a typical pectic polysaccharide with homogalacturonan and rhamnogalacturonan type I regions and arabinogalactan type I and II as side chains. In vitro studies indicated that SJZDP-II-I treatment could significantly enhance the total antioxidant capacity of SW480 cells, resulting from the promoted expressions of antioxidant enzymes and their master regulator PGC-1α, which would be valuable for further research and applications.

Topics: Antioxidants; Cell Culture Techniques; Cell Line; Cell Survival; Drugs, Chinese Herbal; Gastrointestinal Diseases; Humans; Magnetic Resonance Spectroscopy; Molecular Conformation; Molecular Structure; Pectins; Polysaccharides; Spectroscopy, Fourier Transform Infrared; Spleen; Structure-Activity Relationship

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three-dimensional structures and macromolecular compositions of these Golgi stacks, we examined high-pressure frozen/freeze-substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans-Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi-associated vesicles. Margins of trans-Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan-I (PGA/RG-I) are detected in the trans-most cisternae and TGN compartments. LVs produced from TGN compartments (TGN-LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN-LVs containing the XG and PGA/RG-I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans-Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans-cisternae accompanying polysaccharide synthesis with a mathematical model.

Topics: Cell Membrane; Cell Wall; Electron Microscope Tomography; Epitopes; Glucans; Hexuronic Acids; Medicago sativa; Microscopy, Fluorescence; Models, Molecular; Pectins; Plant Roots; Polysaccharides; trans-Golgi Network; Xylans

Native, intact (TrPP) and modified, low-molecular-weight (MTrPP) forms of pectic polysaccharides isolated from turmeric were evaluated for ulcer-preventive potentials in in vitro and in vivo models. Data indicated that MTrPP possessed significantly better ulcer-preventive property than TrPP; inhibiting ulcer scores up to 85%. Results were substantiated by effective muco-protection, H(+),K(+)-ATPase down-regulation, inhibition of H. pylori growth/adherence, higher antioxidant/cytoprotective mechanisms. Structural data indicated TrPP and MTrPP differ in their molecular weights and structural characteristics with different sugar compositions and side chain ratios. MTrPP was rich in galacturonic acid (687mg/g; TrPP-544mg/g) and galactose (52.9%; TrPP-21.7%). Results were substantiated by NMR/FTIR data indicating the presence of homogalacturonan and rhamnogalacturonam-I containing galactans. By virtue of binding to inflammatory marker (galectin-3), galactans may reduce inflammation induced ulcerations. The low molecular weight of MTrPP (155kDa; TrPP-13kDa) may increase its bioavailability than TrPP, thus MTrPP may possess higher antiulcer potential.

Topics: Anti-Ulcer Agents; Antioxidants; Cell Line; Curcuma; Down-Regulation; Galactose; Gastric Mucosa; H(+)-K(+)-Exchanging ATPase; Helicobacter pylori; Hexuronic Acids; Humans; Magnetic Resonance Spectroscopy; Molecular Weight; Pectins; Polysaccharides; Spectroscopy, Fourier Transform Infrared; Stomach Ulcer

Diet and particularly dietary fibres have an impact on the gut microbiome and play an important role in human health and disease. Pectin is a highly consumed dietary fibre found in fruits and vegetables and is also a widely used additive in the food industry. Yet there is no information on the effect of pectin on the human gut microbiome. Likewise, little is known on gut pectinolytic bacteria and their enzyme systems. This study was undertaken to investigate the mechanisms of pectin degradation by the prominent human gut symbiont Bacteroides xylanisolvens.. Transcriptomic analyses of B. xylanisolvens XB1A grown on citrus and apple pectins at mid- and late-log phases highlighted six polysaccharide utilization loci (PUL) that were overexpressed on pectin relative to glucose. The PUL numbers used in this report are those given by Terrapon et al. (Bioinformatics 31(5):647-55, 2015) and found in the PUL database: http://www.cazy.org/PULDB/. Based on their CAZyme composition, we propose that PUL 49 and 50, the most overexpressed PULs on both pectins and at both growth phases, are involved in homogalacturonan (HG) and type I rhamnogalacturonan (RGI) degradation, respectively. PUL 13 and PUL 2 could be involved in the degradation of arabinose-containing side chains and of type II rhamnogalacturonan (RGII), respectively. Considering that HG is the most abundant moiety (>70%) within pectin, the importance of PUL 49 was further investigated by insertion mutagenesis into the susC-like gene. The insertion blocked transcription of the susC-like and the two downstream genes (susD-like/FnIII). The mutant showed strong growth reduction, thus confirming that PUL 49 plays a major role in pectin degradation.. This study shows the existence of six PULs devoted to pectin degradation by B. xylanisolvens, one of them being particularly important in this function. Hence, this species deploys a very complex enzymatic machinery that probably reflects the structural complexity of pectin. Our findings also highlight the metabolic plasticity of B. xylanisolvens towards dietary fibres that contributes to its competitive fitness within the human gut ecosystem. Wider functional and ecological studies are needed to understand how dietary fibers and especially plant cell wall polysaccharides drive the composition and metabolism of the fibrolytic and non-fibrolytic community within the gut microbial ecosystem.

Topics: Bacteroides; Citrus; Dietary Fiber; Genetic Loci; Malus; Mutagenesis; Pectins; RNA, Bacterial; Sequence Analysis, RNA; Transcriptome

Primary cell wall polysaccharides from aqueous extract of buriti fruit pulp (Mauritia flexuosa, an exotic tropical palm) were isolated and characterized. After freeze-thaw and α-amylase treatments, extracted polysaccharides were purified by sequential ultrafiltration through membranes. Two homogeneous fractions were obtained, SBW-100R and SBW-30R (Mw of 126 kDa and 20 kDa, respectively). Monosaccharide composition, methylation and (13)C NMR analysis showed that fraction SBW-100R contained a (1 → 5)-linked arabinan, branched at O-3 and O-2 positions, linked to a type I rhamnogalacturonan. Low amounts of these polymers were also present in fraction SBW-30R according to (13)C NMR analysis and monosaccharide composition. However, a high methyl esterified homogalacturonan (HG) was present in higher proportions. These results reinforce previous findings present in literature data which indicate that pectic polysaccharides are found in high amounts in primary cell walls of palms, which are commelinid monocotyledons.

Topics: Arecaceae; Cell Wall; Fruit; Magnetic Resonance Spectroscopy; Molecular Structure; Monosaccharides; Pectins; Plant Extracts; Polysaccharides

Structurally different pectins were isolated from the wood greenery of Abies sibirica L. by the sequential extraction with water (ASW), HCl solution (pH∼4) (ASA), and NH3 solution (pH∼8.5) (ASN). The GalA/Rha molar ratios for the ASW (15), ASA (8.9), and ASN (3.9) polysaccharides diminish in the order ASW>ASA>ASN, indicating a decrease in homogalacturonans and increase in rhamnogalacturonan I in this series. The ASWH, ASAH, and ASNH homogalacturonans derived by acid hydrolysis of ASW, ASA, and ASN have similar Mw 23.8, 21.1, and 18.9kDa, respectively, corresponding to a mean polymerization degree of 97-122 for the GalA residue. The macromolecule backbone of ASN was represented mainly by moieties of partially methylesterified homogalacturonan and partially 2-O- and/or 3-O-acetylated rhamnogalacturonan I. The carbohydrate side chains of the branched region are primarily made up of terminal, 1,5-O-, 1,3,5-di-O- and 1,2,3,5-tri-O-substituted α-L-Araf residues and terminal, 1,3-O- and 1,3,6-di-O-substituted β-D-Galp residues. The currently known pectin models were refined.

Topics: Abies; Ammonia; Carbohydrate Sequence; Chromatography, Ion Exchange; Hydrochloric Acid; Hydrolysis; Magnetic Resonance Spectroscopy; Pectins; Water

Pectin modified with pH, heat or enzymes, has previously been shown to exhibit anti-cancer activity. However, the structural requirements for modified pectin bioactivity have rarely been addressed. In this study several pectin extracts representing different structural components of pectin were assessed for effects against colon cancer cells. Rhamnogalacturonan I (RGI) extracts reduced proliferation of DLD1 and HCT116 colon cancer cells in a dose- and time-dependent manner. RGI reduced ICAM1 gene expression and siRNA-mediated knockdown of ICAM1 expression decreased cell proliferation providing a potential novel mechanism for the anti-cancer activity of pectin. Structural analysis of bioactive and non-bioactive RGIs suggested that a homogalacturonan component is maybe essential for the anti-proliferative activity, furthering the understanding of the structural requirements for pectin bioactivity.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; HCT116 Cells; Humans; Intercellular Adhesion Molecule-1; Magnetic Resonance Spectroscopy; Pectins; RNA Interference; RNA, Small Interfering

Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I (RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition.

Topics: Antibodies, Monoclonal; Cell Shape; Cell Wall; Cellulose; Chlorophyta; Dinitrobenzenes; Glycoside Hydrolases; Immunohistochemistry; Microarray Analysis; Microtubules; Models, Biological; Pectins; Polysaccharides; Sulfanilamides

Cell wall polysaccharides' occurrences in two internodes of different development stages in M. lutarioriparius stem were analyzed and three major differences between them were identified by cell wall polysaccharide probes. Deposition and modification of cell wall polysaccharides during stem development affect biomass yield of the Miscanthus energy crop. The distribution patterns of cell wall polysaccharides in the 2nd and the 11th internodes of M. lutarioriparius stem were studied using in situ immunofluorescence assay. Crystalline cellulose and xylan were present in most of the stem tissues except phloem, where xyloglucan was the major composition of hemicellulose. The distribution of pectin polysaccharides varied in stem tissues, particularly in vascular bundle elements. Xylogalacturonan, feruloylated-1,4-β-D-galactan and (1,3)(1,4)-β-glucans, however, were insufficient for antibodies binding in both internodes. Furthermore, the distribution of cell wall polysaccharides was differentiated in the two internodes of M. lutarioriparius. The significant differences in the pattern of occurrence of long 1,5-α-L-arabinan chain, homogalacturonan and fucosylated xyloglucans epitope were detected between the two internodes. In addition, the relationships between probable functions of polysaccharides and their distribution patterns in M. lutarioriparius stem cell wall were discussed, which would be helpful to understand the growth characteristics of Miscanthus and identify potential targets for either modification or degradation.

Topics: Cell Wall; Cellulose; Fluorescent Antibody Technique; Pectins; Plant Stems; Poaceae; Polysaccharides

Cell walls are complex structures surrounding plant cells with a composition that varies among species and even within a species between organs, cell types and development stages. For years, cell walls in wheat grains were described as simple walls consisting mostly of arabinoxylans and mixed-linked beta glucans. Proteomic and transcriptomic studies identified enzyme families involved in the synthesis of many more cell wall polysaccharides in the wheat grains. Here we describe the discovery of pectic domains in wheat grain using monoclonal antibodies and enzymatic treatment to degrade the major cell wall polymers. Distinct spatial distributions were observed for rhamnogalacturonan I present in the endosperm and mostly in the aleurone layer and homogalacturonan especially found in the outer layers, and tight developmental regulations were unveiled. We also uncovered a massive deposition of homogalacturonan via large vesicular bodies in the seed coat (testa) beneath a thick cuticle during development. Our findings raise questions about the function of pectin in wheat grain.

Topics: Cell Wall; Endosperm; Organ Specificity; Pectins; Triticum

Arabinogalactan protein 31 (AGP31) is a remarkable plant cell-wall protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls.. Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction.. It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly.. These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in complex supra-molecular scaffolds. Such scaffolds could contribute to the strengthening of cell walls of quickly growing organs such as etiolated hypocotyls.

Topics: Arabidopsis; Arabidopsis Proteins; Brachypodium; Cell Wall; Galactans; Glycosylation; Models, Biological; Mucoproteins; Nicotiana; Pectins; Plant Proteins; Polysaccharides; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Seedlings

Upon hydration, flax seeds secrete mucilages whose content and physico-chemical properties vary according to the genotype and environment. The aim of the work was to investigate the complex genetic relationships between the vegetative period, colour, size and production of seed, the composition (polysaccharides and proteins) and physico-chemical properties of soluble mucilages collected at 28 °C from seeds of 18 lines grown in St Petersburg area. The vegetative period duration was found to impact the size and production of seeds, the yield of mucilages, including the polysaccharides, and the galactosidase enzymes, as well as their composition (mainly the rhamnogalacturonan I moieties) and some of their properties (mainly viscosity). Data allowed to significantly distinguish 6 fibre lines with mucilages enriched in rhamnogalacturonan I, 6 lines with mucilages enriched in arabinoxylan including 5 linseeds and 1 mutated fibre-line, and 5 lines with mucilages enriched in homogalacturonan-like polymer including 4 fibre lines and 1 brown linseed. Seven fibre lines had mucilages particularly rich in galactose. High to very high variability was found for 14 traits. Relatively independent characters (form/shape, protein and galactosidase) were identified and could be combined by breeding, with a focus on mucilage yield, composition and properties. Main-component analyses of line characters showed a large diversity in linseeds mainly due to their different origin but small variation in Russian fibre lines with brown seeds.

Topics: Chromatography, Gel; Factor Analysis, Statistical; Flax; Pectins; Seeds; Viscosity; Xylans

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.. 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.. 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.. 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.

Topics: Carbohydrates; Citrus; Fruit; Hydrogen-Ion Concentration; Molecular Weight; Nitric Acid; Oxalic Acid; Pectins; Rutaceae

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.. Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.. Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.. This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.

Topics: Biological Evolution; Bryopsida; Cell Wall; Pectins; Plant Stomata; Polysaccharides

Six pectic polysaccharides were obtained from white ginseng (GPW-1 and GPW-2), red ginseng (GPR-1 and GPR-2, steamed ginseng at 100°C) and steamed ginseng (GPS-1 and GPS-2, steamed ginseng at 120°C) by combination of water extraction, ion-exchange and gel permeation chromatographies. Based on the data from monosaccharide composition and (13)C NMR analysis, GPW-1, GPR-1 and GPS-1 were identified as type-I rhamnogalacturonan (RG-I)-rich pectins, GPW-2, GPR-2 and GPS-2 were homogalacturonan (HG)-rich pectins with different degrees of methyl-esterification. Remarkably, GalA increased with the increase of processing temperatures in these six fractions, which might be caused by the transformation of esterified GalA into un-esterified form during heat processing. In vivo animal experiments showed that GPs exhibited significant antiohyperglycemic and antioxidant activities in alloxan-induced diabetic mice, and the effects increased with the processing temperature, with the most potent activity in GPS.

Topics: Animals; Diabetes Mellitus, Experimental; Hot Temperature; Hypoglycemic Agents; Male; Mice; Panax; Pectins

Cooking time decreases when beans are soaked first. However, the molecular basis of this decrease remains unclear. To determine the mechanisms involved, changes in both pectic polysaccharides and cell wall enzymes were monitored during soaking. Two cultivars and one breeding line were studied.. Soaking increased the activity of the cell wall enzymes rhamnogalacturonase, galactanase and polygalacturonase. Their activity in the cell wall was detected as changes in chemical composition of pectic polysaccharides. Rhamnose content decreased but galactose and uronic acid contents increased in the polysaccharides of soaked beans. A decrease in the average molecular weight of the pectin fraction was induced during soaking. The decrease in rhamnose and the polygalacturonase activity were associated (r = 0.933, P = 0.01, and r = 0.725, P = 0.01, respectively) with shorter cooking time after soaking.. Pectic cell wall enzymes are responsible for the changes in rhamnogalacturonan I and polygalacturonan induced during soaking and constitute the biochemical factors that give bean cell walls new polysaccharide arrangements. Rhamnogalacturonan I is dispersed throughout the entire cell wall and interacts with cellulose and hemicellulose fibres, resulting in a higher rate of pectic polysaccharide thermosolubility and, therefore, a shorter cooking time.

Topics: beta-Galactosidase; Cell Wall; Cooking; Cotyledon; Galactans; Galactose; Glycoside Hydrolases; Molecular Weight; Pectins; Phaseolus; Plant Proteins; Polygalacturonase; Rhamnose; Seeds; Solubility; Time Factors; Uronic Acids; Water

Intervessel pits act as safety valves that prevent the spread of xylem embolism. Pectin-calcium crosslinks within the pit membrane have been proposed to affect xylem vulnerability to cavitation. However, as the chemical composition of pit membranes is poorly understood, this hypothesis has not been verified. Using electron microscopy, immunolabeling, an antimonate precipitation technique, and ruthenium red staining, we studied the distribution of selected polysaccharides and calcium in the pit membranes of four angiosperm tree species. We tested whether shifts in xylem vulnerability resulting from perfusion of stems with a calcium chelating agent corresponded with the distribution of pectic homogalacturonans (HG) and/or calcium within interconduit pit membranes. No HG were detected in the main part of intervessel pit membranes, but were consistently found in the marginal membrane region known as the annulus. Calcium colocalized with HG in the annulus. In contrast to intervessel pits, the membrane of vessel-ray pits showed a high pectin content. The presence of two distinct chemical domains, the annulus and the actual pit membrane, can have substantial implications for pit membrane functioning. We propose that the annulus could affect the observed shift in xylem vulnerability after calcium removal by allowing increased pit membrane deflection.

Topics: Antibody Specificity; Calcium; Epitopes; Esterification; Glucans; Magnoliopsida; Methylation; Pectins; Ruthenium Red; Species Specificity; Staining and Labeling; Xylans; Xylem

The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development.. Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment.. These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana.

Topics: Cells, Cultured; Epitopes; Gene Expression Regulation, Plant; Musa; Pectins

Pectins are viewed as multiblock cobiopolymers of different pectic polysaccharides, notably, homogalacturonan (HG) and rhamnogalacturonan I (RG I). Furthermore, on the basis of HGs isolated from different (pectins from) dicot cell walls, HG is supposed to have an average degree of polymerization (DP) of approximately 100 irrespective of the plant source. To validate or invalidate these suppositions, pectins from both monocot (pineapple and banana) and dicot (yellow passion fruit and lemon) cell walls were examined. The results show that all the extracted pectins comprise HGs as well as type I and II arabinogalactan side chain-containing RGs I, but of significantly (p < 0.05) different relative proportions; lemon pectin being the richest in HGs, followed by yellow passion fruit pectin. The HG building blocks of each pectin are homogeneous with respect to the molecular size but have a significantly (p < 0.05) reduced length in monocot pectins (59-67) compared to dicot ones (93-102). Lemon pectin displayed the highest degree of esterification (DE), viscosity-average molecular weight (M(v)), and gelling ability, whereas with similar DEs and a higher M(v), banana pectin exhibited a lower gelling ability than yellow passion fruit pectin. It is concluded that both the HG amount and DP strongly influence the gelling properties of pectin.

Topics: Ananas; Citrus; Esterification; Gels; Musa; Passiflora; Pectins; Polymers

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.

Topics: Carbohydrate Sequence; Cell Wall; Fruit; Glucans; Monosaccharides; Pectins; Polysaccharides; Sapotaceae; Xylans

A combination of xylogalacturonan (XGA), homogalacturonan, and rhamnogalacturonan was extracted from watermelon fruit cell walls with 0.1 M NaOH. In contrast to the resistance of xylogalacturonans from most other sources to endopolygalacturonase (EPG), about 50% of the extracted XGA could be converted into oligosaccharides by EPG digestion with a commercial EPG from Megazyme International. The oligosaccharides were fractionated by ion-exchange chromatography, and their structures were investigated by mass spectrometry and NMR spectroscopy. The smallest oligosaccharide was beta-D-Xylp-(1-->3)-alpha-D-GalAp-(1-->4)-alpha-D-GalAp-(1-->4)-alpha-D-GalAp-(1-->4)-GalAp. The most abundant was beta-D-Xylp-(1-->3)-alpha-D-GalAp-(1-->4)-alpha-D-GalAp-(1-->4)(beta-D-Xylp-(1-->3)-alpha-D-GalAp-(1-->4))-alpha-D-GalAp-(1-->4)-alpha-D-GalAp-(1-->4)-GalAp. Given that the nonreducing ends of the oligosaccharides often were xylosylated GalA residues, and that fungal EPG digests homogalacturonans between the third and fourth GalA bound to the enzyme, it appears that EPG can accommodate a xylosylated GalA in the site that binds the fourth GalA. Since all of the oligosaccharides characterized had three unsubstituted GalA residues at their reducing ends, the enzyme appears not to accommodate xylosylated residues in the first three sugar-binding sites. Thus, XGA regions with fewer than three unsubstituted residues between branch points will be resistant to EPG. The EPG-susceptible XGA was not recovered from cell walls prepared using phosphate buffer for the homogenization of the watermelon tissue, probably because it was degraded by endogenous watermelon EPG and lost during isolation of the walls. Use of Tris-buffered phenol during wall isolation to prevent enzyme action caused some amidation of GalA residues with Tris.

Topics: Antidiarrheals; Carbohydrate Conformation; Carbohydrate Sequence; Cell Wall; Chromatography, Ion Exchange; Citrullus; Hexuronic Acids; Hydrolysis; Molecular Sequence Data; Oligosaccharides; Pectins; Polygalacturonase; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity

The aim of this paper was to further elucidate the structure and the immunomodulating properties of the pectic polymer GOA2, previously isolated from Glinus oppositifolius. Enzymatic treatment of GOA2 by endo-alpha-d-(1 --> 4)-polygalacturonase led to the isolation of three pectic subunits, GOA2-I, GOA2-II, and GOA2-III, in addition to oligogalacturonides. GOA2-I was shown to consist of 1,2-linked Rhap and 1,4-linked GalpA in an approximately 1:1 ratio, and NMR-analysis showed that the monomers were linked together in a strictly alternating manner. The galactose units in GOA2-I were found as terminal-, 1,3-, 1,6-, 1,4-, 1,3,4-, and 1,3,6-linked residues, while the arabinofuranosyl existed mainly as terminal- and 1,5-linked units. A rhamnogalacturonan-I type structure was suggested being the predominant part of GOA2-I. According to linkage analysis GOA2-II and GOA2-III contained glycosidic linkages characteristic for rhamnogalacturonan-II type structures. GOA2 was shown by sedimentation velocity in the analytical ultracentrifuge, to have a broad degree of polydispersity with a mode s(20,w) value of approximately 1.9 S, results reinforced by atomic force microscopy measurements. The polydispersity, as manifested by the proportion of material with s(20,w) > 3 S, decreased significantly with enzyme treatment. The abilities of GOA2, GOA2-I, GOA2-II, and GOA2-III to induce the proliferation of B cells, and to exhibit complement fixing activities were tested. In both test systems, GOA2-I showed significantly greater effects compared to its native pectin GOA2. GOA2-I was in addition shown to exhibit a more potent intestinal immune stimulating activity compared to GOA2. The ability of GOA2 to induce secretion of proinflammatory cytokines was examined. Marked upregulations in mRNA for IL-1beta from rat macrophages and IFN-gamma from NK cells were found.

Topics: Animals; Cell Proliferation; Galactose; Humans; Inflammation; Killer Cells, Natural; Macrophages; Mice; Molluginaceae; Monosaccharides; Pectins; Plant Extracts; Polymers; Rats

Particulate enzyme preparations were prepared from etiolated pea ( Pisum sativum L.) epicotyls and used to assay for 1,4-beta-galactan synthase using UDP-[U-(14)C]galactose. Optimum conditions for 1,4-beta-galactan synthesis were determined. The enzyme products were characterized by selective enzymic degradation, gel permeation chromatography and anion-exchange chromatography. Evidence was obtained for the formation of 1,4-beta-galactan chain attached to a pectic backbone containing both polygalacturonic acid and rhamnogalacturonan I. The results also indicated that part or all of this nascent pectin was present as a complex with xyloglucan.

Topics: beta-Galactosidase; Carbon Radioisotopes; Chromatography, Ion Exchange; Galactans; Glucans; Glycoside Hydrolases; Golgi Apparatus; Pectins; Pisum sativum; Polymers; Polysaccharides; Xylans

Antibodies were used to localise polysaccharide and protein networks in the protoxylem of etiolated soybean (Glycine max L.) hypocotyls. The deposition of glycine-rich proteins (GRPs) starts in the cell corners between protoxylem elements and xylem parenchyma cells. Finally, the GRPs form a network between two mature protoxylem elements. The network also interconnects the ring- and spiral-shaped secondary wall thickenings, as well as the thickenings with the middle lamellae of living xylem parenchyma cells. In addition to the GRP network, a polysaccharide network composed mainly of pectins is involved in the attachment of the secondary wall thickenings to the middle lamellae of xylem parenchyma cells.

Topics: Cell Wall; Fluorescent Antibody Technique; Glucans; Glycine max; Hypocotyl; Microscopy, Electron; Pectins; Plant Proteins; Plant Structures; Polysaccharides; Receptors, Cytoplasmic and Nuclear; Xylans

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants.

Topics: Carbohydrate Sequence; Carbon Isotopes; Chromatography, High Pressure Liquid; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oligosaccharides; ortho-Aminobenzoates; Pectins

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.

Topics: Antibodies, Monoclonal; Arabidopsis; Carboxymethylcellulose Sodium; Cell Wall; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Epitopes; Gold; Hypocotyl; Immunohistochemistry; In Vitro Techniques; Pectins; Plant Epidermis; Polylysine; Polysaccharides

Modifications in cell wall pectic polysaccharides are thought to influence cell-cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo- galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1-->4)-beta-D-galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26-30 days after anthesis), whereas homogalacturonan and pectic (1-->5)-alpha-L-arabinan are present in cotyledon cell walls throughout development. (1-->4)-beta-galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1-->4)-beta-galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1-->4)-beta-galactan appearance, indicated that the cotyledons with the galactan-rich cell wall layer were twice as firm as those with no detectable (1-->4)-beta-galactan.

Topics: Antibodies, Monoclonal; Cell Wall; Chromatography, Affinity; Chromatography, Ion Exchange; Cotyledon; Epitopes; Galactans; Immunohistochemistry; Pectins; Pisum sativum; Time Factors

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.

Topics: Cell Wall; Fusarium; Galactans; Glucans; Gossypium; Pectins; Plant Diseases; Plant Roots; Polysaccharides; Xylans

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.

Topics: Carbohydrate Sequence; Cell Wall; Glucans; Glycoproteins; Golgi Apparatus; Immunohistochemistry; Molecular Sequence Data; Monensin; Pectins; Plant Proteins; Plants; Polysaccharides; Protein Processing, Post-Translational; Xylans