pectins and polygalacturonic-acid

Experimental and theoretical data have been revisited to shed light onto the aspects of hydration and chain expansion of pectic acid (galacturonan) upon charging. The prediction of the variation of the number of solvation water molecules between the two limit ionization states from theoretical calculations was confirmed to a very high accuracy by the corresponding number evaluated form dilatometric measurements. The relevance of hydration to the mechanism of bonding of calcium ions by sodium pectate is discussed. Characterization of polymer expansion has been obtained by calculating the values of the characteristic ratio and/or the persistence length on the respective populations and comparing the theoretical predictions with experimental data. The results show that a charged chain in typical conditions of ionic strength is more expanded than its neutral counterpart, whereas the ideal limit (3

Topics: Molecular Conformation; Pectins; Polyelectrolytes; Water

Homogalacturonan (HG) is the most abundant pectin subtype in plant cell walls. Although it is a linear homopolymer, its modification states allow for complex molecular encoding. HG metabolism affects its structure, chemical properties, mobility and binding capacity, allowing it to interact dynamically with other polymers during wall assembly and remodelling and to facilitate anisotropic cell growth, cell adhesion and separation, and organ morphogenesis. HGs have also recently been found to function as signalling molecules that transmit information about wall integrity to the cell. Here we highlight recent advances in our understanding of the dual functions of HG as a dynamic structural component of the cell wall and an initiator of intrinsic and environmental signalling. We also predict how HG might interconnect the cell wall, plasma membrane and intracellular components with transcriptional networks to regulate plant growth and development.

Topics: Cell Wall; Morphogenesis; Pectins; Plant Development

Pectin is an important component of cell wall polysaccharides and is important for normal plant growth and development. As a major component of pectin in the primary cell wall, homogalacturonan (HG) is a long-chain macromolecular polysaccharide composed of repeated α-1,4-D-GalA sugar units. At the same time, HG is synthesized in the Golgi apparatus in the form of methyl esterification and acetylation. It is then secreted into the plasmodesmata, where it is usually demethylated by pectin methyl esterase (PME) and deacetylated by pectin acetylase (PAE). The synthesis and modification of HG are involved in polysaccharide metabolism in the cell wall, which affects the structure and function of the cell wall and plays an important role in plant growth and development. This paper mainly summarizes the recent research on the biosynthesis, modification and the roles of HG in plant cell wall.

Topics: Cell Wall; Esterification; Pectins; Plant Development; Polysaccharides

Pumpkin polysaccharides have arrested researchers' attention in fields of food supplements for healthy product and traditional Chinese medicine due to their multiple bioactivities with non-toxic and highly biocompatible. This review emphatically summarized recent progresses in the primary and spatial structural features, various bioactivities, structure-to-function associations, different preparation techniques, and absorption characteristics across intestinal epithelial and in vivo bio-distribution of pumpkin polysaccharides. Additionally, current challenges and future trends in development of pumpkin polysaccharides were pointed out. We found that pumpkin polysaccharides were primary structure (e.g. glucan, galactoglucan, galactomannan, galactan, homogalacturonan (HG), and rhamnogalacturonan-Ι (RG-Ι)) and special structure diverse (e.g. hollow helix, linear, and sphere-like) and significant functional foods or therapeutic agents (e.g. oral hypoglycemic agents). Moreover, we found that the molecular weight (Mw), uronic acid, linkage types, and modifications all could affect their bioactivities (e.g. anti-oxidant, anti-coagulant, and anti-diabetic activities), and pumpkin polysaccharides may across intestinal epithelial into the blood reaching to target organs. Collectively, the structures diversity and pharmacological values of pumpkin polysaccharides support their therapeutic potentials and sanitarian functions.

Topics: Antioxidants; Cucurbita; Dietary Supplements; Functional Food; Galactans; Galactose; Glucans; Hypoglycemic Agents; Mannans; Medicine, Chinese Traditional; Pectins; Phytochemicals; Plant Extracts; Polysaccharides; Polysaccharides, Bacterial

Pectate lyase treatment can be an alternative strategy of the chemical processing, which causes severe environmental pollution, and has been broadly studied and applied for diverse industrial applications including textile industry, beverage industry, pulp processing, pectic wastewater pretreatment, and oil extraction. This review gave a brief description of the origins, enzymatic characterizations, structure, and applications of pectate lyases (Pels). Most of the reported pectate lyases are originated from microorganisms with a small number of them from plants and animals. Due to the diverse environments that these microorganisms exist, Pels present diversified features, especially for the range of optimal pH and temperature. The diversified biochemical properties of Pels define their applications in different industries, and the applications of alkaline Pels on cotton bioscouring and ramie degumming in textile industry were focused in this review. This review also discussed the perspectives of the development and applications of Pels. KEY POINTS: • The first review on pectate lyase focusing on biotechnological applications. • Origins, features, structures, applications of pectate lyases reviewed. • Applications of alkaline Pels in textile industry demonstrated. • Perspectives on future development and applications of Pels discussed.

Topics: Cloning, Molecular; Pectins; Polysaccharide-Lyases

According to the 'acid growth theory', cell wall acidification controls cell elongation, therefore plant growth. This notably involves changes in cell wall mechanics through modifications of cell wall polysaccharide structure. Recently, advances in cell biology showed that changes in cell elongation rate can be mediated by the remodeling of pectins, and in particular of homogalacturonans (HGs). Their demethylesterification appears to be a key element controlling the chemistry and the rheology of the cell wall. We postulate that precise and dynamic modulation of extracellular pH plays a central role in the control of HG-modifying enzyme activities, and in particular those of pectin methylesterases and polygalacturonases. We propose that acid growth requires dynamic HG remodeling through the tight control of cell wall pH.

Topics: Carboxylic Ester Hydrolases; Gene Expression Regulation, Plant; Pectins; Plant Proteins

Recent publications have increased our knowledge of how pectin composition and the degree of homogalacturonan methylesterification impact the biochemical and biomechanical properties of plant cell walls, plant development, and plants' interactions with their abiotic and biotic environments. Experimental observations have shown that the relationships between the DM, the pattern of de-methylesterificaton, its effect on cell wall elasticity, other biomechanical parameters, and growth are not straightforward. Working towards a detailed understanding of these relationships at single cell resolution is one of the big tasks of pectin research. Pectins are highly complex polysaccharides abundant in plant primary cell walls. New analytical and microscopy techniques are revealing the composition and mechanical properties of the cell wall and increasing our knowledge on the topic. Progress in plant physiological research supports a link between cell wall pectin modifications and plant development and interactions with the environment. Homogalacturonan pectins, which are major components of the primary cell wall, have a potential for modifications such as methylesterification, as well as an ability to form cross-linked structures with divalent cations. This contributes to changing the mechanical properties of the cell wall. This review aims to give a comprehensive overview of the pectin component homogalacturonan, including its synthesis, modification, regulation and role in the plant cell wall.

Topics: Biomechanical Phenomena; Cell Wall; Esterification; Pectins

Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses.

Topics: Carboxylic Ester Hydrolases; Esterases; Molecular Weight; Pectins; Polygalacturonase

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.. In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.

Topics: Cell Wall; Down-Regulation; Fruit; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Microscopy, Atomic Force; Nanostructures; Pectins; Plant Proteins; Plants; Plants, Genetically Modified; Polygalacturonase; Polysaccharides

Recent progress in the identification and characterization of pectin biosynthetic proteins and the discovery of pectin domain-containing proteoglycans are changing our view of how pectin, the most complex family of plant cell wall polysaccharides, is synthesized. The functional confirmation of four types of pectin biosynthetic glycosyltransferases, the identification of multiple putative pectin glycosyl- and methyltransferases, and the characteristics of the GAUT1:GAUT7 homogalacturonan biosynthetic complex with its novel mechanism for retaining catalytic subunits in the Golgi apparatus and its 12 putative interacting proteins are beginning to provide a framework for the pectin biosynthetic process. We propose two partially overlapping hypothetical and testable models for pectin synthesis: the consecutive glycosyltransferase model and the domain synthesis model.

Topics: Cell Wall; Glycosyltransferases; Golgi Apparatus; Models, Biological; Pectins; Plants

Pectin represents a very complex, heterogeneous family of plant cell wall polysaccharides that play a significant role in plant growth, morphology, development, and plant defense and also serves as a gelling and stabilizing polymer in diverse food and specialty products and has positive effects on human health. In this review functional and structural characteristic of pectin molecule elements and their interconnections are described. Attention is also given to process of commercial production of pectin with special emphasis on composition and physical properties of commercial pectin as a result of the acid extraction.

Topics: Models, Molecular; Pectins; Plant Development

Topics: Acetylation; Cell Wall; Evolution, Molecular; Methylation; Molecular Conformation; Pectins

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

The ability of a plant cell to expand is largely defined by the physical constraints imposed by its cell wall. Accordingly, cell wall properties have to be regulated during development. The pectic polysaccharide homogalacturonan is a major component of the plant primary walls. Biosynthesis and in muro modification of homogalacturonan have recently emerged as key determinants of plant development, controlling cell adhesion, organ development, and phyllotactic patterning. This review will focus on recent findings regarding impact of homogalacturonan content and methyl-esterification status of this polymer on plant life. De-methyl-esterification of homogalacturonan occurs through the action of the ubiquitous enzyme 'pectin methyl-esterase'. We here describe various strategies developed by the plant to finely tune the methyl-esterification status of homogalacturonan along key events of the plant lifecycle.

Topics: Carbohydrate Metabolism; Cell Wall; Esterification; Models, Biological; Pectins; Plant Development; Plants

Topics: Biopolymers; Cell Wall; Molecular Structure; Pectins

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.

Topics: Cell Wall; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Molecular Conformation; Pectins; Plants; Polygalacturonase

New potential uses of pectates in food products have recently stimulated interest in re-evaluating the information available concerning the safety of pectins and pectates as food ingredients. Data relevant to this re-evaluation have been obtained in rats in recent 14-day and 13 wk subchronic feeding studies with sodium pectate. Ames tests and other mutagenicity tests have been conducted with sodium pectate, bleached sodium pectate and mixed sodium/calcium pectate salts. These toxicological studies with pectates have provided further evidence of their safety, and support of the continued GRAS status of pectins and pectate salts.

Topics: Animals; Consumer Product Safety; Food Additives; Humans; Mutagenicity Tests; Pectins

Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Dickeya chrysanthemi; Pectins; Plants; Pseudomonas; Virulence

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

Much progress has been made in recent years on the diagnostic value, Ag specificity, and pathogenic roles of autoantibodies correlated to the development of rheumatoid arthritis (RA) in humans. However, carbohydrate Ag-specific autoantibodies that may also play important roles in RA have largely been ignored. In this article, we report that serum levels of Abs capable of recognizing α1,4-polygalacturonic acid [(PGA); major structural component of pectin] strongly correlate with RA in humans. The measurements of PGA-specific Abs (PGA-Abs) in sera are comparable to rheumatoid factors and anti-cyclic citrullinated peptide Abs as serological diagnostic markers for RA in terms of sensitivity and specificity. Immunohistochemical staining results indicate that the PGA-Abs selectively bound synovial membrane cells and chondrocytes in the joints of both humans and rabbits (but not rodents). Induction of PGA-Abs by s.c. immunization of rabbits with carrier protein-conjugated synthetic PGA led to severe inflammatory reactions (synovial hyperplasia, small vessel proliferation, and inflammatory cell infiltration) in the joints. Injection of affinity purified anti-PGA IgG into the synovial cavity of rabbits resulted in accumulation of proinflammatory cytokines such as TNF-α, IL-8, and IL-1β in synovial fluid, as well as local pathological damage. We conclude that the PGA-cross-reactive moiety represents a major autoantigen in the joints and can be targeted by autoantibodies capable of triggering arthritogenic responses in vivo.

Topics: Adult; Animals; Antibody Specificity; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Chondrocytes; Cross Reactions; Cytokines; Female; Humans; Male; Mice; Mice, Inbred BALB C; Middle Aged; Pectins; Rabbits

The calcium pectinate (CaP) capsule, a novel, colon-specific delivery system, was designed and developed using 5-fluorouracil (5-FU) as a model drug. Technically, CaP capsules were prepared by dipping a glass or stainless steel rod successively into pectin and calcium chloride solutions, followed by subsequent air-drying and coating. In vitro studies showed that the release of 5-FU from CaP capsules markedly increased in the presence of rat cecal contents, and the release characteristic was mainly associated with some capsule parameters such as calcium content, shell thickness, and coat amount. Gamma scintigraphic studies demonstrated that CaP capsules could pass through the stomach and small intestine intact and could release drug in colon. The 5-FU releasing characteristics acquired both from in vitro biomimic dissolution experiments and from healthy volunteers indicated that the newly developed CaP capsule possessed the ideal colon-specific drug delivery characteristic.

Topics: Administration, Oral; Adult; Animals; Antineoplastic Agents; Capsules; Cecum; Colon; Colonic Neoplasms; Drug Delivery Systems; Fluorouracil; Humans; Male; Pectins; Radionuclide Imaging; Rats; Solubility

The aim of the present study was to provide "proof of concept" data in man for novel polysaccharide preparations designed for colonic drug delivery using gamma scintigraphy.. Two placebo calcium pectinate matrix tablet formulations were studied: one contained calcium pectinate and pectin (CaP/P) and was designed to rapidly disintegrate in the ascending colon, the other contained calcium pectinate and guar gum (CaP/GG) and was designed to disintegrate more slowly, releasing its contents throughout the ascending and transverse colon. Both formulations were enteric coated in order to protect them from the stomach. Ten healthy volunteers received either a CaP/P or CaP/GG tablet, in a randomised cross-over study. Transit and disintegration of the radiolabelled formulations was followed by gamma scintigraphy. Rat studies were conducted in order to verify that the expected colonic degradation of the polysaccharide formulations was as a consequence of bacterial enzyme attack.. The in vivo clinical study confirmed the results obtained in the rat and bench in vitro fermentation models; complete tablet disintegration for Formulation CaP/GG appeared to be slower than that of Formulation CaP/P and the time and the location of complete tablet disintegration was more reproducible with Formulation CaP/P compared to Formulation CaP/GG.. These results provide "proof of concept" data for the use of calcium pectinate preparations for drug delivery to the colon and highlight the value of scintigraphy in focusing the development strategy for colonic targeting preparations.

Topics: Adult; Animals; Cross-Over Studies; Drug Delivery Systems; Female; Galactans; Gastrointestinal Transit; Humans; Ileocecal Valve; Isotope Labeling; Male; Mannans; Middle Aged; Pectins; Plant Gums; Radionuclide Imaging; Rats; Stomach; Tablets, Enteric-Coated

1. Eight volunteers received in randomized order two 30 microliters drops of either 1% w/v cyclopentolate hydrochloride or a corresponding amount of cyclopentolate polygalacturonate in saline or in acetate buffer in one eye. Cyclopentolate concentrations in plasma were measured by a radioreceptor assay. 2. Peak plasma drug concentrations of about 3 ng ml-1 occurred within 30 min after all formulations. Occasionally, a second concentration peak in plasma, probably reflecting drug absorption from the gastrointestinal tract, was seen after 2 h. The mean elimination half-life of cyclopentolate was 111 min when all subjects and formulations were considered together. There were no statistically significant differences between the formulations with respect to the time-course of plasma drug concentration. 3. The maximal mydriatic effect was reached within about 15 min and was maintained for several hours, often being 1/3 of its peak value after 30 h. Similarly, an intense cycloplegic response was achieved within a few minutes, the peak changes in the near-point of vision being 9 to 10 dioptres. The cycloplegic response was more intense after one of the polygalacturonate complexes, especially at later time points.

Topics: Adult; Buffers; Cyclopentolate; Excipients; Eye; Female; Half-Life; Humans; Hydrogen-Ion Concentration; Male; Ophthalmic Solutions; Pectins; Pupil

Topics: Anti-Inflammatory Agents; Clinical Trials as Topic; Elasticity; Fibrinolytic Agents; Forearm; Humans; Pectins; Phlebitis; Skin

Postharvest textural changes in fruit are mainly divided into softening and lignification. Loquat fruit could have severe lignification with increased firmness during postharvest storage. Pectin is mainly associated with the postharvest softening of fruit, but some studies also found that pectin could be involved in strengthening the mechanical properties of the plant.. This study focused on characterizing the dynamics of pectin and its complexation in the cell wall of lignified loquat fruit during postharvest storage, and how these changes could influence fruit firmness.. The homogalacturonan (HG) pectin in the cell wall of loquat fruit was identified using monoclonal antibodies. An oligogalacturonide (OG) probe was used to label the egg-box structure formed by Ca. The JIM5 antibody revealed that low-methylesterified HG accumulated in the tricellular junctions and middle lamella of loquat fruit that had severe lignification symptoms. The pectin methylesterase (PME) activity increased during the early stages of storage at 0 °C, and the calcium-pectate content and flesh firmness constantly increased during storage. The OG probe demonstrated the accumulation of egg-box structures at the cellular level. The exogenous injection of PME and Ca. PME-mediated demethylesterification generated large amounts of low-methylesterified HG in the cell wall. This low-methylesterified HG further cross-linked with Ca

Topics: Calcium, Dietary; Cell Wall; Eriobotrya; Pectins

Sugar beet pectins (SBPs) are known for their emulsifying properties, but it is yet unknown which structural elements are most important for functionality. Recent results indicated that the arabinose content has a decisive influence, but the approach applied did not allow causality to be established. In this study, a mostly intact SBP was selectively modified and the obtained pectins were analyzed for their molecular structure and their emulsifying properties. De-esterification only resulted in a moderate increase in droplet size. The length of the pectin backbone only influenced the emulsifying properties when the homogalacturonan backbone was cleaved to a higher extent. By using different arabinan-modifying enzymes, it was demonstrated that both higher portions and chain lengths of arabinans positively influence the emulsifying properties of SBPs. Therefore, we were able to refine the structure-function relationships for acid-extracted SBPs, which can be used to optimize extraction conditions.

Topics: Arabinose; Beta vulgaris; Esterification; Pectins

In male Syrian hamsters fed a synthetic high-fat diet enriched with cholesterol (0.3%), administration of a polysaccharide from birch leaves L-rhamnopyranosyl-6-O-methyl-D-galacturonan (3 g/100 g of diet) resulted in a decrease in total cholesterol levels, mainly due to the LDL fraction, triglycerides, and bile acids in blood serum; the content of triglycerides and cholesterol in the liver also decreased, while excretion of bile acids with feces increased. Thus, the lipid-lowering effect of L-rhamnopyranosyl-6-O-methyl-D-galacturonan is related to its ability to bind bile acids in the intestine and interrupt their enterohepatic circulation.

Topics: Animals; Betula; Bile Acids and Salts; Cholesterol; Cricetinae; Diet, High-Fat; Feces; Liver; Male; Mesocricetus; Pectins; Triglycerides

A nanolipidcarrier (NLC) loaded homogalacturonan enriched pectin (citrus modified pectin, MCP4) hydrogel was designed as a novel colon inflammation site-specific oral delivery system for 6-gingerol (6G) (6G-NLC/MCP4 hydrogel) administration, and its colitis alleviation effect were investigated. 6G-NLC/MCP4 exhibited typical "cage-like" ultrastructure with 6G-NLC embedded in the hydrogel matrix as observed by cryoscanning electron microscope. And due to the homogalacturonan (HG) domain in MCP4 specifically combined with Galectin-3, which is overexpressed in the inflammatory region, the 6G-NLC/MCP4 hydrogel targeted to severe inflammatory region. Meanwhile, the prolonged-release characteristics of 6G-NLC provided sustained release of 6G in severe inflammatory regions. The matrix of hydrogel MCP4 and 6G achieved synergistic alleviation effects for colitis through NF-κB/NLRP3 axis. Specifically, 6G mainly regulated the NF-κB inflammatory pathway and inhibited the activity of NLRP3 protein, while MCP4 regulated the expression of Galectin-3 and peripheral clock gene Rev-Erbα/β to prevent the activation of inflammasome NLRP3.

Topics: Colitis; Galectin 3; Humans; Hydrogels; Inflammasomes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pectins

MYB-bHLH-TTG1 (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat biosynthesis pathways via a multi-tiered regulatory mechanism. The MYB genes include MYB5, MYB23 and TRANSPARENT TESTA2 (TT2), which regulate GLABRA2 (GL2), HOMEODOMAIN GLABROUS2 (HDG2) and TRANSPARENT TESTA GLABRA2 (TTG2). Here, we examine the role of PECTIN METHYLESTERASE INHIBITOR14 (PMEI14) in seed coat mucilage pectin methylesterification and provide evidence in support of multi-tiered regulation of seed coat mucilage biosynthesis genes including PMEI14. The PMEI14 promoter was active in the seed coat and developing embryo. A pmei14 mutant exhibited stronger attachment of the outer layer of seed coat mucilage, increased mucilage homogalacturonan demethylesterification and reduced seed coat radial cell wall thickness, results consistent with decreased PMEI activity giving rise to increased PME activity. Reduced mucilage release from the seeds of myb5, myb23, tt2 and gl2, hdg2, ttg2 triple mutants indicated that HDG2 and MYB23 play minor roles in seed coat mucilage deposition. Chromatin immunoprecipitation analysis found that MYB5, TT8 and seven mucilage pathway structural genes are directly regulated by MYB5. Expression levels of GL2, HDG2, TTG2 and nine mucilage biosynthesis genes including PMEI14 in the combinatorial mutant seeds indicated that these genes are positively regulated by at least two of those six TFs and that TTG1 and TTG2 are major regulators of PMEI14 expression. Our results show that MYB-bHLH-TTG1 complexes regulate mucilage biosynthesis genes, including PMEI14, both directly and indirectly via a three-tiered mechanism involving GL2, HDG2 and TTG2.

Topics: Arabidopsis; Arabidopsis Proteins; DNA-Binding Proteins; Gene Expression Regulation, Plant; Mutation; Pectins; Plant Mucilage; Seeds

The ice crystal morphology formed under a series of amidated pectin gels with various crosslink strengths were investigated. The results showed that as the degree of amidation (DA) increased, pectin chains exhibited shorter homogalacturonan (HG) regions. Highly amidated pectin exhibited a faster gelation rate and a stronger gel micro-network via hydrogen bonds. Based on cryogenic scanning electron microscopy (cryo-SEM), smaller ice crystals were formed in frozen gel with low DA, suggesting that a weaker cross-linked gel micro-network was more effective at inhibiting crystallization. After sublimation, lyophilized gel scaffolds with high crosslink strength displayed less number of pores, high porosity, lower specific surface area, and greater mechanical strength. This study is expected to confirm that the microstructure and mechanical properties of freeze-dried pectin porous materials could be regulated by changing the crosslink strength of pectin chains, which is achieved by increasing the degree of amidation in the HG domains.

Topics: Crystallization; Gels; Ice; Pectins

Homogalacturonan (HG), the most abundant pectic glycan, functions as a cell wall structural and signaling molecule essential for plant growth, development and response to pathogens. HG exists as a component of pectic homoglycans, heteroglycans and glycoconjugates. HG is synthesized by members of the GALACTURONOSYLTRANSFERASE (GAUT) family. UDP-GalA-dependent homogalacturonan:galacturonosyltransferase (HG:GalAT) activity has previously been demonstrated for GAUTs 1, 4 and 11, as well as the GAUT1:GAUT7 complex. Here, we show that GAUTs 10, 13 and 14 are also HG:GalATs and that GAUTs 1, 10, 11, 13, 14 and 1:7 synthesize polymeric HG in vitro. Comparison of the in vitro HG:GalAT specific activities of the heterologously-expressed proteins demonstrates GAUTs 10 and 11 with the lowest, GAUT1 and GAUT13 with moderate, and GAUT14 and the GAUT1:GAUT7 complex with the highest HG:GalAT activity. GAUT13 and GAUT14 are also shown to de novo synthesize (initiate) HG synthesis in the absence of exogenous HG acceptors, an activity previously demonstrated for GAUT1:GAUT7. The rate of de novo HG synthesis by GAUT13 and GAUT14 is similar to their acceptor dependent HG synthesis, in contrast to GAUT1:GAUT7 for which de novo synthesis occurred at much lower rates than acceptor-dependent synthesis. The results suggest a unique role for de novo HG synthesis by GAUTs 13 and 14. The reducing end of GAUT13-de novo-synthesized HG has covalently attached UDP, indicating that UDP-GalA serves as both a donor and acceptor substrate during de novo HG synthesis. The functional significance of unique GAUT HG:GalAT catalytic properties in the synthesis of different pectin glycan or glycoconjugate structures is discussed.

Topics: Arabidopsis; Cell Wall; Glucuronosyltransferase; Glycosyltransferases; Oligosaccharides; Pectins

Mutations in both ArabidopsisXyloglucan Xylosyltransferase1 (XXT1) and Xyloglucan Xylosyltransferase2 (XXT2) result in the absence of the major hemicellulose, xyloglucan (XyG). The growth physiology, tensile strength, and cell wall compositions of etiolated hypocotyls of xxt1/xxt2 double and xxt1/xxt2/xxt5 triple mutants were characterized and compared with those of mur2 and mur3 mutations that alter the side group composition of XyG. Wild type and mur2 demonstrated normal elongation growth, whereas the xxt double and triple mutants and mur3 showed reduced elongation and cell swelling at the base of the hypocotyl at the later stages of elongation. Whereas mur3 hypocotyls had reduced tensile strength, the xxt double and triple mutants had similar tensile strengths as wild type, but reached breaking points at lower strains. Loss of XyG in hypocotyls and rosette leaf cell walls was associated with specific increases in the relative abundance of homogalacturonan and the hemicellulose glucomannan, but not arabinoxylan. The increased homogalacturonan and glucomannan abundance did not result from enhanced expression of their polymer synthases. An analysis of published transcriptomic data indicated that genes encoding some members of the expansin, xyloglucan endotransglucosylase/hydrolase, arabinogalactan-protein, and structural protein families were upregulated in the xxt mutants. We hypothesize that an as yet unknown mechanism exists by which pectin and hemicellulose abundance can be altered other than by increased rates of biosynthesis to maintain a structurally sound cell wall.

Topics: Acetyltransferases; Arabidopsis; Arabidopsis Proteins; Cell Wall; Glucans; Mannans; Pectins; Xylans

The pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map-based cloning and confirmed by genetic analysis (co-segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de-esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de-esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de-esterified homogalacturonan in pollen exine formation.

Topics: Fertility; Gene Expression Regulation, Plant; Pectins; Pollen; Polygalacturonase

Antibiotic-associated diarrhea (AAD) is a common side-effect of antibiotic treatment resulting from an imbalance in the colonic bacteria. The hypothesis of this study is to ask whether polysaccharide from the rhizome of Dioscorea opposita which is recorded as conventional herbs and food for diarrhea treatment in Southeast Asia, may be an active compound against diarrhea induced by antibiotics. To address, firstly, a homogenous polysaccharide, DOP0.2-S-3 was characterized as a homogalacturonan containing linear repeating units of → 4)-α-

Topics: Animals; Diarrhea; Dioscorea; Interleukin-1; Interleukin-6; Mice; Pectins

A pectic polysaccharide (WAP) was isolated from squash and identified as a homogalacturonan with a molecular mass of 83.2 kDa by GPC, monosaccharide composition analysis, FT-IR and NMR spectra. Sulfation modification of WAP was carried out and a sulfated derivative (SWAP) was obtained with a substitution degree of 1.81. The NMR spectrum indicated that the sulfation modification mainly occurred at the C-2 and C-3 positions of galacturonan residues. The binding pattern of SWAP to tau K18 protein was observed in 2D

Topics: Heparin; Pectins; Spectroscopy, Fourier Transform Infrared; Sulfates

One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall's mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed "meshing," which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is-at least in part-composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at -45° and +45° relative to the cell's long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.

Topics: Cell Wall; Cellulose; Electron Microscope Tomography; Onions; Pectins

A homogalacturonan (HG) FPLP obtained from Ficus pumila L. was reported to have anti-diabetic activity but how this is influenced by degree of methyl-esterification (DM) of HG is unknown. To comprehensively analyze the role of DM in hypoglycemic activity in insulin-resistant HepG2 cells, HG derivatives (0 < DM < 100) were prepared from FPLP (DM25) by alkali or methanol acidified with acetyl chloride. Interestingly, a quadratic curve relationship revealed that hypoglycemic effect increased and then decreased with DM, and which was the most pronounced with DM54. DM might regulate activity by altering the intracellular drug concentration through cellular uptake. Furthermore, HG-DMn (0 < n < 100) were dependent on macropinocytosis, while HG-DMn (30 < n < 100) were also dependent on caveolae-mediated endocytosis. For HG, higher lipophilicity, smaller particle size, and more endocytosis mechanisms involved were favorable for cellular uptake, thereby increasing the intracellular drug concentration and enhancing the hypoglycemic activity. This work provides ideas for future investigations on structure-activity relationships.

Topics: Esters; Hep G2 Cells; Humans; Hypoglycemic Agents; Pectins

This study aimed to compare the differences between oral administration and intravenous injection of polygalacturonic acid (PGA) in the regulation of immune and intestinal microflora in ulcerative colitis (UC) mice. PGA was administered orally or intravenously. PGA in the high-dose ig group was the most effective in treating UC by increasing colon length and downregulating disease activity index, histopathological score and proinflammatory cytokine levels. In spleen, the efficacy of PGA on restoring Th17/Treg balance in the high-dose iv group was better than that in the high-dose ig group, the opposite was observed in the lamina propria. The level of colonic IL-17A in the high-dose ig group was lower than that in the high-dose iv group, the opposite was observed for that of colonic IL-10. Western blot and immunohistochemistry analysis revealed that PGA in the high-dose ig group decreased the protein expression of RORγt, and increased that of FOXP3. Furthermore, PGA in the high-dose ig group was more effective than that in the high-dose iv group in improving the intestinal microflora structure. Our results suggest that in immune regulation, oral PGA is more effective in the lamina propria and gut microbiota while intravenous PGA is more effective in the spleen.

Topics: Administration, Oral; Animals; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Gastrointestinal Microbiome; Immunomodulation; Injections, Intravenous; Mice; Pectins; T-Lymphocytes, Regulatory

An extracellular matrix (ECM) mimicking a 3D microenvironment is an essential requirement to achieve desirable repair or regeneration of damaged tissue or organ. In this context, hydrogels may be able to create an appropriate 3D microenvironment. The lack of mechanical stability limits their application. This study prepared and characterized thermosensitive injectable hydrogels based on chitosan and polygalacturonic acid (PgA). A method of producing novel biomimetic polymeric-based injectable hydrogel using hydrothermal assisted hydrolysis is introduced. The synthesized hydrogels showed good compressive stiffness. We have also studied the possible chemistry of the materials in the hydrogel network. The biocompatibility and gelation time of the hydrogel was optimized by adding β-glycerophosphate (βGP) and hydroxyapatite. The synthesized liquid formulation can turn into gel at 37 °C. The biocompatibility for MG63 cells within 3D hydrogels was investigated. Scanning electron microscopy revealed that the PEC fibers are uniformly distributed in the hydrogel matrix. MTT assay and confocal imaging were employed to observe cytotoxicity and proliferation of cells cultured in the hydrogels with and without an osteogenic medium. Alkaline phosphatase activity (ALP) and collagen production in cell-cultured hydrogel were also measured to evaluate osteoblast activity. The cellular responses to various types of hydrogels cultured at a 14-day culture appeared to be superior in the hydrogels with gelatin incorporated and hydrothermally treated PEC fibers. These results indicated that hydrothermal treatment and inclusion of gelatin in the chitosan-βGP hydrogel system enhanced the hydrogel bioactivity and mechanical properties. Overall, improved cellular proliferation, osteogenic differentiation, and stable physical network with uniform distribution of fibrous matrix in-vitro were achieved.

Topics: Chitosan; Gelatin; Hydrogels; Osteogenesis; Pectins; Polyelectrolytes; Tissue Engineering

Homogalacturonan (HG)-type pectin has been considered suitable for the formation of hydrogel, but it is unknown whether the less side chain will limit the properties of hydrogel. The current study successfully obtained low methoxyl HG-type hawthorn pectin (LMHP) from hawthorn by sequential hot water extraction and alkaline de-esterification. The proportion of HG domain, the proportion of rhamnogalacturonan-I (RG-I) domain, molecular weight, and particle size of LMHP were 81.13 %, 4.04 %, 348.43 kDa, and 1386.92 nm, respectively. Compared with commercial citrus pectin (CP), LMHP was more suitable for the preparation of hydrogel. The hydrogel with the densest network structure and relatively stable performance in digestion simulation environment can be obtained as the concentration of LMHP increases to 2.5 %, which is lower than the consumption of commercial citrus pectin. Overall, these findings highlight the potential of hawthorn pectin as a novel hydrogel raw material.

Topics: Crataegus; Esters; Hydrogels; Pectins

A number of nickel complexes of sodium pectate with varied Ni

Topics: Nickel; Oxygen; Pectins; Protons

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.

Topics: Aspergillus oryzae; Carboxylic Ester Hydrolases; Genetic Vectors; Hexuronic Acids; Pectins

Pectic acid/sodium pectate is one of the most widespread hydrocolloid used in the food industry. It is able to form strong ionotropic gels by the addition of ions, in particular, calcium ions. The initial steps of binding Ca

Topics: Calcium; Gels; Ions; Pectins

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Topics: Bacillus subtilis; Bacterial Proteins; Cloning, Molecular; Hexuronic Acids; Metabolic Engineering; Pectins; Pectobacterium carotovorum; Polygalacturonase; Potassium Chloride; Promoter Regions, Genetic

Though numerous studies have focused on the cell wall disassembly of bananas during the ripening process, the modification of homogalacturonan (HG) during fruit development remains exclusive. To better understand the role of HGs in controlling banana fruit growth and ripening, RNA-Seq, qPCR, immunofluorescence labeling, and biochemical methods were employed to reveal their dynamic changes in banana peels during these processes. Most HG-modifying genes in banana peels showed a decline in expression during fruit development. Four polygalacturonase and three pectin acetylesterases showing higher expression levels at later developmental stages than earlier ones might be related to fruit expansion. Six out of the 10 top genes in the Core Enrichment Gene Set were HG degradation genes, and all were upregulated after softening, paralleled to the significant increase in HG degradation enzyme activities, decline in peel firmness, and the epitope levels of 2F4, CCRC-M38, JIM7, and LM18 antibodies. Most differentially expressed alpha-1,4-galacturonosyltransferases were upregulated by ethylene treatment, suggesting active HG biosynthesis during the fruit softening process. The epitope level of the CCRC-M38 antibody was positively correlated to the firmness of banana peel during fruit development and ripening. These results have provided new insights into the role of cell wall HGs in fruit development and ripening.

Topics: Antibodies; Epitopes; Fruit; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Musa; Pectins; Time Factors

Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes' thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.

Topics: Biofuels; Carboxymethylcellulose Sodium; Cellulases; Enzyme Stability; Escherichia coli; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Pectins; Polygalacturonase; Protein Denaturation; Protein Folding; Temperature; Thermodynamics

Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction.. The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris.. Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG.. The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The K. The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

Topics: Cations, Divalent; Cloning, Molecular; Copper; Enzyme Stability; Food Technology; Fruit and Vegetable Juices; Fungal Proteins; Gene Expression; Genetic Vectors; Humans; Hydrogen-Ion Concentration; Iron; Kinetics; Malus; Molecular Weight; Pectins; Pichia; Polygalacturonase; Recombinant Proteins; Silver; Sporothrix; Temperature

Tissue bending is vital to plant development, as exemplified by apical hook formation during seedling emergence by bending of the hypocotyl. How tissue bending is coordinated during development remains poorly understood, especially in plants where cells are attached via rigid cell walls. Asymmetric distribution of the plant hormone auxin underlies differential cell elongation during apical hook formation. Yet the underlying mechanism remains unclear. Here, we demonstrate spatial correlation between asymmetric auxin distribution, methylesterified homogalacturonan (HG) pectin, and mechanical properties of the epidermal layer of the hypocotyl in Arabidopsis. Genetic and cell biological approaches show that this mechanochemical asymmetry is essential for differential cell elongation. We show that asymmetric auxin distribution underlies differential HG methylesterification, and conversely changes in HG methylesterification impact the auxin response domain. Our results suggest that a positive feedback loop between auxin distribution and HG methylesterification underpins asymmetric cell wall mechanochemical properties to promote tissue bending and seedling emergence.

Topics: Arabidopsis; Esterification; Feedback, Physiological; Hypocotyl; Indoleacetic Acids; Methylation; Pectins; Seedlings

This study presents a novel model of homogalacturonan (HG) based on the dissipative particle dynamics (DPD). The model was applied to investigate the mechanism of self-aggregation of low-methoxylated homogalacturonan in aqueous solutions in the absence of cations. The coarse-grained model provided new insights into the structural features of HG aggregates and networks in aqueous solutions. Depending on the properties and concentration of polysaccharides, two major patterns of self-assembly were observed for HG - ellipsoidal aggregates and a continuous three-dimensional network. Simulations showed that a decrease in the degree of dissociation of HG results in a higher rate of self-aggregation, as well as facilitating the formation of larger assemblies or thicker nanofilaments depending on the type of final self-assembly. Simulations of polysaccharides of different chain lengths suggested the existence of a structural threshold for the formation of a spatial network for HG consisting of less than 35 GalA units.

Topics: Calibration; Carboxylic Acids; Cations; Hexuronic Acids; Hydrogen Bonding; Hydrogen-Ion Concentration; Molecular Structure; Particle Size; Pectins; Polysaccharides; Water

Changes in the cell wall of Japanese radish due to heating at 100 °C or 117 °C for 3 h were examined. Signals in

Topics: Cell Wall; Cooking; Hot Temperature; Magnetic Resonance Spectroscopy; Pectins; Pressure; Raphanus; Rhamnogalacturonans; Water

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

Topics: Adjuvants, Immunologic; Animals; Immunization; Mice; Ovalbumin; Pectins; Portulaca

GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae  with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

Topics: Acetates; Arabidopsis; Ascomycota; Cyclopentanes; Disease Resistance; Gene Expression Regulation, Plant; Gossypium; Lignin; Oxylipins; Pectins; Phylogeny; Plant Diseases; Plant Immunity; Plant Proteins; Plants, Genetically Modified; Salicylic Acid; Transcription Factors

Antiseptic wound ointments are widely used to treat dermal wounds that are microbially contaminated. Polygalacturonic acid (PG)+caprylic acid (CAP) is a novel combination that has been shown to eradicate biofilms. We developed a novel PG+CAP ointment and compared the biofilm eradication capability and cytotoxicity of PG+CAP with that of commercially available antiseptic wound ointments. We used a well-established biofilm model to quantitatively assess the eradication of organisms following exposure to the wound ointments for 2 hours. PG+CAP ointment completely eradicated

Topics: Animals; Anti-Infective Agents, Local; Biofilms; Candida albicans; Caprylates; Cell Line; Methicillin-Resistant Staphylococcus aureus; Mice; Ointments; Pectins; Pseudomonas aeruginosa

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

Topics: Adhesiveness; Arabidopsis; Arabidopsis Proteins; Calcium; Carboxylic Ester Hydrolases; Cross-Linking Reagents; Esterification; Genes, Plant; Membrane Proteins; Mutation; Nucleotide Motifs; Pectins; Phenotype; Plant Epidermis; Plant Mucilage; Protein Binding; Repressor Proteins; Seeds; Transcription Factors, General

Plant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus.. Rice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes.. Four R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

Topics: China; India; Oryza; Pectins; Plant Diseases; Rhizoctonia

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 contribution of ATR-FTIR spectroscopy to study cell wall polysaccharides (CWPs) was carefully investigated. The region 1800-800 cm

Topics: Cell Wall; Cellulose; Galactans; Monosaccharides; Pectins; Plants; Polysaccharides; Principal Component Analysis; Spectroscopy, Fourier Transform Infrared

Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes.. Here, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size.. These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

Topics: Arabidopsis; Arabidopsis Proteins; Calcium; Cell Wall; Cellulose; Esterification; Galactans; Glucuronosyltransferase; Monosaccharides; Pectins; Phenotype; Polysaccharides; Seeds

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

The intestinal fermentability of pectic polysaccharides is largely determined by its molecular size. In this study, fermentation properties of enzymatic-modified apple pectin (AP) and homogalacturonans (HG) with high, medium and low molecular weight (Mw) were evaluated by in vitro fermentation model, and their structural changes were also investigated. Results showed that Mw, monosaccharide contents and molecular linearity of the AP hydrolysates were reduced after microbial degradation. On the other hand, culture media supplemented with low-Mw AP (60,300 g/mol) and low-Mw HG (861 g/mol) exhibited lower pH (5.1 and 5.7, respectively) and produced higher total short-chain fatty acid contents (SCFA, 230.40 mmol/L and 187.19 mmol/L, respectively). However, reduced trends in abundance of the pectinolytic microorganisms Faecalibacterium and Eubacterium were showed as Mw of the HG decreased, whereas growth of the SCFA-producer genera Bifidobaacterium, Megasphaera and Allisonella were improved. This work confirmed that low-Mw pectin and homogalacturonan generated more beneficial metabolites, developing structure-microbiota-gut health relationship.

Topics: Adult; Bacteria; Fatty Acids, Volatile; Feces; Female; Fermentation; Gastrointestinal Microbiome; Humans; Male; Malus; Molecular Weight; Pectins

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 oomycete

Topics: Copper; Mixed Function Oxygenases; Models, Molecular; Oxidation-Reduction; Pectins; Phytophthora infestans; Plant Diseases; Plant Leaves; Polysaccharides; Protein Conformation; Protein Domains; Solanum lycopersicum; Solanum tuberosum; Virulence Factors

Biological activity of incomplete degradation products of polygalacturonic acid (IDPP) is closely related to its molecular weight and molecular weight distribution. Therefore, it is necessary to provide a reliable quantitative characterization method for evaluating these types of bioproducts. A novel method was established in this work for the quantitative characterization of IDPP based upon ethanol fractional precipitation. IDPP was fractionated into several fractions with high recovery (>95%), and the average molecular weights of each fraction was in descending order with the increase of ethanol concentration. Oligosaccharides (polymerization degree: 2-20) could be effectively harvested from the polygalacturonic acid enzymatic hydrolysate by ethanol precipitation. Moreover, the developed method had good repeatability and could also be applied to quantify enzymatic hydrolysis products of citrus-derived pectin polysaccharides. In conclusion, this paper provides a simple, accurate method for the quantitative characterization of IDPP and a strategy for the extraction of oligosaccharides.

Topics: Chromatography, High Pressure Liquid; Ethanol; Hydrolysis; Molecular Weight; Oligosaccharides; Pectins; Polysaccharides

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function.

Topics: Cell Wall; Epitopes; Pectins; Plant Stomata; Vigna; Zea mays

Microbial valorization of plant biomass is a key target in bioeconomy. A promising candidate for consolidated bioprocessing is the dimorphic fungus Ustilago maydis. It harbors hydrolytic enzymes to degrade biomass components and naturally produces valuable secondary metabolites like itaconic acid, malic acid or glycolipids. However, hydrolytic enzymes are mainly expressed in the hyphal form. This type of morphology should be prevented in industrial fermentation processes. Genetic activation of these enzymes can enable growth on cognate substrates also in the yeast form. Here, strains were engineered for growth on polygalacturonic acid as major component of pectin. Besides activation of intrinsic enzymes, supplementation with heterologous genes for potent enzymes was tested. The presence of an unconventional secretion pathway allowed exploiting fungal and bacterial enzymes. Growth of the engineered strains was evaluated by a recently developed method for online determination of residual substrates based on the respiration activity. This enabled the quantification of the overall consumed substrate as a key asset for the assessment of the enzyme degradation potential even on polymeric substrates. Co-fermentation of endo- and exo-polygalacturonase overexpression strains resulted in efficient growth on polygalacturonic acid. In the future, the approach will be extended to establish efficient degradation and valorization of pectin.

Topics: Amino Acid Sequence; Biomass; Computational Biology; Fermentation; Hyphae; Organ Specificity; Pectins; Plants; Sequence Alignment; Ustilago

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

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as 'Picual' undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in 'Picual' olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

Topics: Arabinose; Cell Wall; Esterification; Fruit; Galactans; Galactose; Mucoproteins; Olea; Pectins; Plant Proteins; Polysaccharides

The process by which plant cells expand and gain shape has presented a challenge for researchers. Current models propose that these processes are driven by turgor pressure acting on the cell wall. Using nanoimaging, we show that the cell wall contains pectin nanofilaments that possess an intrinsic expansion capacity. Additionally, we use growth models containing such structures to show that a complex plant cell shape can derive from chemically induced local and polarized expansion of the pectin nanofilaments without turgor-driven growth. Thus, the plant cell wall, outside of the cell itself, is an active participant in shaping plant cells. Extracellular matrix function may similarly guide cell shape in other kingdoms, including Animalia.

Topics: Arabidopsis; Cell Shape; Cell Wall; Cotyledon; Methylation; Molecular Imaging; Pectins; Plant Cells; Plant Development; Plant Epidermis

The polysaccharide pectin is a major component of the plant cell wall. The pectic glycan homogalacturonan (HG) is a proportionally small but important component of a specialized seed cell wall called mucilage. HG is synthesized in a highly methylesterified form, and, following secretion, is de-methylesterified by pectin methylesterases (PMEs). The degree of methylesterification of HG determines the structural and functional properties of pectin, but how methylesterification is regulated remains largely unknown. Here, we identified two BEL1-Like homeodomain (BLH) transcription factors, BLH2 and BLH4, as positive regulators of HG de-methylesterification in Arabidopsis (

Topics: Arabidopsis Proteins; Cell Wall; Gene Expression Regulation, Plant; Homeodomain Proteins; Pectins; Protein Binding; Seeds; Transcription Factors

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

Mealiness is one of the most important textural failure of apple fruit and four patterns of mealiness involving five apple cultivars were identified as the rapid, moderate, slow and none, requiring 3, 7, 14, 49 days at 25 °C, respectively. In comparison with the non-mealy 'Fuji' apple, parenchyma cells of mealy apples became detached and remained intact. Highly methyl-esterified homogalacturonan was strongly immunolabeled in the cell wall of slow and non-mealy apples. The mobility of water was enhanced in the cell wall during mealiness. Principal components analysis of FTIR spectra discriminated the cell wall materials (CWM) based on the mealiness progress. Heavy loss of CWM and its water-insoluble fractions but limited increase of water-soluble fractions, and the increase of crystalline micelles of CWM were closely associated with the mealiness progress. Overall, the occurrence of mealiness might attribute to structural, physical and biochemical modifications of CWM during tissue senescence.

Topics: Cell Wall; Fruit; Malus; Meals; Pectins

The current study is pertaining to develop a novel wound dressing, comprising natural biologically absorbable materials for wound healing In-vivo. Wound dressing is composed of Polygalacturonic acid, Hyaluronic acid embedded silver nanoparticles, which is further fabricated to form nanofibrous mat, using electrospinning. Silver nanoparticles was prepared using PGA. AgNPs in this formula will serve as an antioxidant and anti-inflammatory that protect cells from destructive effect of elevated ROS and accelerate wound healing. The physical performance and water contact angle for nanofiber was evaluated. The produced nanofiber was characterized by Fourier-transform infrared (FTIR), scanning electron microscopy and thermal analysis. Also, the embedded AgNPs was also characterized by UV-vis spectroscopy and TEM. The nanofiber mates embedded AgNPs was applied to the wounded site of albino rats in-vivo. Histopathological assessment for the wound was fully performed. Also, the antimicrobial activity for the fabricated wound dressing was evaluated against gram+ve and gram -ve bacteria.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Bandages; Hyaluronic Acid; Male; Metal Nanoparticles; Nanofibers; Pectins; Rats; Silver; Wound Healing

Topics: Bacterial Proteins; Carboxylic Ester Hydrolases; Paenibacillus; Pectins; Substrate Specificity

Exotic fruits and their co-products may be valuable sources of antioxidant dietary fibres (DF) which are useful for food industry and human health. In this study, we aimed to characterize DF obtained from guavira fruit pomace and investigate its antioxidant potential employing TEAC assay as well as a cell model. The DF were chemically characterized as containing arabinan, highly-methoxylated homogalacturonan and arabinogalactan. The DF-containing fraction (CPW) presented ABTS free radical scavenger activity. MTT and DCFH-DA assay were performed to assess, respectively, changes in cell viability and the potential intracellular antioxidant activity against H

Topics: Animals; Antioxidants; Cell Survival; Dietary Fiber; Fibroblasts; Fruit; Galactans; Hydrogen Peroxide; Mice; NIH 3T3 Cells; Oxidative Stress; Pectins; Polysaccharides

How the plant cell wall expands and forms shapes is a long-standing mystery. Traditional thought is that turgor pressure drives these processes. However, a recent study by Haas and colleagues shows for the first time that the expansion of pectin homogalacturonan nanofilaments drives morphogenesis without turgor pressure in plant epidermal cells.

Topics: Cell Wall; Epidermal Cells; Morphogenesis; Pectins; Plant Cells

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

Topics: Carboxylic Ester Hydrolases; Cell Wall; Gene Expression Regulation, Plant; Germination; Nicotiana; Oryza; Pectins; Plant Leaves; Plant Proteins; Pollen Tube; Polyphenols; Transcriptome

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

Passion fruit peel (PFP) is a by-product from the fruit processing industry, accounting for approximately 50 % of the fruit weight. It is well known for its health properties, although few studies evaluated its rheological properties. PFP polysaccharides (PFPP) contain a high methoxyl pectin (HMP), specifically a 70 % methyl-esterified homogalacturonan. Flow behaviour analysis of PFPP (with or without sucrose) revealed a shear-thinning non-Newtonian behaviour. Dynamic oscillatory tests showed a weak gel-like behaviour, even without sucrose addition. Moreover, under simulated pasteurization process PFPP maintained its gel structure. Taken together we demonstrated that PFPP has divergent behaviour from commercial HMP, since it does not require sucrose or low pH to form gel. The present work reinforces the use of PFP as a source of soluble dietary fibres and pectins, providing its alternative application as a rheological modifier in a wide range of products, including those with low sugar.

Topics: Dietary Fiber; Elastic Modulus; Fruit; Gels; Humans; Hydrogen-Ion Concentration; Passiflora; Pasteurization; Pectins; Phase Transition; Polysaccharides; Shear Strength; Waste Products

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (

Topics: Arabidopsis; Arabidopsis Proteins; Cell Adhesion; Cell Wall; Cellulose; Dinitrobenzenes; Gene Expression Regulation, Plant; Hypocotyl; Methyltransferases; Microtubules; Mutation; Pectins; Plant Cells; Plants, Genetically Modified; Sulfanilamides; Uronic Acids

In this study, the beneficial effects of a homogalacturonan(HG)-type pectic polysaccharide from Ficus pumila L. fruits (FPLP) in obese mice were investigated. The 17-week FPLP treatment effectively attenuated obesity, as mainly demonstrated by the reductions of body weight, serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in high-fat diet (HFD)-induced obese mice. The decreased Firmicutes to Bacteroidetes abundance ratio, enriched Akkermansia, and reduced Blautia abundance suggested that FPLP ameliorated the HFD-induced gut dysbiosis. FPLP also influenced the levels of metabolites altered upon HFD feeding, including increases in myristoleic acid and pentadecanoic acid levels. The correlation studies indicated that FPLP ameliorated HFD-induced rise in TC and LDL-C levels through regulating gut microbial community and their associated metabolites. In conclusion, this study extends our understanding of the relationships among gut microbiota (Akkermansia and Blautia), metabolites (myristoleic acid and pentadecanoic acid), HG-type pectin and its TC- and LDL-C- lowering functions.

Topics: Akkermansia; Animals; Bacteroidetes; Body Weight; Diet, High-Fat; Dysbiosis; Ficus; Firmicutes; Fruit; Gastrointestinal Microbiome; Gastrointestinal Tract; Male; Mice, Inbred C57BL; Obesity; Pectins; Polysaccharides; Population Dynamics

Drought causes the excessive abscission of flowers in yellow lupine, leading to yield loss and serious economic consequences in agriculture. The structure that determines the time of flower shedding is the abscission zone (AZ). Its functioning depends on the undisturbed auxin movement from the flower to the stem. However, little is known about the mechanism guiding cell-cell adhesion directly in an AZ under water deficit. Therefore, here, we seek a fuller understanding of drought-dependent reactions and check the hypothesis that water limitation in soil disturbs the natural auxin balance within the AZ and, in this way, modifies the cell wall structure, leading to flower separation. Our strategy combined microscopic, biochemical, and chromatography approaches. We show that drought affects indole-3-acetic acid (IAA) distribution and evokes cellular changes, indicating AZ activation and flower abortion. Drought action was manifested by the accumulation of proline in the AZ. Moreover, cell wall-related modifications in response to drought are associated with reorganization of methylated homogalacturonans (HG) in the AZ, and upregulation of pectin methylesterase (PME) and polygalacturonase (PG)-enzymes responsible for pectin remodeling. Another symptom of stress action is the accumulation of hemicelluloses. Our data provide new insights into cell wall remodeling events during drought-induced flower abscission, which is relevant to control plant production.

Topics: Carboxylic Ester Hydrolases; Cell Wall; Chromatography, Gas; Droughts; Flowers; Gene Expression Regulation, Plant; Indoleacetic Acids; Lupinus; Mass Spectrometry; Pectins; Plant Proteins; Polygalacturonase; Polysaccharides; Proline; Water

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.

Topics: Arabidopsis; Arabidopsis Proteins; Botrytis; Carboxylic Ester Hydrolases; Cyclopentanes; Defensins; Escherichia coli; Ethylenes; Gene Expression; Isoenzymes; Oxylipins; Pectins; Plant Diseases; Promoter Regions, Genetic; Recombinant Proteins; Saccharomycetales

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [

Topics: Acetyl Coenzyme A; Acetylation; Acetyltransferases; Arabidopsis; Arabidopsis Proteins; Ascomycota; Botrytis; Cell Wall; Cellulose; Disease Resistance; Mutation; Pectins; Phylogeny; Plant Diseases; Plant Leaves; Plants, Genetically Modified

The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M.. The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent K. The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C.. The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.

Topics: Biotechnology; Enzyme Stability; Fruit and Vegetable Juices; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; Pectins; Penicillium; Polygalacturonase; Temperature

How cell wall elasticity, plasticity, and time-dependent extension (creep) relate to one another, to plant cell wall structure and to cell growth remain unsettled topics. To examine these issues without the complexities of living tissues, we treated cell-free strips of onion epidermal walls with various enzymes and other agents to assess which polysaccharides bear mechanical forces in-plane and out-of-plane of the cell wall. This information is critical for integrating concepts of wall structure, wall material properties, tissue mechanics and mechanisms of cell growth. With atomic force microscopy we also monitored real-time changes in the wall surface during treatments. Driselase, a potent cocktail of wall-degrading enzymes, removed cellulose microfibrils in superficial lamellae sequentially, layer-by-layer, and softened the wall (reduced its mechanical stiffness), yet did not induce wall loosening (creep). In contrast Cel12A, a bifunctional xyloglucanase/cellulase, induced creep with only subtle changes in wall appearance. Both Driselase and Cel12A increased the tensile compliance, but differently for elastic and plastic components. Homogalacturonan solubilization by pectate lyase and calcium chelation greatly increased the indentation compliance without changing tensile compliances. Acidic buffer induced rapid cell wall creep via endogenous α-expansins, with negligible effects on wall compliances. We conclude that these various wall properties are not tightly coupled and therefore reflect distinctive aspects of wall structure. Cross-lamellate networks of cellulose microfibrils influenced creep and tensile stiffness whereas homogalacturonan influenced indentation mechanics. This information is crucial for constructing realistic molecular models that define how wall mechanics and growth depend on primary cell wall structure.

Topics: Cell Wall; Cellulase; Cellulose; Elasticity; Fungal Proteins; Glycoside Hydrolases; Microfibrils; Microscopy, Atomic Force; Onions; Pectins; Plant Cells; Polysaccharide-Lyases; Polysaccharides; Tensile Strength

Topics: Cell Wall; Embryophyta; Esterification; Ferns; Pectins; Vigna; Zea mays

Expansins are cell wall proteins associated with several processes, including changes in the cell wall during ripening of fruit, which matches softening of the fruit. We have previously reported an increase in expression of specific expansins transcripts during softening of

Topics: Cell Wall; Cellulose; Fragaria; Fruit; Gene Expression Regulation, Plant; Glucans; Ligands; Molecular Dynamics Simulation; Pectins; Plant Proteins; Protein Conformation; Xylans

During the storage of apples, apple softening is one of the main problems. Sodium silicate has been used to enhance disease resistance and maintain quality of fruits. In the present study, apple fruit (cv. Golden delicious) were treated with 100 mmol L. The results indicated that 100 mmol L. Delaying apple softening by sodium silicate treatment is closely related to the inhibition of the activity of cell wall-degrading enzymes and weight loss. © 2018 Society of Chemical Industry.

Topics: Cell Wall; Cellulase; Food Preservation; Food Preservatives; Fruit; Malus; Methyltransferases; Pectins; Plant Proteins; Quality Control; Silicates

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

Brown-rot fungi are the wood-decay basidiomycetes and have ability to break down plant cell wall carbohydrates. It has been suggested that degradation of pectin is important for the initial stages of brown rot. We purified an endo-polygalacturonase (FpPG28A) from the brown-rot fungus Fomitopsis palustris, analysis of the predicted amino acid sequence indicated that FpPG28A belongs to GH family 28. The highest activity of purified FpPG28A was observed at 60 °C in 50 mM sodium acetate buffer (pH 5.0); this activity was highly specific for polygalacturonic acid chains. However, calcium polygalacturonate gel was not degraded by FpPG28A under those optimal conditions. We observed that calcium polygalacturonate gel was readily degraded by the enzyme in the oxalate buffer. Furthermore, the thermostability of FpPG28A was elevated in oxalate buffer at pH 3.0. These results indicated that oxalate has an important role in the degradation of woody pectin by FpPG28A.

Topics: Amino Acid Sequence; Cloning, Molecular; Coriolaceae; Fungal Proteins; Oxalates; Pectins; Polygalacturonase; Wood

Tomato (

Topics: Cell Wall; CRISPR-Cas Systems; Enzymes; Esterification; Fruit; Galactans; Gene Expression Regulation, Plant; Gene Silencing; Mutation; Pectins; Plant Proteins; Plants, Genetically Modified; Solanum lycopersicum

In the present study, oxido-reductive degradation of diazo dye, Direct Red 23, has been carried out by Ziziphus mauritiana peroxidases (specific activity 17.6 U mg

Topics: Adsorption; Anilides; Azo Compounds; Gas Chromatography-Mass Spectrometry; Longitudinal Studies; Oxidation-Reduction; Pectins; Peroxidase; Temperature; Water Pollutants, Chemical; Water Purification; Ziziphus

A possible transcription factor TLP2 was identified to be involved in the regulation of HG biosynthesis in Arabidopsis seed mucilage. TLP2 can translocate into nucleus from plasma membrane by interacting with NF-YC3. The discovery of TLP2 gene function can further fulfill the regulatory network of pectin biosynthesis in Arabidopsis thaliana. Arabidopsis seed coat mucilage is an excellent model system to study the biosynthesis, function and regulation of pectin. Rhamnogalacturonan I (RG-I) and homogalacturonan (HG) are the major polysaccharides constituent of the Arabidopsis seed coat mucilage. Here, we identified a Tubby-like gene, Tubby-like protein 2 (TLP2), which was up-regulated in developing siliques when mucilage began to be produced. Ruthenium red (RR) staining of the seeds showed defective mucilage of tlp2-1 mutant after vigorous shaking compared to wild type (WT). Monosaccharide composition analysis revealed that the amount of total sugars and galacturonic acid (GalA) decreased significantly in the adherent mucilage (AM) of tlp2-1 mutant. Immunolabelling and dot immunoblotting analysis showed that unesterified HG decreased in the tlp2-1 mutant. Furthermore, TLP2 can translocate into nucleus by interacting with Nuclear Factor Y subunit C3 (NF-YC3) to function as a transcription factor. RNA-sequence and transactivation assays revealed that TLP2 could activate UDP-glucose 4-epimerase 1 (UGE1). In all, it is concluded that TLP2 could regulate the biosynthesis of HG possibly through the positive activation of UGE1.

Topics: Arabidopsis; Arabidopsis Proteins; F-Box Proteins; Gene Expression Regulation, Plant; Hexuronic Acids; Mutation; Pectins; Phenotype; Plant Mucilage; Plants, Genetically Modified; Polysaccharides; Seeds; Sequence Analysis, RNA; Transcription Factors; Transcriptional Activation; Uridine Diphosphate Glucose Dehydrogenase

Collenchyma cells occur widely in eudicotyledons and provide mechanical support for growing organs. At maturity, the cells are elongated and have thick, non-lignified walls, which in celery contain cellulose and pectic polysaccharides, together with xyloglucans and heteroxylans and heteromannans. A previous study suggested that at least some of the collenchyma cell wall in celery is laid down after expansion has stopped and is thus secondary. In the present study, we re-examined this. We used chemical analysis and immunomicroscopy to determine changes in the polysaccharide compositions of these walls during development. Additionally, solid-state NMR spectroscopy was used to examine changes in polysaccharide mobilities during development.. We showed the collenchyma walls are deposited only during cell expansion, i.e. they are primary walls. During cell-wall development, analytical and immunomicroscopy studies showed that within the pectic polysaccharides there were no overall changes in the proportions of homogalacturonans, but there was a decrease in their methyl esterification. There was also a decrease in the proportions of the (1 → 5)-α-L-arabinan and (1 → 4)-β-D-galactan side chains of rhamnogalacturonan I. The proportions of cellulose increased, and to a lesser extent those of xyloglucans and heteroxylans. Immunomicroscopy showed the homogalacturonans occurred throughout the walls and were most abundant in the middle lamellae and middle lamella junctions. Although the (1 → 4)-β-D-galactans occurred only in the rest of the walls, some of the (1 → 5)-α-L-arabinans also occurred in the middle lamellae and middle lamella junctions. During development, the location of the xyloglucans changed, being confined to the middle lamellae and middle lamella junctions early on, but later occurred throughout the walls. The location of the heteroxylans also changed, occurring mostly in the outer walls in young cells, but were more widely distributed in mature cells. Solid-state NMR spectroscopy showed that particularly cellulose, but also homogalacturonans, decreased in mobility during development.. Our studies showed that celery collenchyma cell walls are primary and that during their development the polysaccharides undergo dynamic changes. Changes in the mobilities of cellulose and homogalacturonans were consistent with the cell walls becoming stiffer as expansion ceases.

Topics: Apium; Cell Wall; Cellulose; Magnetic Resonance Spectroscopy; Microscopy, Fluorescence; Pectins; Plant Leaves; Polysaccharides

Stomatal pores are vital for the diffusion of gasses into and out of land plants and are, therefore, gatekeepers for photosynthesis and transpiration. Although much published literature has described the intercellular signaling and transcriptional regulators involved in early stomatal development, little is known about the cellular details of the local separation between sister guard cells that give rise to the stomatal pore or how formation of this pore is achieved. Using three-dimensional (3D) time-lapse imaging, we found that stomatal pore formation in Arabidopsis (

Topics: Arabidopsis; Models, Biological; Osmotic Pressure; Pectins; Plant Stomata; Time-Lapse Imaging

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

Grafting is a technique widely used in horticulture. The processes involved in grafting are diverse, and the technique is commonly employed in studies focusing on the mechanisms that regulate cell differentiation or response of plants to abiotic stress. Information on the changes in the composition of the cell wall that occur during the grafting process is scarce. Therefore, this study was carried out for analyzing the composition of the cell wall using Arabidopsis hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological and cellular changes that accompany autografting of Arabidopsis hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union.. During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either "closed" or "open". Only three cell wall epitopes, namely: LM19 (un/low-methyl-esterified homogalacturonan), JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration.. To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during Arabidopsis grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectin-extensin interaction but also due to its origin. The findings presented here implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material.

Topics: Arabidopsis; Cell Wall; Epitopes; Esterification; Glycoproteins; Hypocotyl; Pectins; Plant Proteins

To investigate the relationship between anti-α-1,4-D-polygalacturonic acid (PGA) antibodies, particularly immunoglobulin (Ig)A, and Henoch-Schönlein purpura (HSP) in children.. This observational case-control study investigated PGA-IgA, PGA-IgG, and PGA/PGA-IgA circulating immune complex (PGA/PGA-IgA CIC) in paediatric patients with HSP versus controls. Children with HSP were also evaluated for food specific IgG and food intolerance. Between-group differences in anti-PGA antibodies were analysed.. Serum PGA-IgA and PGA-IgG levels were significantly increased in patients with acute HSP ( n = 251) versus those with urticaria ( n = 48), acute respiratory infections ( n = 95), surgical controls ( n = 53) and neonates ( n = 92). PGA/PGA-IgA CIC levels were also significantly higher in the acute HSP group versus surgical control and neonate groups. Levels of PGA/PGA-IgA CIC and PGA-IgA were significantly correlated ( r = 0.997), and PGA-IgA showed high diagnostic specificity for HSP. No statistically significant differences were observed in PGA-IgA and PGA-IgG between various degrees of food intolerance in children with HSP.. Increased anti-PGA antibodies, particularly PGA-IgA and PGA/PGA-IgA CIC, were significantly associated with acute HSP in children. Food intolerance was not found to be associated with increased anti-PGA antibodies in children with HSP.

Topics: Adolescent; Autoantibodies; Case-Control Studies; Child; Child, Preschool; Female; Humans; IgA Vasculitis; Immunoglobulin A; Immunoglobulin G; Infant; Male; Pectins; Prognosis

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

Because they suck phloem sap and act as vectors for phytopathogenic viruses, aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has been described as a defensive element for plants during infections with phytopathogens. However, its role during aphid infestation remains unexplored. Using immunofluorescence assays and biochemical approaches, the HG methylesterification status and associated modifying enzymes during the early stage of Arabidopsis (

Topics: Animals; Aphids; Arabidopsis; Arabidopsis Proteins; Gene Expression Regulation, Plant; Pectins

The endo-polygalacturonase enzyme (endoPG: EC 3.2.1.15) plays an important role in the fruit juice and wine industries, so the development of new tools for the quantitative and qualitative analysis of its enzymatic action is necessary. In this work, we report the development of a simple, fast and practical method that did not use any chemical reagent to identify and evaluate the action of the endoPG enzyme, produced by the yeast Kluyveromyces marxianus CCT3172, using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy combined with principal component analysis-linear discriminant analysis (PCA-LDA). This method evaluated the action of the endoPG enzyme on the polygalacturonic acid (PGA) substrate at 5 different times (0, 10, 15, 20 and 30 minutes), and at each time interval the samples were analyzed by ATR-FTIR. It was demonstrated that there was clear segregation between the samples that were and that were not subjected to the action of the endoPG enzyme, and it was also possible to distinguish the samples that were subjected to different incubation times with the enzyme. Through PCA-LDA it was possible to obtain wavelengths that are biomarkers for this enzymatic reaction and the observed changes as a function of hydrolysis duration were found to be in agreement with the breakdown of the glycosidic chain (1011 cm

Topics: Catalysis; Colorimetry; Discriminant Analysis; Hydrolysis; Kinetics; Kluyveromyces; Pectins; Polygalacturonase; Principal Component Analysis; Spectroscopy, Fourier Transform Infrared

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

Topics: Catalytic Domain; Crystallography, X-Ray; Glycoside Hydrolases; Models, Molecular; Pectins; Phylogeny; Polysaccharide-Lyases; Protein Conformation; Tryptophan; Yersinia enterocolitica

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

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (

Topics: Arabidopsis; Biomechanical Phenomena; Cell Wall; Cellulose; Cotyledon; Esterification; Pectins; Plant Leaves

Passion fruit rind (PFR) represents 90% of the total fruit weight and is wasted during juice processing. Passion fruit rind is known to contain flavonoids and pectin. An alternative use for this fruit juice industrial residue is to obtain these compounds. This study aimed to verify the influence of pressurized solvent extraction (PSE) or ultrasound assisted extraction (UAE) of flavonoid and pectin in a sequential process.. The PSE using ethanol at 60:40 (v/v) yielded a total polyphenol content of 4.67 g GAE kg. With this study it can be concluded that mixtures of alcohols with water favor the extraction of bioactive compounds of passion fruit peel. Both PSE and UAE were effective in sequentially extracting flavonoids and pectin. The preferred solvent is ethanol due to its lower toxicity. © 2017 Society of Chemical Industry.

Topics: Ethanol; Flavonoids; Fruit; Glucosides; Hexuronic Acids; Luteolin; Passiflora; Pectins; Polyphenols; Pressure; Solvents; Ultrasonics

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 review of literature and patents shows that enhancing the polygalacturonase (PG) production and activity are still required to fulfill the increasing demands.. A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular PG enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized.. Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 μmol/min, respectively.. This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications.

Topics: Aspergillus flavus; Food Industry; Hydrogen-Ion Concentration; Kinetics; Metals; Patents as Topic; Pectins; Polygalacturonase

Our previous study isolated a natural high-methoxyl homogalacturonan (HRWP-A) from Hippophae rhamnoides and showed antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of HRWP-A were further investigated. Results showed that HRWP-A could recover the body condition and activated macrophage in Cyclophosphamide (CTX)-induced immunosuppressed mice. Further, we investigated the possible mechanism underlying the effects of HRWP-A on mouse peritoneal macrophages. qPCR and western blot revealed that HRWP-A upregulated the expression of TLR4 mRNA in vitro. This process was accompanied by a clear increase in MyD88 expression and p-IκB-α, but these effects were largely abrogated by pretreatment with anti-TLR4 antibodies. The effects of HRWP-A on macrophage NO, IL-1β and IL-6 production were also inhibited by anti-TLR4 antibodies and were greatly influenced by the NF-κB inhibitor PDTC. Moreover, HRWP-A failed to induce the production of NO, IL-1β and IL-6 in peritoneal macrophages prepared from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, suggesting the involvement of the TLR4 molecule in HRWP-A-mediated macrophage activation. These results may have important implications for our understanding of the structure-activity relationship of immunopotentiating polysaccharides from medicinal herbs.

Topics: Animals; Antineoplastic Agents; Cyclophosphamide; Fruit; Gene Expression Regulation, Neoplastic; Hippophae; Humans; Immunologic Factors; Interleukin-1beta; Interleukin-6; Macrophage Activation; Macrophages; Mice; Myeloid Differentiation Factor 88; Nitric Oxide; Pectins; Signal Transduction; Structure-Activity Relationship; Toll-Like Receptor 4

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

A total of 1940 isolates from gut samples of 60 bumblebees representing Bombus pascuorum, Bombus terrestris, Bombus lucorum and Bombus lapidarius was collected and identified through state-of the-art taxonomic methods. The bacterial species diversity in these Bombus species exceeded that suggested by phylotype analysis through 16S rRNA amplicon sequencing, and revealed that B. pascuorum and B. terrestris had a unique microbiota composition, each. Representatives of most phylotypes reported earlier and detected in the present study were effectively isolated, and included several novel bacterial taxa and species reported for the first time in the bumblebee gut. Isolates were screened in pectin degradation assays and growth inhibition assays against the honeybee pathogens Paenibacillus larvae, Melissococcus plutonius and Ascosphaera apis and the bumblebee parasite Crithidia bombi. While inhibitory activity against each of these pathogens was observed, only one single culture was able to degrade pectin and polygalacturonic acid in vitro. The availability of accurately identified microbial isolates will facilitate future evaluation of the functional potential of the bumblebee gut microbiota.

Topics: Animals; Bacteria; Bees; Biological Control Agents; Crithidia; Gastrointestinal Microbiome; Paenibacillus larvae; Pectins; RNA, Ribosomal, 16S

Artemisia afra (Jacq. Ex. Willd), is an indigenous plant in South Africa and other parts of the African continent, where it is used as traditional medicine mostly for respiratory conditions. The objective of this study was to investigate the structural features of the polysaccharides from the leaves of this plant, as well as the biological activities of the polysaccharide fractions against the complement assay. Leaves of Artemisia afra were extracted sequentially with organic solvents (dichloromethane and methanol), 50% aqueous ethanol, and water at 50 and 100°C respectively. The polysaccharide extracts were fractionated by ion exchange chromatography and the resulting fractions were tested for biological activity against the complement fixation assay. Active fractions were further fractionated using gel filtration. Monosaccharide compositions and linkage analyses were determined for the relevant fractions. Polysaccharides were shown to be of the pectin type, and largely contain arabinogalactan, rhamnogalacturonan and homogalacturonan structural features. The presence of arabinogalactan type II features as suggested by methylation analysis was further confirmed by the ready precipitation of the relevant polysaccharides with the Yariv reagent. An unusual feature of some of these polysaccharides was the presence of relatively high levels of xylose as one of its monosaccharide constituents. Purified polysaccharide fractions were shown to possess higher biological activity than the selected standard in the complement assay. Digestion of these polysaccharides with an endo-polygalacturonase enzyme resulted in polymers with lower molecular weights as expected, but still with biological activity which exceeded that of the standard. Thus on the basis of these studies it may be suggested that immunomodulating properties probably contribute significantly to the health-promoting effects of this medicinal plant.

Topics: Artemisia; Complement Fixation Tests; Galactans; Pectins; Plant Extracts; Plant Leaves; Plants, Medicinal; Polysaccharides; South Africa; Structure-Activity Relationship

The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells.

Topics: Cell Shape; Cell Wall; Ferns; Glucans; Microscopy, Electron, Transmission; Pectins; Plant Leaves; Polysaccharides; Vigna; Zea mays

The most notable and unique property of pectin is the ability to form gel; thus, many biological applications of pectin are based on its gelation properties. Pectin isolated from different plant cell walls may differ in molecular structure and distribution pattern, which may result in different gelling and function properties. In this work, we investigated the chemical characteristics, gelation properties, and biological application of calcium pectate (CaP) prepared using apple (AP) or citrus pectin (CP). These two types of pectins exhibited similar molecular parameters and glycosidic bone structure; however, there was a difference in the composition proportion of single monosaccharide. In addition, it was found that it was relatively easier to form CaP beads with CP compared with AP. Moreover, CP exhibited a higher binding capability with Ca

Topics: Calorimetry; Cell Line; Cell Proliferation; Cell Survival; Chemical Phenomena; Citrus; Magnetic Resonance Spectroscopy; Malus; Microspheres; Molecular Weight; Pectins; Phytochemicals; Spectroscopy, Fourier Transform Infrared; Thermodynamics

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

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.

Topics: Carboxylic Ester Hydrolases; Cell Wall; Coenzyme A Ligases; Gene Expression Regulation, Plant; Microscopy, Electron, Transmission; Pectins; Plant Proteins; Plants, Genetically Modified; Populus; Promoter Regions, Genetic; Xylem

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Topics: Biofuels; Biomass; Boron; Calcium; Cell Wall; Crops, Agricultural; Glucuronosyltransferase; Panicum; Pectins; Plants, Genetically Modified; Populus; Sugars

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

Sedum dendroideum, popularly known in Brazil as balsam, is traditionally used as a wound healing agent, to treat gastritis, and several other health problems. Some studies have shown that plant polysaccharides may have gastroprotective properties.. Considering the popular use of S. dendroideum and the gastroprotective activity of polysaccharides, the objective of this work was to obtain, to characterize, and to evaluate the gastroprotective activity of a polysaccharide fraction from this plant.. Polysaccharides of S. dendroideum were extracted with water by infusion, fractionated by freeze-thawing process and dialyzed at a 100 kDa cut-off membrane, and characterized by monosaccharide composition and NMR analysis. The gastroprotective activity of the pectic polysaccharide fraction RSBAL was evaluated in the ethanol-induced ulcer model in rats, followed by determination of the mucus and glutathione levels in the gastric tissue.. RSBAL was constituted by a homogalacturonan and a homogalacturonan branched by side chains of arabinans and type II arabinogalactans. It reduced ethanol-induced gastric ulcers in rats, preserving mucus and glutathione levels in the stomach.. This study demonstrated that polysaccharides could be related to the pharmacological activity of S. dendroideum.

Topics: Animals; Anti-Ulcer Agents; Brazil; Ethanol; Female; Gastric Mucosa; Glutathione; Pectins; Plant Extracts; Plant Leaves; Plants, Medicinal; Polysaccharides; Protective Agents; Rats, Wistar; Sedum; Stomach Ulcer

Pectins are plant polysaccharides used in food industry as gelling and stabilizing agents. This study investigated the ability of pectins to improve survival of probiotic species Lactobacillus fermentum PCC, L. reuteri RC-14, L. rhamnosus LGG and L. paracasei F-19 in simulated gastric solution in relationship to their structural and physical properties. Electrostatic interactions between pectins and bacteria were evaluated by the Zeta-potential approach. Bacterial survival was assessed by flow cytometry and plate counting. L. fermentum PCC and L. reuteri RC-14 were more resistant to gastric conditions; their survival rate was further improved in the presence of five out of ten tested pectins. Additionally, two of the pectins had a positive effect on viability of the less resistant L. rhamnosus LGG and L. paracasei F-19. The beneficial effect was generally observed for the high-methoxylated pectins, indicating that substituted polygalacturonic acid in the backbone is essential for bacterial protection. Other pectin features associated with improved survival, included less negative Zeta-potential, higher molecular weight, as well as lower values of hydrodynamic sizes, viscosity and degree of branching. The study indicates that pectins have a potential to protect probiotic bacteria through the gastro-intestinal transit and identifies the features linked to their functionality.

Topics: Gastric Juice; Gastrointestinal Microbiome; Hydrodynamics; Lactobacillus; Microbial Viability; Particle Size; Pectins; Probiotics; Solubility; Viscosity

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

Cellulose microfibrils are crucial for many of the remarkable mechanical properties of primary cell walls. Nevertheless, many structural features of cellulose microfibril organization in cell walls are not yet fully described. Microscopy techniques provide direct visualization of cell wall organization, and quantification of some aspects of wall microstructure is possible through image processing. Complementary to microscopy techniques, scattering yields structural information in reciprocal space over large sample areas. Using the onion epidermal wall as a model system, we introduce resonant soft X-ray scattering (RSoXS) to directly quantify the average interfibril spacing. Tuning the X-ray energy to the calcium L-edge enhances the contrast between cellulose and pectin due to the localization of calcium ions to homogalacturonan in the pectin matrix. As a consequence, RSoXS profiles reveal an average center-to-center distance between cellulose microfibrils or microfibril bundles of about 20 nm.

Topics: Calcium; Cell Wall; Cellulose; Microfibrils; Models, Biological; Onions; Pectins; X-Rays

Aluminum toxicity limits the plant growth by inducing inhibition of root elongation. Although several mechanisms have been proposed regarding the phytotoxic effects of aluminum on inhibition of root elongation; the primary causes of aluminum induced root inhibition and its mitigation by boron (B) are still elusive. The present study was carried out to explore the mechanisms of B induced mitigation of aluminum toxicity and to investigate the changes in well wall structure under aluminum toxicity coupled with the techniques of confocal laser microscope, lumogallion and transmission electron microscope. The results revealed that aluminum toxicity severely hampered the root elongation and plant biomass. Moreover, alteration in subcellular structure were observed under aluminum toxicity, however, such negative effects were further exacerbated with B deficiency. Aluminum toxicity indicated disorganized distribution of HG (homogalacturonan) epitopes with higher accumulation of apoplastic aluminum. Nevertheless, B supply improved root elongation, and reduced the aluminum uptake. Taken together, it is concluded that B application can reduce aluminum toxicity and improve root elongation by decreasing Al

Topics: Aluminum; Benzenesulfonates; Boron; Cell Wall; Epitopes; Microscopy, Confocal; Pectins; Plant Roots; Poncirus; Soil; Spectroscopy, Fourier Transform Infrared

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

In Cicer arietinum, as in several plant species, the β-galactosidases are encoded by multigene families, although the role of the different proteins is not completely elucidated. Here, we focus in 2 members of this family, βIII-Gal and βIV-Gal, with high degree of amino acid sequence identity (81%), but involved in different developmental processes according to previous studies. Our objective is to deepen in the function of these proteins by establishing their substrate specificity and the possible alterations caused in the cell wall polysaccharides when they are overproduced in Arabidopsis thaliana by constructing the 35S::βIII-Gal and 35S::βIV-Gal transgenic plants. βIII-Gal does cause visible alterations of the morphology of the transgenic plant, all related to a decrease in growth at different stages of development. FTIR spectroscopy and immunological studies showed that βIII-Gal causes changes in the structure of the arabidopsis cell wall polysaccharides, mainly a reduction of the galactan side chains which is compensated by a marked increase in homogalacturonan, which allows us to attribute to galactan a role in the control of the architecture of the cell wall, and therefore in the processes of growth. The 35S::βIV-Gal plants do not present any phenotypic changes, neither in their morphology nor in their cell walls. In spite of the high sequence homology, our results show different specificity of substrate for these proteins, maybe due to other dissimilar characteristics, such as isoelectric points or the number of N-glycosylation sites, which could determine their enzymatic properties and their distinct action in the cell walls.

Topics: Arabidopsis; beta-Galactosidase; Cell Wall; Chromosome Mapping; Cicer; Fluorescent Antibody Technique; Galactans; Pectins; Plant Proteins; Plants, Genetically Modified; Quantitative Trait Loci; Spectroscopy, Fourier Transform Infrared

Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (

Topics: Arabidopsis; Arabidopsis Proteins; Glucuronosyltransferase; HEK293 Cells; Humans; Models, Biological; Molecular Structure; Pectins; Static Electricity; Substrate Specificity; Uridine Diphosphate Sugars

Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation in situ. Here we present a tool for efficient real-time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium-mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real-time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures.

Topics: Arabidopsis; Calcium; Cell Wall; Pectins; Pollen Tube

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.

Topics: Aspergillus; Carbon; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Thin Layer; Citrus sinensis; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Enzyme Stability; Ethanol; Fungal Proteins; Hexuronic Acids; Hydrogen-Ion Concentration; Isoelectric Focusing; Kinetics; Metals; Molecular Weight; Pectins; Polygalacturonase; Sulfur Dioxide; Temperature; Time Factors

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed

Topics: Amino Acid Sequence; Cell Count; Cell Cycle; Cell Death; Cell Wall; Cloning, Molecular; Esterification; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Models, Biological; Mutation; Oryza; Pectins; Phenotype; Phylogeny; Plant Development; Plant Leaves; Polysaccharide-Lyases; Reactive Oxygen Species; Transcriptome

Homogalacturonan (HG) is the main component of pectins. HG methylesterification has recently emerged as a key determinant controlling cell attachment, organ formation, and phyllotaxy. However, whether and how HG methylesterification affects intercellular metabolite transport has rarely been reported. Here, we identified and characterized knockout mutants of the rice (

Topics: Carbon Dioxide; Cell Communication; Cell Wall; Esterification; Genes, Reporter; Golgi Apparatus; Methyltransferases; Mutation; Oryza; Pectins; Phenotype; Plant Proteins; Plant Vascular Bundle; Plants, Genetically Modified; Seeds; Subcellular Fractions; Sucrose

The change of pectin epitopes during procambium-cambium continuum development was investigated by immunolocalization in poplar. The monoclonal antibody JIM5 labels homogalacturonan (HGA) with a low degree of esterification, and the monoclonal antibody JIM7 labels HGA with a high degree of methyl-esterification. Arabinan, rather than galactan, and HGA with low degree of esterification were located in the cell walls of procambial, while HGA with a low degree of esterification was located in the tangential walls, and galactan was located in both the tangential and radial walls of procambial, yet nearly no arabinan was located in the tangential walls of the cambial cells. The changes in pectin distribution took place when periclinal divisions appeared within a procambial trace. The distribution difference of pectin epitopes was also present in procambium-cambium derivatives. The arabinan existed in all cell walls of primary xylem, but was absent from the tangential walls of secondary xylem cells. The galactan existed only in mature primary phloem. Furthermore, 19 pectin methylesterases (PMEs) genes were identified by RNA sequencing, six genes presented highly differentially and were supposed to be involved in the cell wall esterification process. The results provide direct evidence of the dynamic changes of pectin epitopes during the development of the procambium-cambium continuum in poplar.

Topics: Antibodies, Monoclonal; Cambium; Cell Wall; Epitopes; Gene Expression Regulation, Plant; Genes, Plant; Multigene Family; Pectins; Phylogeny; Populus

Significant cellulose-pectin interactions in plant cell walls have been reported recently based on 2D

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Cellulose; Hexuronic Acids; Mutation; Pectins; Polygalacturonase

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

Pectin is the main soluble fiber in apples or citruses. It may be fermented by gut microbiota to metabolites showing local intestinal and systemic effects. A wide range of beneficial effects of dietary pectin includes impacts on the redox milieu and microbiota profile. We prepared pectin-derived oligosaccharides (apple (APDO) and citrus) and polygalacturonic acid-derived oligosaccharides, using alkaline hydrolysis by hydrogen peroxide, and analyzed them by Fourier Transform Infrared spectrometry. Furthermore, we analyzed the effects of pectin-derived oligosaccharides on hydroxyl radical (HO)-generating Fenton reaction using electron paramagnetic resonance spin-trapping spectroscopy, and the effects on the growth of Escherichia coli and Staphylococcus aureus in the presence of dietary-relevant HO-generating system (iron+ascorbate). The oligosaccharides react with HO radical to produce carbon dioxide radical anion (CO

Topics: Anions; Ascorbic Acid; Carbon Dioxide; Electron Spin Resonance Spectroscopy; Escherichia coli; Free Radicals; Hydrogen Peroxide; Hydroxyl Radical; Iron; Malus; Oligosaccharides; Pectins; Staphylococcus aureus

While polycarboxylates and hydroxyl-acid complexes have long been known to be photoactive, simple carboxylate complexes which lack a significant LMCT band are not typically strongly photoactive. Hence, it was somewhat surprising that a series of reports demonstrated that materials synthesized from iron(III) and polysaccharides such as alginate (poly[guluronan-co-mannuronan]) or pectate (poly[galacturonan]) formed photoresponsive materials that convert from hydrogels to sols under the influence of visible light. These materials have numerous potential applications in areas such as photopatternable materials, materials for controlled drug delivery, and tissue engineering. Despite the near-identity of the functional units in the polysaccharide ligands, the reactivity of iron(III) hydrogels can depend on the configuration of some chiral centers in the sugar units and in the case of alginate the guluronate to mannuronate block composition, as well as pH. Here, using temperature- and field-dependent transmission Mössbauer spectroscopy, we show that the dominant iron compound detected for both the alginate and pectate gels displays features typical of a polymeric (Fe

Topics: Alginates; Coordination Complexes; Hydrogels; Iron Compounds; Light; Magnetic Phenomena; Nanoparticles; Particle Size; Pectins; Spectroscopy, Mossbauer

The aim of this work was to develop a polyelectrolyte complex-based hemostatic dressing made from chitosan and polygalacturonic acid. Porous dressings were fabricated by ultrasonication of chitosan and alginate solutions followed by freeze-drying. Since chitosan has inherent hemostatic properties, and polygalacturonic acid is anti-inflammatory in nature, it was desired to combine these two polymers to develop an effective hemostatic dressing, which may also promote wound healing. Porous structure of the bandages was observed using field-emission scanning electron microscope. Blood clotting behavior was studied using whole blood clotting assay. Plasma recalcification time, prothrombin time, and activated partial thromboplastin time were also determined to study the mechanism of clotting. The dressings were found to accelerate clotting rates and showed increased thrombin activity with an increase in chitosan concentration.

Topics: Animals; Anti-Inflammatory Agents; Bandages; Blood Coagulation; Chitosan; Freeze Drying; Goats; Hemostasis; Hemostatics; Pectins; Polyelectrolytes; Wound Healing

Vegetables are eaten as part of a healthy diet throughout the world, and some are also applied topically as a traditional medicine. We evaluated the innate immunostimulating activities of hot water extracts of various vegetables using the silkworm muscle contraction assay system, and found that broccoli, Brassica oleracea var. italica, contains a strong innate immunostimulant. We purified the innate immunostimulant from broccoli, and characterized the chemical structure by chemical analyses and NMR spectroscopy. The innate immunostimulant comprised galacturonic acid, galactose, glucose, arabinose, and rhamnose, and had a pectic-like polysaccharide structure. To determine the structural motif involved in the innate immunostimulating activity, we modified the structure by chemical and enzymatic treatment, and found that the activity was attenuated by pectinase digestion. These findings suggest that a pectic-like polysaccharide purified from broccoli has innate immune-stimulating activity, for which the polygalacturonic acid structure is necessary.

Topics: Adjuvants, Immunologic; Animals; Arabinose; Bombyx; Brassica; Galactose; Glucose; Hexuronic Acids; Immunity, Innate; Larva; Magnetic Resonance Spectroscopy; Molecular Structure; Muscle Contraction; Pectins; Plant Extracts; Polysaccharides; Rhamnose

The viscoelastic mechanical properties of water-rich plant tissues are fundamental for many aspects of organ physiology and plant functioning. These properties are determined partly by the water in cellular vacuole and partly by the mechanical properties of the cell wall, the latter varying according to the composition and organization of its polysaccharides. In this study, relationships between the viscoelastic properties of apple cortex parenchyma tissue and cell wall pectin, hemicelluloses, and cellulose structures were studied by infusing the tissue with selected sets of purified enzymes in a controlled osmoticum. The results showed that tissue elasticity and viscosity were related, and controlled to variable extents by all the targeted polysaccharides. Among them, pectic homogalacturonan domains, crystalline cellulose, and fucosylated xyloglucan were revealed as being of prime importance in determining the viscoelastic mechanical properties of apple cortex tissue.

Topics: Biomechanical Phenomena; Cell Wall; Cellulose; Elasticity; Glucans; Hydrolases; Malus; Mesophyll Cells; Models, Biological; Pectins; Polysaccharides; Viscosity; Water; Xylans

Topics: Acetylation; Animals; Antibodies, Bacterial; Antibody Formation; Disease Models, Animal; Immunization, Secondary; Immunogenicity, Vaccine; Immunoglobulin G; Immunologic Memory; Mice; Pectins; Polysaccharides, Bacterial; Salmonella typhi; Typhoid Fever; Typhoid-Paratyphoid Vaccines; Vaccines, Synthetic

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.

Topics: Animals; Coleoptera; Enterobacteriaceae; Genome Size; Genome, Bacterial; Pectins; Symbiosis

The composition of pomegranate peel, the main by-product during pomegranate processing, and some of the characteristics of the water-soluble pectins were investigated. Four tunisian pomegranate peels were subjected to hot aqueous extractions (86°C, 80min, 20mM nitric acid). Pomegranate peels yielded between 6.8% and 10.1% pectins. The extracted pectins were low methylated and were characterized by the predominance of homogalacturonan regions. Principal component analysis applied on FT-IR spectral data in the region between 4000 and 650cm(-1) differentiated the samples according to their degree of methylation. At pH 3, in the presence of 0.7% pectin, all solutions showed a rapid gel formation with G'>G″. With decreasing temperature from 90°C to 10°C, G' increased to reach a plateau at 10°C. The variation in the pectin gel formation between varieties was attributed to difference in pectin characteristics particularly the hydrodynamic volume and the neutral sugar content.

Topics: Lythraceae; Pectins; Spectroscopy, Fourier Transform Infrared

The relationship between pectin structure and the antimicrobial activity of nisin-loaded pectin particles was examined. The antimicrobial activity of five different nisin-loaded pectin particles, i.e., nisin-loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0-7.0 by agar-diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin-loaded pectin particles. Nisin-loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin-loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin-loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245-251, 2017.

Topics: Anti-Infective Agents; Arthrobacter; Bacillus subtilis; Esterification; Food Preservation; Hydrogen-Ion Concentration; Nisin; Pectins

Aspartic proteases are a class of proteolytic enzymes with conserved aspartate residues, which are implicated in protein processing, maturation, and degradation. Compared with yeast and animals, plants possess a larger aspartic protease family. However, little is known about most of these enzymes. Here, we characterized two Arabidopsis (Arabidopsis thaliana) putative glycosylphosphatidylinositol (GPI)-anchored aspartic protease genes, A36 and A39, which are highly expressed in pollen and pollen tubes. a36 and a36 a39 mutants display significantly reduced pollen activity. Transmission electron microscopy and terminal-deoxynucleotidyl transferase-mediated nick end labeling assays further revealed that the unviable pollen in a36 a39 may undergo unanticipated apoptosis-like programmed cell death. The degeneration of female gametes also occurred in a36 a39 Aniline Blue staining, scanning electron microscopy, and semi in vitro guidance assays indicated that the micropylar guidance of pollen tubes is significantly compromised in a36 a39 A36 and A39 that were fused with green fluorescent protein are localized to the plasma membrane and display punctate cytosolic localization and colocalize with the GPI-anchored protein COBRA-LIKE10. Furthermore, in a36 a39, the abundance of highly methylesterified homogalacturonans and xyloglucans was increased significantly in the apical pollen tube wall. These results indicate that A36 and A39, two putative GPI-anchored aspartic proteases, play important roles in plant reproduction in Arabidopsis.

Topics: Apoptosis; Arabidopsis; Arabidopsis Proteins; Aspartic Acid Proteases; Cell Membrane; Chromosome Segregation; Crosses, Genetic; Genetic Complementation Test; Germination; Glucans; Green Fluorescent Proteins; Mutation; Ovule; Pectins; Phenotype; Pollen; Pollen Tube; Pollination; Proteolysis; Seeds; Subcellular Fractions; Xylans

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

Topics: Adhesiveness; Arabidopsis; Arabidopsis Proteins; Cell Shape; Cell Wall; Flowers; Genotype; Membrane Lipids; Models, Biological; Mutation; Palmitic Acids; Pectins; Plant Epidermis; Spectroscopy, Fourier Transform Infrared

Homogalacturonans (HGs) are polysaccharide copolymers of galacturonic acid and its methylesterified counterpart. The inter- and intramolecular distributions of the methylesterifed residues are vital behavior-determining characteristics of a sample's structure, and much experimental effort has been directed to their measurement. While many techniques are able to measure the sample-averaged degree of methylesterification (DM), the measurement of inter- and intramolecular charge distributions are challenging. Here, molecular dynamics (MD) simulations are used to calculate the electrophoretic mobilities of HGs that have different amounts and distributions of charges placed along the backbone. The simulations are shown to capture experimental results well, even for low-DM samples that possess high charge densities. In addition, they illuminate the role that local counterion condensation can play in the determination of the electrophoretic mobility of heterogeneous blocky polyelectrolytes that cannot be adequately described by a single chain-averaged charge spacing.

Topics: Electrophoresis, Capillary; Molecular Dynamics Simulation; Pectins; Polymers

Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli. The mature BacPelA exhibited maximum activity at pH 10.5 and 70 °C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2 U mg

Topics: Bacillus clausii; Biotransformation; Boehmeria; Cloning, Molecular; Enzyme Stability; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Pectins; Polysaccharide-Lyases; Recombinant Proteins; Temperature

Topics: Analysis of Variance; Animals; Antibiosis; Cattle; Colony Count, Microbial; DNA, Bacterial; Food Microbiology; Gram-Positive Bacteria; Hydrogen-Ion Concentration; Latilactobacillus sakei; Listeria monocytogenes; Meat; Nitrates; Nitrogen; Pectins; Peptones; Phosphates; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis; Temperature

Oligogalacturonides (OGs) are pectic fragments derived from the partial degradation of homogalacturonan in the plant cell wall and able to elicit plant defence responses. Recent methodological advances in the isolation of OGs from plant tissues and their characterization have confirmed their role as bona fide plant Damage-Associated Molecular Patterns. Here, we describe the methods for the isolation of OGs from Arabidopsis leaf tissues and for the characterization of OG structure and biological activity.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Pectins; Peptides; Plant Immunity; Plant Leaves; Protein Conformation

Tomato fruit texture depends on histology and cell wall architecture, both under genetic and developmental controls. If ripening related cell wall modifications have been well documented with regard to softening, little is known about cell wall construction during early fruit development. Identification of key events and their kinetics with regard to tissue architecture and cell wall development can provide new insights on early phases of texture elaboration. In this study, changes in pectin and hemicellulose chemical characteristics and location were investigated in the pericarp tissue of tomato (Solanum lycopersicon var Levovil) at four stages of development (7, 14 and 21day after anthesis (DPA) and mature green stages). Analysis of cell wall composition and polysaccharide structure revealed that both are continuously modified during fruit development. At early stages, the relative high rhamnose content in cell walls indicates a high synthesis of rhamnogalacturonan I next to homogalacturonan. Fine tuning of rhamnogalacturonan I side chains appears to occur from the cell expansion phase until prior to the mature green stage. Cell wall polysaccharide remodelling also concerns xyloglucans and (galacto)glucomannans, the major hemicelluloses in tomato cell walls. In situ localization of cell wall polysaccharides in pericarp tissue revealed non-ramified RG-I rich pectin and XyG at cellular junctions and in the middle lamella of young fruit. Blocks of non-methyl esterified homogalacturonan are detected as soon as 14 DPA in the mesocarp and remained restricted to cell corner and middle lamella whatever the stages. These results point to new questions about the role of pectin RGI and XyG in cell adhesion and its maintenance during cell expansion.

Topics: Cell Wall; Epitopes; Fluorescent Antibody Technique; Fruit; Glucans; Organ Size; Pectins; Polysaccharides; Solanum lycopersicum; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Xylans

Topics: Biotechnology; Cloning, Molecular; Enzyme Stability; Fruit and Vegetable Juices; Hydrogen-Ion Concentration; Kinetics; Pectins; Penicillium; Pichia; Polygalacturonase; Tropical Climate

Starfruit (Averrhoa carambola L.) is an edible tropical fruit, which is usually consumed as a fresh table fruit or as fruit juice. It also exhibits various pharmacological activities. In this study, polysaccharides were extracted with boiling water and purified by freeze-thawing and Fehling treatments. After purification steps, a homogenous fraction was obtained. It was analyzed by sugar composition, gel permeation chromatography, methylation, and two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy analyses. It comprised arabinose (Ara), galactose (Gal), and galacturonic acid (GalA) in a molar ratio of 12.3:1.7:86.0. Methylation and NMR spectroscopy analyses showed that it contained a substituted galacturonan composed of (1→4)-linked α-D-Galp A units branched at O-2 by (1→5)-linked α-L-Araf and terminal α-L-Araf and α-D-Galp A units. The effect of PFSCW (10-300mg/kg, i.p.) on nocifensive behavior induced by intraplantar injection of formalin in mice was evaluated. The fraction demonstrated antinociceptive and anti-inflammatory properties, suggesting that it may be useful in therapeutic intervention for the management of inflammatory pain.

Topics: Analgesics; Animals; Anti-Inflammatory Agents; Averrhoa; Disease Models, Animal; Female; Magnetic Resonance Spectroscopy; Mice; Molecular Structure; Pectins; Plant Extracts; Polysaccharides

We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* < 0.1) than for zinc and barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).

Topics: Cations, Divalent; Pectins; Thermodynamics; Water

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

The kinetics of oxidation of alginate (Alg) and pectate (Pec) carbohydrate biopolymers was studied by spectrophotometry in aqueous perchloric and sulfuric acid solutions at fixed ionic strengths and temperature. In both acids, the reactions showed a first order dependence on [Ce(IV)], whereas the orders with respect to biopolymer concentrations are less than unity. In perchloric acid, the reactions exhibited less than unit orders with respect to [H(+)] whereas those proceeded in sulfuric acid showed negative fractional-first order dependences on [H(+)]. The effect of ionic strength and dielectric constant was studied. Probable mechanistic schemes for oxidation reactions were proposed. In both acids, the final oxidation products were characterized as mono-keto derivatives of both biopolymers. The activation parameters with respect to the slow step of the mechanisms were computed and discussed. The rate laws were derived and the reaction constants involved in the different steps of the mechanisms were calculated.

Topics: Alginates; Biopolymers; Cerium; Glucuronic Acid; Hexuronic Acids; Kinetics; Osmolar Concentration; Oxidation-Reduction; Pectins; Perchlorates; Solutions; Sulfuric Acids; Temperature

Pectins are major components of plant primary cell walls. They include homogalacturonans (HGs), which are the most abundant pectin and can be the target of apoplastic enzymes like pectin methylesterases (PMEs) that control their methylesterification level. Several PMEs are expressed in the seed coat of Arabidopsis thaliana, particularly in mucilage secretory cells (MSCs). On the basis of public transcriptomic data, seven PME genes were selected and checked for their seed-specific expression by quantitative reverse transcription PCR. Of these, PME58 presented the highest level of expression and was specifically expressed in MSCs at the early stages of seed development. pme58 mutants presented two discrete phenotypes: (i) their adherent mucilage was less stained by ruthenium red when compared to wild-type seeds, but only in the presence of EDTA, a Ca(2+)chelator; and (ii) the MSC surface area was decreased. These phenotypes are the consequence of an increase in the degree of HG methylesterification connected to a decrease in PME activity. Analysis of the sugar composition of soluble and adherent mucilage showed that, in the presence of EDTA, sugars of adherent mucilage were more readily extracted in pme58 mutants. Immunolabelling with LM19, an antibody that preferentially recognizes unesterified HGs, also showed that molecular interactions with HGs were modified in the adherent mucilage of pme58 mutants, suggesting a role of PME58 in mucilage structure and organization. In conclusion, PME58 is the first PME identified to play a direct role in seed mucilage structure.

Topics: Arabidopsis; Arabidopsis Proteins; Carboxylic Ester Hydrolases; DNA, Bacterial; Esterification; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Genes, Plant; Mutagenesis, Insertional; Mutation; Pectins; Phenotype; Plant Mucilage; Promoter Regions, Genetic; Seeds

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

The formation of fibrous tissue is part of the natural healing response following a laminectomy. Severe scar tissue adhesion, known as epidural fibrosis, is a common cause of failed back surgery syndrome. In this study, by combining the advantages of drug treatment with a physical barrier, an ibuprofen-conjugated crosslinkable polygalacturonic acid and hyaluronic acid hydrogel was developed for epidural fibrosis prevention. Conjugation was confirmed and measured by 1D(1)H NMR spectroscopy.In vitroanalysis showed that the ibuprofen-conjugated polygalacturonic acid-hyaluronic acid hydrogel showed low cytotoxicity. In addition, the conjugated ibuprofen decreased prostaglandin E2production of the lipopolysaccharide-induced RAW264.7 cells. Histological data inin vivostudies indicated that the scar tissue adhesion of laminectomized male adult rats was reduced by the application of our ibuprofen-conjugated polygalacturonic acid-hyaluronic acid hydrogel. Its use also reduced the population of giant cells and collagen deposition of scar tissue without inducing extensive cell recruitment. The results of this study therefore suggest that the local delivery of ibuprofenviaa polygalacturonic acid-hyaluronic acid-based hydrogel reduces the possibility of epidural fibrosis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cicatrix; Drug Carriers; Epidural Space; Hydrogel, Polyethylene Glycol Dimethacrylate; Ibuprofen; Laminectomy; Male; Mice; Pectins; Postoperative Complications; Rats; Rats, Sprague-Dawley; RAW 264.7 Cells; Tissue Adhesions; Wound Healing

The vascular cambium is a lateral meristem which can differentiate into secondary phloem and xylem. The secondary growth of woody plants resulting from vascular cambium activity has been a focus of considerable attention, but the quantitative relationships between cambial activity and secondary xylem formation have been little studied. Our analysis of cytological changes in the cambium of Chinese fir (Cunninghamia lanceolata), revealed a significant positive correlation between vascular cambium cell numbers and cambium zone width through the seasonal cycle. Cambium cell numbers and the cambium cell radial diameter were closely related to xylem formation. Immuno-labeling showed that de-esterified homogalacturonan and (1-4)-β-d-galactan epitopes were highly abundant in cell walls of dormant-stage cambium, whereas high methylesterified homogalacturonan was strongly labeled in the active stage. Raman spectroscopy detected significant changes in the chemical composition of cell walls during the active-dormant stage transition. More pectin and less monolignols occurred in radial cell walls than in tangential walls during the dormant stage, but no significant changes were found in other stages, indicating that pectin accumulation facilitates cell wall expansion, with cambium activity transition. Our quantitative analysis of the relationship between cambial activity and xylem formation, as well as the cell wall modification during the active stage provides useful information about cambial characteristics and xylogenesis.

Topics: Cambium; Cell Wall; Cunninghamia; Pectins; Phloem; Polysaccharides; Seasons; Xylem

A combined approach, using a carbohydrate microarray as a support for genomic data, has revealed subtle plant cell-wall remodelling during Tuber melanosporum and Corylus avellana interaction. Cell walls are involved, to a great extent, in mediating plant-microbe interactions. An important feature of these interactions concerns changes in the cell-wall composition during interaction with other organisms. In ectomycorrhizae, plant and fungal cell walls come into direct contact, and represent the interface between the two partners. However, very little information is available on the re-arrangement that could occur within the plant and fungal cell walls during ectomycorrhizal symbiosis. Taking advantage of the Comprehensive Microarray Polymer Profiling (CoMPP) technology, the current study has had the aim of monitoring the changes that take place in the plant cell wall in Corylus avellana roots during colonization by the ascomycetous ectomycorrhizal fungus T. melanosporum. Additionally, genes encoding putative plant cell-wall degrading enzymes (PCWDEs) have been identified in the T. melanosporum genome, and RT-qPCRs have been performed to verify the expression of selected genes in fully developed C. avellana/T. melanosporum ectomycorrhizae. A localized degradation of pectin seems to occur during fungal colonization, in agreement with the growth of the ectomycorrhizal fungus through the middle lamella and with the fungal gene expression of genes acting on these polysaccharides.

Topics: Ascomycota; Carbohydrate Metabolism; Cell Wall; Corylus; Gene Expression Profiling; Mycorrhizae; Pectins; Plant Roots; Transcriptome

Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity.

Topics: Enzyme Stability; Enzymes, Immobilized; Food Handling; Fruit; Fruit and Vegetable Juices; Glyoxylates; Hydrogen-Ion Concentration; Pectins; Polygalacturonase; Prunus domestica; Sepharose; Vitis

Polysaccharide-based nanocomplexes, intended for simultaneous encapsulation and controlled release of 5-Fluorouracil (5-FU) and Temozolomide (TMZ) were developed via the complexation method using chitosan, alginic and polygalacturonic acid. Investigation focused on the influence of polysaccharides on the properties of the system and amelioration of the stability of the drugs, in particular TMZ. The dimensions of particles and their ζ-potential were found to range between 100 and 200nm and -25 to +40mV, respectively. Encapsulation efficiency varied from 16% to over 70%, depending on the given system. The influence of pH on the release and co-release of TMZ and 5-FU was evaluated under different pH conditions. The stability of the loaded drug, in particular TMZ, after release was evaluated and confirmed by LC-MS analysis. Results suggested that the amount of loaded drug(s) and the release rate is connected with the weight ratio of polysaccharides and the pH of the media. One-way ANOVA analysis on the obtained data revealed no interference between the drugs during the encapsulation and release process, and in particular no hydrolysis of TMZ occurred suggesting that CS-ALG and CS-PGA would represent interesting carriers for multi-drug controlled release and drugs protection.

Topics: Alginates; Antimetabolites, Antineoplastic; Antineoplastic Agents, Alkylating; Chitosan; Dacarbazine; Delayed-Action Preparations; Drug Carriers; Drug Combinations; Drug Compounding; Drug Liberation; Drug Stability; Fluorouracil; Glucuronic Acid; Hexuronic Acids; Nanoparticles; Pectins; Prodrugs; Temozolomide

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Cyclopentanes; Flowers; Gene Expression Regulation, Plant; Indoleacetic Acids; Inflorescence; Meristem; Mutation; Oxylipins; Pectins; Plant Diseases; Pseudomonas syringae; Salicylic Acid; Sequence Analysis, RNA; Transcription Factors

Immunomodulatory drugs are available to maintain immune homeostasis but some have undesirable side effects. Six oligo- and poly-saccharides were assessed for their pro- and anti-inflammatory responses in two in vitro model systems, the monocytic THP-1 cell line and human whole blood cultures (HWBC). The compounds were first characterised for their molecular mass and physical properties. Following incubation with lipopolysaccharide (LPS) or the compounds, cytokine and chemokine secretion was assayed in both models and intracellular TNF-α was measured by flow cytometry in HWBC cell sub-populations. LPS, inulin, galacturonan, heteroglycan and fucoidan demonstrated pro-inflammatory properties and intracellular TNF-α expression was increased in the monocytes of HWBC. Mannan and xyloglucan did not elicit any significant responses. Inulin induced maximum cytokine secretion and heteroglycan induced maximum chemokine secretion in HWBC. This study emphasises the potential of inulin and heteroglycan as potential immunomodulatory therapeutics and that HWBC had a greater and more varied response in comparison to THP-1 cells.

Topics: Cell Line; Computer Simulation; Drug Discovery; Homeostasis; Humans; Immunologic Factors; Interleukin-8; Inulin; Lipopolysaccharides; Monocytes; Pectins; Polysaccharides; Tumor Necrosis Factor-alpha

The root cap covers the tip of the root and functions to protect the root from environmental stress. Cells in the last layer of the root cap are known as border cells, or border-like cells (BLCs) in Arabidopsis (Arabidopsis thaliana). These cells separate from the rest of the root cap and are released from its edge as a layer of living cells. BLC release is developmentally regulated, but the mechanism is largely unknown. Here, we show that the transcription factor NIN-LIKE PROTEIN7 (NLP7) is required for the proper release of BLCs in Arabidopsis. Mutations in NLP7 lead to BLCs that are released as single cells instead of an entire layer. NLP7 is highly expressed in BLCs and is activated by exposure to low pH, a condition that causes BLCs to be released as single cells. Mutations in NLP7 lead to decreased levels of cellulose and pectin. Cell wall-loosening enzymes such as CELLULASE5 (CEL5) and a pectin lyase-like gene, as well as the root cap regulators SOMBRERO and BEARSKIN1/2, are activated in nlp7-1 seedlings. Double mutant analysis revealed that the nlp7-1 phenotype depends on the expression level of CEL5 Mutations in NLP7 lead to an increase in susceptibility to a root-infecting fungal pathogen. Together, these data suggest that NLP7 controls the release of BLCs by acting through the cell wall-loosening enzyme CEL5.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Adhesion; Cell Wall; Cellulose; Fusarium; Gene Expression Regulation, Plant; Hydrogen-Ion Concentration; Mutation; Pectins; Plant Cells; Plant Roots; Plants, Genetically Modified; Soil Microbiology; Stress, Physiological; Transcription Factors

Cotton fibers arise through highly anisotropic expansion of a single seed epidermal cell. We obtained evidence that apical cell wall synthesis occurs through examining the tips of young elongating Gossypium hirsutum (Gh) and G. barbadense (Gb) fibers. We characterized two tip types in Gh fiber (hemisphere and tapered), each with distinct apical diameter, central vacuole location, and distribution of cell wall components. The apex of Gh hemisphere tips was enriched in homogalacturonan epitopes, including a relatively high methyl-esterified form associated with cell wall pliability. Other wall components increased behind the apex including cellulose and the α-Fuc-(1,2)-β-Gal epitope predominantly found in xyloglucan. Gb fibers had only one narrow tip type featuring characters found in each Gh tip type. Pulse-labeling of cell wall glucans indicated wall synthesis at the apex of both Gh tip types and in distal zones. Living Gh hemisphere and Gb tips ruptured preferentially at the apex upon treatment with wall degrading enzymes, consistent with newly synthesized wall at the apex. Gh tapered tips ruptured either at the apex or distantly. Overall, the results reveal diverse cotton fiber tip morphologies and support primary wall synthesis occurring at the apex and discrete distal regions of the tip.

Topics: Cell Wall; Cotton Fiber; Cryoelectron Microscopy; Epitopes; Flowers; Glucans; Gossypium; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Pectins; Plant Cells; Polysaccharides; Xylans

Cell-to-cell adhesion in plants is mediated by the cell wall and the presence of a pectin-rich middle lamella. However, we know very little about how the plant actually controls and maintains cell adhesion during growth and development and how it deals with the dynamic cell wall remodeling that takes place. Here we investigate the molecular mechanisms that control cell adhesion in plants. We carried out a genetic suppressor screen and a genetic analysis of cell adhesion-defective Arabidopsis thaliana mutants. We identified a genetic suppressor of a cell adhesion defect affecting a putative O-fucosyltransferase. Furthermore, we show that the state of cell adhesion is not directly linked with pectin content in the cell wall but instead is associated with altered pectin-related signaling. Our results suggest that cell adhesion is under the control of a feedback signal from the state of the pectin in the cell wall. Such a mechanism could be necessary for the control and maintenance of cell adhesion during growth and development.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Adhesion; Cell Wall; Fucosyltransferases; Genes, Plant; Genetic Testing; Golgi Apparatus; Models, Biological; Mutation; Pectins; Signal Transduction; Substrate Specificity; Suppression, Genetic

In vitro effects of acetylated pectin (OP) isolated from cacao pod husks (Theobroma cacao L.), its partially deacetylated and de-esterified form (MOP), and a commercial homogalacturonan (PG) were investigated on murine peritoneal macrophages. MOP stood out among the studied pectins. After 48h of incubation, compared with the control group, it was able to promote significant macrophage morphological differentiation from resident to activated stage and also stimulated nitric oxide production, which reached a level of 85% of that of LPS stimulus. In the presence of the highest tested concentration of MOP (200μg·mL

Topics: Acetylation; Animals; Cacao; Female; Gene Expression; Inflammation; Interleukin-10; Interleukin-12; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Mice; Nitric Oxide; Pectins; Primary Cell Culture; Tumor Necrosis Factor-alpha

The purpose of this work was to reveal the structural changes of cell wall polysaccharides' fractions during tomato fruit development by analysis of spectral data. Mature green and red ripe tomato fruit were taken into consideration. The FT-IR spectra of water soluble pectin (WSP), imidazole soluble pectin (ISP) and diluted alkali soluble pectin (DASP) contained bands typical for pectins. Whereas for KOH fraction spectra bands typical for hemicelluloses were present. The FT-IR spectra showed the drop down of esterification degree of WSP and ISP polysaccharides during maturation. The changes in polysaccharides structure revealed by spectra were the most visible in the case of pectic polysaccharides. The WSP and DASP fraction pectins molecules length were shortened during tomato maturation and ripening. Whereas the ISP fraction spectra analysis showed that this fraction contained rhamnogalacturonan I, but also for red ripe was rich in pectic galactan comparing with ISP fraction from mature green.

Topics: Cell Wall; Fruit; Pectins; Polysaccharides; Principal Component Analysis; Solanum lycopersicum; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman

This study investigated the degradation kinetics and structural properties of pectin with combining ultrasound and pectinase treatment. Ultrasound at an intensity of 4.5WmL(-1) and a time of 10min significantly enhanced the enzymatic degradation of pectin weight-average molecular weight (Mw). The degradation kinetics model of pectin followed 1/Mwt-1/Mw0=kt, suggesting the randomness of the degradation process. Synergistic effects of ultrasound and pectinase were observed at 20-60°C and were more effective at lower temperatures. Furthermore, the degree of methoxylation (DM) of sonoenzymolysis pectin significantly decreased whereas the degree of acetylation (DAc) remained unchanged compared to the original and enzymolysis pectin. Simultaneous functions of ultrasound and pectinase caused severe decomposition in pectin homogalacturonan (HG) regions without altering the monosaccharides types, configurations and glycoside linkages of the pectin samples. The complex polymeric structures of pectin transformed into smaller units with simpler branches and shorter chains after sonoenzymolysis reactions.

Topics: Acetylation; Chromatography, High Pressure Liquid; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Microscopy, Atomic Force; Molecular Weight; Monosaccharides; Nanostructures; Pectins; Polygalacturonase; Temperature; Ultrasonics

Increase in the number of cancer related deaths has made the study on developing new drugs and treatments essential. One of the main aims in developing new therapies is to use natural resources which have the ability to induce apoptosis. Pectin is one of these natural compounds, a complex polysaccharide found in apples with anti-cancer properties. The aim of this study was to examine anti-cancer properties of pectic acid both in vitro in 4T1 breast cancer cells and in vivo using an animal model of breast cancer.. MTT cell proliferation assays, double fluorescence staining (acridine orange/ethidium bromide) and cell cycle analysis were employed to measure apoptosis in vitro. 4T1 cells were implanted into female BALB/c mice for in vivo studies. Then tumor volumes, histological analysis and immunohistochemical staining of P53 and tunnel test were applied to evaluate apoptosis in tumors.. The results of in vitro studies showed that concentration of 0.1% of pectic acid could induce apoptosis, inhibit cell growth (p<0.001) and reduce cell attachment, fragmented chromatin, and membrane blebbing as well as blocking the sub-G1 phase (p<0.001). In addition, in vivo studies showed that pectic acid could inhibit the progression of tumors through over-expression of P53 and increasing the number of apoptotic cells.. Our results demonstrated that pectic acid, a natural component of apple, can prevent metastasis in both cancer cell lines and primary tumors. This potential effect is mainly due to its ability to induce apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Adhesion; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin Assembly and Disassembly; Dose-Response Relationship, Drug; Female; Fruit; Malus; Mice, Inbred BALB C; Pectins; Phytotherapy; Plants, Medicinal; Time Factors; Tumor Burden; Tumor Suppressor Protein p53; Up-Regulation

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Topics: Carboxylic Ester Hydrolases; Cell Wall; Computational Biology; Gene Expression Regulation, Bacterial; Genome, Bacterial; Oryza; Pectins; Plant Diseases; Polygalacturonase; Polysaccharide-Lyases; Xanthomonas

Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent T

Topics: Amino Acid Sequence; Catalysis; Catalytic Domain; Cations; Kinetics; Molecular Dynamics Simulation; Mutagenesis; Pectins; Penicillium; Polygalacturonase; Protein Conformation; Thermotolerance

Cell adhesion occurs primarily at the level of middle lamella which is mainly composed by pectin polysaccharides. These can be degraded by cell wall degrading enzymes (CWDEs) during developmental processes to allow a controlled separation of plant cells. Extensive cell wall degradation by CWDEs with consequent cell separation is performed when protoplasts are isolated from plant tissues by using mixtures of CWDEs. We have evaluated whether modification of pectin affects cell separation and protoplast isolation. Arabidopsis plants overexpressing the pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2, and Arabidopsis pme3 plants, mutated in the gene encoding pectin methylesterase 3, showed an increased efficiency of isolation of viable mesophyll protoplasts as compared with Wild Type Columbia-0 plants. The release of protoplasts was correlated with the reduced level of long stretches of de-methylesterified homogalacturonan (HGA) present in these plants. Response to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplasts.

Topics: Arabidopsis; Carboxylic Ester Hydrolases; Cell Wall; Esterification; Mesophyll Cells; Pectins; Protoplasts; Stress, Physiological; Transfection

A novel pectate lyase of a deep subseafloor bacterium, Georgenia muralis strain JAM 3H7-3 (JCM19733), was purified to homogeneity from a culture broth by an anion exchange chromatography, followed by heat treatment of the enzyme solution at 60 °C for 30 min, and a gel filtration in the presence of SDS. The purified enzyme (Pel-S2) had a molecular mass of ~51 kDa by SDS-PAGE and ~75 kDa by gel filtration. In contrast, without heat treatment, the purified enzyme in SDS sample buffer was found to consist of 23- and 23.5-kDa polypeptides by SDS-PAGE. The enzyme was gradually inactivated by heat treatment with and without SDS in parallel with a shift of polypeptides molecular masses from 23 and 23.5 to 51 kDa on SDS-PAGE. Pel-S2 degraded pectate optimally at pH 10 in a glycine buffer and temperature of 50 °C. The enzyme showed relatively broad substrate specificity toward pectic acid and pectin.

Topics: DNA, Bacterial; Glycine; Gram-Positive Bacteria; Hot Temperature; Hydrogen-Ion Concentration; Pectins; Peptides; Polysaccharide-Lyases; Protein Structure, Tertiary; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Substrate Specificity; Water Microbiology

Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.

Topics: Amino Acid Sequence; Bacillus; Bacterial Proteins; Boehmeria; Cloning, Molecular; DNA Primers; DNA, Bacterial; Enzyme Stability; Escherichia coli; Half-Life; Hydrogen-Ion Concentration; Molecular Sequence Data; Pectins; Plant Gums; Polysaccharide-Lyases; Sequence Alignment; Substrate Specificity; Temperature

The composition of homogalacturonans (HGs) in the ovule and the female gametophyte cell walls was shown to be rearranged dynamically during sexual reproduction of H. orientalis. In angiosperms, homogalacturonans (HGs) play an important role in the interaction between the male gametophyte and the pistil transmitting tract, but little is known about the participation of these molecules at the final stage of the progamic phase and fertilization. The aim of our study was to perform immunocytochemical localization of highly (JIM7 MAb) and weakly (JIM5 MAb) methyl esterified and Ca(2+)-associated HG (2F4 MAb) in the ovule and female gametophyte cells of Hyacinthus orientalis before and after fertilization. It was found that pollination induced the rearrangement of HG in (1) the micropylar canal of the ovule, (2) the filiform apparatus of the synergids, and (3) the region of fusion between sperm cells and their target cells. Fertilization led to further changes in pectin composition of these three regions of the ovule. A new cell wall was synthesized around the zygote with a characteristic pattern of localization of all examined HG fractions, which we called "sporoderm-like". The developing endosperm prepared for cellularization by synthesizing highly methyl-esterified HG, which was stored in the cytoplasm. Pollination- and fertilization-induced changes in the composition of the HG in the micropyle of the ovule and the apoplast of female gametophyte cells are discussed in the context of: (1) micropylar pollen tube guidance, (2) preparation of the egg cell and the central cells for fusion with sperm cells, and (3) the polyspermy block.

Topics: Endosperm; Hyacinthus; Immunohistochemistry; Microscopy, Fluorescence; Ovule; Pectins; Pollen; Pollen Tube; Pollination; Seeds; Time Factors

Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60 °C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca(2+) was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96 IU mg(-1) by the addition of 0.1 mM Ca(2+), whereas the K m values were decreased from 0.681 to 0.514 mg ml(-1). Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16 %) to modest (27.37 %) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site.

Topics: Amino Acid Substitution; Calcium; Catalytic Domain; DNA Mutational Analysis; DNA, Fungal; Enzyme Activators; Enzyme Stability; Glycosylation; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Pectins; Polysaccharide-Lyases; Protein Sorting Signals; Sequence Analysis, DNA; Temperature; Volvariella

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

Topics: Bacillus subtilis; Bacterial Proteins; Calcium; Catalytic Domain; Cloning, Molecular; Enzyme Stability; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Inclusion Bodies; Kinetics; Pectins; Plasmids; Polysaccharide-Lyases; Protein Binding; Protein Conformation; Protein Refolding; Recombinant Proteins; Solubility; Temperature; Urea

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48-/- pollen grains. In contrast, the PME activity was lower in pme48-/-, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48-/- with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca(2+) necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

Topics: Arabidopsis; Arabidopsis Proteins; Calcium; Carboxylic Ester Hydrolases; Culture Media; Esterification; Gene Expression Regulation, Plant; Germination; Homozygote; Mutation; Organ Specificity; Pectins; Phenotype; Pollen; Pollen Tube; Reverse Transcriptase Polymerase Chain Reaction

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy.

Topics: Antibodies; Arabidopsis; Arabidopsis Proteins; Biomechanical Phenomena; Carboxylic Ester Hydrolases; Cell Size; Esterification; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Mutagenesis, Insertional; Pectins; Phenotype; Plant Mucilage; Plants, Genetically Modified; Seeds

Visible-light responsive gels were prepared from two plant-origin polyuronic acids (PUAs), alginate and pectate, coordinated to Fe(III) ions. Comparative quantitative studies of the photochemistry of these systems revealed unexpected differences in the photoreactivity of the materials, depending on the polysaccharide and its composition. The roles that different functional groups play on the photochemistry of these biomolecules were also examined. Mannuronic-rich alginates were more photoreactive than guluronic acid-rich alginate and than pectate. The microstructure of alginates with different mannuronate-to-guluronate ratios changed with polysaccharide composition. This influenced the gel morphology and the photoreactivity. Coordination hydrogel beads were prepared from both Fe-alginate and Fe-pectate. The beads were stable carriers of molecules as diverse as the dye Congo Red, the vitamin folic acid, and the antibiotic chloramphenicol. The photoreactivity of the hydrogel beads mirrored the photoreactivity of the polysaccharides in solution, where beads prepared with alginate released their cargo faster than beads prepared with pectate. These results indicate important structure-function relationships in these systems and create guidelines for the design of biocompatible polysaccharide-based materials where photoreactivity and controlled release can be tuned on the basis of the type of polysaccharide used and the metal coordination environment.

Topics: Alginates; Delayed-Action Preparations; Glucuronic Acid; Hexuronic Acids; Hydrogels; Iron; Light; Materials Testing; Pectins; Polysaccharides

Hyaluronic acid-based hydrogels can reduce postoperative adhesion. However, the long-term application of hyaluronic acid is limited by tissue mediated enzymatic degradation. To overcome this limitation, we developed a polygalacturonic acid and hyaluronate composite hydrogel by Schiff's base crosslinking reaction. The polygalacturonic acid and hyaluronate composite hydrogels had short gelation time (less than 15 s) and degraded by less than 50 % in the presence of hyaluronidase for 7 days. Cell adhesion and migration assays showed polygalacturonic acid and hyaluronate composite hydrogels prevented fibroblasts from adhesion and infiltration into the hydrogels. Compared to hyaluronate hydrogels and commercial Medishield™ gels, polygalacturonic acid and hyaluronate composite hydrogel was not totally degraded in vivo after 4 weeks. In the rat laminectomy model, polygalacturonic acid and hyaluronate composite hydrogel also had better adhesion grade and smaller mean area of fibrous tissue formation over the saline control and hyaluronate hydrogel groups. Polygalacturonic acid and hyaluronate composite hydrogel is a system that can be easy to use due to its in situ cross-linkable property and potentially promising for adhesion prevention in spine surgeries.

Topics: Adjuvants, Immunologic; Animals; Compressive Strength; Dura Mater; Hardness; Hyaluronic Acid; Hydrogels; Male; Pectins; Rats; Rats, Sprague-Dawley; Tissue Adhesions; Treatment Outcome

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

Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30min and substrate concentration is 5g/L. For polygalacturonase, the sample should be adjusted to have 0.3-0.8U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor×0.687U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined.

Topics: Aspergillus niger; Biotechnology; Citrus; Enzyme Assays; Fermentation; Kinetics; Pectins; Polygalacturonase; Reproducibility of Results; Substrate Specificity; Trichoderma

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

While it is kno3wn that complex tissues with specialized functions emerged during land plant evolution, it is not clear how cell wall polymers and their structural variants are associated with specific tissues or cell types. Moreover, due to the economic importance of many flowering plants, ferns have been largely neglected in cell wall comparative studies.. To explore fern cell wall diversity sets of monoclonal antibodies directed to matrix glycans of angiosperm cell walls have been used in glycan microarray and in situ analyses with 76 fern species and four species of lycophytes. All major matrix glycans were present as indicated by epitope detection with some variations in abundance. Pectic HG epitopes were of low abundance in lycophytes and the CCRC-M1 fucosylated xyloglucan epitope was largely absent from the Aspleniaceae. The LM15 XXXG epitope was detected widely across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection in ground tissues. Mannan epitopes were found to be associated with the development of mechanical tissues. We provided the first evidence for the presence of MLG in leptosporangiate ferns.. The data sets indicate that cell wall diversity in land plants is multifaceted and that matrix glycan epitopes display complex spatio-temporal and phylogenetic distribution patterns that are likely to relate to the evolution of land plant body plans.

Topics: Antibodies, Monoclonal; Cell Wall; Epitopes; Ferns; Fluorescent Antibody Technique, Indirect; Galactans; Glucans; Mannans; Microarray Analysis; Organ Specificity; Pectins; Phloem; Phylogeny; Plant Extracts; Polysaccharide-Lyases; Polysaccharides; Xylans

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human β1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.

Topics: Arabidopsis; Cell Wall; Epitopes; Galactosyltransferases; Genetic Engineering; Glycosylation; Humans; Hydrogen Peroxide; Mucoproteins; N-Acetyllactosamine Synthase; Nicotiana; Pectins; Phenotype; Plant Leaves; Plant Proteins; Plants, Genetically Modified; Polysaccharides; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The structural role of pectins in plant primary cell walls is not yet well understood because of the complex and disordered nature of the cell wall polymers. We recently introduced multidimensional solid-state nuclear magnetic resonance spectroscopy to characterize the spatial proximities of wall polysaccharides. The data showed extensive cross peaks between pectins and cellulose in the primary wall of Arabidopsis (Arabidopsis thaliana), indicating subnanometer contacts between the two polysaccharides. This result was unexpected because stable pectin-cellulose interactions are not predicted by in vitro binding assays and prevailing cell wall models. To investigate whether the spatial contacts that give rise to the cross peaks are artifacts of sample preparation, we now compare never-dried Arabidopsis primary walls with dehydrated and rehydrated samples. One-dimensional (13)C spectra, two-dimensional (13)C-(13)C correlation spectra, water-polysaccharide correlation spectra, and dynamics data all indicate that the structure, mobility, and intermolecular contacts of the polysaccharides are indistinguishable between never-dried and rehydrated walls. Moreover, a partially depectinated cell wall in which 40% of homogalacturonan is extracted retains cellulose-pectin cross peaks, indicating that the cellulose-pectin contacts are not due to molecular crowding. The cross peaks are observed both at -20 °C and at ambient temperature, thus ruling out freezing as a cause of spatial contacts. These results indicate that rhamnogalacturonan I and a portion of homogalacturonan have significant interactions with cellulose microfibrils in the native primary wall. This pectin-cellulose association may be formed during wall biosynthesis and may involve pectin entrapment in or between cellulose microfibrils, which cannot be mimicked by in vitro binding assays.

Topics: Arabidopsis; Carbon-13 Magnetic Resonance Spectroscopy; Cell Wall; Cellulose; Desiccation; Magnetic Resonance Spectroscopy; Pectins; Proton Magnetic Resonance Spectroscopy; Time Factors; Water

Low-methylesterified homogalacturonans have been suggested to play a role in the binding and immobilization of Pb in CW. Using root apices of hybrid aspen, a plant with a high phytoremediation potential, as a model, we demonstrated that the in situ distribution pattern of low-methylesterified homogalacturonan, pectin epitope (JIM5-P), reflects the pattern of Pb occurrence. The region which indicated high JIM5-P level corresponded with "Pb accumulation zone". Moreover, JIM5-P was especially abundant in cell junctions, CWs lining the intercellular spaces and the corners of intercellular spaces indicating the highest accumulation of Pb. Furthermore, JIM5-P and Pb commonly co-localized. The observations indicate that low-methylesterified homogalacturonan is the CW polymer that determines the capacity of CW for Pb sequestration. Our results suggest a promising directions for CW modification for enhancing the efficiency of plant roots in Pb accumulation, an important aspect in the phytoremediation of soils contaminated with trace metals.

Topics: Antibodies, Monoclonal; Biodegradation, Environmental; Biomarkers; Esterification; Lead; Pectins; Plant Roots; Populus; Soil Pollutants

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.

Topics: Arabidopsis; Arabidopsis Proteins; Binding Sites; Carboxylic Ester Hydrolases; Enzyme Inhibitors; Hydrogen-Ion Concentration; Hypocotyl; Molecular Docking Simulation; Multiprotein Complexes; Pectins; Recombinant Proteins; Substrate Specificity

The effects of plant products on cancer cells has become a field of major importance. Many substancesmay induce apoptosis in anti-cancer treatment. Pectins, a family of complex polysaccharides, and their degradation products may for exasmple exert apoptotic effects in cancer cells. Apples and citrus fruits are the main sources of pectin which can be applied for anti-cancer research. The present study concerned an intact form of pectic-oligoshaccharide named pectic acid (poly galactronic acid).. Inhibition of cell proliferation assays (MTT), light microscopy, fluorescence microscopy (acridin orange/ethidium bromide), DNA fragmentation tests, cell cycle analysis, annexin PI and Western blotting methods were applied to evaluate apoptosis.. The results indicated that pectic acid inhibited cell growth and reduced cell attachment after 24h incubation. This did not appear to be due to necrosis, since morphological features of apoptosis were detected with AO/EB staining and cell cycling was blocked in the sub-G1 phase. Annexin/PI and DNA fragmentation findings indicated that apoptosis frequency increased after 24h incubation with pectic acid. In addition, the data showed pectic acid induced caspase-dependent apoptosis.. These data indicate that apple pectic acid without any modification could trigger apoptosis in MDA-MB-231 human breast cancer cells and has potential to improve cancer treatment as a natural product.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Female; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Malus; Pectins

Our previous study isolated an anti-fatigue polysaccharide (HRWP) from the Hippophae rhamnoides berry. In this study, using ion-exchange chromatography and gel filtration chromatography in turn, a water-soluble homogenous polysaccharide HRWP-A was isolated from HRWP. Structural analysis determined that HRWP-A was a polysaccharide with repeating units of (1→4)-β-d-galactopyranosyluronic residues, of which 85.16% were esterified with methyl groups. An antitumor activity assay showed that HRWP-A could significantly inhibit the Lewis lung carcinoma (LLC) growth in tumor-bearing mice. Further experiments suggested that the antitumor effect of HRWP-A might be mediated through immunostimulating activity, as it enhances the lymphocyte proliferation, augments the macrophage activities, as well as promoting NK cell activity and CTL cytotoxicity in tumor-bearing mice. To our knowledge, this is the first report on a natural antitumor high-methoxyl homogalacturonan pectin from the H. rhamnoides berry-a compound that acts as a potential immunostimulant and anticancer adjuvant.

Topics: Animals; Antineoplastic Agents; Body Weight; Carbon-13 Magnetic Resonance Spectroscopy; Carcinoma, Lewis Lung; Concanavalin A; Cytotoxicity, Immunologic; Fruit; Hippophae; Immunologic Factors; Killer Cells, Natural; Lipopolysaccharides; Macrophages; Male; Mice, Inbred C57BL; Nitric Oxide; Pectins; Phagocytosis; Proton Magnetic Resonance Spectroscopy; Spectroscopy, Fourier Transform Infrared; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha

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

In the root apoplasm, V(V) and V(IV) toxicity can be alleviated through redox and complexation reactions involving phenolic substances and the polyuronic components. In such context we report the role of polygalacturonic acid (PGA) on the reducing activity of caffeic acid (CAF) towards V(V). The redox reaction was particularly effective at pH 2.8 leading to the formation of oxidation products with redox activity towards V(V). An o-quinone was identified as the first product of the reaction which is further involved in the formation of CAF dimers. At pH ≥ 3.6 the redox activity decreased and a yield in V(IV) equal to 38, 31, 21 and 14% was found at pH 3.6, 4.0. 5.0 and 6.0 respectively compared with that obtained at pH 2.8. The redox reaction was faster in the presence of PGA and a higher yield of V(IV) was found in the 4.0-6.0 pH range with respect to the CAF-V(V) binary system. The higher efficiency of the redox reaction in the presence of PGA was related with the ability of PGA to bind V(IV). The biological significance of the redox reaction between CAF and V(V), as well as the role of PGA in such reaction, was established "in vivo" using triticale plants. Results showed that PGA reduced significantly the phytotoxic effects of the V(V)-CAF system.

Topics: Caffeic Acids; Environmental Pollutants; Environmental Pollution; Inactivation, Metabolic; Oxidation-Reduction; Pectins; Plant Roots; Plants; Vanadates

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.

Topics: Botrytis; Cell Membrane; Culture Media; Cytoplasmic Vesicles; Gene Expression; Gene Expression Profiling; Gene Knockout Techniques; Genome, Fungal; Hexuronic Acids; Membrane Transport Proteins; Nicotiana; Pectins; Real-Time Polymerase Chain Reaction; Solanum lycopersicum; Virulence

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

Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2-day-old culture filtrates by gel filtration and ion-exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate-polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide mass fingerprinting (matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) fragment ion analysis). The pattern of substrate degradation analyzed by thin layer chromatography confirmed the mode of action of the enzyme as an endopolygalacturonase. High activity of the polygalacturonase against polygalacturonic acid was observed between 4 and 6 of pH, and between 30 and 50 °C, being 5 and 50 °C the optimum pH and temperature, respectively. The enzyme was fully stable at pH 5 for 120 min and 30 °C and sensible to the presence of some metal ions. This information would contribute to understand the most favorable environmental conditions for establishment of the disease.

Topics: Argentina; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Fusarium; Hydrogen-Ion Concentration; Molecular Weight; Pectins; Plant Diseases; Polygalacturonase; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Triticum

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1-99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5-10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64-81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75-90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis.

Topics: Animals; Bacterial Adhesion; Bacterial Load; Caco-2 Cells; Cecum; Cell Line; Chickens; Dietary Fiber; Dietary Supplements; Enterocytes; Epithelial Cells; Humans; Ileum; Intestinal Mucosa; Liver; Pectins; Plantago; Polysaccharides; Poultry Diseases; Salmonella enteritidis; Salmonella typhimurium; Spleen; Swine

Pectin methylesterases (PMEs) hydrolyze the methylester groups that are found on the homogalacturonan (HG) chains of pectic polysaccharides in the plant cell wall. Plant and bacterial PMEs are especially interesting as the resulting de-methylesterified (carboxylated) sugar residues are found to be arranged contiguously, indicating a so-called processive nature of these enzymes. Here we report the results of continuum electrostatics calculations performed along the molecular dynamics trajectory of a PME-HG-decasaccharide complex. In particular it was observed that, when the methylester groups of the decasaccharide were arranged in order to mimic the just-formed carboxylate product of de-methylesterification, a net unidirectional sliding of the model decasaccharide was subsequently observed along the enzyme's binding groove. The changes that occurred in the electrostatic binding energy and protein dynamics during this translocation provide insights into the mechanism by which the enzyme rectifies Brownian motions to achieve processivity. The free energy that drives these molecular motors is thus demonstrated to be incorporated endogenously in the methylesterified groups of the HG chains and is not supplied exogenously.

Topics: Biocatalysis; Biochemical Phenomena; Carboxylic Ester Hydrolases; Erwinia; Hydrolysis; Models, Molecular; Motion; Pectins; Static Electricity; Substrate Specificity; Thermodynamics

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

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.

Topics: Calcium; Cell Adhesion; Cell Wall; Cellulose; Charophyceae; Edetic Acid; Epitopes; Microarray Analysis; Models, Biological; Pectins; Polygalacturonase; Polysaccharide-Lyases

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

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km = 14.7 ± 7.6 mg mL(-1). Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km = 2.2 ± 0.5 mg mL(-1). XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km = 7.4 ± 2.0 mg mL(-1)) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.

Topics: Agaricales; Endo-1,4-beta Xylanases; Fungal Proteins; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Pectins

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50°C. It is stable in the range pH 5.0-7.0 and below 50°C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, (1)H NMR of polymer and LC-HILIC-MS(n) after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.

Topics: Acetylation; Bacillus; Enzyme Stability; Esterases; Esterification; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Pectins; Substrate Specificity; Temperature

Studies on angiosperm plants have shown that homogalacturonan present in the extracellular matrix of pistils plays an important role in the interaction with the male gametophyte. However, in gymnosperms, knowledge on the participation of HG in the pollen-ovule interaction is limited, and only a few studies on male gametophytes have been reported. Thus, the aim of this study was to determine the distribution of HG in male gametophytes and ovules during their interaction in Larix decidua Mill. The distribution of HG in pollen grains and unpollinated and pollinated ovules was investigated by immunofluorescence techniques using monoclonal antibodies that recognise high methyl-esterified HG (JIM7), low methyl-esterified HG (JIM5) and calcium cross-linked HG (2F4). All studied categories of HG were detected in the ovule. Highly methyl-esterified HG was present in the cell walls of all cells throughout the interaction; however, the distribution of low methyl-esterified and calcium cross-linked HG changed during the course of interaction. Both of these categories of HG appeared only in the apoplast and the extracellular matrix of the ovule tissues, which interact with the male gametophyte. This finding suggests that in L. decidua, low methyl-esterified and calcium cross-linked HG play an important role in pollen-ovule interaction. The last category of HG is most likely involved in adhesion between the pollen and the ovule and might provide an optimal calcium environment for pollen grain germination and pollen tube growth.

Topics: Antibodies, Monoclonal; Cell Wall; Epitopes; Esterification; Extracellular Matrix; Fluorescent Antibody Technique; Larix; Ovule; Pectins; Pollen; Pollen Tube; Pollination

The crystallinity, allomorph content, and mesoscale ordering of cellulose produced by Gluconacetobacter xylinus cultured with different plant cell wall matrix polysaccharides were studied with vibrational sum frequency generation (SFG) spectroscopy and X-ray diffraction (XRD). Crystallinity and ordering were assessed as the intensity of SFG signals in the CH/CH2 stretch vibration region (and confirmed by XRD), while Iα content was assessed by the relative intensity of the OH stretch vibration at 3240 cm(-1). A key finding is that the presence of xyloglucan in the culture medium greatly reduced Iα allomorph content but with a relatively small effect on cellulose crystallinity, whereas xylan resulted in a larger decrease in crystallinity with a relatively small decrease in the Iα fraction. Arabinoxylan and various pectins had much weaker effects on cellulose structure as assessed by SFG and XRD. Homogalacturonan with calcium ion reduced the SFG signal, evidently by changing the ordering of cellulose microfibrils. We propose that the distinct effects of matrix polysaccharides on cellulose crystal structure result, at least in part, from selective interactions of the backbone and side chains of matrix polysaccharides with cellulose chains during the formation of the microfibril.

Topics: Carbohydrate Conformation; Cell Wall; Cellulose; Crystallization; Crystallography, X-Ray; Glucans; Gluconacetobacter xylinus; Pectins; Plant Cells; Vibration; Xylans

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

Two natural homogalacturonan (HG) pectins (MW ca. 20 kDa) were isolated from green tea based on their immunomodulatory activity. The crude tea polysaccharides (TPS1 and TPS2) were obtained from green tea leaves by hot water extraction and followed by 40% and 70% ethanol precipitation, respectively. Two homogenous water soluble polysaccharides (TPS1-2a and TPS1-2b) were obtained from TPS1 after purification with gel permeation, which gave a higher phagocytic effect than TPS2. A combination of composition, methylation and configuration analyses, as well as NMR (nuclear magnetic resonance) spectroscopy revealed that TPS1-2a and TPS1-2b were homogalacturonan (HG) pectins consisting of a backbone of 1,4-linked α-D-galacturonic acid (GalA) residues with 28.4% and 26.1% of carboxyl groups as methyl ester, respectively. The immunological assay results demonstrated that TPS1-2, which consisted mainly of HG pectins, showed phagocytosis-enhancing activity in HL-60 cells.

Topics: Cell Line; Humans; Immunologic Factors; Methylation; Pectins; Phagocytosis; Tea

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.. Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).. Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.. The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.

Topics: Antibodies, Monoclonal; Cell Wall; Epitopes; Esterification; Glucans; Glycoproteins; Immunohistochemistry; Mucoproteins; Orobanchaceae; Pectins; Plant Proteins; Polysaccharides; Xylans; Xylem

Duckweed is potentially an ideal biofuel feedstock due to its high proportion of cellulose and starch and low lignin content. However, there is little detailed information on the composition and structure of duckweed cell walls relevant to optimising the conversion of duckweed biomass to ethanol and other biorefinery products. This study reports that, for the variety and batch evaluated, carbohydrates constitute 51.2% (w/w) of dry matter while starch accounts for 19.9%. This study, for the first time, analyses duckweed cell wall composition through a detailed sequential extraction. The cell wall is rich in cellulose and also contains 20.3% pectin comprising galacturonan, xylogalacturonan, rhamnogalacturonan; 3.5% hemicellulose comprising xyloglucan and xylan, and 0.03% phenolics. In addition, essential fatty acids (0.6%, α-linolenic and linoleic/linoelaidic acid) and p-coumaric acid (0.015%) respectively are the most abundant fatty acids and phenolics in whole duckweed.

Topics: Araceae; Cell Wall; Cellulose; Chemical Fractionation; Pectins; Polysaccharides

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

A bio-inspired coating consisting of pectin (polygalacturonic acid) and cationic cellulose nanofibers were successfully produced by the layer-by-layer method. The build-up and the morphology of the resulting coatings were studied with spectroscopic ellipsometry and atomic force microscopy, respectively. The coating was able to survive the exposure of a simulated gastric fluid, but was partially degraded upon exposure to pectinase enzyme, which simulate the action of the microbial symbionts present in the human colon. Prior to exposure, the oxygen permeability coefficient of the coating (0.033 ml(STP)mmm(-2)day(-1)atm(-1) at 23°C and 20% RH) was in the same order of magnitude as for ethylene vinyl alcohol films (0.001-0.01 ml(STP)mmm(-2)day(-1)atm(-1)). However, after exposure to the mimicked gastrointestinal (GI) tract conditions, the contribution of coating to the overall barrier properties was not measurable.

Topics: Cellulose; Drug Delivery Systems; Nanofibers; Oxygen; Pectins; Surface Properties

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

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

Topics: Arabidopsis; Cell Wall; Chitin; Desmidiales; Extracellular Matrix; Metal Nanoparticles; Microarray Analysis; Microscopy, Electron, Transmission; Molecular Probes; Molecular Structure; Oligosaccharides; Optical Imaging; Pectins; Plant Root Cap

In this work, novel modified nanoclays were used to mineralize hydroxyapatite (HAP) mimicking biomineralization in bone. This in situ HAPclay was further incorporated into chitosan/polygalacturonic acid (Chi/PgA) scaffolds and films for bone tissue engineering. Differences in microstructure of the scaffolds were observed depending on the changes in processing of in situ HAPclay with ChiPgA biopolymer system. Response of human mesenchymal stem cells (hMSCs) on these scaffolds and films was studied using imaging and assays. SEM micrographs indicate that hMSCs were able to adhere to ChiPgA/in situ HAPclay scaffolds and phase contrast images indicated formation of mineralized nodules on ChiPgA/in situ HAPclay films in absence of osteogenic supplements used for differentiation of hMSCs. The formation of mineralized nodules by hMSCs was confirmed by positive staining of the nodules by Alizarin Red S dye. Viability and differentiation assays showed that ChiPgA/in situ HAPclay scaffolds were favorable for viability and differentiation of hMSCs. Unique two-stage cell seeding experiments were performed as a strategy to enhance tissue formation by hMSCs on ChiPgA/in situ HAPclay composite films. This work showed that biomaterials based on ChiPgA/in situ HAPclay composites can be used for bone tissue engineering applications and in situ nanoclay-HAP system mediates osteoinductive and osteoconductive response from hMSCs.

Topics: Alkaline Phosphatase; Bentonite; Biocompatible Materials; Biomimetic Materials; Biopolymers; Cell Differentiation; Cells, Cultured; Chitosan; Durapatite; Extracellular Matrix; Humans; Materials Testing; Mesenchymal Stem Cells; Minerals; Nanostructures; Osteogenesis; Pectins; Tissue Scaffolds

We previously demonstrated that pectin-protein complex (PPC) isolated from white cabbage adsorbs the β-glucuronidase (βG) enzyme of E. coli. Concurrently, we discovered a significant increase in βG activity in the presence of PPC. The aim of this study is to identify the structural components of PPC that are responsible for βG adsorption and activation. PPC was isolated from white cabbage using a saline solution containing hydrochloric acid (pH 1.5) at 37 °C for 4 h. PPC proteins were precipitated by aqueous 10% (m/v) trichloroacetic acid to yield the pectin-protein fractions PPC1 and PPC2. PPC was digested using 1,4-α-d-galacturonase, yielding the PPC6 fraction. Partial acid hydrolysis of PPC revealed the galacturonan fraction, PPC3, to be the core of the macromolecule. The purified PPC4 and PPC5 fractions were isolated from PPC by ion-exchange chromatography on DEAE-cellulose. βG activity and its adsorption in the PPC fractions were studied in vitro. Crystalline cellulose was used as a control. This study found that the PPC3 fraction (the galacturonan core) does not adsorb βG and does not affect its activity. The adsorption of βG in the PPC samples is inversely proportional to the degree of methyl esterification of its carbohydrate component. The PPC4 and PPC5 fractions adsorb the highest proportion of βG (51.2% and 54%, respectively). The stimulation of βG enzyme activity is directly proportional to the protein content of the PPC sample. The PPC and PPC1 samples have the greatest ability to increase βG activity (57.6% and 52.1%, respectively).

Topics: Adsorption; Brassica; Chemical Precipitation; Enzyme Activation; Escherichia coli; Glucuronidase; Hydrolysis; Pectins; Peptide Fragments; Plant Proteins

Polygalacturonic acid (PGA) hydrogel cross-linked via disulfide bonds was synthesized using a thiol oxidation reaction. PGA was grafted with cysteine to yield thiolated PGA (denoted PGAcys). Per gram, PGA-conjugated cysteine was 725 ± 77 μmol, and the degree of modification was 16.24 %. A PGAcys hydrogel film was fabricated under physiological conditions, with gel content 91.6 % and water content 43.3 %. The PGAcys hydrogel was used as a drug carrier for rosmarinic acid (RA) (denoted PGAcys/RA) and to prevent postsurgical adhesion. The in vitro dynamic release behavior of RA from the PGAcys hydrogel was analyzed. The profiles showed that 80 % of the total RA was released from the hydrogel within 15 min, followed by zero-order kinetic release. Animal implant studies showed that PGAcys and PGAcys/RA hydrogel films reduced adhesion incidence by over 90 %, significantly higher than did Hyaluronate/Carboxymethylcellulose (analogous Seprafilm™) (42 %). The PGAcys/RA hydrogel film also reduced the early inflammatory reaction.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cinnamates; Cross-Linking Reagents; Depsides; Diffusion; Disulfides; Drug Implants; Hydrogels; Materials Testing; Pectins; Rats; Rats, Sprague-Dawley; Rosmarinic Acid; Tissue Adhesions; Treatment Outcome

The effect of poly-L-lysine (PLL) molecular weight and structure on pH stability of polygalacturonic acid (PGaLA)-based multilayer films is studied over a pH cycle 7.0-1.6-7.0. The multilayer assembled with the lowest molecular weight PLL (1 kDa) showed the largest pH response. Only 12% of the mass remained and a preferential loss of PLL was observed. Extensive structural reorganisation of the layer as the pH was increased was due to the PGaLA reionisation leading to extensive net loss of hydrated mass. The multilayers assembled with the higher molecular weight linear PLLs (10 kDa, 200 kDa) showed loss of about 50% of their initial polymer mass. The multilayer assembled with the dendrimer (22 kDa) showed a stronger response to pH compared to the linear higher molecular weight PLLs. Over the pH cycle a loss of about 60% polymer mass and a decrease in the film thickness was observed. Despite having a reduced density at pH 1.6, the density substantially recovered to 0.54 g mL(-1) on return to pH 7.0.

Topics: Hydrogen-Ion Concentration; Molecular Structure; Pectins; Polylysine; Spectroscopy, Fourier Transform Infrared; Stereoisomerism

The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the role of dynamics of substrates in the enzymatic activity of PME from Erwinia chrysanthemi, a processive enzyme that catalyzes the hydrolysis of methylester groups from the galacturonic acid residues of homogalacturonan chains, the major component of pectin. Extensive molecular dynamics simulations of this PME in complex with decameric homogalacturonan chains possessing different degrees and patterns of methylesterification show how the carbohydrate substitution pattern governs the dynamics of the substrate in the enzyme's binding cleft, such that substrate dynamics represent a key prerequisite for the PME biological activity. The analyses reveal that correlated rotations around glycosidic bonds of monosaccharide subunits at and immediately adjacent to the active site are a necessary step to ensure substrate processing. Moreover, only substrates with the optimal methylesterification pattern attain the correct dynamical behavior to facilitate processive catalysis. This investigation is one of the few reported examples of a process where the dynamics of a substrate are vitally important.

Topics: Amino Acid Sequence; Binding Sites; Carbohydrate Sequence; Carboxylic Ester Hydrolases; Erwinia; Molecular Dynamics Simulation; Molecular Sequence Data; Monosaccharides; Pectins; Substrate Specificity

A galacturonan fragment was obtained from a 38% DM commercial apple pectin by endopolygalacturonase digestion followed by separation using ion exchange chromatography. By NMR, MALDI-TOF MS and chemical analysis the structure of this oligomer was found to be a tetramer of galacturonic acid with a single methyl ester on the GalA next to the reducing end residue. Assignment of all the (13)C and (1)H chemical shifts for this oligomer will be helpful in determining the structure of more complex pectin fragments.

Topics: Esterification; Pectins

To enable structural characteristics of individual cell wall polysaccharides from rapeseed (Brassica napus) meal (RSM) to be studied, polysaccharide fractions were sequentially extracted. Fractions were analysed for their carbohydrate (linkage) composition and polysaccharide structures were also studied by enzymatic fingerprinting. The RSM fractions analysed contained pectic polysaccharides: homogalacturonan in which 60% of the galacturonic acid residues are methyl-esterified, arabinan branched at the O-2 position and arabinogalactan mainly type II. This differs from characteristics previously reported for Brassica campestris meal, another rapeseed cultivar. Also, in the alkali extracts hemicelluloses were analysed as xyloglucan both of the XXGG- and XXXG-type decorated with galactosyl, fucosyl and arabinosyl residues, and as xylan with O-methyl-uronic acid attached. The final residue after extraction still contained xyloglucan and remaining (pectic) polysaccharides next to cellulose, showing that the cell wall matrix of RSM is very strongly interconnected.

Topics: Brassica napus; Carbohydrate Sequence; Cell Wall; Chromatography, Ion Exchange; Enzyme Assays; Galactans; Glucans; Hexuronic Acids; Molecular Sequence Data; Pectins; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Xylans

Two homogeneous water-soluble polysaccharides (TPSR4-2B and TPSR4-2C) were obtained from preinfused green tea. Their average molecular weights were estimated to be 41 kDa and 28 kDa, respectively. A combination of composition, methylation, and configuration analysis, as well as NMR spectroscopy, indicated that both TPSR4-2B and TPSR4-2C were poly-(1-4)-α-d-galactopyranosyluronic acid in which 30.5 ± 0.3% and 28.3 ± 0.5%, respectively, of uronic acid existed as methyl ester. Two sulfated derivatives (Sul-R4-2B and Sul-R4-2C) from TPSR4-2B and TPSR4-2C were prepared after sulfation with a 2:1 chlorosulfonic acid-pyridine ratio. The anticomplementary assay showed that Sul-R4-2B and Sul-R4-2C demonstrated a stronger inhibitory effect on the complement activation through the classic pathway, compared to that of heparin. Preliminary mechanism studies by using complement component depleted-sera indicated that both Sul-R4-2B and Sul-R4-2C selectively interact with C1q, C1r, C1s, C2, C5, and C9 but not with C3 and C4. The relationship between DS and the anticomplementary activity of sulfated derivatives of homogalacturonans showed that low sulfated derivatives of homogalacturonans also exhibited potent anticomplementary effect, which might greatly reduce the side effects related to heparin and oversulfated chondroitin sulfate, such as anticoagulant activity and allergic-type reaction. These results suggested that sulfated derivatives of homogalacturonans might be promising drug candidates for therapeutic complement inhibition.

Topics: Animals; Camellia sinensis; Complement Activation; Complement Inactivator Proteins; Complement System Proteins; Humans; Molecular Structure; Pectins; Plant Extracts; Sheep; Tea

In this study, raw wheat bran (R-WB), a type of waste biomass (WB) was treated with Pectinase PL (P-WB), and the properties (yield percentage, carboxy group surface concentration, the solution pH, and specific surface area) of R-WB and P-WB were investigated. The surface concentration of carboxy groups on R-WB (3.56 mmol/g) was greater than that of P-WB (2.11 mmol/g). In contrast, the specific surface area of P-WB (24.98 m²/g) was greater than that of R-WB (3.25 m²/g). In addition, the adsorption of cadmium and lead ions to WB was evaluated. Adsorption of the heavy-metal ions reached equilibrium within 9 h, and the experimental data was fitted to a pseudo-second-order model. More heavy-metal ions were adsorbed onto R-WB than onto P-WB. The correlation coefficient between the amount of ions adsorbed and the number of carboxy groups or pectin exceeded 0.884 and 0.975, respectively. This study indicated that wheat bran was useful for the removal of cadmium or lead ions from aqueous solutions. The adsorption mechanism of cadmium and lead ions to WB was associated with presence of carboxy group in pectin.

Topics: Adsorption; Cadmium; Carboxylic Acids; Chemical Phenomena; Dietary Fiber; Food Handling; Ions; Lead; Pectins; Polygalacturonase; Solutions; Time Factors; Waste Products; Wastewater; Water; Water Purification

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca²⁺) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue-tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.

Topics: Calcium; Carboxylic Ester Hydrolases; Cell Wall; Esterification; Fruit; Gene Expression; Organ Specificity; Pectins; Plant Epidermis; Plant Proteins; Polygalacturonase; Solanum lycopersicum

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS-PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0-5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3h. The addition of Mn(2+), K(+), Zn(2+), Ca(2+) and Al(3+) inhibited the enzyme activity; it increased in the presence of Mg(2+) and Cu(2+) ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K(m) and V(max) values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T(650) from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.

Topics: Aspergillus niger; Chromatography, Gel; Copper; Enzyme Stability; Fruit; Fungal Proteins; Hydrogen-Ion Concentration; Kinetics; Magnesium; Malus; Molecular Weight; Pectins; Phenols; Plant Extracts; Polygalacturonase; Psidium; Substrate Specificity; Sulfhydryl Compounds

The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.

Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Biomechanical Phenomena; Carboxylic Ester Hydrolases; Cell Size; Cell Wall; Enzyme Activation; Esterification; Flowers; Gene Expression Regulation, Plant; Genes, Plant; Germination; Microscopy, Electron, Scanning; Pectins; Phenotype; Plants, Genetically Modified; Seeds; Uronic Acids

We examined the redox effects of UV irradiation on cell wall isolates from Pisum sativum leaves, and polygalacturonic and galacturonic acid, in the presence of hydrogen peroxide. For this purpose, electron paramagnetic resonance spectroscopy and two spin-traps (DEPMPO and BMPO), capable of differentiating between various free radicals, were applied. Systems were exposed to UV-B (maximum emission at 312 nm) and UV-A (352 nm) for 10 min (6 J m(-2) s(-1)). Cell wall isolates exposed to UV in the presence of hydrogen peroxide, produced hydroxyl radical, carbon dioxide radical and superoxide. The production of superoxide was observed for cell wall isolates, polygalacturonic acid (in the presence and in the absence of calcium) and galacturonic acid, and it was diminished upon superoxide dismutase supplementation. The production is at least partially based on the reaction of hydroxyl radicals with (poly)galacturonic acid having carbon dioxide radicals as a products. Acting as a strong reducing agent, carbon dioxide radical reacts with molecular oxygen to produce superoxide. The results presented here shed a new light on: (1) the redox-modulating role of cell wall; (2) the production of superoxide in the extracellular compartment; (3) the mechanisms involved in translating UV stress into molecular signaling and (4) some other UV-related phenomena in plants, such as CO(2) emission.

Topics: Cell Wall; Oxidation-Reduction; Pectins; Pisum sativum; Pyrroles; Spin Labels; Superoxides; Ultraviolet Rays

A cost-effective biosorbent was prepared by a green chemical modification process from muskmelon peel by saponification with alkaline solution of Ca(OH)2. Its adsorption behavior for lead ions was investigated and found to exhibit excellent adsorption properties. Results showed that the optimal equilibrium pH range for 100% adsorption is from 4 up to 6.4. Adsorption equilibrium was attained within 10 min. The adsorption process can be described well by Langmuir model and pseudo-second-order kinetics equation, respectively. The maximum adsorption capacity for lead ions was found to be 0.81 mol/kg. Pectic acid contained in the muskmelon peel is the main factor responsible for the uptake of lead ions onto the gel, and the chemical modification process presented in this study can be assumed effective to prepare other similar biomaterials. The large adsorption capacity and the fast adsorption rate indicated that chemically saponified muskmelon peel gel in present study has great potential to be used as a cost-effective adsorbent for the removal of lead ions from the water.

Topics: Adsorption; Calcium Hydroxide; China; Cucumis melo; Drinking Water; Fruit; Hydrogen-Ion Concentration; Kinetics; Lead; Pectins; Solutions; Temperature; Water Pollutants, Chemical; Water Purification

In contrast to the damaging effects of high doses, low dose radiation (UV, gamma) has been reported to provoke constructive changes in plants. However, the mechanisms by which plants recognize and respond to low dose radiation are not understood. We have shown recently that polygalacturonic acid, cell wall polysaccharide, converts the highly reactive product of radiation - hydroxyl radical into superoxide which may be further dismutated to hydrogen peroxide. Superoxide has been proposed to act as a signaling molecule, while hydrogen peroxide is known to be the key species in redox signaling cascades which are involved in the regulation of various physiological processes. Hence we propose that polygalacturonic acid may operate as radiation-signaling convertor. The outlined principles of radiation-sensing could also be valid for mammalian cells, with some other molecules mediating the conversion.

Topics: Hydroxyl Radical; Oxidation-Reduction; Pectins; Signal Transduction; Superoxides

The interactions between procyanidins and pectic compounds are of importance in food chemistry. Procyanidins with low (9) and high (30) average degrees of polymerization (DP9 and DP30) were extracted from two cider apple varieties. Commercial apple and citrus pectins, as well as three pectin subfractions (homogalacturonans, partially methylated homogalacturonans with degree of methylation 30 and 70) at 30 mM galacturonic acid equivalent, were titrated with the two procyanidin fractions (at 30 mM (-)-epicatechin equivalent) by isothermal titration calorimetry and UV-vis spectrophotometry. Slightly stronger affinities were recorded between commercial apple or citrus pectins and procyanidins of DP30 (Ka = 1460 and 1225 M(-1) respectively, expressed per monomer units) compared to procyanidins of DP9 (Ka = 1240 and 1085 M(-1), respectively), but stoichiometry and absorbance maxima differed between apple and citrus pectins. It was proposed that methylated homogalacturonans interacted with procyanidins DP30 mainly through hydrophobic interactions. The stronger association was obtained with the longer procyanidin molecules interacting with highly methylated pectins.

Topics: Catechin; Citrus; Flavonoids; Food Handling; Fruit; Hexuronic Acids; Malus; Methylation; Pectins; Polymerization; Proanthocyanidins

Imbibed seeds of the Arabidopsis thaliana accession Djarly are affected in mucilage release from seed coat epidermal cells. The impaired locus was identified as a pectin methylesterase inhibitor gene, PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), specifically expressed in seed coat epidermal cells at the time when mucilage polysaccharides are accumulated. This spatio-temporal regulation appears to be modulated by GLABRA2 and LEUNIG HOMOLOG/MUCILAGE MODIFIED1, as expression of PMEI6 is reduced in mutants of these transcription regulators. In pmei6, mucilage release was delayed and outer cell walls of epidermal cells did not fragment. Pectin methylesterases (PMEs) demethylate homogalacturonan (HG), and the majority of HG found in wild-type mucilage was in fact derived from outer cell wall fragments. This correlated with the absence of methylesterified HG labeling in pmei6, whereas transgenic plants expressing the PMEI6 coding sequence under the control of the 35S promoter had increased labeling of cell wall fragments. Activity tests on seeds from pmei6 and 35S:PMEI6 transgenic plants showed that PMEI6 inhibits endogenous PME activities, in agreement with reduced overall methylesterification of mucilage fractions and demucilaged seeds. Another regulator of PME activity in seed coat epidermal cells, the subtilisin-like Ser protease SBT1.7, acts on different PMEs, as a pmei6 sbt1.7 mutant showed an additive phenotype.

Topics: Arabidopsis; Arabidopsis Proteins; Carboxylic Ester Hydrolases; Cell Wall; Esterification; Methylation; Mutation; Pectins; Phenotype; Plant Epidermis; Plant Mucilage; Plants, Genetically Modified; Seeds; Subtilisins

Pulp from peaches contained polygalacturonic acid and arabinogalactan as main polysaccharides, which were isolated and characterised. The polygalacturonic acid (AE-CWI) contained 95% GalA and its (13)C NMR spectrum showed signals at δ 98.9, 78.0, 71.4, 69.1, 68.4, and 175.1 from C-1, C-4, C-5, C-3, C-2, and C-6 respectively, from (1→4)-linked α-GalpA units. Methylation-MS analysis of carboxy-reduced material (AE-CWI-CR) gave 90% of 2,3,6-Me(3)-galactitol acetate. The arabinogalactan (AE-AG) was composed mainly of Ara (41%) and Gal (50%) and was characterised (methylation analysis and (13)C NMR) as a type II-arabinogalactan. It induced peritoneal macrophage activation in mice, ~70% of cells treated with this fraction (1-50 μg/mL) having morphology of activated cells. However, NO production in macrophages treated with AE-AG was not affected. This suggests a new biological activity for peach polysaccharides.

Topics: Animals; Galactans; Macrophages, Peritoneal; Male; Mice; Molecular Structure; Pectins; Plant Extracts; Prunus

A quartz crystal microbalance with dissipation monitoring (QCMD) has been used to monitor the adsorption and structure of lysozyme monolayers and multilayers, and poly-L-lysine (PLL)-polygalacturonic acid (PGalA) multilayers at a solid-liquid interface using freshly-cleaved mica as a substrate. QCMD measurements were complemented with atomic force microscopy (AFM). AFM images revealed that lysozyme formed incomplete monolayers and provided a basis for calculation of the thickness of the protein film. Comparative studies of adsorption onto standard and mica-coated quartz crystals showed higher areal mass adsorption and a longer-time adsorption process for mica-coated quartz crystals. Simultaneous AFM images and QCMD data were obtained for lysozyme, linear PLL-PGalA and 7 nm PLL dendrimer-PGalA multilayers. The layer-by-layer deposited multilayer films exhibited viscoelastic properties and their growth followed a non-linear regime, associated with the PLL diffusion in and out of the film formation for linear PLL-PGalA films. For the PLL 7 nm dendrimer-PGalA films the AFM images revealed marked changes in surface roughness during layer by layer deposition: these changes influence the interpretation of the QCMD data and provide additional information on the growth and structure of the multilayers.

Topics: Animals; Chickens; Microscopy, Atomic Force; Muramidase; Pectins; Polylysine; Quartz Crystal Microbalance Techniques

Pectic acids participate in the transport of heavy metal ions in the root apoplasm by establishing interactions that can lead to their partial or total immobilization. The ions accumulated can be mobilized by phenolic compounds and organic acids of the root exudates. In this context, we tested, in aqueous phase, the ability of malic acid and esculetine (ESC) to mobilize the Cu(II) ions accumulated in a Ca-polygalacturonate matrix (Ca-PGA) used as a model of the root apoplasm. The results show that at pH 5.0 and 6.0 malic acid mobilizes about 22% and 34% of the Cu(II) accumulated, respectively, whereas ESC about 12% and 25%. ESC was found to cause the reduction of Cu(II) to Cu(I) with formation of ESC oxidation products. The study of the Cu(II)-ESC binary system evidenced that one molecule of ESC reduces one Cu(II) ion with formation of semiquinonic radicals that couple to form two dimers. The Cu(II) reduction by ESC was found faster in the presence of malic acid.

Topics: Biopolymers; Calcium; Copper; Malates; Molecular Structure; Oxidation-Reduction; Pectins

Condensed tannins are a group of polyphenols that are associated with the astringency sensation, as they readily interact and precipitate salivary proteins. As this interaction is affected by carbohydrates, the aim of this work was to study the effect of some carbohydrates used in the food industry [arabic gum (AG), pectin, and poligalacturonic acid (PGA)] on the salivary proteins/grape seed procyanidins interaction. This was assessed monitoring the salivary proteins that remain soluble in the presence of condensed tannins with the addition of carbohydrates (HPLC) and analysis of the respective precipitates (SDS-PAGE). The results show that pectin was the most efficient in inhibiting protein/tannin precipitation, followed by AG and PGA. The results suggest that pectin and PGA exert their effect by formation of a ternary complex protein/polyphenol/carbohydrate, while AG competes with proteins for tannin binding (competition mechanism). The results also point out that both hydrophilic and hydrophobic interactions are important for the carbohydrate effects.

Topics: Down-Regulation; Gum Arabic; Humans; Pectins; Proanthocyanidins; Protein Binding; Salivary Proteins and Peptides

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

Topics: Biological Transport; Calcium; Cell Size; Cell Wall; Chara; Egtazic Acid; Pectins

This study examined the potential involvement of polygalacturonase (PG) in commercial pickling cucumbers on gradual softening of refrigerated and pasteurized fresh pack pickle products that adversely affects their market-life. PG activity was detected in all sources of commercial pickling cucumbers and refrigerated pickle spears, and activity was detected in some sources of pasteurized spears. Polygalacturonic acid hydrolysis determined from changes in fluidity, reducing groups and oligogalacturonides indicated involvement of both exo- and endo-PG. Treatment of pasteurized pickle mesocarp tissues with concentrated extracts from pickling cucumbers resulted in rapid softening and alteration of pectic substances. D-values for thermal inactivation of pickling cucumber PG at 75, 80, 85, and 90 °C were 22.0, 19.5, 14.5, and 4.2 min, respectively, indicating that residual activity would be expected in commercially pasteurized pickle products. It is concluded that residual cucumber PG could be responsible for softening of processed pickle products, and it is suspected that variations in textural quality of given products are caused by differences in residual types and levels of native PG.. Since enzyme extracts from commercial pickling cucumbers were demonstrated to be capable of softening pickle tissues and that PG activity was present in some processed fresh pack products, methods that inactivate or eliminate the enzyme(s) should reduce softening of products during storage and marketing.

Topics: Chemical Phenomena; Cucumis sativus; Enzyme Stability; Food Packaging; Food, Preserved; Fruit; Hot Temperature; Hydrolysis; Kinetics; Mechanical Phenomena; Missouri; Oligosaccharides; Pasteurization; Pectins; Plant Proteins; Polygalacturonase; Quality Control; Refrigeration

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

Topics: Methyl Ethers; Models, Molecular; Pectins; Polygalacturonase

Using successive extractions with water and 0.7% aqueous ammonium oxalate, pectic polysaccharides were isolated from the following plants growing in the arid climate of Mongolia (Gobi): saxaul Haloxylon ammodendron Maxim., rhubarb Rheum nanum Sievers, Nitraria sibirica Pall., Peganum harmala L. and almond Amygdalus mongolica Maxim. The data obtained exhibited the primary synthesis of the cell wall pectic polysaccharides but not the middle lamellae water-soluble pectins in plants growing in the dry climatic zone. Both α-(1→4)-D-galacturonan and α-(1→4)-D-galacturonan, which was substituted with methyl groups, were found to be backbone of pectins. The L-arabinofuranose residues were identified as the main components of ramified regions. The pectins from almond differed from other pectins due to a high arabinose content. The data from NMR spectroscopy and methylation analyses demonstrated that pectic polysaccharides from almond included terminal, (1→5)-, (1→3)-linked and 3,5-substituted L-arabinofuranose residues and a small terminal D-galactopyranose and 2,5- and 2,3,5-substituted L-arabinofuranose residue content. The pectic polysaccharides were found to decrease the absorption of ovalbumin (OVA) in the blood from the gut lumen. The serum OVA level was lower in mice fed with OVA mixed with the pectins compared with the control group, which was administered OVA alone.

Topics: Administration, Oral; Animals; Arabinose; Cell Wall; Desert Climate; Galactose; Hydrolysis; Intestinal Absorption; Magnetic Resonance Spectroscopy; Methylation; Mice; Mongolia; Ovalbumin; Pectins; Plants; Species Specificity

Secondary cell walls, which contain lignin, have traditionally been considered essential for the mechanical strength of the shoot of land plants, whereas pectin, which is a characteristic component of the primary wall, is not considered to be involved in the mechanical support of the plant. Contradicting this conventional knowledge, loss-of-function mutant alleles of Arabidopsis thaliana PECTIN METHYLESTERASE35 (PME35), which encodes a pectin methylesterase, showed a pendant stem phenotype and an increased deformation rate of the stem, indicating that the mechanical strength of the stem was impaired by the mutation. PME35 was expressed specifically in the basal part of the inflorescence stem. Biochemical characterization showed that the activity of pectin methylesterase was significantly reduced in the basal part of the mutant stem. Immunofluorescence microscopy and immunogold electron microscopy analyses using JIM5, JIM7, and LM20 monoclonal antibodies revealed that demethylesterification of methylesterified homogalacturonans in the primary cell wall of the cortex and interfascicular fibers was suppressed in the mutant, but lignified cell walls in the interfascicular and xylary fibers were not affected. These phenotypic analyses indicate that PME35-mediated demethylesterification of the primary cell wall directly regulates the mechanical strength of the supporting tissue.

Topics: Arabidopsis; Arabidopsis Proteins; Carboxylic Ester Hydrolases; Cell Wall; Genetic Complementation Test; Inflorescence; Molecular Sequence Data; Mutation; Pectins; Phenotype; Plant Stems; Stress, Mechanical

The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome.

Topics: Arabidopsis; Cell Wall; Host-Pathogen Interactions; Immunohistochemistry; Lipopolysaccharides; Pectins; Plant Diseases; Plant Epidermis; Plant Roots; Ralstonia solanacearum; Seedlings; Xylem

By using surface plasmon resonance (SPR) technology, the kinetics of the interaction of various fungal endopolygalacturonases (EPGs) (13 EPGs) with Phaseolus vulgaris (bean) PGIP2 was carried out to determine whether or not there is any interaction between polygalacturonases-inhibiting protein (PGIP) and EPG. The effect of polygalacturonic acid (PGA) on these interactions was also evaluated. The results show that all EPGs evaluated bind to PGIP2, except for AnPGb and the strength of the interaction depends on the EPG/PGIP2 pairing. Further, the presence of PGA has a moderate to strong effect on the EPG/PGIP2 interaction and the strength of the effect is dependent on the exact EPG/PGIP2 pairing. The differences in affinity in the absence and presence of PGA, suggest a certain level of cooperativity. These results indicate a three-component complex similar to that observed for the heparin-ATIII-thrombin, the FGF-FGFR-heparin, or the hedgehog-interference hedgehog-heparan complexes. This data points to an architecture in which the inhibitor binds at a location distant from the substrate binding site. Furthermore, we applied differential proteolysis mass spectrometry (DPMS) to study the location of the binding site between EPG and PGIP2. DPMS studies indicate that PGIP2 does not bind AnPGII, AnPGa, and AnPGc directly over the active site but instead binds on the face opposite to the active site, creating an allosteric interaction.

Topics: Fungi; Kinetics; Mass Spectrometry; Pectins; Phaseolus; Plant Proteins; Polygalacturonase; Protein Binding; Protein Interaction Mapping; Proteolysis; Surface Plasmon Resonance

A water-soluble polysaccharide CSPS-2B-2 with a molecular mass of 8.8 kDa, was obtained from the fruits of Capparis spinosa L. Chemical and NMR spectral analysis verified CSPS-2B-2 was a linear poly-(1-4)-α-D-galactopyranosyluronic acid in which 12.9±0.4% of carboxyl groups existed as methyl ester and 2.6±0.1% of D-GalpA residues were acetylated. A sulfated derivative Sul-2B-2 with a sulfation degree of 0.88±0.02 was prepared via the substitution of C-2 and/or C-3 of GalpA residues in CSPS-2B-2. Bioassay on the complement and coagulation system demonstrated that Sul-2B-2 (CH(50): 3.5±0.2 μg/mL) had a stronger inhibitory effect on the activation of complement system through the classic pathway than that of heparin (CH(50): 8.9±0.3 μg/mL). Interestingly, Sul-2B-2 at low dose even middle dose (for example 52 μg/mL) had no effect on coagulation system, which was totally different from heparin. Thus, our observation indicated that Sul-2B-2 was more efficient than heparin in inhibiting the activation of the complement system through classical pathway and exhibiting a relatively less anti-coagulant activity. These results suggested that the sulfated derivative Sul-2B-2 prepared from the homogalacturonan in the fruits of Capparis spinosa L, might be a promising drug candidate in case of necessary therapeutic complement inhibition.

Topics: Animals; Anticoagulants; Blood Coagulation; Capparis; Chromatography; Complement Inactivating Agents; Complement System Proteins; Erythrocytes; Fruit; Heparin; Magnetic Resonance Spectroscopy; Pectins; Plant Extracts; Rabbits; Sheep; Sulfuric Acid Esters

• Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. • Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. • Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. • This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.

Topics: Animals; Arabidopsis; Cell Wall; Epitopes; Esterification; Feeding Behavior; Female; Giant Cells; Glucans; Mannans; Pectins; Plant Diseases; Plant Roots; Polysaccharides; Tylenchoidea; Xylans; Xylem

Accumulating evidence shows that hydrogen sulfide (H(2)S) plays various physiological roles in plants, such as seed germination, root organogenesis, abiotic stress tolerance, and senescence of cut flowers. However, whether H(2)S participates in the regulation of ripening and senescence in postharvest fruits remains unknown. In the present study, the effect of H(2)S on postharvest shelf life and antioxidant metabolism in strawberry fruits was investigated. Fumigation with H(2)S gas released from the H(2)S donor NaHS prolonged postharvest shelf life of strawberry fruits in a dose-dependent manner. Strawberry fruits fumigated with various concentrations of H(2)S sustained significantly lower rot index, higher fruit firmness, and kept lower respiration intensity and polygalacturonase activities than controls. Further investigation showed that H(2)S treatment maintained higher activities of catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase and lower activities of lipoxygenase relative to untreated controls. H(2)S also reduced malondialdehyde, hydrogen peroxide, and superoxide anion to levels below control fruits during storage. Moreover, H(2)S treatment maintained higher contents of reducing sugars, soluble proteins, free amino acid, and endogenous H(2)S in fruits. We interpret these data as indicating that H(2)S plays an antioxidative role in prolonging postharvest shelf life of strawberry fruits.

Topics: Antioxidants; Carbohydrates; Food Preservation; Food Preservatives; Fragaria; Fruit; Hydrogen Sulfide; Pectins; Plant Proteins; Time Factors

Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1), was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246). Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

Topics: Arabidopsis; Arabidopsis Proteins; Arabinose; Cell Adhesion; Cloning, Molecular; Galactose; Golgi Apparatus; Pectins

Pectate (polygalacturonic acid) acts as a chelator to bind calcium and form cross-links that hold adjacent pectate polymers and thus plant cell walls together. When under tension from turgor pressure in the cell, the cross-links appear to distort and weaken. New pectate supplied by the cytoplasm is undistorted and removes wall calcium preferentially from the weakened bonds, loosening the wall and accelerating cell expansion. The new pectate now containing the removed calcium can bind to the wall, strengthening it and linking expansion to wall deposition. But new calcium needs to be added as well to replenish the calcium lost from the vacated wall pectate.  A recent report demonstrated that growth was disrupted if new calcium was unavailable.  The present addendum highlights this conclusion by reviewing an experiment from before the chelation chemistry was understood. Using cell wall labeling, a direct link appeared between wall expansion and wall deposition. Together, these experiments support the concept that newly supplied pectate has growth activity on its way to deposition in the wall. Growth rate is thus controlled by signals affecting the rate of pectate release. After release, the coordination of expansion and deposition arises naturally from chelation chemistry when polymers are under tension from turgor pressure. 

Topics: Cell Wall; Chara; Pectins

Plant-parasitic cyst nematodes form a specialized feeding site, termed a syncytium, in the roots of host plants. Monoclonal antibodies to defined glycans, in addition to a cellulose-binding module, were used to characterize the cell walls of a functioning syncytia in situ. Cell walls of syncytia were found to contain cellulose, xyloglucan and mannan. Analysis of the pectin network revealed syncytial cell walls are abundant in homogalacturonan, which was heavily methyl-esterified. Arabinan was also detected and the results suggest the cell walls of syncytia are highly flexible.

Topics: Arabidopsis; Cell Wall; Cellulose; Giant Cells; Glucans; Mannans; Pectins; Plant Roots; Xylans

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N(2)-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N(2) fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N(2)-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.

Topics: Bacterial Proteins; Biomass; Bradyrhizobium; Brazil; DNA, Intergenic; Epitopes; Fabaceae; Likelihood Functions; Microscopy, Electron, Transmission; Nitrogen; Oxidoreductases; Pectins; Phylogeny; Plant Roots; RNA, Ribosomal, 16S; Soil; Soil Microbiology; Time Factors

Clarification of citrus juice is a severe quality defect related to pectin methylesterase (PME) activity. PME activity and calcium chelation of pectic acid as well as other physical interactions of cloud components influence cloud stability. The cloud stability and physical properties of pulp-free, fresh juice with and without ammonium oxalate (AO) at pH 4.0 and pH 5.5 was evaluated.. The only juice to clarify in the 3-week study was the sample without AO at pH 4.0. Particle size analysis showed that the samples at pH 4.0 were larger than those at pH 5.5, and samples at pH 5.5 had a more negative zeta potential than samples at pH 4.0. Furthermore, cloud particle size increased and then decreased prior to the onset of clarification.. Ammonium oxalate prevented sedimentation via calcium pectate cross-bridges and subsequent clarification. Interaction of cloud constituents, change in particle size with pH and change in particle size with storage time suggest that, in addition to electrostatic attraction and calcium binding, cloud particles associate and dissociate via non-covalent, non-electrostatic interactions.

Topics: Beverages; Calcium; Citrus sinensis; Food Technology; Fruit; Hydrogen-Ion Concentration; Oxalates; Particle Size; Pectins; Solubility

The ultimate goal of bone tissue engineering is to develop bony tissues on tissue engineered constructs that mimic the native bone. Nanoscale characterization of in vitro generated bony tissues on engineered scaffolds is essential to understanding both the physical and mechanical characteristics of the engineered bone. Bone nodule formation, a typical early indicator of bone formation was observed on chitosan-polygalacturonic acid-hydroxyapatite (Chi-PgA-HAP) nanocomposite films without the use of differentiating media. Thus, the Chi-PgA-HAP substrates designed are osteoinductive and provide an appropriate microenvironment for cell organization and tissue regeneration. Imaging using atomic force microscopy revealed several levels of hierarchical structures of bone in the bone nodules, consisting of mineralized collagen fibers, fibrils and mineral deposits in extrafibrillar spaces. The nanoscale elastic properties of the collagen and mineral crystals were found to be in close agreement with the experimental and simulations results on natural bone reported in the literature. Fourier transform infrared spectroscopy experiments indicate the presence of collagen and biological apatite in bone nodules exhibiting the characteristics of newly precipitated, immature bone. Collectively, our structural, chemical, and mechanical analyses support the conclusion that synthetic bone nodules mimic the hierarchy of natural bone.

Topics: Bone and Bones; Chitosan; Durapatite; Microscopy, Atomic Force; Nanocomposites; Osteoblasts; Pectins; Spectroscopy, Fourier Transform Infrared; Tissue Engineering

In this report, the localization and spatial distribution of two categories of pectin, high and low methylesterified, on the background of dynamic in loosely bound calcium (Ca(2+)) in Haemanthus hollow style were studied before and after pollination. In the style transmitting tract of unpollinated pistil, mainly high-methylesterified pectins were present, both in the transmitting tract epidermis and in the style canal. After pollination, an increase in the level of two investigated categories of pectin was observed, but the amount of high-methylesterified one in each period of time analyzed was permanently higher. Locally, in the regions of the style canal penetrated by pollen tubes, process of pectin de-esterification was initiated. However, pollination caused an increase of loosely bound Ca(2+) level in the style transmitting tract, this process appears to be not linked with pectin de-esterification and possible Ca(2+) release after the lysis of Ca(2+) cross-linked de-esterified pectin. Instead, it seems to be based on Ca(2+) exocytosis from the transmitting tract epidermis cells providing a source of Ca(2+) for pollen tubes growing in Haemanthus hollow style.

Topics: Calcium; Esterification; Exocytosis; Flowers; Immunohistochemistry; Liliaceae; Pectins; Plant Epidermis; Pollen; Pollination; Vacuoles

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.

Topics: Cycadopsida; Larix; Mucoproteins; Ovule; Pectins; Plant Proteins

The effect of shading on xylem hydraulic traits and xylem anatomy was studied in hybrid poplar (Populus trichocarpa x deltoides, clone H11-11). Hydraulic measurements conducted on stem segments of 3-month-old saplings grown in shaded (SH) or control light (C) conditions indicated that shading resulted in more vulnerable and less efficient xylem. Air is thought to enter vessels through pores in inter-vessel pit membranes, thereby nucleating cavitation. Therefore, we tested if the ultrastructure and/or chemistry of pit membranes differed in SH and C plants. Transmission electron micrographs revealed that pit membranes were thinner in SH, which was paralleled by lower compound middle lamella thickness. Immunolabelling with JIM5 and JIM7 monoclonal antibodies surprisingly indicated that pectic homogalacturonans were not present in the mature pit membrane regardless of the light treatment. Porosity measurements conducted with scanning electron microscopy were significantly affected by the method used for sample dehydration. Drying through a gradual ethanol series seems to be a better alternative to drying directly from a hydrated state for pit membrane observations in poplar. Scanning electron microscopy based estimates of pit membrane porosity probably overestimated real porosity as suggested by the results from the 'rare pit' model.

Topics: Cell Membrane; Cell Wall; Light; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Pectins; Plant Leaves; Populus; Porosity; Xylem

The effect of pH on the stability of layer-by-layer deposited polygalacturonic acid (PGalA)-based multilayer films prepared with the polycations poly-L-lysine, chitosan, and lysozyme is studied. The response was characterized using a quartz crystal microbalance, dual polarization interferometry, and Fourier transform infrared spectroscopy which probe multilayer thickness, density, polymer mass (composition and speciation), and hydration. All multilayers showed irreversible changes in response to pH change becoming thinner due to the partial disassembly. Preferential loss of the polycation (50-80% w/w) and relative small losses of PGaLA (10-35% w/w) occurred. The charge density on the polycation has a strong influence on the response to the acid cycle. Most of the disassembly takes place at the pH lower that pK(a) of PGaLA, indicating that this factor was crucial in determining the stability of the films. The pH challenge also revealed a polycation-dependent shift to acid pH in the PGaLA pK(a).

Topics: Chitosan; Hydrogen-Ion Concentration; Membranes, Artificial; Muramidase; Pectins; Polylysine

The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase.

Topics: Aspergillus; Base Sequence; Batch Cell Culture Techniques; Galactose; Genetic Vectors; Isoelectric Point; Molecular Sequence Data; Pectins; Polygalacturonase; Saccharomyces cerevisiae

Symptom development of Pierce's disease (PD) in grapevine (Vitis vinifera) depends largely on the ability of the bacterium Xylella fastidiosa to use cell wall-degrading enzymes (CWDEs) to break up intervessel pit membranes (PMs) and spread through the vessel system. In this study, an immunohistochemical technique was developed to analyze pectic and hemicellulosic polysaccharides of intervessel PMs. Our results indicate that PMs of grapevine genotypes with different PD resistance differed in the composition and structure of homogalacturonans (HGs) and xyloglucans (XyGs), the potential targets of the pathogen's CWDEs. The PMs of PD-resistant grapevine genotypes lacked fucosylated XyGs and weakly methyl-esterified HGs (ME-HGs), and contained a small amount of heavily ME-HGs. In contrast, PMs of PD-susceptible genotypes all had substantial amounts of fucosylated XyGs and weakly ME-HGs, but lacked heavily ME-HGs. The intervessel PM integrity and the pathogen's distribution in Xylella-infected grapevines also showed differences among the genotypes. In pathogen-inoculated, PD-resistant genotypes PM integrity was well maintained and Xylella cells were only found close to the inoculation site. However, in inoculated PD-susceptible genotypes, PMs in the vessels associated with bacteria lost their integrity and the systemic presence of the X. fastidiosa pathogen was confirmed. Our analysis also provided a relatively clear understanding of the process by which intervessel PMs are degraded. All of these observations support the conclusion that weakly ME-HGs and fucosylated XyGs are substrates of the pathogen's CWDEs and their presence in or absence from PMs may contribute to grapevine's PD susceptibility.

Topics: Coated Pits, Cell-Membrane; Genotype; Glucans; Immunity, Innate; Microscopy, Electron, Scanning; Pectins; Plant Diseases; Plant Immunity; Polysaccharides; Vitis; Xylans; Xylella

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ∼10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).

Topics: Biocatalysis; Carboxylic Ester Hydrolases; Citrus; Enzyme Stability; Hot Temperature; Hydrogen-Ion Concentration; Models, Chemical; Models, Theoretical; Molecular Structure; Pectins; Plant Proteins

Polygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition by polygalacturonase-inhibiting proteins (PGIPs) from tomato stems. In cultures of wild-type strain ToUdk2, slimy (s), retarded slimy (rs) and non-slimy (ns) colonies appeared. The conversion of the 's' into the 'rs' colony form coincided with the beginning of PG production. PG activity of the PC strain increased about 5 h earlier (at 6 hpi), and was up to 35 times higher in media supplemented with two different tomato stem extracts or polygalacturonic acid, compared to the wild-type at 6 hpi, and generally 4-8 times higher across test media and time. By hydrophobic interaction chromatography (HIC), fluorophor-assisted carbohydrate-polyacrylamid-gel electrophoresis (FACE-PAGE) and mass spectrometry analyses, endo-PG PehA, exo-PGs PehB and PehC were identified. PGs of the PC mutant consisted mainly of endo-PG. The increased PG production after supplementing the medium with tomato cell wall extract was reflected by a higher activity of exo-PGs for both strains. Total PGs (endo-PG and exo-PGs) activities were inhibited by PGIPs of tomato stem extracts. PGIP activity was concentration dependent, constitutively present, and not related to resistance nor susceptibility of tomato recombinant inbred lines to R. solanacearum. The proteinaceous character of the inhibiting component was inferred from ammonium sulphate precipitation. For the first time a plant PGIP activity against a bacterial pathogen is reported. Observations support that endo- and exo-PG synthesis is governed by a sensitive regulatory network, which, in interaction with PGIP and cell wall degradation products, leads to generation or avoidance of elicitor-active oligomers, and, thus, may contribute to the development of the compatible or incompatible interaction.

Topics: Bacterial Proteins; Cell Wall; Host-Pathogen Interactions; Mutation; Pectins; Plant Diseases; Plant Immunity; Plant Proteins; Plant Stems; Polygalacturonase; Ralstonia solanacearum; Solanum lycopersicum

Plant cell walls are complex structures that offer structural and mechanical support to plant cells and are ultimately responsible for plant architecture and form. Pectins are a large group of complex polysaccharides of the plant cell wall that are made in the Golgi and secreted to the wall. The methylesterification of pectins is believed to be an important factor for the dynamic properties of the cell wall. Here, we report on a protein of unknown function discovered using an extensive proteomics analysis of cotton Golgi. Through bioinformatic analyses, we identified the ortholog of such protein, here named cotton Golgi-related 3 (CGR3) in Arabidopsis and found that it shares conserved residues with S-adenosylmethionine methyltransferases. We established that CGR3 is localized at the Golgi apparatus and that the expression of the CGR3 gene is correlated with that of several cell wall biosynthetic genes, suggesting a possible role of the protein in pectin modifications. Consistent with this hypothesis, immunofluorescence microscopy with antibodies for homogalacturonan pectins (HG) indicated that the cell walls of cgr3 knockout mutants and plants overexpressing CGR3 are decreased and increased in HG methylesterification, respectively. Our results suggest that CGR3 plays a role in the methylesterification of homogalacturonan in Arabidopsis.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Cloning, Molecular; Esterification; Golgi Apparatus; Molecular Sequence Data; Pectins; Protein Transport

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

Abscission zones (AZ) are sites where leaves and other organs are shed. Investigating the AZ by classical biochemical techniques is difficult due to its small size and because the surrounding tissue is not involved in abscission. The goals of this study were to determine whether AZ cell walls are chemically unique from the other cells of the petiole, perhaps making them more susceptible to enzymatic degradation during abscission and to identify which cell wall polysaccharides are degraded during abscission.. A battery of antibodies that recognize a large number of cell wall polysaccharide and glycoprotein epitopes was used to probe sections of the Impatiens leaf AZ at several time points in the abscission process.. Prior to abscission, the walls of the AZ cells were found to be similar in composition to the walls of the cells both proximal and distal to the AZ. Of all the epitopes monitored, only the highly de-esterified homogalacturonans (HG) of the middle lamellae were found to be reduced post-abscission and only at the plane of separation. More highly esterified homogalacturonans, as well as other pectin and xyloglucan epitopes were not affected. Furthermore, cellulose, as detected by an endoglucanase-gold probe and cellulose-binding module staining, was unaffected, even on the walls of the cells facing the separation site.. In the leaf abscission zone of Impatiens, wall alterations during abscission are strictly limited to the plane of separation and involve only the loss of highly de-esterified pectins from the middle lamellae.

Topics: Cell Wall; Cellulose; Esterification; Glucans; Impatiens; Pectins; Plant Leaves; Polysaccharides; Xylans

A polysaccharide, PGA4-3b, with an average molecular weight of 8.9kDa estimated by high-performance gel-permeation chromatography (HPGPC), was isolated from radix of Platycodon grandiflorum (Jacq.) A. DC. Using monosaccharide analysis, methylation analysis and NMR spectroscopy, PGA4-3b was elucidated to be a linear poly-(1→4)-α-d-galactopyranosyluronic acid that contains no methyl ester groups. Partial acid hydrolysis of PGA4-3b yielded a series of poly- or oligogalacturonic acids with different degrees of polymerization (DP), that is, 4-3bde, 4-3bde-O-1, 4-3bde-O-2, 4-3bde-O-3, and 4-3bde-O-4. Cell tube formation inhibition tests with human microvascular endothelial cells (HMEC) for antiangiogenesis analysis showed that 4-3bde-O-1 and 4-3bde-O-2, the fractions with higher molecular weights, could inhibit tube formation, while the native PGA4-3b and low molecular weight fraction 4-3bde-O-3 and 4-3bde-O4 are ineffective. Moreover, 4-3bde-O-2 with DP 5-10 impaired cell tube formation in a dose-dependent way, suggesting its potential to be developed as an anti-angiogenesis drug. This is the first time oligogalacturonic acids are reported to show an anti-angiogenesis effect.

Topics: Angiogenesis Inhibitors; Cell Line; Humans; Magnetic Resonance Spectroscopy; Neovascularization, Physiologic; Oligosaccharides; Pectins; Platycodon

Pectins are complex polysaccharides that are essential components of the plant cell wall. In this study, a novel putative Arabidopsis S-adenosyl-L-methionine (SAM)-dependent methyltransferase, termed QUASIMODO 3 (QUA3, At4g00740), has been characterized and it was demonstrated that it is a Golgi-localized, type II integral membrane protein that functions in methylesterification of the pectin homogalacturonan (HG). Although transgenic Arabidopsis seedlings with overexpression, or knock-down, of QUA3 do not show altered phenotypes or changes in pectin methylation, this enzyme is highly expressed and abundant in Arabidopsis suspension-cultured cells. In contrast, in cells subjected to QUA3 RNA interference (RNAi) knock-down there is less pectin methylation as well as altered composition and assembly of cell wall polysaccharides. Taken together, these observations point to a Golgi-localized QUA3 playing an essential role in controlling pectin methylation and cell wall biosynthesis in Arabidopsis suspension cell cultures.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Cell Wall; Cells, Cultured; Evolution, Molecular; Golgi Apparatus; Methylation; Methyltransferases; Molecular Sequence Data; Pectins; Phylogeny; Protein Transport; Sequence Alignment

Beta-expansins accumulate to high levels in grass pollen, a feature apparently unique to grasses. These proteins, which are major human allergens, facilitate pollen tube penetration of the maize stigma and style (the silk). Here we report that treatment of maize silk cell walls with purified β-expansin from maize pollen led to solubilization of wall matrix polysaccharides, dominated by feruloyated highly substituted glucuronoarabinoxylan (60%) and homogalacturonan (35%). Such action was selective for cell walls of grasses, and indicated a target preferentially found in grass cell walls, probably the highly substituted glucuronoarabinoxylan. Several tests for lytic activities by β-expansin were negative and polysaccharide solubilization had weak temperature dependence, which indicated a non-enzymatic process. Concomitant with matrix solubilization, β-expansin treatment induced creep, reduced the breaking force and increased the plastic compliance of wall specimens. From comparisons of the pH dependencies of these processes, we conclude that matrix solubilization was linked closely to changes in wall plasticity and breaking force, but not so closely coupled to cell wall creep. Because matrix solubilization and increased wall plasticity have not been found with other expansins, we infer that these novel activities are linked to the specialized role of grass pollen β-expansins in promotion of penetration of the pollen tube through the stigma and style, most likely by weakening the middle lamella.

Topics: Allergens; Cell Wall; Pectins; Plant Proteins; Pollen; Xylans; Zea mays

Although many highly successful weed species use a ballistic seed dispersal mechanism, little is known about the mechanics of this process. Bittercress (Cardamine hirsuta) siliques are morphologically similar to Arabidopsis siliques, but they can project their seeds up to 5 m, while Arabidopsis seeds are dispersed by gravity. Comparison of these species should enable us to determine which structures might be responsible for ballistic seed dispersal.. Sections of Arabidopsis and bittercress siliques were immunolabeled with antibodies raised against a variety of polysaccharide epitopes.. In bittercress, the second endocarp layer (enB) of the valve had strongly asymmetrical cell wall thickenings, whereas the analogous cells in Arabidopsis were reinforced symmetrically and to a lesser extent. Additionally, an accumulation of mucilaginous pectins was found between the first and second endocarp (enA and enB) layers in the bittercress valve that was not present in Arabidopsis. However, in both species, highly de-esterified homogalacturonan was lost in the dehiscence zone (at the carpel/replum interface) as the siliques matured, thus allowing for separation of the valve at maturity.. Ballistic seed dispersal in bittercress may involve the contraction of the outer pericarp tissue against the highly asymmetrically thickened enB cells, which are hypothesized to bend in one direction preferentially. The stress generated by the differential drying of the inner and outer layers of the valve is released suddenly as the adhesion between the cells of the dehiscence zone is lost, leading to a rapid coiling of the valve and dispersal of the seeds.

Topics: Antibodies; Arabidopsis; Biomechanical Phenomena; Cardamine; Cell Wall; Epitopes; Immunohistochemistry; Microscopy, Electron, Transmission; Pectins; Plant Cells; Polysaccharides; Seed Dispersal; Seeds; Species Specificity; Stress, Mechanical

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

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.

Topics: Alkalies; Bacillus subtilis; Batch Cell Culture Techniques; Biomass; Biotechnology; Carbon; Culture Media; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression; Genes, Bacterial; Glycerol; Isopropyl Thiogalactoside; Pectins; Plasmids; Polysaccharide-Lyases; Recombination, Genetic; Temperature; Time Factors

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.

Topics: Colorimetry; Daucus carota; Pectins; Plant Extracts; Plant Proteins; Polygalacturonase; Ruthenium Red

Polysaccharide acid (PSA) based devices (consisting of alginic acid and polygalacturonic acid) were investigated for the detection of contaminating microorganisms. PSA-CaCl(2) hydrogel systems were compared to systems involving covalent cross-linking of PSA with glycidylmethacrylate (PSA-GMA) which was confirmed with Fourier Transformed Infrared (FTIR) analysis. Incubation of PSA-CaCl(2) and PSA-GMA beads loaded with Alizarin as a model ingredient with trigger enzymes (polygalacturonases or pectate lyases) or bacteria lead to a smoothening of the surface and exposure of Alizarin according to Environmental Scanning Electron Microscopy (ESEM) analysis. Enzyme triggered release of Alizarin was demonstrated for a commercial enzyme preparation from Aspergillus niger and with purified polygalacturonase and pectate lyase from S. rolfsii and B. pumilus, respectively. In contrast to the PSA-CaCl(2) beads, cross-linking (PSA-GMA beads) restricted the release of Alizarin in absence of enzymes. There was a linear relation between release of Alizarin (5-348 μM) and enzyme activity in a range of 0-300 U ml(-1) dosed. In addition to enzymes, both PSA-CaCl(2) and PSA-GMA beads were incubated with Bacillus subtilis and Yersinia entercolitica as model contaminating microorganism. After 72 h, a release between 10 μM and 57 μM Alizarin was detected. For protection of the hydrogels, an enzymatically modified PET membrane was covalently attached onto the surface. This lead to a slower release and improve long term storage stability based on less than 1% release of dye after 21 days. Additionally, this allowed simple detection by visual inspection of the device due to a colour change of the white membrane to orange upon enzyme triggered release of the dye.

Topics: Anthraquinones; Aspergillus niger; Bacillus subtilis; Biosensing Techniques; Biotechnology; Calcium Chloride; Culture Media; Epoxy Compounds; Hydrogels; Methacrylates; Microscopy, Electron, Scanning; Microspheres; Pectins; Polygalacturonase; Polysaccharide-Lyases; Yersinia enterocolitica

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Glucuronosyltransferase; Golgi Apparatus; Immunoprecipitation; Models, Biological; Multiprotein Complexes; Pectins; Protein Binding; Protein Processing, Post-Translational; Proteomics; Substrate Specificity

In vitro experiments showed that calcium pectate added to the culture medium produces a dose-dependent prebiotic effect on lacto-and bifidobacteria cultures and on non-pathogenic strain of Escherichia coli. Calcium pectate produced a pronounced bacteriostatic effect on Candida albicans strain; the effects was more pronounced in a concentration of 4%.

Topics: Anti-Infective Agents; Bifidobacterium; Candida albicans; Escherichia coli; Pectins; Polysaccharides

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Topics: Cations; Cell Culture Techniques; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Extracellular Space; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Kinetics; Metals; Molecular Weight; Paecilomyces; Pectins; Polygalacturonase; Substrate Specificity; Temperature

Around 25% of proteins in living organisms are membrane proteins that perform many critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins are extracted from the lipid bilayer and solubilized with a detergent for biochemical characterization; however, their solubilization is an empirical technique and sometimes insufficient quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid synthase localized at the plant Golgi membrane was enhanced by up to 9.9-fold upon addition of spermidine to the solubilization buffer. These additives also enhanced the solubilization of other plant membrane proteins localized in other organelles such as the endoplasmic reticulum and plasma membrane as well as that of an animal Golgi-localized membrane protein. Thus, addition of alkylamines and polyamines to solubilization buffer is a generally applicable method for effective solubilization of membrane proteins. The mechanism of the enhancement of solubilization is discussed.

Topics: Amines; Animals; Cattle; Cell Membrane; Electron Transport Complex IV; gamma-Glutamyltransferase; Ligases; Membrane Proteins; NADH Dehydrogenase; Pectins; Plant Proteins; Polyamines; Solubility

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.

Topics: Arabidopsis; Aspergillus niger; Biofuels; Biomass; Carboxylic Ester Hydrolases; Cell Wall; Cellulose; DNA Primers; Genetic Vectors; Hypocotyl; Nicotiana; Pectins; Plant Cells; Plant Leaves; Plant Physiological Phenomena; Plant Proteins; Polygalacturonase; Polysaccharides; RNA, Plant; Tissue Engineering

In flax hypocotyls, cadmium-induced reorientation of growth coincides with marked changes in homogalacturonan (HGA) epitopes that were recognized by JIM7 and JIM5 antibodies in the external tangential wall of the epidermis. In the present study, LM7 and 2F4 monoclonal antibodies were used, in addition to JIM5 and JIM7, to extend the investigation on the methyl-esterification pattern of HGA within various domains of the cortical tissues, including the cortical parenchyma where cell cohesion is crucial.. The PATAg (periodic acid thiocarbohydrazide-silver proteinate) test was applied to ultrathin sections so that the polysaccharides could be visualized and the ultrastructure studied. The monoclonal LM7, JIM5 and JIM7 antibodies that recognize differently methyl-esterified HGA were used. The monoclonal 2F4 antibody that is specific to a particular polygalacturonic acid conformation induced by a given calcium to sodium ratio was also applied. After immunogold labelling, the grids were stained with uranyl-acetate, the samples were observed using a transmission electron microscope and the gold particles were counted.. In the presence of cadmium, the increase of LM7 labelling in external tangential wall of the epidermis, together with a decrease of JIM7 labelling, suggested a specific role for randomly partially de-esterified HGA to counteract the radial swelling stress. Enhanced JIM5 and 2F4 labelling in the junctions of the inner tissues indicated that the presence of blockwise de-esterified HGA might oppose cell separation.. The response of the hypocotyl to cadmium stress was to adapt the structure of the wall of cortical tissues by differently modulating the methyl-esterification pattern of HGA in various domains.

Topics: Cadmium; Cell Wall; Esterification; Pectins

Biosorption by cheaply and abundantly available materials such as citrus peels can be a cost efficient method for removing heavy metals from wastewater. To investigate the role pectin plays in metal binding by citrus peels, native orange peels, protonated peels, depectinated peels, and extracted pectic acid were compared. Kinetic experiments showed that equilibrium was achieved in 1h. The 1st-order model was more effective in describing the kinetics than the 2nd-order model. Titrations showed two acidic sites with pK(a) values around 4 (carboxyl) and 10.5 (hydroxyl), respectively. The pH dependent surface charge was described well by a two-site model. Sorption isotherms were best modeled assuming a 1:2 binding stoichiometry, followed by the Langmuir and the Freundlich model. The binding capacity was highest for pectic acid (2.9 mequiv./g) followed by protonated peels and native peels, being lowest for depectinated peels (1.7 mequiv./g). This showed the importance of pectin in metal binding by citrus peels. However, even depectinated peels were still good sorbents which still provided carboxyl groups that were involved in metal binding. FTIR spectra confirmed the presence of carboxyl and hydroxyl groups in all materials and their involvement in metal binding.

Topics: Adsorption; Binding Sites; Biodegradation, Environmental; Cadmium; Carboxylic Acids; Citrus sinensis; Kinetics; Metals; Pectins

Sodium montmorillonite (Na-MMT) clay was modified with three different unnatural amino acids in order to design intercalated clay structures that may be used for bone biomaterials applications. Prior work on polymer-clay nanocomposites (PCNs) has indicated the effect of the appropriate choice of modifiers on enhancing properties of PCNs. Our X-ray diffraction results indicate an increase in the d-spacing of Na-MMT clay after it was modified with the three unnatural amino acids. Transmission Fourier transform infrared spectroscopy experiments were carried out on the unmodified and modified MMT clay samples to study the molecular interactions between the amino acids used as modifiers and the Na-MMT clay. Cell culture experiments showed that the Na-MMT clay modified with the three amino acids was biocompatible as were the modified clay-incorporated films of chitosan/polygalacturonic acid/hydroxyapatite.

Topics: Aluminum Silicates; Amino Acids; Bentonite; Biocompatible Materials; Cell Culture Techniques; Chitosan; Clay; Durapatite; Humans; Materials Testing; Nanocomposites; Nanostructures; Osteoblasts; Pectins; Polymers; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

In this work, we have investigated osteoblast adhesion, proliferation and differentiation on nanocomposites of chitosan, polygalacturonic acid (PgA) and hydroxyapatite. These studies were done on both two- and three-dimensional (scaffold) samples. Atomic force microscopy experiments showed nanostructuring of film samples. Scaffolds were prepared by freeze-drying methods. The mechanical response and porosity of the scaffolds were also determined. The compressive elastic modulus and compressive strength were determined to be around 0.9 and 0.023 MPa, respectively, and the porosity of these scaffolds was found to be around 97 per cent. Human osteoblast cells were used to study their adhesion, proliferation and differentiation. Optical images were collected after different intervals of time of seeding cells. This study indicated that chitosan/PgA/hydroxyapatite nanocomposite films and scaffolds promote cellular adhesion, proliferation and differentiation. The formation of bone-like nodules was observed after 7 days of seeding cells. The nodule size continues to increase with time, and after 20 days the size of some nodules was around 735 microm. Scanning electron microscope images of nodules showed the presence of extracellular matrix. The alizarin red S staining technique was used to confirm mineralization of these nodules.

Topics: Biocompatible Materials; Bone and Bones; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Durapatite; Elasticity; Electrolytes; Humans; Nanocomposites; Nanotechnology; Osteoblasts; Pectins; Pressure; Stress, Mechanical

Synthesis of heat shock proteins (HSPs) in response to heat shock (HS) is essential for thermotolerance. The effect of a Ca(2+) chelator, EGTA, was investigated before a lethal HS treatment in soybean (Glycine max) seedlings with acquired thermotolerance induced by preheating. Such seedlings became non-thermotolerant with EGTA treatment. The addition of Ca(2+), Sr(2+) or Ba(2+) to the EGTA-treated samples rescued the seedlings from death by preventing the increased cellular leakage of electrolytes, amino acids, and sugars caused by EGTA. It was confirmed that EGTA did not affect HSP accumulation and physiological functions but interfered with the recovery of HS-released Ca(2+) concentration which was required for thermotolerance. Pectin methylesterase (PME, EC 3.1.1.11), a cell wall remodelling enzyme, was activated in response to HS, and its elevated activity caused an increased level of demethylesterified pectin which was related to the recovery of the HS-released Ca(2+) concentration. Thus, the recovery of HS-released Ca(2+) in Ca(2+)-pectate reconstitution through PME activity is required for cell wall remodelling during HS in soybean which, in turn, retains plasma membrane integrity and co-ordinates with HSPs to confer thermotolerance.

Topics: Adaptation, Physiological; Calcium; Carboxylic Ester Hydrolases; Egtazic Acid; Esterification; Glycine max; Heat-Shock Response; Models, Biological; Organelles; Pectins; Polygalacturonase; Protein Stability; Protein Transport; Seedlings; Solubility; Spectrophotometry, Atomic; Staining and Labeling; Temperature

Harpins are extracellular glycine-rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380-amino-acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non-host), PopW induced a rapid tissue collapse via a heat-stable but protease-sensitive HR-eliciting activity. PopW has an N-terminal harpin domain (residues 1-159) and a C-terminal pectate lyase (PL) domain (residues 160-366); its HR-eliciting activity depends on its N-terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB-dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW-deficient mutant retained the ability of wild-type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall-associated, hrpB-dependent, two-domain harpin that is conserved across the R. solanacearum species complex.

Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Blotting, Western; Cell Wall; Conserved Sequence; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Nicotiana; Pectins; Phylogeny; Plant Leaves; Polysaccharide-Lyases; Protein Binding; Protein Transport; Ralstonia solanacearum; Sequence Alignment; Sequence Homology, Amino Acid; Subcellular Fractions; Virulence

An alkaline pectate lyase, Bsp165PelA, was purified to homogeneity from the culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme showed a specific activity as high as 1000 U/mg and had optimum activity at pH 11.5 and 50 degrees . It was composed of a single polypeptide chain with a molecular of 42 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 6.0. It could efficiently depolymerize polygalacturonate and pectin. Characterization of product formation revealed unsaturated digalacturonate and trigalacturonate as the main product. The pectate lyase gene (pelA) contained an open reading frame (ORF) of 1089 bp, encoding a 36-amino acids signal peptide and a mature protein of 326 amino acids with a calculated molecular mass of 35.943 Da. The deduced amino acid sequence from the pelA ORF exhibited significant homology to those of known pectate lyases in polysaccharide lyase family 1. Some conserved active-site amino acids were found in the deduced amino acid sequence of Bsp165PelA. Ca2+ was not required for activity on pectic substrates.

Topics: Amino Acid Sequence; Bacillus; Base Sequence; Cloning, Molecular; DNA, Bacterial; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Pectins; Polymerase Chain Reaction; Polysaccharide-Lyases

Antiinflammatory (antiexudative, antiproliferative) activity of calcium pectate was revealed by tests on the mice leg carrageenan-induced edema and cotton-ball granuloma models. It was also established that this polysaccharide produced an anesthetic effect comparable with that of indomethacin on the model of acetate-induced convulsions in mice.

Topics: Anesthetics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Edema; Female; Granuloma; Indomethacin; Mice; Pectins; Rats; Rats, Wistar

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.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Carboxylic Ester Hydrolases; Electrophoresis, Capillary; Esterification; Esters; Methylation; Models, Theoretical; Pectins; Substrate Specificity

Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The purified enzymes exhibited optimum activity at 60 degrees C, and the half-time of thermoinactivation at this temperature was approximately 30 min. Pel-22 had a low specific activity compared with the other two enzymes. However, it displayed high affinity for the substrate, about 2.5-fold higher than those of Pel-66 and Pel-90. The optimum pHs were 7.5 for Pel-22 and 8.0 for Pel-66 and Pel-90. The three enzymes trans-eliminated polygalacturonate in a random manner to generate two long oligogalacturonides, as well as trimers and dimers. A synergistic effect was observed between Pel-22 and Pel-66 and between Pel-22 and Pel-90, but not between Pel-90 and Pel-66. The Pels were also strongly active on highly methylated pectins (up to 60% for Pel-66 and Pel-90 and up to 75% for Pel-22). Fe(2+) was found to be a better cofactor than Ca(2+) for Pel-22 activity, while Ca(2+) was the best cofactor for Pel-66 and Pel-90. The amino acid sequences deduced from the cloned genes showed the characteristics of Pels belonging to Family 1. The pel-66 and pel-90 genes appear to be very similar, but they are different from the pel-22 gene. The characterized enzymes form two groups, Pel-66/Pel-90 and Pel-22; members of the different groups might cooperate to depolymerize pectin during the fermentation of cocoa beans.

Topics: Bacillus; Cacao; Calcium; Cations, Divalent; Chromatography; Cloning, Molecular; Coenzymes; DNA, Bacterial; Enzyme Stability; Hot Temperature; Hydrogen-Ion Concentration; Iron; Molecular Sequence Data; Pectins; Polysaccharide-Lyases; Protein Stability; Recombinant Proteins; Seeds; Sequence Analysis, DNA

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l(-1) urea solution, the flocculating activity was enhanced to 720 U ml(-1) in 36 h. High cell density (2.12 g l(-1)) and flocculating activity (820 U ml(-1)) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l(-1), and residual urea decreased to 0.03 g l(-1). Even higher flocculating activity of 920 U ml(-1) and biomass of 3.26 g l(-1) were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.

Topics: Biomass; Bioreactors; Corynebacterium glutamicum; Culture Media, Conditioned; Fermentation; Flocculation; Genetic Engineering; Glucose; Pectins; Sucrose; Urea

Two pectate lyases were identified from Paenibacillus amylolyticus 27C64; both enzymes demonstrated activity on methylated pectin in addition to polygalacturonic acid. PelA is in a subclass of the pectate lyase family III. PelB shows some features of pectate lyase family I but is highly divergent.

Topics: Calcium Chloride; Coenzymes; DNA, Bacterial; Enzyme Stability; Hydrogen-Ion Concentration; Molecular Sequence Data; Paenibacillus; Pectins; Polysaccharide-Lyases; Sequence Analysis, DNA; Temperature

The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.

Topics: Anticoagulants; Magnetic Resonance Spectroscopy; Pectins; Spectrophotometry, Infrared; Sulfonic Acids; Sulfur

How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.

Topics: Animals; Antibodies, Monoclonal; beta-Mannosidase; Cell Wall; Esterification; Fluorescent Antibody Technique, Direct; Magnoliopsida; Male; Mannans; Pectins; Pinus; Rats; Rats, Wistar

With emphasis on thermal behavior in presence of different pH conditions and salts, the kinetic and thermodynamic parameters of purified polygalacturonase (PG) of E. carotovora subsp. carotovora (Ecc) BR1 were studied since characterization of an enzyme is significant in the context of burgeoning biotechnological applications. Thermodynamic parameters for polygalacturonic acid hydrolysis by purified PG were, deltaH* = 7.98 kJ/mol, deltaG* = 68.86 kJ/mol, deltaS*= -194.48 J/mol/K, deltaG(E-S) = -1.04 kJ/mol and deltaG(E-T) = -8.96 kJ/mol. Its turnover number (k(cat)) was 21/sec. Purified PG was stable in 20-50 degrees C temperature range and was deactivated at 60 degrees C and 70 degrees C. Thermodynamic parameters (deltaH*, deltaG*, deltaS*) for irreversible inactivation of PG at different temperatures (30-60 degrees C) were determined, where effectiveness of various salts and different pH (4-8) individually for thermal stability of PG were characterized. The efficacy of various salts for thermal stability of PG was in the following order: MgCl₂ >BaCl₂ >KCl >CaCl₂ >NaCl. Present work projects biochemical, thermodynamics of substrate hydrolysis as well as thermal stabilization parameters of PG from Ecc.

Topics: Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Stability; Hot Temperature; Isoelectric Focusing; Kinetics; Pectins; Pectobacterium carotovorum; Polygalacturonase; Thermodynamics

Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.

Topics: Cell Wall; Cellulose; Endo-1,4-beta Xylanases; Glycoside Hydrolases; Nicotiana; Pectins; Pisum sativum; Plant Proteins; Plants; Polysaccharide-Lyases; Polysaccharides; Receptors, Cell Surface; Triticum; Xylans

Apoplastic Ca(2+) concentration controls membrane permeability, cell wall stabilization, and cell integrity; however, little is known about its role in thermotolerance in plants. Here, we report that the acquired thermotolerance of etiolated rice seedlings (Oryza sativa) was abolished by an exogenously supplied Ca(2+) chelator, EGTA, related to increased cellular content leakage during heat shock (HS) treatment. Thermotolerance was restored by the addition of Ca(2+) during EGTA incubation. Pectin methylesterase (EC 3.1.1.11), a cell-wall remodeling enzyme, was activated in response to HS, and its elevated activity was related to the recovery of the HS-released Ca(2+) concentration. EGTA interfered with the capability of HS to increase oscillation of [Ca(2+)]cyt content. We assume that heat-activated PME activity is involved in cell-wall-localized Ca(2+). The removal of apoplastic Ca(2+) might participate in HS signaling to induce HS protein expression and cell-wall remodeling to retain plasma membrane integrity, prevent cellular content leakage and confer thermoprotection.

Topics: Adaptation, Physiological; Calcium; Calcium Signaling; Carboxylic Ester Hydrolases; Cytosol; Egtazic Acid; Heat-Shock Response; Oryza; Pectins; Seedlings; Temperature

In order to better understand the processes that regulate the accumulation in the apoplasm of heavy metals and their mobilization by the plant metabolites it is essential to study the mechanisms that regulate the interactions between metal ions and pectins. In such a context, the sorption of Cd(II), Zn(II), Cu(II) and Pb(II) from single and multi-metal solutions, by a Ca-polygalacturonate gel with a degree of esterification of 18.0 (PGAM(1)) and 65.5% (PGAM(2)) was studied in the 3.0-6.0 pH range in the presence of CaCl(2) 2.5mM. The sorption of Cr(III) from single metal solution was also considered. The results show that the amount of each metal ion sorbed increases with increasing the initial metal ion concentration and pH. The data from the single metal solution tests show that at pH 6.0 the affinity of the metal ions towards the PGAM(1) matrix follows the order: Cr(III)>Cu(II)≅Pb(II)≫Zn(II)≅Cd(II). The simultaneous sorption of the bivalent metal ions by the PGAM(1) gels indicates that Pb(II) is selectively sorbed. The FT-IR spectra show that the carboxylate groups are mainly responsible for the metal ion coordination. The ability of PGAM(2) to accumulate Cr(III), Cu(II), and Pb(II) was lower than that found in the PGAM(1) systems whereas the sorption of Zn(II) and Cd(II) was negligible.

Topics: Calcium; Cations; Gels; Hydrogen-Ion Concentration; Metals, Heavy; Pectins; Plant Roots; Solutions

In membrane bioreactors (MBRs) for wastewater treatment, membrane fouling, particularly biofouling caused by soluble microbial products (SMP), is a nuisance problem causing decreases in permeation flux. In a previous study, we identified primary biofoulants of microfiltration (MF) membranes in SMP as polysaccharides containing uronic acids that undergo inter- and intramolecular ionic cross-linking by polyvalent cations, forming a gelatinous mass that clogs membrane pores. In the present study, we therefore attempted to isolate biofoulant-degrading microorganisms from activated sludge on a polygalacturonic acid-overlaid agar medium and evaluate their efficiency for preventing biofouling of MF membranes. Among the isolates, the fungal strain HO1 identified as Phialemonium curvatum degraded 30% of polysaccharides containing uronic acids into smaller molecules in a SMP solution containing a high concentration of saccharides after 30 days of cultivation. Microfiltration tests using a laboratory-scale submerged MBR indicated that the filtration resistance of this degraded SMP solution was lower than that of the control SMP solution without fungal inoculation. Importantly, accumulation of gelatinous mass on the membrane responsible for biofouling was avoided in the SMP solution augmented with P. curvatum HO1 during the microfiltration test. This is the first report to describe a new method for avoiding biofouling of MBRs by microbial degradation of primary biofoulants.

Topics: Ascomycota; Biodegradation, Environmental; Biofouling; Bioreactors; Pectins; Ultrafiltration; Uronic Acids; Waste Disposal, Fluid

PGA/OGA/PF represent apoplastic signaling molecules implicated in the control of gene expression and the activity of enzymes involved in defense regulation. However, the underlying mechanisms behind such processes are lacking. Here we unequivocally show using EPR spectroscopy with DEPMPO spin-trap capable of differentiating between •OH and •O(2)(-) that PGA and PF can produce •O(2)(-) by transforming •OH. The potential physiological implications of this unique property are discussed. We propose that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H(2)O(2) being a downstream secondary messenger.

Topics: Electron Spin Resonance Spectroscopy; Enzymes; Gene Expression Regulation, Plant; Oxidation-Reduction; Pectins; Plants; Spin Trapping; Superoxides

A pronounced antianemic effect of water-soluble Na,Ca,Fe-polygalacturonate is demonstrated. The drug in solution is more effective than in the solid form; it promotes absorption of nutrient calcium and iron. During this treatment, consumed calcium does not impair iron absorption.

Topics: Anemia; Animals; Calcium; Erythrocyte Count; Female; Hemoglobins; Iron; Pectins; Rats; Sodium; Solubility; Water

Ultraviolet (UV) radiation has recently been demonstrated to drive an aerobic production of methane (CH(4)) from plant tissues and pectins, as do agents that generate reactive oxygen species (ROS) in vivo independently of UV. As the major building-blocks of pectin do not absorb solar UV found at the earth's surface (i.e. >280 nm), we explored the hypothesis that UV radiation affects pectin indirectly via generation of ROS which themselves release CH(4) from pectin. Decreasing the UV absorbance of commercial pectin by ethanol washing diminished UV-dependent CH(4) production, and this was restored by the addition of the UV photosensitizer tryptophan. Certain ROS scavengers [mannitol, a hydroxyl radical ((*)OH) scavenger; 1,4-diazabicyclo[2.2.2] octane; and iodide] strongly inhibited UV-induced CH(4) production from dry pectin. Furthermore, pectin solutions emitted CH(4) in darkness upon the addition of (*)OH, but not superoxide or H(2)O(2). Model carbohydrates reacted similarly if they possessed -CH(3) groups [e.g. methyl esters or (more weakly) acetyl esters but not rhamnose]. We conclude that UV evokes CH(4) production from pectic methyl groups by interacting with UV photosensitizers to generate (*)OH. We suggest that diverse processes generating (*)OH could contribute to CH(4) emissions independently of UV irradiation, and that environmental stresses and constitutive physiological processes generating ROS require careful evaluation in studies of CH(4) formation from foliage.

Topics: Esters; Free Radical Scavengers; Hydroxyl Radical; Methane; Pectins; Photosensitizing Agents; Plants; Reactive Oxygen Species; Ultraviolet Rays

The production of enzymes and the colonization of leaves by Leucoagaricus gongylophorus were investigated to further understand the digestive interactions of leaf-cutting ant colonies. The enzymes detected were indicative of a saprophytic origin of this fungus, producing all the enzymes necessary for plant tissue breakdown. Enhanced activities of certain enzymes in the fungus garden extracts may be due to the particular behaviour of the adult worker ants that concentrate fungal acquired enzymes in the rectal fluid and subsequently defaecate these enzymes onto the leaves. The production of chitinases by the fungus may be an ancestral vestige of lower attines, and may have a role as agonists of invading microbes. Growth of the fungus on plant cell wall medium resulted in highest enzyme activity against pectin, reflecting the fact that polygalacturonans comprise the main matrix of the primary plant cell wall. SEM shows that L. gongylophorus does not form specialized structures for cell wall penetration, but gains access to the inner plant tissue at the cut edges of the leaf fragments. Enzymes secreted by the fungus were compared to those seen in larval and adult leaf-cutting ants, demonstrating the inter-dependence of the symbiotic relationship between the ants and their fungi.

Topics: Animals; Ants; Chitinases; Fungi; Host-Parasite Interactions; Hydrolysis; Pectins; Plant Leaves; Symbiosis

The capacity of four xylan-directed probes (carbohydrate-binding modules CfCBM2b-1-2 and CjCBM15; monoclonal antibodies LM10 and LM11) to recognize xylan polysaccharides in primary and secondary cell walls of tobacco stem sections has been determined. Enzymatic removal of pectic homogalacturonan revealed differential recognition of xylans in restricted regions of cortical primary cell walls. Monoclonal antibody binding to these exposed xylans was more sensitive to xylanase action than carbohydrate-binding module (CBM) binding. In contrast, the recognition of xylans by CBMs in secondary cell walls of the same organ was more sensitive to xylanase action than the recognition of xylans by the monoclonal antibodies. A methodology was developed to quantify indirect immunofluorescence intensities, and to evaluate xylanase impacts. The four xylan probes were also used to detect xylan populations in chromatographic separations of solubilized cell wall materials from tobacco stems. Altogether, these observations reveal the heterogeneity of the xylans in plant cell walls. They indicate that although CBM and antibody probes can exhibit similar specificities against solubilized polymers, they can have differential capacities for xylan recognition in muro, and that the access of molecular probes and enzymes to xylan epitopes/ligands also varies between primary and secondary cell walls that are present in the same organ.

Topics: Cell Wall; Chromatography, Ion Exchange; Endo-1,4-beta Xylanases; Enzyme-Linked Immunosorbent Assay; Microscopy, Fluorescence; Molecular Probes; Nicotiana; Pectins; Plant Stems; Substrate Specificity; Xylans

Three novel rat monoclonal antibodies, designated LM18, LM19 and LM20, were isolated from screens for binding to Arabidopsis thaliana seed coat mucilage. The binding of these antibodies to mucilage subject to enzyme and high pH pre-treatments and to a series of model homogalacturonan-rich pectins with defined levels of methyl-esterification indicated their recognition of pectic homogalacturonan epitopes. The binding capacities of these monoclonal antibodies to cell walls in sections of tobacco stem pith parenchyma were also differentially sensitive to equivalent treatments with high pH buffers and pectate lyase. The epitopes bound by these antibodies display some similarities and some differences to the epitopes recognized by the previously isolated and established pectic homogalacturonan probes JIM5 and JIM7.

Topics: Adhesives; Animals; Antibodies, Monoclonal; Arabidopsis; Epitopes; Pectins; Polysaccharides; Rats; Seeds

The structural characterization of branched rhamnogalacturonans (RGs) requires the availability of methods that selectively cleave the Rhap-(1-->4)-alpha-GalAp linkage and thereby generate oligosaccharide fragments that are suitable for mass spectrometric and NMR spectroscopic analyses. Enzymic cleavage of this linkage is often ineffective, especially in highly branched RGs. Therefore, we have developed an improved chemical fragmentation method based on beta-elimination of esterified 4-linked GalpA residues. At least 85% of the carboxyl groups of the GalA residues in Arabidopsis thaliana seed mucilage RG is esterified using methyl iodide or 3-iodopropanol in Me(2)SO containing 8% water and 1% tetrabutylammonium fluoride. However, beta-elimination fragmentation at pH 7.3 and 120 degrees C is far more extensive with hydroxypropyl-esterified RG than with methyl-esterified RG. The non-reducing 4-deoxy-beta-l-threo-hex-4-enepyranosyluronic acid residue formed by the beta-elimination reaction is completely removed by treatment with aqueous N-bromosuccinimide, thereby simplifying the structural characterization of the chemically generated oligoglycosyl fragments. This newly developed procedure was used to selectively fragment the branched RG from peppergrass seed mucilage. The products were characterized using MALDI-TOF mass spectrometry, glycosyl residue composition analysis, and 1 and 2D NMR spectroscopy. Our data show that the most abundant low-molecular weight fragments contained a backbone rhamnose residue substituted at O-4 with a single sidechain, and suggest that peppergrass seed mucilage RG is composed mainly of the repeating unit 4-O-methyl-alpha-d-GlcpA-(1-->4)-beta-d-Galp-(1-->4)-[-->4)-alpha-d-GalpA-(1-->2)-]-alpha-l-Rhap-(1-->.

Topics: Arabidopsis; Bromosuccinimide; Carbohydrate Sequence; Iodine; Lepidium sativum; Nuclear Magnetic Resonance, Biomolecular; Pectins; Polysaccharides; Potassium Iodide; Seeds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Pectins are a major component of plant cell walls and have numerous roles in plant growth and development. Extracted pectins are widely used as functional food ingredients in products including ice creams, jams, jellies and milk drinks. Although all are based on a galacturonan-rich backbone, pectins are an immensely diverse family of polysaccharides, the functional properties of which are dictated by their fine structures. Understanding the biological roles of pectins and optimizing their industrial usage requires a detailed knowledge of their diversity and spatial and temporal distributions, and microarray technology is a promising tool for high throughput pectin analysis. This article discusses the technical aspects of the production of pectin microarrays and explores their current and potential future uses in the context of recent work in the field.

Topics: Antibodies; Cell Wall; Food Technology; Microarray Analysis; Pectins; Polysaccharides; Technology, Pharmaceutical

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

The role of Fe(III) stored at the soil-root interface in the accumulation of arsenate and the influence of citric acid on the As(V) mobility were investigated by using Ca-polygalacturonate networks (PGA). The results indicate that in the 2.5-6.2 pH range Fe(III) interacts with As(V) leading to the sorption of As(V) on Fe(III) precipitates or Fe-As coprecipitates. The FT-IR analysis of these precipitates evidenced that the interaction produces Fe(III)-As(V) inner-sphere complexes with either monodentate or bidentate binuclear attachment of As(V) depending on pH. In the 3.0-6.0 pH range, As(V) diffuses freely through the polysaccharidic matrix that was found to exert a negligible reducing action towards As(V). At pH 6.0 citric acid is able to mobilize arsenate from the As-Fe-PGA network through the complexation of the Fe(III) polyions that leads to the release of As(V).

Topics: Adsorption; Arsenates; Arsenites; Calcium; Citric Acid; Ferric Compounds; Hydrogen-Ion Concentration; Pectins; Plant Roots; Soil

The interaction of covalently coupled hyaluronic acid, alginic acid, and pectic acid with proteins, cells (hematopoietic KG1a and Jurkat cells), and marine organisms (algal zoospores and barnacle cypris larvae) is compared. In contrast to cells and proteins for which such polysaccharide coatings are known for their antiadhesive properties, marine algal spores and barnacle cyprids were able to colonize the surfaces. Of the three polysaccharides, hyaluronic acid showed the lowest settlement of both Ulva zoopores and barnacles. Photoelectron spectroscopy reveals that the polysaccharide coatings tend to bind bivalent ions, such as calcium, from salt water. Such pretreatment with a high salinity medium significantly changes the protein and hematopoietic cell resistance of the surfaces. Complexation of bivalent ions is therefore considered as one reason for the decreased resistance of polysaccharide coatings when applied in the marine environment.

Topics: Animals; Calcium; Cell Adhesion; Hematopoietic Stem Cells; Humans; Hyaluronic Acid; Jurkat Cells; Marine Biology; Pectins; Polysaccharides; Proteins; Spores; Surface Properties; Thoracica; Ulva

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray-based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.

Topics: Amino Acid Substitution; Carboxylic Ester Hydrolases; Data Interpretation, Statistical; Dickeya chrysanthemi; Microarray Analysis; Pectins; Peptide Library; Reproducibility of Results; Sequence Analysis, Protein

Capillary electrophoresis has been used to characterize samples of the copolymeric anionic polysaccharides homo- and rhamno-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.

Topics: Chromatography, High Pressure Liquid; Electrophoresis, Capillary; Esterification; Osmolar Concentration; Pectins; Polymers; Temperature

Triggering of defences by microbes has mainly been investigated using single elicitors or microbe-associated molecular patterns (MAMPs), but MAMPs are released in planta as complex mixtures together with endogenous oligogalacturonan (OGA) elicitor. We investigated the early responses in Arabidopsis of calcium influx and oxidative burst induced by non-saturating concentrations of bacterial MAMPs, used singly and in combination: flagellin peptide (flg22), elongation factor peptide (elf18), peptidoglycan (PGN) and component muropeptides, lipo-oligosaccharide (LOS) and core oligosaccharides. This revealed that some MAMPs have additive (e.g. flg22 with elf18) and even synergistic (flg22 and LOS) effects, whereas others mutually interfere (flg22 with OGA). OGA suppression of flg22-induced defences was not a result of the interference with the binding of flg22 to its receptor flagellin-sensitive 2 (FLS2). MAMPs induce different calcium influx signatures, but these are concentration dependent and unlikely to explain the differential induction of defence genes [pathogenesis-related gene 1 (PR1), plant defensin gene 1.2 (PDF1.2) and phenylalanine ammonia lyase gene 1 (PAL1)] by flg22, elf18 and OGA. The peptide MAMPs are potent elicitors at subnanomolar levels, whereas PGN and LOS at high concentrations induce low and late host responses. This difference might be a result of the restricted access by plant cell walls of MAMPs to their putative cellular receptors. flg22 is restricted by ionic effects, yet rapidly permeates a cell wall matrix, whereas LOS, which forms supramolecular aggregates, is severely constrained, presumably by molecular sieving. Thus, MAMPs can interact with each other, whether directly or indirectly, and with the host wall matrix. These phenomena, which have not been considered in detail previously, are likely to influence the speed, magnitude, versatility and composition of plant defences.

Topics: Arabidopsis; Bacteria; Calcium Signaling; Cell Wall; Gene Expression Regulation, Plant; Genes, Plant; Pectins; Peptides; Plant Diseases; Reactive Oxygen Species; Receptors, Pattern Recognition

Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Galactans; Glucans; Hexosyltransferases; Hexuronic Acids; Immunohistochemistry; Mutation; Pectins; Phenotype; Plant Roots; Spectroscopy, Fourier Transform Infrared; Xylans

Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection with root growth inhibition induced by Al.. An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differing in Al resistance.. In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall. However, treatment with 50 microm Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 % root growth inhibition in 'B73', an Al-sensitive cultivar, resulted in faint and irregular distribution of both epitopes. In 'Nongda3138', an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 microm Al, 9 h). Altered distribution of both epitopes was also observed when of roots were exposed to 50 microm LaCl(3) for 24 h, resulting in 40 % inhibition of root growth.. Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in maize.

Topics: Aluminum; Cell Wall; Epitopes; In Vitro Techniques; Microscopy, Confocal; Pectins; Plant Roots; Soil Pollutants; Zea mays

Calcium pectinate gel (CPG) micrometer-sized beads (microbeads) containing insulin, as a model amphoteric protein, were prepared by ionotropic gelation technique together with an air compressor. The influences of phosphate buffer, pH as well as calcium and pectin concentrations of cross-linking solution on the characteristics and release profiles of microbeads were investigated. With the aid of compressed air flow, the mean diameters of beads were successfully decreased to micron-sized. The results showed that all the factors investigated greatly affected the entrapment efficiencies and release profiles of the microbeads. Suitable formulation concentrations should be considered and great care should be taken to maintain the pH of working solutions at or close to isoelectric point of protein loaded during the whole preparation process. Hence, CPG microbeads of perfect spherical shape, uniform sizes, enhanced mechanical strength, good entrapment efficiencies and delayed release profiles were prepared for a load of amphoteric protein and peptide drugs, without any use of organic solvents or harsh ingredients. Therefore, CPG microbeads could be a promising carrier for oral controlled-release systems of amphoteric protein and peptide drugs.

Topics: Drug Delivery Systems; Gels; Insulin; Microscopy, Electron, Scanning; Microspheres; Pectins

Papaya (Carica papaya) is a climacteric fruit that undergoes dramatic pulp softening. Fruits sampled at three different conditions (natural ripening or after exposition to ethylene or 1-methylcyclopropene) were used for the isolation of cell wall polymers to find changes in their degradation pattern. Polymers were separated according to their solubility in water, CDTA, and 4 M alkali, and their monosaccharide compositions were determined. Water-soluble polymers were further characterized, and their increased yields in control and ethylene-treated fruit, in contrast to those that were treated with 1-MCP, indicated a strong association between fruit softening and changes in the cell wall water-soluble polysaccharide fraction. The results indicate that the extensive softening in the pulp of ripening papayas is a consequence of solubilization of large molecular mass galacturonans from the pectin fraction of the cell wall. This process seems to be dependent on the levels of ethylene, and it is likely that the releasing of galacturonan chains results from an endo acting polygalacturonase.

Topics: Carica; Cell Wall; Food Handling; Molecular Weight; Pectins; Solubility

Cellulose synthase-like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell-wall polymers. The CSL D sub-family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in-depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map-based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub-family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C- or N-terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype-rescued transgenic plants expressing OsCSLD4-GUS under the control of its own promoter. Two phenotype-altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell-wall formation and plant growth.

Topics: Amino Acid Sequence; Cell Wall; Cloning, Molecular; DNA, Plant; Gene Expression Regulation, Plant; Genes, Plant; Glucosyltransferases; Golgi Apparatus; Molecular Sequence Data; Oryza; Pectins; Plant Proteins; Plants, Genetically Modified; Sequence Alignment; Sequence Analysis, DNA; Xylans

In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling.. CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0.5 mm Cd(NO(3))(2). PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples.. In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18.. The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling.

Topics: Cadmium; Carboxylic Ester Hydrolases; Cell Wall; Flax; Gene Expression; Hypocotyl; Isoenzymes; Pectins; Peroxidase

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (approximately tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

Topics: Arabidopsis; Cell Wall; Cellulose; Glucans; Hypocotyl; Models, Theoretical; Pectins; Plants, Genetically Modified; Tensile Strength; Xylans

Pectin is composed of complex polysaccharides that can inhibit cancer metastasis and proliferation with no evidence of toxicity. In the present study, the apoptotic and necrotic effects of pectic acid (PA) on the rat pituitary GH3/B6 tumor cells has been investigated.. GH3/B6 cells were cultured in the Ham's F12 medium enriched with 15% horse serum and 2.5% fetal bovine serum for 3 days. Then, they were treated by various amounts of PA in different periods (6, 24 and 48 hours). Bromocriptine was used as positive control and the cell viability was detected by MTT test. The nuclear morphology of cells was explored by florescent stains including acridine orange (AO)/ethidium bromide (EB). In addition, percentages of apoptotic and necrotic cells were studied with triphosphate nick-end labeling (TUNEL) assay, cell cycle analysis and propidium iodide (PI) staining.. Long-term incubation with PA results in increased cell death and DNA damage as detected by MTT assay and AO/EB staining. TUNEL assay showed that PA (100 microg/ml to 1 mg/ml) could induce apoptosis in a dose-dependent manner, while higher concentrations of PA (2.5 and 5 mg/ml) induced necrosis which was confirmed by PI staining. Furthermore, cell cycle analysis indicated that PA induced sub G1 events, and DNA fragmentation was also correlated with the number of the apoptotic cells.. It can be concluded that PA is responsible for apoptosis in the rat pituitary tumor cells. Therefore, one may suggest that this group of polysaccharides can be used in treatment of pituitary tumors.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; DNA Fragmentation; In Situ Nick-End Labeling; Microscopy, Fluorescence; Necrosis; Nucleosomes; Pectins; Pituitary Neoplasms; Rats

Calcium pectate chemistry was reported to control the growth rate of cells of Chara corallina, and required turgor pressure (P) to do so. Accordingly, this chemistry should account for other aspects of growth, particularly the ability of plants to compensate for brief exposure to low P, that is, to 'store' growth. Live Chara cells or isolated walls were attached to a pressure probe, and P was varied. Low P caused growth to be inhibited in live cells, but when P returned to normal (0.5 MPa), a flush of growth completely compensated for that lost at low P for as long as 23-53 min. This growth storage was absent in isolated walls, mature cells and live cells exposed to cold, indicating that the cytoplasm delivered a metabolically derived growth factor needing P for its action. Because the cytoplasm delivered pectate needing P for its action, pectate was supplied to isolated walls at low P as though the cytoplasm had done so. Growth was stored while otherwise none occurred. It was concluded that a P-dependent cycle of calcium pectate chemistry not only controlled growth rate and new wall deposition, but also accounted for stored growth.

Topics: Cell Survival; Cell Wall; Chara; Models, Biological; Pectins; Temperature; Time Factors

The large production of acidic oligosaccharides was investigated by non-enzymatic depolymerization of polygalacturonic acid (PGA) using free hydroxyl radical hydrolysis process from H2O2/copper (II) system. A large amount of oligogalacturonides (OGAs) with degrees of polymerization up to 6 were fractionated, and characterized by ESI-Q/TOF-MASS spectrometry and 1H NMR spectroscopy. This efficient production of uronic oligosaccharides from PGA constitutes an original process to produce bioactive compounds in large scale up.

Topics: Hydroxyl Radical; Magnetic Resonance Spectroscopy; Mass Spectrometry; Oligosaccharides; Pectins; Plants

Determine the susceptibility of forage chicory (Cichorium intybus L.) to degradation by ruminal fibrolytic bacteria and measure the effects on cell-wall pectic polysaccharides.. Large segments of fresh forage chicory were degraded in vitro by Lachnospira multiparus and Fibrobacter succinogenes, but not by Ruminococcus flavefaciens or Butyrivibrio hungatei. Cell-wall pectins were degraded extensively (95%) and rapidly by L. multiparus with a simultaneous release of uronic acids and the pectin-derived neutral monosaccharides arabinose, galactose and rhamnose. Fibrobacter succinogenes also degraded cell-wall pectins extensively, but at a slower rate than L. multiparus. Immunofluorescence microscopy using monoclonal antibodies revealed that, after incubation, homogalacturonans with both low and high degrees of methyl esterification were almost completely lost from walls of all cell types and from the middle lamella between cells.. Only two of the four ruminal bacteria with pectinolytic activity degraded fresh chicory leaves, and each showed a different pattern of pectin breakdown. Degradation was greatest for F. succinogenes which also had cellulolytic activity.. The finding of extensive removal of pectic polysaccharides from the middle lamella and the consequent decrease in particle size may explain the decreased rumination and the increased intake observed in ruminants grazing forage chicory.

Topics: Animals; Arabinose; Bacteria; Cell Wall; Cichorium intybus; Colony Count, Microbial; Fluorescent Antibody Technique; Galactose; Pectins; Rhamnose; Rumen; Uronic Acids

Pectic acid extracted from plants increases the secretion of prolactin (PRL) when injected intravenously into ewes or fed to rats. Fragments of ewe hypophysis and lactating rabbit mammary gland incubated in vitro in the presence of pectic acid secreted more PRL and caseins compared to the controls. However, it is not known whether pectic acid directly stimulates PRL secretion in pituitary or interference of factors from hypophysis is required for this process.. GH3/B6 cells, a clonal strain of rat pituitary, were cultured and incubated with pectic acid (2.5-100 microg/mL). The integrity of cells was examined under pectic acid treatment microscopically. Controls or pectic acid treated cells were assayed for their ability to produce PRL. The PRL was assayed by Western-blotting and Radioimmunoassay.. pectic acid did not have any significant effect on the viability of cells. After being incubated with pectic acid, the cells started to become circular and protuberant shape. The maximum stimulation and PRL secretion occurred at 100 microg/mL concentration within 30 min of incubation with pectic acid.. pectic acid could stimulate the release of PRL in GH3/B6 cells in the short-term incubation. This result suggested that pectic acid is a non-toxic agent that could directly stimulate PRL secretion in pituitary cells without any interference of hypophysis.

Topics: Animals; Blotting, Western; Cell Line; Cell Shape; Cell Survival; HEPES; Pectins; Pituitary Gland; Prolactin; Rabbits; Radioimmunoassay; Rats; Thyrotropin-Releasing Hormone

Complete assignment of (1)H and (13)C NMR of six hexagalactopyranuronic acids with varying degree and pattern of methyl esterification is reported. The NMR experiments were run at room temperature using approximately 2mg of sample making this method convenient for studying the structure of homogalacturonan oligosaccharides.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Pectins; Reference Standards

Plant organs are produced from meristems in a characteristic pattern. This pattern, referred to as phyllotaxis, is thought to be generated by local gradients of an information molecule, auxin. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth. A major cell-wall component is the linear alpha-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion. HG is deposited in the cell wall in a highly (70%-80%) methyl-esterified form and is subsequently de-methyl-esterified by pectin methyl-esterases (PME, EC 3.1.1.11). PME activity is itself regulated by endogenous PME inhibitor (PMEI) proteins. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth. Here, we show that the formation of flower primordia in the Arabidopsis shoot apical meristem is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell walls. In addition, experimental perturbation of the methyl-esterification status of pectins within the meristem dramatically alters the phyllotactic pattern. These results demonstrate that regulated de-methyl-esterification of pectins is a key event in the outgrowth of primordia and possibly also in phyllotactic patterning.

Topics: Arabidopsis; Body Patterning; Carboxylic Ester Hydrolases; Cell Wall; Esterification; Genes, Plant; Indoleacetic Acids; Meristem; Models, Biological; Multigene Family; Mutation; Pectins

Course prophylactic administration and single therapeutic treatment with calcium pectate improved resistance of rat gastroduodenal mucosa to ethanol-induced ulcers, prednisolone-induced ulcers, and H. Shay ulceration of the gastric mucosa.

Topics: Animals; Anti-Ulcer Agents; Ethanol; Female; Gastric Mucosa; Intestinal Mucosa; Male; Pectins; Peptic Ulcer; Rats; Treatment Outcome

A simple, rapid and straightforward procedure for identification and determination of intracellular and extracellular activity of aminopeptidases employing synthetic substrates beta-naphtylamides of L-Ala, L-Phe, and L-Tyr was used. Poppy cells (Papaver somniferum L.) permeabilized by Tween 80 were immobilized via crosslinking by glutaraldehyde. Glutaraldehyde immobilized poppy cells lost their viability and demonstrated significantly lower aminopeptidase activities than untreated control cells probably due to a damage to the enzyme active centre. Poppy cells immobilized by pectate and alginate have retained high activity of studied aminopeptidases. The culture medium (without cells) used for the identification and determination of extracellular enzyme activities retained 20-21%, whereas intracellular activities were estimated to be 79-80% of total enzyme activity. Thus the intracellular specific activity was 1.00-1.07 higher.

Topics: Alanine; Alginates; Amides; Aminopeptidases; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Extracellular Space; Glucose; Glutaral; Intracellular Space; Papaver; Pectins; Phenylalanine; Polysorbates; Tyrosine

Two fractions of peroxidase activity, cationic Px-cat and anionic Px-ani, were isolated and partially purified (143.5- and 5.49-fold, respectively) from homogenate of spring cabbage heads. Optimum pH for both fractions is 6.0; however, Px-cat is almost equally active at neutral pH (7.0) while Px-ani reveals high activity in more acidic pHs (with 60% of maximum activity at pH 3.0). Optimal temperature for both fractions was 40 degrees C. Px-ani possessed much higher thermal stability at 40-50 degrees C (60% of remaining activity after 144h of incubation) than Px-cat. The peroxidases remained fully active during 4 weeks of storage at 4 degrees C. Kinetic studies revealed that Px-cat and Px-ani had lower apparent Km values for ABTS (0.0377 and 0.0625mM) and o-dianisidine (0.357 and 0.286mM) than for guaiacol (6.41 and 13.89mM). The best substrate for Px-cat was pyrogallol and for Px-ani-o-dianisidine. Px-cat immobilized on polyanionic PyBA-modified carbon electrode was found to produce linear repetitive signals upon consecutive additions of hydrogen peroxide during at least 1-week period and to work effectively under buffered and non-buffered conditions. These properties were comparable with those of commercially available horseradish peroxidase. Stability of the hybrid bioelectrocatalytic film and low costs of extraction and partial purification of Px-cat make it a highly promising enzyme for practical applications, including construction of bioelectrodes.

Topics: Binding Sites; Brassica; Catalysis; Electrochemistry; Enzyme Activation; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Pectins; Peroxidases; Substrate Specificity; Temperature

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

Response surface methodology was used to optimize bead preparation conditions, including CaCl(2) concentration (X(1)), hydroxypropylmethylcellulose concentration (X(2)), and bead-hardening time (X(3)), for the sustained-release of catechin from the calcium pectinate gel beads reinforced with liposomes and hydroxypropylmethylcellulose into simulated gastric fluid (SGF) and intestinal fluid (SIF). The optimized values of X(1), X(2), and X(3) were found to be 5.82%, 0.08%, and 10.29min, respectively. The beads prepared according to the optimized conditions released only about half of the entrapped catechin into SGF while most of the entrapped catechin was released into SIF after 24h incubation.

Topics: Camellia; Catechin; Chemistry, Pharmaceutical; Chemistry, Physical; Delayed-Action Preparations; Drug Carriers; Equipment Design; Gels; Models, Chemical; Models, Statistical; Pectins; Surface Properties; Technology, Pharmaceutical; Time Factors

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.

Topics: Cannabis; Cell Wall; Cellulose; Flax; Fluorescent Antibody Technique; Immunohistochemistry; Mucoproteins; Pectins; Phloem; Plant Proteins; Plant Stems; Xylans

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence factors. In this study, sequence and mutational analysis has demonstrated that Xcc pehA encodes the major polygalacturonase, a member of family 28 of the glycosyl hydrolases. Using the 5' RACE (rapid amplification of cDNA ends) method, the pehA transcription initiation site was mapped at 102 nt downstream of a Clp (cAMP receptor protein-like protein)-binding site. Transcriptional fusion assays showed that pehA transcription is greatly induced by polygalacturonic acid, positively regulated by Clp and RpfF (an enoyl-CoA hydratase homologue which is required for the synthesis of cis-11-methyl-2-dodecenoic acid, a low-molecular-mass diffusible signal factor), subjected to catabolite repression, which is independent of Clp or RpfF, and repressed under conditions of oxygen limitation or nitrogen starvation. Our findings extend previous work on Clp and RpfF regulation to show that they both influence the expression of pehA in Xcc.

Topics: Artificial Gene Fusion; Bacterial Proteins; Base Sequence; beta-Galactosidase; Endopeptidase Clp; Enoyl-CoA Hydratase; Gene Expression Regulation, Bacterial; Genes, Reporter; Molecular Sequence Data; Pectins; Polygalacturonase; Promoter Regions, Genetic; Transcription Initiation Site; Xanthomonas campestris

* The observation that plants produce methane (CH4) under aerobic conditions has caused considerable controversy among the scientific community and the general public. It led to much discussion and debate not only about its contribution to the global CH4 budget but also about the authenticity of the observation itself. Previous results suggested that methoxyl groups of the abundant plant structural component pectin might play a key role in the in situ formation process of CH4. Here, this effect is investigated using an isotope labelling study. * Polysaccharides, pectin and polygalacturonic acid, with varying degrees of trideuterium-labelled methyl groups in the methoxyl moieties, were investigated for CH4 formation under UV irradiation and heating. * A strong deuterium signal in the emitted CH4 was observed from these labelled polysaccharides. * Results clearly demonstrate that ester methyl groups of pectin can serve as a precursor of CH4, supporting the idea of a novel chemical route of CH4 formation in plants under oxic environmental conditions.

Topics: Atmosphere; Deuterium; Esterification; Hot Temperature; Isotope Labeling; Light; Methane; Pectins; Plants

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

Topics: Arabidopsis; Motion; Mutation; Pectins; Pliability; Rheology

With the aim to verify if Fe(III) ions accumulated in a network of Ca-polygalacturonate (PGA) may promote the oxidation of caffeic acid (CAF) the interaction at pH 5.0 between CAF and Fe(III) ions trapped in a PGA was studied. The sorption kinetics evidenced a great affinity of CAF towards the Fe-PGA matrix. Chromatographic tests showed that the interaction leads to the formation of products which can be considered as CAF oligomers characterized by FT-IR spectra similar to those of natural humic acids. Tests carried out under nitrogen suggest that at pH 5.0 oxygen does not affect the nature of these oxidation products. Oxygen was hypothesized to exert a direct action on the redox process by oxidizing the Fe(II) ions, produced by oxidation of CAF, to Fe(III) thus regenerating oxidizing sites. A possible mechanism of formation of the polymers was proposed that implies that the CAF oxidation leads to highly reactive species such as semiquinones which give rise, by an oxidative coupling reaction, to the formation of oligomers that can aggregate through secondary bonds to produce more complex structures as those that characterize humic acids.

Topics: Benzoquinones; Caffeic Acids; Cations; Humic Substances; Hydrogen-Ion Concentration; Iron; Oxidation-Reduction; Oxygen; Pectins

A galabiose disaccharide building block was synthesized by an efficient pectinase cleavage of polygalacturonic acid and subsequent chemical functional group transformations. Besides the disaccharide, the corresponding trisaccharide was also obtained and modified. The compounds were subsequently conjugated to dendrimers with up to eight end groups using 'click' chemistry. The compounds were evaluated as inhibitors of adhesion of the pathogen Streptococcus suis in a hemagglutination assay and strong inhibition was observed for the tetra- and octavalent galabiose compound with MIC values in the low nanomolar range. The corresponding octavalent trisaccharide was a ca. 20-fold weaker inhibitor.

Topics: Carbohydrate Sequence; Cell Adhesion; Dendrimers; Disaccharides; Dose-Response Relationship, Drug; Hemagglutination Inhibition Tests; Molecular Sequence Data; Molecular Structure; Pectins; Polygalacturonase; Sensitivity and Specificity; Streptococcus suis

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

Detailed structures of the active polysaccharides extracted from the leaf and stem cell walls and mucilage of Dendrobium huoshanense are determined by using various techniques, including chromatographic, spectroscopic, chemical, and enzymatic methods. The mucilage polysaccharide exhibits specific functions in activating murine splenocytes to produce several cytokines including IFN-gamma, IL-10, IL-6, and IL-1alpha, as well as hematopoietic growth factors GM-CSF and G-CSF. However, the deacetylated mucilage obtained from an alkaline treatment fails to induce cytokine production. The structure and bioactivity of mucilage components are validated by further fractionation. This is the first study that provides clear evidence for the structure and activity relationship of the polysaccharide in D. huoshanense.

Topics: Animals; Arabinose; Carbohydrate Sequence; Cell Proliferation; Cell Wall; Chemical Fractionation; Cytokines; Dendrobium; Medicine, Chinese Traditional; Mice; Molecular Sequence Data; Pectins; Pentoses; Plant Leaves; Plant Stems; Plants, Medicinal; Polysaccharides; Spleen; Xylans

The zinc-binding activity of different water-soluble pectin compounds varying according to their degree of esterification and of insoluble calcium pectate beads in aqueous solution was studied in a batch sorption system. The zinc uptake by all pectin compounds was highest within the pH range from 4.0 to 7.0. The binding capacities and rates of zinc ions by pectin compounds were evaluated. The Langmuir, Freundlich, and BET sorption models were applied to describe the isotherms and constants. Sorption isotherm data could be well interpreted by the Langmuir equation. The results obtained through the study suggest that pectin compounds are favorable sorbents. The largest number of zinc ions are bound by pectin with the degree of esterification close to zero. Therefore, it can be concluded that low-esterified pectins are more effective substances for elimination of zinc ions from aqueous disposals.

Topics: Adsorption; Chemistry, Physical; Colorimetry; Hydrogen-Ion Concentration; Materials Testing; Models, Statistical; Pectins; Solutions; Surface Properties; Temperature; Time Factors; Water; Zinc

Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.. Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types.. These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.

Topics: Animals; Antibodies, Monoclonal; Cell Wall; Cotyledon; Enzyme-Linked Immunosorbent Assay; Epitopes; Fluorescent Antibody Technique; Glucans; Nasturtium; Nicotiana; Oligonucleotide Array Sequence Analysis; Pectins; Pisum sativum; Plant Stems; Polysaccharide-Lyases; Rats; Seeds; Solubility; Tamarindus; Xylans

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of L: (+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce L: (+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.

Topics: Biotechnology; Cells, Immobilized; Culture Media; Fermentation; Lactic Acid; Lacticaseibacillus casei; Lactose; Milk Proteins; Pectins; Whey Proteins

Understanding the adsorption properties of polysaccharides in terms of substrate affinity, kinetics, and layer structure is of paramount importance in numerous industrial and natural systems. The structural growth of the layers of two model polysaccharides--sodium alginate and polygalacturonic acid (PGA)--was characterized by quartz crystal microbalance with dissipation and atomic force microscopy. Monitoring the variations in frequency and dissipation energy provides insights into both the average adsorbed mass and the viscoelastic properties of the adsorbed layer of polyelectrolytes along with the associated ions and water molecules. Both polysaccharides had similar adsorption patterns with increasing ionic strengths and showed significant complexation of calcium ions. In the presence of calcium, the alginate gel layer exhibited significant swelling with an increasing concentration of monovalent salt that the PGA gel layer did not manifest. Basing our discussion on the "egg-box model", we interpreted these different swelling behaviors as resulting from differences in the complexation modes of the two polysaccharides. The dimerization of the polymers by cross-linking and the weaker dimer-dimer associations play major roles in the sensitivity of the polysaccharide gel matrix to high salt concentration environments.

Topics: Adsorption; Alginates; Calcium; Calcium Chloride; Crystallization; Electrolytes; Gels; Glucuronic Acid; Hexuronic Acids; Ions; Kinetics; Macromolecular Substances; Microscopy, Atomic Force; Molecular Conformation; Pectins; Polysaccharides; Potassium

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Fractionation; Cell Wall; Crosses, Genetic; Glucosyltransferases; Glycosyltransferases; Immunohistochemistry; Mutation; Pectins; Phenotype; Plant Stems; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Xylans; Xylem

A number of bacteria in the family Enterobacteriaceae harbor the genes comprising well-developed pectinolytic pathways (e.g. Erwinia sp.) or abridged versions of this pathway (e.g. Yersinia sp.). One of the most enigmatic components present in some of these pathways is a small gene that encodes a predicted secreted protein of approximately 160 amino acid residues with unknown function. This protein shows distant amino acid sequence similarity over its entire length to galactose-specific family 32 carbohydrate-binding modules (CBMs). Here we demonstrate the ability of the Yersinia enterocolitica example, here called YeCBM32, to bind polygalacturonic acid containing components of pectin. This binding is selective for highly polymerized galacturonic acid and shows a complex mode of polysaccharide recognition. The high resolution X-ray crystal structure (1.35 A) shows YeCBM32s overall structural similarity to galactose specific CBMs and conserved binding site location but reveals a substantially different binding site topology, which likely reflects its unique polymeric and acidic ligand. The results suggest the possibility of a unique role for YeCBM32 in polygalacturonic acid transport.

Topics: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Models, Biological; Models, Molecular; Molecular Sequence Data; Pectins; Periplasmic Proteins; Protein Binding; Sequence Homology, Amino Acid; Yersinia enterocolitica

Xylem hydraulic conductivity (K(s)) in stems of tobacco (Nicotiana tabacum) wild-type SR1 was compared to that of PG7 and PG16, two transgenic lines with increased levels of expression of the gene encoding the Aspergillus niger endopolygalacturonase (AnPGII). Activity of AnPGII removes in planta blocks of homogalacturonan (HG) with deesterified carboxyls, thus increasing the degree of neutrality of pectins. The effect of K+ was tested in increasing stem K(s) using model plants with more neutral polysaccharides in primary walls and, hence, in intervessel pit membranes. K(s) measured with deionized water was compared to that with KCl solutions at increasing concentrations (DeltaK(s), %). Plants transformed for HG degree of neutrality showed a dwarfed phenotype, but DeltaK(s) did not differ among the three experimental groups. The ion-mediated hydraulic effect saturated at a KCl concentration of 25 mm in SR1 plants. All the three tobacco lines showed DeltaK(s) of around +12.5% and +17.0% when perfused with 10 and 25 mm KCl, respectively. Because modification of HG content did not influence ion-mediated hydraulic enhancement, we suggest that pectin components other than HG, like rhamnogalacturonan-I and/or rhamnogalacturonan-II, might play important roles in the hydrogel behavior of pit membranes.

Topics: Ions; Nicotiana; Pectins; Water; Xylem

Changes in homogalacturonans (HGs) and enzymes degrading them have been investigated during cotton (Gossypium hirsutum L.) cotyledon expansion. Using an in vivo assay for pectin-degrading enzymes that involves fluorescent labeled oligomers of GalA as substrate and capillary electrophoresis for product analysis, we found that endo- and exo-polygalacturonases are present in the cotyledon extracellular spaces, and there are dramatic changes in the levels of both activities as the cotyledons change their rate of expansion. Capacity for endo-polygalacturonase activity was highest during the initial stages of cotyledon expansion. However, for exo-polygalacturonase activity it was highest in the later stages of expansion. Cell walls were prepared from 3-, 5-, and 7-day-old cotton cotyledons and treated with liquid HF at -23 degrees C. This treatment cleaves the glycosidic linkages of most neutral sugars in the walls without degrading HGs. HGs with a relatively high degree of esterification can then be solubilized with water, and those with low esterification can be solubilized with concentrated imidazole buffer. The majority of HGs were obtained in the water extracts. The degrees of esterification were 57%, 47%, and 47% in water extracts and 34%, 25%, and 27% in imidazole extracts, in 3-, 5-, and 7-day-old cotton cotyledons, respectively. Using a PA100 ion-exchange column, the members of a GalA homologous series up to approximately 70 residues can be separated. The results from HG molecular length distribution analysis indicated that the HG at 3 days was on average shorter than that in the older cotyledons, perhaps reflecting the higher level of endo-polygalacturonase activity at this stage of more rapid growth.

Topics: Carbohydrate Metabolism; Cell Fractionation; Cell Wall; Cotyledon; Esterification; Glycoside Hydrolases; Gossypium; Pectins; Polygalacturonase

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.

Topics: Arabidopsis; Arabidopsis Proteins; Fluorescent Antibody Technique; Golgi Apparatus; Green Fluorescent Proteins; Methyltransferases; Microscopy, Confocal; Pectins; Spectroscopy, Fourier Transform Infrared

With the aim to investigate the role of the polyuronic components in the accumulation of iron and phosphate at the soil-root interface, the interactions of Ca-polygalacturonates (PGAs) with Fe(III) and P ions and of Fe(III)-Ca-polygalacturonates (Fe-PGAs) with P ions were studied at pH 4.7. The role of citric, malic and pyruvic acids in the mobilization of Fe(III) and P, in the presence and absence of Ca(II) 2.5mM, was also investigated. The sorption kinetics evidenced that P diffuses freely through the calcium polysaccharidic matrix whereas Fe(III) accumulates as an hydroxypolymer. The sorption kinetics of P by the Fe-PGA indicated that the amount of P sorbed increases with increasing its initial concentration up to a constant value equal to 0.98micromol/3.87micromolmg(-1) of Fe(III)-polymer trapped. The FT-IR spectra of the P-Fe-PGA systems, show bands attributable to P-O(H) stretching vibrations. The study of systems with a constant initial P amount and varying Fe(III) amounts allowed to hypothesize that phosphate settles down inside holes formed by the carboxylate groups of galacturonic units. Citric and malic acids showed to be active in the mobilization of both Fe and P whereas pyruvic acid appeared inactive.

Topics: Carbohydrate Conformation; Citric Acid; Iron; Kinetics; Malates; Pectins; Phosphates; Plant Roots; Pyruvic Acid; Soil; Time Factors; Uronic Acids

The underlying mechanisms governing nonenzymatic pectin and pectate degradation during thermal treatment have not yet been fully elucidated. This study determined the extent of nonenzymatic degradation due to beta-elimination, acid hydrolysis, and demethylation during prolonged heating of citrus pectins and its influence on physicochemical properties. Solutions of citrus pectins, buffered from pH 4.0 to 8.5, were heated at 75, 85, 95, and 110 degrees C for 0-300 min. Evolution of methanol and formation of reducing groups and unsaturated uronides were monitored during heating. Molecular weight and viscosity changes were determined through size exclusion chromatography and capillary viscometry, respectively. Results showed that at pH 4.5, the activation energies of acid hydrolysis, beta-elimination, and demethylation are 95, 136, and 98 kJ/mol, respectively. This means that at this pH, acid hydrolysis occurs more rapidly than beta-elimination. Furthermore, the rate of acid hydrolysis is diminished by higher levels of methyl esterification. Also, citrus pectin (93% esterified) degrades primarily via beta-elimination even under acidic conditions. Acid hydrolysis and beta-elimination caused significant reduction in relative viscosity and molecular weight.

Topics: Citrus; Esterification; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Methylation; Pectins

A technically simple and inexpensive discontinuous turbidity assay for qualitative and/or quantitative assessments of polygalacturonic acid depolymerase activity is presented. The enzyme reaction is initiated by the addition of enzyme preparation to a reaction mixture containing 0.02% polygalacturonic acid (PGA) in acetate buffer. The progress of the reaction is monitored by terminating aliquots of the reaction mixture (via heat treatment at appropriate times), subsequently adding poly(diallyldimethylammonium chloride) (PDADMAC) for turbidity development (approximately 30 min), and then measuring the turbidity (typically at 420 nm) of the resulting PGA-PDADMAC complex-containing solution. PGA depolymerase activity causes a decrease in the observed turbidity of PGA-PDADMAC-containing solutions because the stability of the interpolyelectrolyte PGA-PDADMAC complex is a function of the degree of polymerization of the PGA. The rate of turbidity change is herein shown to be proportional to a relatively wide range of enzyme concentrations. The assay is demonstrated using a commercial pectinase preparation and tomato fruit extracts.

Topics: Fruit; Kinetics; Nephelometry and Turbidimetry; Pectins; Polyethylenes; Polygalacturonase; Quaternary Ammonium Compounds; Solanum lycopersicum

Bacteriophage phi29 is a small, well-characterized dsDNA virus that infects Bacillus subtilis. The anti-receptor of phi29 consists of oligomers of the 854-residue protein gp12 and plays an essential role in infection initiation by binding to the receptor on the host cell surface. Oligomers of gp12 exhibit a narrow spindle-shaped configuration 15 nm in length as revealed by electron microscopy and thus are potentially useful nanoscale tools, building blocks, or motor arms. To understand the mechanism of viral infection initiation and to provide a basis for engineering recombinant gp12 for nanotechnology applications, we have initiated structural and bioinformatics studies of gp12. We report here the growth of crystals of gp12 that diffract to 3.0 A resolution. The space group is P3(1)21 or P3(2)21 with unit cell lengths of a = 84.4 A and c = 167.6 A. The asymmetric unit is predicted to contain one gp12 molecule and 32% solvent (VM = 1.8 A3/Da). Domain boundary analysis revealed that gp12 may harbor three domains besides a 24 residue auto-cleave region. The N-terminal half of gp12 contains a domain with about 400 residues that held 44% sequence identity to endopolygalacturonase, a fungal glycosyl hydrolase that catalyzes hydrolysis of the polygalacturonic acid alpha1-4 glycosidic linkage found in plant cell walls. Interestingly, the cell wall of Bacillus subtilis contains a polysaccharide component made from two sugar monomers, N-acetylmuramic acid and N-acetylglucosamine, which resemble alpha-galacturonic acid in that they possess a six-membered pyranose ring. Hence, polygalacturonic acid of plant cell walls and peptidoglycan of bacterial cell walls may offer a similar topography in relation to the polysaccharides. These results suggest a function for gp12 as a cell-wall degrading enzyme in addition to its role in recognition of the host receptor.

Topics: Amino Acid Sequence; Bacillus Phages; Bacillus subtilis; Cell Wall; Computational Biology; Crystallization; Glycoside Hydrolases; Hydrolysis; Molecular Sequence Data; Nanoparticles; Nanotechnology; Pectins; Recombinant Proteins; Sequence Homology, Amino Acid; X-Ray Diffraction

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

S-adenosylmethionine (SAM) is the substrate used in the methylation of homogalacturonan (HGA) in the Golgi apparatus. SAM is synthesized in the cytosol, but it is not currently known how it is then transported into the Golgi. In this study, we find that HGA methyltransferase is present in Golgi-enriched fractions and that its catalytic domain faces the lumen of this organelle. This suggests that SAM must be imported into the Golgi. We performed uptake experiments using [methyl-(14)C]SAM and found that SAM is incorporated into the Golgi vesicles, resulting in the methylation of polymers that are sensitive to pectinase and pectin methylesterase but not to proteases. To avoid detecting the transfer reaction, we also used [carboxyl-(14)C]SAM, the uptake of which into Golgi vesicles was found to be sensitive to temperature, detergents, and osmotic changes, and to be saturable with a K(m) of 33 microm. Double-label uptake experiments using [methyl-(3)H]SAM and [carboxyl-(14)C]SAM also revealed a time-dependent increase in the (3)H to (14)C ratio, suggesting that upon transfer of the methyl group, the resulting S-adenosylhomocysteine is not accumulated in the Golgi. SAM incorporation was also found to be inhibited by S-adenosylhomocysteine, whereas UDP-GalA, UDP-GlcA, and acetyl-CoA had no effect. DIDS, a compound that inhibits nucleotide sugar transporters, also had little effect upon SAM incorporation. Interestingly, the combination of UDP-GalA + acetyl-CoA or UDP-GlcA + acetyl-CoA produced a slight increase in the uptake of SAM. These results support the idea that a SAM transporter is required for HGA biosynthesis.

Topics: Biological Transport; Golgi Apparatus; Methylation; Pectins; Pisum sativum; S-Adenosylmethionine; Time Factors

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.

Topics: Buffers; Cholic Acids; Chromatography, Gel; Enzyme Activation; Fabaceae; Kinetics; Pectins; Protein Binding; Solubility

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

Topics: Arabidopsis; Arabidopsis Proteins; Benzamides; Glucosyltransferases; Glucuronidase; Nicotiana; Pectins; Pentosyltransferases; Plants, Genetically Modified; Xylans

The effects of water-soluble Na-,Fe-,Co-,Cu-polygalacturonate on hemopoiesis were studied in laboratory animals of various species, age, and functional groups. The compound had a strong positive effect on hemopoiesis, which manifested in an increase in hemoglobin concentration and erythrocyte count and rapid recovery of blood parameters after acute blood loss.

Topics: Age Factors; Animals; Cobalt; Copper; Erythrocyte Count; Female; Hematopoiesis; Hemoglobins; Iron; Male; Mice; Pectins; Rats; Sex Factors; Sodium; Species Specificity

Recent work showed that polygalacturonate (pectate) chemistry controlled the growth rate of the large-celled alga Chara corallina when turgor pressure (P) was normal (about 0.5 MPa). The mechanism involved calcium withdrawal from the wall by newly supplied pectate acting as a chelator. But P itself can affect growth rate. Therefore, pectate chemistry was investigated at various P. A pressure probe varied P in isolated walls, varying the tension on the calcium pectate cross-links bearing the load of P. When soluble pectate was newly supplied, the wall grew irreversibly but the pectate was inactive below a P of 0.2 MPa, indicating that tension was required in the existing wall before new pectate acted. It was suggested that the tension distorted some of the wall pectate (the dominant pectin), weakening its calcium cross-links and causing the calcium to be preferentially lost to the new pectate, which was not distorted. The preferential loss provided a molecular mechanism for loosening the wall structure, resulting in faster growth. However, the resulting relaxation of the vacated wall pectate would cause calcium to be exchanged with load-bearing calcium pectate nearby, auto-propagating throughout the wall for long periods. There is evidence for this effect in isolated walls. In live cells, there is also evidence that auto-propagation is controlled by binding the newly supplied pectate (now calcium pectate) to the wall and/or by additional Ca(2+) entering the wall structure. A tension-dependent cycle of pectate chemistry thus appeared to control growth while new wall was deposited as a consequence.

Topics: Cell Wall; Chara; Pectins; Pressure

This study has investigated the potential of immersion coating calcium containing pellet cores first with pectin, and then with two different cross-linkers, calcium or chitosan. The interaction between pectin and calcium, and between pectin and chitosan, are believed to slow down the drug release, and thereby, the coated pellets might possibly be used for colon specific drug delivery. Both the calcium coated pellets and the chitosan coated pellets had a reduced drug release compared to uncoated pellets in 0.1M HCl (1 h) and phosphate buffer pH 6.8 (4 h). The most successful combination had a drug release of only 17% during the entire test period in comparison to the uncoated pellets that had a drug release of 80%. When chitosan was used as a cross-linker, a higher reduction in drug release was obtained than by using calcium as the cross-linker. For the pellets coated with pectin in combination with chitosan, the type of pectin with a degree of methoxylation (DM) of 35 was superior to the pectin type with DM 17. The drug release was further slowed down by choosing a type of chitosan with a high degree of deacetylation (Dda) 89% and by coating at low concentrations (0.1%) in the immersion solution.

Topics: Calcium; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Gastrointestinal Agents; Pectins; Solubility; Time Factors

Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear beta(1,4) galactan and linear alpha(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.

Topics: Arabidopsis; Arabidopsis Proteins; Carbohydrate Epimerases; Cell Wall; Electrophoresis, Polyacrylamide Gel; Galactans; Hydrolases; Pectins; Plant Leaves; Plant Roots; Plant Stems; Polysaccharides; Seeds

A method was developed to selectively methyl esterify and then cleave GalA residues in pectic polysaccharides. The method was optimized using a rhamnogalacturonan (RG) from Arabidopsis mucilage as a model compound. The carboxyl group of the GalA residues in the RG was selectively methyl esterified using tetrabutylammonium fluoride and iodomethane in Me(2)SO containing 8% water. A 1D HMQC NMR method to determine the degree of methyl esterification was developed using (13)C-iodomethane as the methylating agent. The methyl-esterified pectins were fragmented by beta-elimination in 0.2M sodium borate, pH7.3, at 125 degrees C. The resulting oligoglycosyl fragments, which contain a nonreducing 4-deoxy-beta-l-threo-hex-4-enepyranosyluronic acid residue, were characterized using MALDI-TOF mass spectrometry, monosaccharide composition analysis, and 1D and 2D (1)H and (13)C NMR spectroscopy. Application of this method to branched RG from potato generated low-molecular-weight fragments containing two residues from the RG backbone and a single side chain. In contrast, the fragments obtained when RG is treated with RG lyase contain a minimum of four backbone residues. The chemical method thus facilitates the release and structural characterization of the side-chain structures of RG obtained from various plant sources. The method also provides a convenient method for generating fully or partially methyl-esterified homogalacturonans.

Topics: Arabidopsis; Carbohydrate Conformation; Carbohydrate Sequence; Cell Wall; Esterification; Hexuronic Acids; Molecular Sequence Data; Pectins; Polymers; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0-10 degrees C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 degrees C with a very small activation volume of -1.1 ml/mol. At 10 degrees C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme-substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme-substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.

Topics: Cold Temperature; Cryptococcus; Enzyme Activation; Hydrolysis; Kinetics; Oceans and Seas; Pectins; Polygalacturonase; Pressure; Seawater

The aim of this study was to obtain improved strains of pectinolytic yeasts adapted to the conditions of an industrial fermentation process, which was continuously operated to convert citrus molasses into ethanol.. The starter yeast of the industrial fermentation process was a commercial baker's yeast, which was capable of growing without forming any secretion halo of pectinase activity on solid medium. Nevertheless, isolates showing secretion of pectinolytic activity on plates were obtained from the fermentation process. The secretion of pectin-degrading activity by isolates on plates was repressed by galactose and improved as the result of colony aging on polygalacturonic acid plates at 30 degrees C. Liquefaction of polygalacturonate gels as well as the splitting of the pectin-degrading activity into a wall-linked and a supernatant fraction were also observed when the starter yeast was propagated under agitation in liquid medium containing pectin.. Isolates capable of secreting pectinolytic activity on plates were predominant at the end of the citrus molasses fermentation. Nevertheless, the sizes of the secretion haloes on plates were not necessarily an indication of the levels of pectinolytic activity secreted in the liquid medium.. Improved pectinolytic strains of Saccharomyces can be used as a source of pectinases for a variety of applications. This organism also participates in plant deterioration processes.

Topics: Citrus; Culture Media; Fermentation; Food Microbiology; Galactose; Glucose; Molasses; Pectins; Polygalacturonase; Saccharomyces cerevisiae

Clostridium stercorarium F-9 pectate lyase Pel9A is a modular enzyme composed of two hypothetical family-9 catalytic modules of the polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. In this study, we constructed and characterized CM9-1 and CM9-2 polypeptides as rCM9-1 and rCM9-2 respectively. Both of them, like the full-length Pel9A, required the Ca2+ ion for their enzyme activities and showed high activity toward polygalacturonic acid but lower activity toward pectin. The specific activity of rCM9-2 was three times higher than that of rCM9-1 and rCM9-2 by itself efficiently catalyzed the depolymerization reaction of polygalacturonic acid into monosaccharide as the major product. It was found that rCM9-1 and rCM9-2 adsorbed to polygalacturonic acid and pectin on native affinity PAGE analysis, suggesting that they contain an independent carbohydrate-binding module separable from a catalytic module or consist of a catalytic module with a binding affinity for pectic substrates.

Topics: Calcium; Catalysis; Cations, Divalent; Clostridium; Methylation; Pectins; Polysaccharide-Lyases; Protein Binding; Recombinant Proteins; Solubility; Substrate Specificity

Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction.

Topics: Arabidopsis Proteins; Binding Sites; Calcium; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Mutagenesis, Site-Directed; Mutation; Pectins; Protein Binding; Protein Kinases; Protein Structure, Tertiary; Recombinant Proteins; Saccharomyces cerevisiae; Sodium

Galacturonan, the main constituent of the backbone (core) of the comaruman macromolecule, a pectin from the marsh cinquefoil Comarum palustre L., was obtained on partial acid hydrolysis of the pectin. Using atomic force microscopy and methylation analysis of the galacturonan, the backbone of the comaruman macromolecule was shown to contain branches as side chains consisting of alpha-1,4-linked residues of D-galactopyranosyl uronic acid attached to the 2- and 3-positions of the galacturonic acid residues of the core, in addition to linear regions of alpha-1,4-D-galacturonan. A few side chains appear to attach to 2,3-positions of the D-galacturonic acid residues.

Topics: Carbohydrate Conformation; Galactose; Hexuronic Acids; Hydrolysis; Methylation; Microscopy, Atomic Force; Pectins; Polysaccharides; Potentilla

Pectin, a normal constituent of cell walls, caused growth rates to accelerate to the rates in living cells when supplied externally to isolated cell walls of Chara corallina. Because this activity was not reported previously, the activity was investigated. Turgor pressure (P) was maintained in isolated walls or living cells using a pressure probe in culture medium. Pectin from various sources was supplied to the medium. Ca and Mg were the dominant inorganic elements in the wall. EGTA or pectin in the culture medium extracted moderate amounts of wall Ca and essentially all the wall Mg, and wall growth accelerated. Removing the external EGTA or pectin and replacing with fresh medium returned growth to the original rate. A high concentration of Ca2+ quenched the accelerating activity of EGTA or pectin and caused gelling of the pectin, physically inhibiting wall growth. Low pH had little effect. After the Mg had been removed, Ca-pectate in the wall bore the longitudinal load imposed by P. Removal of this Ca caused the wall to burst. Live cells and isolated walls reacted similarly. It was concluded that Ca cross-links between neighbouring pectin molecules were strong wall bonds that controlled wall growth rates. The central role of Ca-pectate chemistry was illustrated by removing Ca cross-links with new pectin (wall "loosening"), replacing vacated cross-links with new Ca2+ ("Ca2+-tightening"), or adding new cross-links with new Ca-pectate that gelled ("gel tightening"). These findings establish a molecular model for growth that includes wall deposition and assembly for sustained growth activity.

Topics: Calcium; Cell Fractionation; Cell Wall; Chara; Culture Media; Egtazic Acid; Magnesium; Models, Biological; Pectins

The theoretical model devised in the previous paper (Donati, I.; Benegas, J. C.; Cesàro, A.; Paoletti, S. Biomacromolecules 2006, 7 (5), 1587-1596) for the description of ion-induced chain aggregation is here applied to the case of chain dimerization of poly(galacturonate) in the presence of calcium ions. Particular attention has been directed toward the initial stage of dimer formation [i.e., in the low regime of calcium-to-polymer ratio (Rj)]. Circular dichroism (CD) data allowed evaluation of the fraction, theta, of calcium ions bound within chain dimers according to the "egg-box"-model. The theoretical model was able to reproduce satisfactorily the total molar enthalpy variation experimentally determined; the contributions of affinity (specificity in territorial condensation) and chemical bonding of calcium counterions to the thermodynamic properties of the system (i.e., enthalpy and entropy) were calculated. The intrinsic molar enthalpy of bonding, DeltaH(bond,0), displayed a peculiar sigmoid dependence on Rj. In particular, its decrease toward more negative values was interpreted as stemming from a (cooperative) calcium-induced conformational change that accompanies pectate chain pairing upon junction formation. The calculated pKin of instability of the Ca-(GalA-)4 complex was 10.80, in very good agreement with the corresponding value reported for the Ca-EDTA complex (i.e., 10.96). Significant contributions to the complex stability were the enthalpy of ion pairing (DeltaH(ionpairing,bond) = -5.1 kcal (mol calcium)-1, in good agreement with the value reported for calcium-EDTA: approximately -5.4 kcal (mol calcium)-1), and the entropy of desolvation (DeltaS(desolv,bond) = 43.7 cal mol-1 K-1, well within the range of values reported for calcium-EDTA: 42-57 cal mol-1 K-1).

Topics: Calcium; Electrolytes; Kinetics; Pectins; Thermodynamics

A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl(2), 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20-23 mg l(-1) and 0.5-2 mg l(-1), respectively). The maximum lysozyme yield achieved was about 23 mg l(-1) by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl(2)). Response surface methodology was used to investigate the effect of pH and water activity (a(w)). The best medium pH was 4.5-5.0, and bead a(w) for optimum lysozyme yield was 0.94, regardless of polymer type.

Topics: Alginates; Aspergillus niger; Bioreactors; Calcium Chloride; Cloning, Molecular; Culture Media; Fermentation; Glucuronic Acid; Hexuronic Acids; Hydrogen-Ion Concentration; Muramidase; Pectins

The most commonly used anti-adhesion device for separation and isolation of wounded tissues after surgery is the polymeric film. In this study, a new anti-adhesion membrane based on polygalacturonic acid (PGA) has been synthesized, and its biocompatibility and anti-adhesion capabilities evaluated. The PGA film was reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to obtain a cross-linked PGA film with an 86% gel content and a 47% water content when immersed in aqueous solution. This PGA-EDC film did not show any evidence of cytotoxic effects since it did not induce any significant increase in cytoplasmic LDH release from the L929 cells in contact with it. When implanted into rats, the PGA-EDC film exhibited a most promising anti-adhesion potential with only 1 out of 21 rats operated not forming any tissue adhesion. This anti-adhesion potency is significantly higher than that found for Seprafilm and untreated rats where 11 out of 21 and 18 out of 21 operated rats, respectively, formed tissue adhesions. The implanted PGA-EDC film did not elicit any acute inflammatory reaction based on the results of histological examination and peritoneal fluid leukocytes analysis. The newly developed PGA-EDC film thus has a great potential for future use in clinical applications.

Topics: Absorbable Implants; Adhesiveness; Animals; Bandages; Biocompatible Materials; Cell Adhesion; Cell Line; Cross-Linking Reagents; Ethyldimethylaminopropyl Carbodiimide; Fibroblasts; Hyaluronic Acid; Membranes, Artificial; Mice; Pectins; Rats; Rats, Sprague-Dawley; Wound Healing; Wounds, Penetrating

Wall-associated kinase 1 (WAK1) is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell walls. In order to characterize further the interaction of WAK1 with pectin, a 564 bp DNA sequence corresponding to amino acids 67-254 of the extracellular domain of WAK1 from Arabidopsis thaliana was cloned and expressed as a soluble recombinant peptide in yeast. Using enzyme-linked immunosorbent assays (ELISA), we show that peptide WAK(67-254) binds to polygalacturonic acid (PGA), oligogalacturonides, pectins extracted from A. thaliana cell walls and to structurally related alginates. Our results suggest that both ionic and steric interactions are required to match the relatively linear pectin backbone. Binding of WAK(67-254) to PGA, oligogalacturonides and alginates occurred only in the presence of calcium and in ionic conditions promoting the formation of calcium bridges between oligo-and polymers (also known as 'egg-boxes'). The conditions inhibiting the formation of calcium bridges (EDTA treatment, calcium substitution, high NaCl concentrations, depolymerization and methylesterification of pectins) also inhibited the binding of WAK(67-254) to calcium-induced egg-boxes. The relevance of this non-covalent link between WAK(67-254) and cell wall pectins is discussed in terms of cell elongation, cell differentiation and host-pathogen interactions.

Topics: Arabidopsis; Arabidopsis Proteins; Calcium; Cell Differentiation; Cell Wall; Enzyme-Linked Immunosorbent Assay; Esterification; Membrane Proteins; Pectins; Protein Binding; Protein Kinases; Protein Structure, Tertiary; Recombinant Proteins; Signal Transduction

Crude alpha-glucosidase from Baker's yeast was immobilized in polygalacturonic acid beads and coated with chitosan. Chemical and physical characterization were performed by using p-nitrophenyl-alpha-D-glucopyranoside (pNPG) as an artificial substrate. Operation, thermal, pH, and strorage stabilities of the free and immobilized enzyme were also examined. The stabilities of immobilized enzyme were found to be better than that of the free enzyme. Furthermore, the hydrolysis rate of the chitosan coated alpha-glucosidase polygalacturonic acid beads were studied. In conclusion, the enzyme beads appear to have good characteristics and offer the prospect that this system may find application in enzyme immobilization, in addition to controlled drug release studies.

Topics: alpha-Glucosidases; Chitosan; Coated Materials, Biocompatible; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Materials Testing; Pectins; Temperature

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Delta and mod1Deltamod2Delta strains showed a severe defect in the growth yield, although the mod2Delta strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

Topics: Alcohol Oxidoreductases; Carboxylic Ester Hydrolases; Electrophoresis, Polyacrylamide Gel; Gene Deletion; Gene Expression; Methanol; Pectins; Peroxisomes; Pichia; RNA, Fungal; RNA, Messenger; Transcription, Genetic

Structure and immunological characteristics of the pectic arabinogalactan Vk2a (previously reported as Vk100A2a) from the roots of Vernonia kotschyana Sch. Bip. ex Walp. were investigated after enzymatic digestion of the galacturonan moiety and the side chains of the rhamnogalacturonan structure of Vk2a. endo-alpha-D-(1-->4)-Polygalacturonase digestion released the high molecular weight 'hairy region' (Vk2a-HR) and oligogalacturonides. Vk2a-HR consisted of GalA (4-linked) and Rha (2- or 2,4-linked) in a 1:1 ratio, with 60% of Rha branched at C-4. The Rha located in the rhamnogalacturonan core was branched randomly by Gal units. Vk2a-HR was rich in neutral sugars such as Araf 5- (12.2%) and 3,5-substituted (12.8%) and terminally- (14.1%) linked and Gal 4- (13.0%), 3- (0.9%), 6- (2.2%) and 3,6- (1.1%) substituted. Arabinans with chain lengths up to 11 units were identified. Araf residues were attached to C-3 of alpha-L-(1-->5)-Araf chains and to C-4 of Gal residues. Single Gal units and chains of beta-D-(1-->6)-linked galacto di- to penta-saccharides were attached to a beta-D-(1-->3)-galactan core. All the enzyme resistant fractions expressed potent complement fixation and induction of B-cell mitogenic activity, and the present study indicates that there may be several and possibly structurally different active sites involved in the bioactivity of Vk2a. The bioactive sites may be located both in the more peripheral parts of the molecule but also in the inner core of the 'hairy region' or in larger enzyme-resistant chains.

Topics: Animals; Binding Sites; Carbohydrate Conformation; Carbohydrates; Chromatography, Gel; Chromatography, High Pressure Liquid; Complement System Proteins; Erythrocytes; Galactans; Galactose; Gas Chromatography-Mass Spectrometry; Humans; Ions; Mass Spectrometry; Methylation; Mice; Mice, Inbred C3H; Mitogens; Oligosaccharides; Pectins; Plant Roots; Polysaccharides; Sheep; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship

Alpha-(1,4)-galacturonosyltransferases (GalATs) catalyze the addition of (1,4)-linked alpha-D-galacturonosyl residues onto the nonreducing end of homogalacturonan chains. The nucleotide-sugar donor for the enzymatic reaction is uridine diphospho-D-galactopyranosyluronic acid (UDP-D-GalpA). Many GalAT activity assays are based on the incorporation of D-[(14)C]GalpA from UDP-D-[(14)C]GalpA onto exogenously added homogalacturonan acceptors. Reactions based on this method can be time-consuming because multiple labor-intensive centrifugations and washes with organic solvents are required to remove the unincorporated UDP-D-[(14)C]GalpA from the (14)C-labeled products. Here we report the development of an alternative GalAT filter assay based on the ability of homogalacturonan to bind to cetylpyridinium chloride (CPC). GalAT assay reaction products made using radish (Raphanus sativus) microsomal membranes or solubilized proteins from tobacco (Nicotiana tabacum L. cv. Samsun) and Arabidopsis thaliana (cv. Columbia) were spotted onto Whatman 3MM paper treated with 2.5% (w/v) CPC. Unincorporated UDP-D-[(14)C]GalpA was selectively removed from the filters by washing with 150-250 mM NaCl. The versatility of this assay is demonstrated by using it to identify GalAT activity in fractions obtained during the partial purification of tobacco GalAT by SP Sepharose cation exchange chromatography and by detecting the GalAT-catalyzed incorporation of D-[(14)C]GalpA onto endogenous acceptors from Arabidopsis membranes.

Topics: Arabidopsis; Filtration; Glucuronosyltransferase; Glycosyltransferases; Membrane Proteins; Microsomes; Nicotiana; Pectins; Plant Proteins; Plant Roots

Watermelon cells after permeabilization in Tween 80 were immobilized by glutaraldehyde without any carrier. Cells immobilized by cross-linking demonstrated significantly lower aminopeptidase activities than untreated cells. Pectate and alginate hydrogels were used successfully for immobilization of watermelon cells which retained activities of some aminopeptidases. A simple and rapid procedure for determination of extracellular aminopeptidases was developed using a synthetic substrate. p-Nitroanilides of amino acids were used as substrates for the determination of the extracellular and intracellular enzymatic activities. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activities, whereas intracellular activities were estimated from the cell suspension.

Topics: Alginates; Aminopeptidases; Anilides; Cell Membrane Permeability; Cell Survival; Cells, Immobilized; Citrullus; Glutaral; Hydrogels; Pectins; Polysorbates; Time Factors

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan alpha-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of beta-1-4-D-xylan synthase, beta-1-4-D-galactan synthase and beta-glucan synthase II activities were also measured in microsomal membranes. Of these, only beta-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on alpha-1,4-D-galacturonosyltransferase and beta-1,4-D-xylan synthase activities.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Gene Expression; Hexosyltransferases; Monosaccharides; Pectins; Pentosyltransferases; Plant Stems; Uronic Acids

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.

Topics: Agaricales; Amino Acid Sequence; Blotting, Western; Crystallization; Crystallography, X-Ray; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Molecular Weight; Pectins; Plasmids; Polygalacturonase; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization

The zygomycete Rhizopus oryzae sb is a very efficient organism for retting of flax, the initial microbiological step in the process of making linen. An extracellular polygalacturonase, when isolated could perform retting, and therefore probably is the key component in the retting system of R. oryzae. This was purified and characterized. The purified enzyme has a molecular mass of 37,436 Da from mass spectrometric determination, an isoelectric point of 8.4, and has non-methylated polygalacturonic acid as its preferred substrate. Peptide sequences indicate that the enzyme belongs to family 28, in similarity with other polygalacturonases (EC. 3.2.1.15). It contains, however an N-terminal sequence absent in other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium Ralstonia solanacearum. The biochemical background for the superior retting efficiency of R. oryzae sb is discussed.

Topics: Amino Acid Sequence; Fungal Proteins; Industrial Microbiology; Molecular Sequence Data; Molecular Weight; Pectins; Polygalacturonase; Rhizopus; Sequence Alignment; Substrate Specificity

Thermal analysis (TG-DTA) and FT-IR spectroscopy have been performed on calcium-pectate membranes to investigate their structure and the consequent variation caused by aluminium sorption. Calcium-polygalacturonate (Ca-PG) membranes, model systems of the soil-root interface, were exposed to aluminium solutions at different concentrations (25-800 microM). Three different pHs (3.50, 4.00 and 4.50) were chosen to study the influence of different aluminium species, such as [Al(H2O)6]3+, [Al(OH)(H2O)5]2+ and [Al(OH)2(H2O)(4)]+, on the structure of the Ca-PG membrane. The DTA profiles and FT-IR spectra showed how aluminium sorption induces structural modifications leading to a reorganisation of the chain aggregates and a weakening of the structure. Higher pH, that is, 4.00 and 4.50, and thus hydrolytic aluminium species and related higher calcium content maintain a more regular structure than at pH 3.50. At pH 3.50, both the effect of [Al(H2O)6]3+ and a major calcium release had a greater impact and thus induced a greater weakening of the structure.

Topics: Aluminum; Calorimetry, Differential Scanning; Hydrogen-Ion Concentration; Membranes, Artificial; Pectins; Spectroscopy, Fourier Transform Infrared

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.

Topics: Antibodies, Monoclonal; Cell Growth Processes; Cell Wall; Cytokinesis; Endosomes; Enzyme-Linked Immunosorbent Assay; Epitopes; Glucans; Immunohistochemistry; Microscopy, Electron; Pectins; Plant Roots; Triticum; Xylans; Zea mays

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.

Topics: Alkalies; Arabidopsis; Arabidopsis Proteins; Calcium; Cell Adhesion; Cell Wall; Cells, Cultured; Chelating Agents; Culture Media; Immunohistochemistry; Intercellular Junctions; Mutation; Pectins; Polysaccharides

The Clostridium stercorarium F-9 pel9A gene encodes a pectate lyase Pel9A consisting of 1,240 amino acids with a molecular weight of 135,171. The mature form of Pel9A is a modular enzyme composed of two family-9 catalytic modules of polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. Pel9A showed an overall sequence similarity to the hypothetical pectate lyase PelX of Bacillus halodurans (sequence identity 53%), and CM9-2 showed moderate sequence similarities to some pectate lyases of family 9. Sequence identity between CM9-1 and CM9-2 was 21.3%. The full-length Pel9A lacking the N-terminal signal peptide was expressed, purified, and characterized. The enzyme required Ca(2+) ion for its enzyme activity and showed high activity toward polygalacturonic acid but lower activity toward pectin, indicating that Pel9A is a pectate lyase. Immunological analysis using an antiserum raised against the purified enzyme indicated that Pel9A is constitutively synthesized by C. stercorarium F-9.

Topics: Calcium; Catalytic Domain; Cloning, Molecular; Clostridium; Kinetics; Pectins; Polysaccharide-Lyases; Sequence Homology; Substrate Specificity

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.

Topics: Amino Acid Sequence; Bacillus; Cloning, Molecular; Dickeya chrysanthemi; DNA, Bacterial; Enzyme Inhibitors; Enzyme Stability; Escherichia coli; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Pectins; Polysaccharide-Lyases; Protein Sorting Signals; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sugar Acids; Temperature; Trisaccharides

Several molluscs have been shown to alternate between a non-adhesive trail mucus and a similar gel that forms a strong glue. The major structural difference between the two secretions is the presence of specific proteins in the adhesive mucus. The present study identifies similar proteins from the glue of the slug Arion subfuscus and the land snail Helix aspersa. To investigate the role played by these proteins in adhesion, the proteins were isolated from the adhesive mucus of different molluscs and added to commercial polymer solutions. The effect was observed qualitatively, and quantified using a dynamic rheometer. The isolated proteins triggered gelling or visible stiffening of agar, pectin and polygalacturonic acid. The effect was stronger on more negatively charged polymers. The effect of the proteins was concentration dependent with an optimal concentration of 1-1.5 mg ml(-1), and was weakened when their structure changed. Other proteins and carbohydrates found in the adhesive mucus had no clear mechanical effect on gels. These findings show that the addition of these proteins to large, anionic polymers plays a central role in the formation of a glue from a mucus-like secretion. Such a mechanism may be common among invertebrates, and it may guide biomimetic approaches in the development of glues and gels.

Topics: Adhesiveness; Agar; Animals; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Gels; Helix, Snails; Mucus; Pectins; Polymers; Proteins; Rheology; Snails

Extracted tomato polygalacturonase was purified by cation-exchange chromatography (and gel filtration) and characterized for molar mass, isoelectric point, as well as optimal pH for polygalacturonase activity. The enzymatic reaction of purified tomato polygalacturonase on polygalacturonic acid as substrate was investigated during a combined high-pressure/temperature treatment in a temperature range of 25 degrees to 80 degrees C and in a pressure range of 0.1 to 500 MPa at pH 4.4 (the pH of tomato-based products). The optimal temperature for initial tomato polygalacturonase activity in the presence of polygalacturonic acid at atmospheric pressure is about 55 degrees to 60 degrees C. The optimal temperature for initial tomato polygalacturonase activity during processing shifted to lower values at elevated pressure as compared with atmospheric pressure, and the catalytic activity of pure tomato polygalacturonase decreased with increasing pressure, which was mostly pronounced at higher temperatures. The elution profiles of the degradation products on high-performance anion-exchange chromatography indicated that for both thermal and high-pressure treatment all oligomers were present in very small amounts in the initial stage of polygalacturonase activity. The amounts of monomer and small oligomers increased with increasing incubation times, whereas the amount of larger oligomers decreased due to further degradation.

Topics: Enzyme Activation; Enzyme Stability; Pectins; Polygalacturonase; Pressure; Solanum lycopersicum; Substrate Specificity; Temperature

The kinetics of both malolactic fermentation in Chardonnay wine by encapsulating Lactobacillus casei cells in pectate gel and lyophilized Oenococcus oeni culture has been carried out. The influence of acidity, sulfur dioxide content, and organic acid content on the malolactic activity of the bacteria has been controlled. Encapsulated bacteria degraded 30%, of malic acid in white wine, deacidifying it from pH 3.15 to 3.40, whereas the lyophilized culture degraded 48% of malic acid, deacidifying from pH 3.15 to 3.60. The degree of conversion of malic acid in wine by the encapsulated cells was twice as high as that obtained by the free Lactobacillus casei cells. The operational stability of calcium pectate gel capsules was 6 months. It has been proved that the encapsulated biocatalyst increases the rate of fermentation, and induces the fermentation to take place at high ethanol concentrations. The proposed encapsulated biocatalyst is an attractive material for industrial applications in continuous winemaking processes.

Topics: Capsules; Fermentation; Kinetics; Lactates; Lacticaseibacillus casei; Pectins; Wine

Bacterial cells Nocardia tartaricans with cis-epoxysuccinate hydrolase activity were entrapped in hardened calcium pectate gel by a commercial high performance encapsulator. This enzyme (in a single step reaction with no formation of side products) was used to hydrolyze disodium cis-epoxysuccinate to a pure enantiomer--disodium L-(+)-tartrate. Activities of this enzyme were determined using flow calorimetry. The validity of this method was corroborated by HPLC and isotachophoresis. The immobilized biocatalyst has activity (75.8 U/mgdry) able to convert disodium cis-epoxysuccinate to disodium tartrate at 94% yield in 5.5h. Immobilization of N. tartaricans in hardened calcium pectate gel beads had a positive effect on the activity of cis-epoxysuccinate hydrolase, storage stability, yield, and time of bioconversion.

Topics: Calorimetry; Enzyme Stability; Enzymes, Immobilized; Hydrolases; Kinetics; Microspheres; Nocardiaceae; Pectins; Succinates; Tartrates

A study on the determination and standardization of endopolygalacturonase (EPG) activity is reported, with emphasis on the influence of the degree of substrate esterification using pure yeast EPG. Differences in the results, depending on how the EPG activity unit was defined, are described and discussed. From a theoretical analysis of the expressions established, a general equation for expressing EPG activity in standard international units was obtained, together with the proportional coefficient for each of the substrates studied. It was observed that for a wide range of enzyme concentrations good linear correlations were obtained. Analysis of the comparison ratio (CR) values calculated revealed that these do not differ significantly, except for low-methoxyl apple pectin, confirming the validity of the general expression obtained for pectins with different degrees of esterification. The anomalous CR value found for low-methoxyl (LM) apple pectin is discussed.

Topics: Esterification; Malus; Mathematics; Pectins; Polygalacturonase; Substrate Specificity; Viscosity

The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated. Sucrose was chosen as a carbon source for REA-11 production. Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination. A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ml. The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best. Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control. In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kappaLa of 100 h(-1). REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0-6.5. Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants. In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant.

Topics: Carbon; Corynebacterium; Flocculation; Hydrogen-Ion Concentration; Nitrogen; Pectins; Sucrose; Temperature; Urea; Waste Disposal, Fluid; Zea mays

Insulin-loaded calcium pectinate nanoparticles were prepared as a potential colonic delivery system by ionotropic gelation with calcium ions. The effects of pectin molecular weight (Mv) and formulation pH on the characteristics of the nanoparticles were evaluated. Commercial pectins, LM101 and LM104, with respective degrees of esterification of 36% and 28%, were depolymerized by mechanical milling to give Mv ranging from 89 to 5.6 kDa. Milled pectins did not yield nanoparticles with significantly different mean diameter and insulin association efficiency (AE) compared to nanoparticles of unmilled pectins. LM104 nanoparticles had smaller variation in mean size than the LM101 nanoparticles. Formulation pH significantly influenced the AE and stability of the nanoparticles. Increasing the pH from 2 to 3 enhanced the AE by three-fold, from 32.76% to 93.31%, at an insulin loading concentration of 80 U/mL. This increase in AE was correlated to the charge density on the pectin molecules as a function of pH. Subsequent release of associated insulin from the nanoparticles was dependent on the extent of dilution of the nanoparticle dispersion and the pH of the dissolution medium. Cross-flow filtration could be used to separate the nanoparticles from unassociated ions and molecules, without compromising the characteristics of the nanoparticles.

Topics: Calcium; Fluoresceins; Hydrogen-Ion Concentration; Insulin; Microspheres; Molecular Weight; Nanotechnology; Pectins; Technology, Pharmaceutical

The objective of this study was to develop a floatable multiparticulate system with potential for intragastric sustained drug delivery. Cross-linked beads were made by using calcium and low methoxylated pectin (LMP), which is an anionic polysaccharide, and calcium, LMP, and sodium alginate. Beads were dried separately in an air convection type oven at 40 degrees C for 6 hours and in a freeze dryer to evaluate the changes in bead characteristics due to process variability. Riboflavin (B-2), tetracycline (TCN), and Methotrexate (MTX) were used as model drugs for encapsulation. Ionic and nonionic excipients were added to study their effects on the release profiles of the beads. The presence of noncross linking agents in low amounts (less than 2%) did not significantly interfere with release kinetics. For an amphoteric drug like TCN, which has pH dependent solubility, three different pHs (1.5, 5.0, and 8.0) of cross-linking media were used to evaluate the effects of pH on the drug entrapment capacity of the beads. As anticipated, highest entrapment was possible when cross-linking media pH coincided with least drug solubility. Evaluation of the drying process demonstrated that the freeze-dried beads remained buoyant over 12 hours in United States Pharmacopeia (USP) hydrochloride buffer at pH 1.5, whereas the air-dried beads remained submerged throughout the release study. Confocal laser microscopy revealed the presence of air-filled hollow spaces inside the freeze dried beads, which was responsible for the flotation property of the beads. However, the release kinetics from freeze dried beads was independent of hydrodynamic conditions. Calcium-pectinate-alginate beads released their contents at much faster rates than did calcium-pectinate beads (100% in 10 hours vs. 50% in 10 hours). It appears that the nature of cross-linking, drying method, drug solubility, and production approach are all important and provide the opportunity and potential for development of a gastroretentive drug delivery system.

Topics: Alginates; Chemistry, Pharmaceutical; Delayed-Action Preparations; Excipients; Gastrointestinal Transit; Glucuronic Acid; Hexuronic Acids; Hydrogen-Ion Concentration; Methotrexate; Pectins; Riboflavin

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

Topics: Arabidopsis; Aspergillus niger; Base Sequence; DNA Primers; Nicotiana; Pectins; Plant Stems; Plants, Genetically Modified; Polygalacturonase; Recombinant Proteins

The present study focuses on the application of immobilization technology to enzymic sugar syntheses. The paper describes an improved silica-alginate matrix established for entrapment and encapsulation. The replacement of alginate with pectate provided enhanced chemical resistance of the matrix, which allows the use of 1% (w/v) polyphosphate in reaction mixtures. Polylysine, a reagent for silica condensation, was replaced by a much cheaper alternative, namely polyethyleneimine. The proposed design was applied in the production of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid (CMP-sialic acid) by immobilized recombinant enzymes or Escherichia coli cells containing overexpressed enzymes. A comparison between these two strategies was made. On the basis of the results we conceptualized a system to synthesize sialyloligosaccharides by using a biocatalyst entrapped in calcium pectate-silica gel beads.

Topics: Cell Culture Techniques; Cells, Immobilized; Coated Materials, Biocompatible; Cytidine Monophosphate N-Acetylneuraminic Acid; Enzymes, Immobilized; Escherichia coli; Oxo-Acid-Lyases; Pectins; Recombinant Proteins; Silica Gel; Silicon Dioxide

To screen industrially important extracellular enzymes from the newly isolated alkalophilic bacilli and to characterize them by phenotypic and 16S-internal transcribed spacer (ITS) rDNA restriction pattern analysis.. Three different environmental samples, soil, leather and horse faeces, were collected within the province of Izmir. Isolates grown on Horikoshi-I medium for 24 h at 37 degrees C were screened for extracellular enzyme activity by using eight different substrates: birchwood xylan, carboxymethylcellulose, casein, citrus pectin, polygalacturonic acid, soluble starch, and Tween 20 and 80. In total, 115 extracellular enzyme-producing bacilli were obtained. Casein was hydrolysed by 78%, soluble starch by 67%, citrus pectin by 63%, polygalacturonic acid by 62%, Tween 20 by 34%, birchwood xylan by 16%, Tween 80 by 12%, and carboxymethylcellulose by 3% of the isolates. The isolates were differentiated into 19 distinct homology groups by the 16S-ITS rDNA restriction pattern analysis.. Eight different extracellular enzyme activities were determined in 115 endospore forming bacilli. The largest 16S-ITS rDNA homology group (HT1) included 36% of the isolates, 98% of which degraded casein, polygalacturonic acid, pectin and starch.. This study is the first report on the characterization of the industrial enzyme-producing alkalophilic bacilli by 16S-ITS rDNA restriction fragment length polymorphism (RFLP). Restriction profiles of 64% of the isolates were found to be different from those of five reference strains used.

Topics: Animals; Bacillus; Caseins; Deoxyribonucleases, Type II Site-Specific; DNA, Bacterial; DNA, Ribosomal Spacer; Feces; Horses; Hydrogen-Ion Concentration; Pectins; Peptide Hydrolases; Phenotype; Phylogeny; Polymorphism, Restriction Fragment Length; Sequence Homology, Nucleic Acid; Soil Microbiology; Starch; Temperature; Turkey

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

In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins. Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, homogalacturonan, from wheat leaf cell walls. Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis. The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses. Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer homogalacturonan fraction. The near absence of rhamnosyl residues proved that the isolated homogalacturonan fraction was free of rhamnogalacturonans. If HF-solvolysis was performed at -23 degrees C, the resulting homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall. The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains. We can thus isolate native homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.

Topics: Cell Wall; Ether; Hydrofluoric Acid; Imidazoles; Immunohistochemistry; Pectins; Triticum; Water

Bacillus sp. DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin supplements. Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 micro l/g dry substrate) with distilled water at 75% moisture level, after 36 h of incubation at 37 degrees C.

Topics: Bacillus; Carbon; Culture Media; Dietary Fiber; Fermentation; Nitrogen; Pectins; Polygalacturonase; Vitamins

The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40 degrees C, respectively, these being around 0.5 U higher and 5 degrees C lower than those shown by the native enzyme. The K(m) value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.

Topics: Culture Media; Food Industry; Hydrogen-Ion Concentration; Pectins; Polygalacturonase; Recombinant Proteins; Saccharomyces cerevisiae; Schizosaccharomyces; Temperature; Viscosity

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

Potato (Solanum tuberosum) cultivars differ quantitatively in their responses to mechanical stress including the ability to synthesize melanin pigments in tuber tissues. Investigations into the cellular events induced by mechanical stress on tuber tissues have shown that an early cellular response is a significant and rapid synthesis of superoxide radicals. This burst of radical production distinctively displays a reproducible biphasic pattern over time with peaks of generation at 2 and 5 h. A concomitant consequence of the generation of these free radicals is elevated levels of oxidatively modified tuber proteins. Both radical generation and protein modification vary between cultivars but both are directly proportional to the amount of melanin pigments produced. Cell-free extracts of mechanically stressed tissues, pectic fragments, and scission products generated from cell walls are able to induce superoxide generation in non-stressed tissues, indicating the participation of a biologically active factor that induces a further a phase of radical synthesis.

Topics: Adaptation, Physiological; Cell Wall; Hydrogen Peroxide; Melanins; Oxidative Stress; Pectins; Pigments, Biological; Plant Stems; Polysaccharides; Signal Transduction; Solanum tuberosum; Stress, Mechanical; Superoxides

The water solubility of pectin was successfully decreased by cross-linking with increasing amounts of epichlorohydrin in the reaction media. The initial molar ratios of epichlorohydrin/ galacturonic acid monomer in the reaction mixtures were 0, 0.37, 0.56, 0.74, 1.00, 1.47, and 2.44. The resulting epichlorohydrin cross-linked pectins were thus referred to as C-LP0, C-LP37, C-LP56, C-LP75, C-LP100, C-LP150, and C-LP250, respectively. Methoxylation degrees ranged from 60.5 +/- 0.9% to 68.0 +/- 0.6%, and the effective cross-linking degrees, determined by quantification of the hydroxyl anions consumed during the reaction, were 0, 17.8, 26.0, 38.3, 46.5, 53.5, and 58.7%. respectively. After incubating the different cross-linked pectins (0.5% w/v) in 25 mL of 0.05 M acetate-phosphate buffer (pH 4.5), containing 50 microL of Pectinex Ultra SP-L (pectinolytic enzymes), between 60 and 80% of the pectin osidic bounds were broken in less than 1 hr. Moreover, increasing the cross-linking degree only resulted in a weak slowing on the enzymatic degradation velocity.

Topics: Carbohydrate Sequence; Chemistry, Pharmaceutical; Colon; Cross-Linking Reagents; Drug Compounding; Drug Delivery Systems; Drug Stability; Enzymes; Epichlorohydrin; Molecular Sequence Data; Pectins; Solubility; Time Factors

Eight cold-adapted, polygalacturonase-producing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofilobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2 degrees C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14 degrees C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be optimally active at 40 degrees C and pH 5, and that from the Cryptococcus strains at 50 degrees C and pH 4. The polygalacturonase from C. macerans started to lose activity after 1 h of incubation at 40 degrees C, while that from the other strains had already lost activity at 30 degrees C. All the strains except C. aquaticus produced isoenzymes of polyglacturonase. In addition to polygalacturonase, the Cystofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase.

Topics: Carbon; Cold Temperature; Cryptococcus; Culture Media; DNA; DNA, Ribosomal; Hydrogen-Ion Concentration; Pectins; Polygalacturonase; Polymerase Chain Reaction; Protein Isoforms; Temperature; Time Factors; Yeasts

The bacterial wilt pathogen Ralstonia solanacearum produces three extracellular polygalacturonases (PGs): PehA, PehB, and PehC. All three PGs hydrolyze pectin's polygalacturonic acid backbone, but each releases different reaction products. PehA and PehB contribute significantly to pathogen virulence, probably by facilitating root invasion and colonization. To determine the collective contribution of PGs to virulence and saprophytic survival, we cloned, characterized, and mutated the R. solanacearum pehC gene, which encodes a distinctive monogalacturonate-releasing exo-PG. The virulence of a pehC mutant on tomato was indistinguishable from that of its wild-type parent; thus, this exo-PG alone does not contribute significantly to wilt pathogenesis. Unexpectedly, a completely PG-deficient triple pehA/B/C mutant was slightly more virulent than a pehA/B mutant. PehC may degrade galacturonide elicitors of host defense, thereby protecting the pathogen from plant antimicrobial responses. A galacturonate transporter gene, exuT, is immediately downstream of pehC and the two genes are co-transcribed. It has been hypothesized that galacturonic acid released by PGs from plant cell walls nourishes bacteria during pathogenesis. To separate the pectolytic and nutrient-generating roles of the PGs, we made an exuT mutant, which still produces all three isozymes of PG but cannot uptake PG degradation products. This exuT mutant had wild-type virulence on tomato, demonstrating that metabolism of galacturonic acid does not contribute significantly to bacterial success inside the plant.

Topics: Amino Acid Sequence; Bacterial Proteins; DNA, Bacterial; Glycoside Hydrolases; Gram-Negative Aerobic Rods and Cocci; Hexuronic Acids; Membrane Proteins; Molecular Sequence Data; Mutation; Operon; Pectins; Sequence Analysis, DNA; Virulence

Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit. A biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 was proposed. Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid. The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 microM UDP-glucose. In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts. Furthermore, the activities of UDP-glucose pyrophosphorylase (R2=0.97), UDP-galactose epimerase (R2=0.75) and UDP-galactose dehydrogenase (R2=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources. Therefore, the biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase. Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.

Topics: Corynebacterium; Culture Media; Flocculation; Pectins; Uridine Diphosphate Galactose; Uridine Diphosphate Glucose; Uridine Diphosphate Sugars

A colorimetric bioassay has been developed enabling separation of wood pulp qualities based on the quantity of methyl-esterified galacturonan exposed on fiber surfaces. Thermomechanical pulp (TMP) and chemi-TMP (CTMP) had, respectively, 3.1 and 0.7 microg galacturonan on the surface per mg wood pulp. The presence of galacturonan on the surface of TMP and CTMP was further confirmed by immunogold localization.

Topics: Colorimetry; Immunoassay; Methylation; Pectins; Sensitivity and Specificity; Wood

A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies.

Topics: Antibodies, Monoclonal; Antibody Specificity; Binding, Competitive; Enzyme-Linked Immunosorbent Assay; Epitopes; Haptens; Methylation; Pectins

Combinations of Eudragit RS and deesterified pectin, polygalacturonic acid (PGA), or its potassium and sodium salts, when applied as a film coat, has a potential value as a colon-specific delivery system. Dispersions of PGA in Eudragit RS were used as the film former for coating of 5-aminosalicylic acid (5-ASA) tablet cores. Drug release behavior was assessed, in vitro, under simulating conditions in term of pH and time to in vivo during their transit to the colon. Negligible drug release occurred during first 5 hr where the coated tablets were in the stomach and small intestine. After that, the pectinolytic enzymes were added into the pH 6.8 medium to simulate the in vivo condition where there is the digestion of bacteria in the colon. The release of 5-ASA from the coated tablets occurred linearly as a function of time. Drug release depended on the composition of the mixed film, as well as the ratio of Eudragit RS to PGA or its salts. The highest drug release from the coated tablets of about 40% was obtained when the ratio of Eudragit RS to potassium salt of PGA was 2.5 to 1. Drug release profiles seemed to conform to the mechanism involving the osmotically driven release and formation of channels in the film caused by dissolution of PGA salts. Channel formation was, in most cases, activated by the presence of pectinolytic enzymes, showing that the PGA in the mixed film was subjected to enzymic breakdown. In conclusion, PGA could be used as an additive in Eudragit RS films to control the release of colonic delivery system.

Topics: Acrylic Resins; Colon; Drug Delivery Systems; Mesalamine; Pectins; Tablets, Enteric-Coated

A method for the quantitative determination of oligogalacturonic acids (OGAs) by on-line high-performance liquid chromatographic (HPLC) separation and mass spectrometric detection via an electrospray interface (ESI-MS) without additional desalting steps was developed. Saturated OGAs up to a degree of polymerization (dp) of 3 were quantified by comparison with reference compounds. The calibration plots showed high linearity (R(2)>0.99), and the detection limits for dp 1, 2, and 3 were 11, 28, and 6 ng per injection, respectively. Non- and partially methyl-esterified OGAs with a dp of 3 and 4 were calculated semi-quantitatively as dp 3. The analytical system was used for the quantification of OGAs of digests obtained by incubation of polygalacturonic acid, pectin, and carrot pomace with commercial enzyme preparations. Furthermore, methyl-esterified OGAs up to a dp of 12 containing up to 4 methyl esters were detected in a pectin digest.

Topics: Cellulases; Chromatography, High Pressure Liquid; Daucus carota; Oligosaccharides; Pectins; Polygalacturonase; Spectrometry, Mass, Electrospray Ionization

The electrophoretic migration in polyacrylamide gels of oligogalacturonic acids (OGAs) derivatized by a fluorophore (2-aminoacridone) was studied. We found conditions such that OGAs can be separated up to a degree of polymerization (DP) of 40. The migration was dependent on degree of methylation and DP, because the OGA mobility relies on the charge of the galacturonic acid residues. Since both methylated and unmethylated oligosaccharides can be resolved, polysaccharide analysis using carbohydrate gel electrophoresis (PACE) is a powerful method for studying the fingerprint of pectin hydrolysis. It can be used to characterize endopolygalacturonase (Endo-PG) tolerance of methylation. Furthermore, using an Endo-PG that can distinguish low and highly methylated pectin, PACE can be used to investigate the blockwise or nonblockwise distribution of methylation of polygalacturonic acid. We show that the method can be applied to crude cell wall preparations of Arabidopsis inflorescence stems. Using chemical deesterification before or after Endo-PG digestion, we show that in the Arabidopsis cell wall, the pectins have both nonesterified and highly esterified regions.

Topics: Arabidopsis; Cell Wall; Electrophoresis, Polyacrylamide Gel; Methylation; Pectins; Polysaccharides

A high-performance size-exclusion chromatography-evaporative light scattering detector method was used to separate, detect and quantify galacturonic acid (GA) oligomers. In 40 mM acetic acid GA monomer, dimer and trimer could be separated with baseline resolution but polygalacturonic acid (PGA) precipitated and could not be eluted from the column. An NH4OAc, pH 3.7, buffer was developed as the eluent which separated GA oligomers as well as PGA and pectin without precipitation. Linear calibration curves for mono-, di- and tri-GA were produced with this buffer which could be used to estimate masses of tetra-, penta- and hexa-GA, as well as 19mer and 20mer.

Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Light; Pectins; Scattering, Radiation

Plants possess an efficient nonself surveillance system triggering induced disease resistance mechanisms upon molecular recognition of microbial invaders. Successful pathogens have evolved strategies to evade or counteract these mechanisms, e.g., by the generation of suppressors. Pectic fragments produced during host cell wall degradation can act as endogenous suppressors of the hypersensitive response in wheat leaves. We have isolated and characterized homogalacturonans from cell walls of two wheat cultivars susceptible to the stem rust fungus, Puccinia graminis f. sp. tritici, namely cvs. Prelude and Marquis, and from near-isogenic lines of both cultivars containing the Sr5-gene for hypersensitive rust resistance. Two independent approaches were used to compare their methyl esterification: i) immunochemistry using the monoclonal antibodies JIM5, JIM7, PAM1, and LM7 and ii) chromatography of oligogalacturonides representing stretches of contiguous nonmethyl-esterified GalA residues. The results clearly indicate a significant difference in the homogalacturonans from susceptible and resistant wheat lines. The difference can best be explained by assuming a nonrandom and more blockwise distribution of the methyl esters in the homogalacturonans of susceptible wheat cultivars as compared with a presumably more random distribution in the near-isogenic resistant lines. Possible consequences of this difference for the enzymatic generation of endogenous suppressors are discussed.

Topics: Antibodies, Monoclonal; Basidiomycota; Cell Wall; Esterification; Pectins; Plant Diseases; Triticum

Topics: Acetylation; Animals; Antigens, Bacterial; Chemical Phenomena; Chemistry, Physical; Glycoconjugates; Humans; Hydrazines; Immunochemistry; Immunodiffusion; In Vitro Techniques; Ion Exchange; Mice; Molecular Structure; Pectins; Phenylhydrazines; Polylysine; Polysaccharides, Bacterial; Salmonella typhi

The objective of this study is to determine the influence of aluminum sorption on a calcium-polygalacturonate (Ca-PG) network used as a soil-root interface model. The Ca-PG network is exposed to aluminum solutions at different concentrations (25-800 microM) at pH 3.50. High concentrations lead to a release of calcium (80%) and aluminum becomes the predominant reticulating cation of the polygalacturonic chains. The FTIR spectra show how aluminum sorption induces shifts of the characteristic bands of carbohydrates in the spectral regions of 1700-1400 and 1200-800 cm(-1), which are enhanced by decreasing intensities. This might be induced by a weakening of the metal-PG complex through conformational variations of the structure. Scanning electron micrographs also show a collapse of the fibrillar structure of Ca-PG that is due to aluminum sorption. This structural rearrangement suggests that the soil-root interface could modify its functionality, affecting the transport of metal ions (nutrients) across the interface and consequently through the cell membranes.

Topics: Aluminum; Calcium; Models, Biological; Pectins; Spectroscopy, Fourier Transform Infrared

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides and have a pectin-like structure. The results of a structural analysis of SSPS by using polygalacturonase (PGase) and rhamnogalacturonase (RGase) clarified that the main backbone consisted of galacturonan (GN) and rhamnogalacturonan (RG), which were composed of the diglycosyl repeating unit, -4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-. The side chains of beta-1,4-galactans, branched with fucose and arabinose residues, were linked to the C-4 side of rhamnose residues in the RG regions. The degree of polymerization (dps) of GN, which linked the RG regions together, was estimated to be about 4-10 residues, and some were modified with xylose residues on the C-3 side of the galacturonates. The dps of GN at the reducing end of SSPS was estimated to be about 7-9 residues. Moreover, the fragment of the basic structure of the RG region, -[4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-]2-, some of which had long-chain beta-1,4-galactans branched on the C-4 side of rhamnose residues, were liberated from SSPS by the RGase treatment. The dps of the galactan side chain was estimated to be about 43-47 residues by an analysis of the digestion products from the beta-galactosidase treatment.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Glycine max; Glycoside Hydrolases; Methylation; Molecular Sequence Data; Monosaccharides; Pectins; Solubility; Spectrometry, Mass, Fast Atom Bombardment

Amide exchange mass spectrometry (MS) was used to study the enzyme endopolygalacturonase II (EPG-II) from Aspergillus niger as it binds to an oligosaccharide substrate. A localized decrease in the level of deuterium incorporation in EPG-II of the EPG-II-oligosaccharide complex relative to that of the free EPG-II identified the location of substrate contact, which is in agreement with published site specific mutation studies. In addition, when bound with substrate, regions of EPG-II remote from the substrate binding site became exposed to the solvent, as revealed by an increase in the amount of incorporated deuterium, indicating a conformational change in the enzyme. Fluorescence experiments were performed to provide additional evidence for an altered conformation of EPG-II as a result of substrate binding. This novel application of amide exchange-MS to the study of protein-carbohydrate binding has, for the first time, described in detail the conformational changes associated with EPG-II when it binds a substrate. Amide exchange-MS was also used to study the interactions of EPG-II and the polygalacturonase inhibitor protein (PGIP). Mass spectral data of the EPG-II-oligosaccharide complex in the presence of Phaseolus vulgaris PGIP indicate that the inhibitor contacts EPG-II at a site remote from the substrate binding cleft, and is restricting the conformational changes of EPG-II. Fluorescence experiments also revealed that upon binding of PGIP, the conformational changes mentioned above for the EPG-II-substrate complex are minimized. These results, together with previously reported data, point to a location on EPG-II for interaction with PGIP as well as a possible mechanism for noncompetitive inhibition of EPG-II.

Topics: Amides; Aspergillus niger; Binding, Competitive; Enzyme Inhibitors; Fungal Proteins; Macromolecular Substances; Mass Spectrometry; Pectins; Phaseolus; Plant Proteins; Polygalacturonase; Protein Conformation; Spectrophotometry, Ultraviolet; Substrate Specificity

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, contains a neutral endopolygalacturonase (endoPG) subfamily of genes that was previously isolated. We report here that pg2, a member of this subfamily, is early and strongly expressed during the first steps of pathogenesis of sunflower cotyledons. The corresponding protein, PG2, was produced in the heterologous Kluyveromyces lactis system and purified. Characterization of the recombinant enzyme revealed a narrow pH activity curve with an optimal pH of 4.5. Hydrolysis of polygalacturonic acid by PG2 resulted in the accumulation of oligomers ranging from 2- to 9-mer. This degradation profile indicates a random attack on the polymer and demonstrates an endo-mode of action. These results provide evidence that pg2 contributes to the infection process during the early phase of host colonization.

Topics: Ascomycota; Blotting, Northern; Chromatography, Thin Layer; Gene Expression Regulation, Fungal; Helianthus; Hydrogen-Ion Concentration; Isoelectric Point; Kluyveromyces; Pectins; Plant Diseases; Polygalacturonase; Recombinant Proteins; Temperature; Transcription, Genetic

Two exo-polygalacturonases (EC 3.2.1.67) were purified from a commercial Aspergillus niger enzyme preparation by ammonium sulfate precipitation, preparative electrofocusing, anion-exchange and size-exclusion chromatographies. The enzymes had molar masses of 82 kDa (exo-PG1) and 56 kDa (exo-PG2). Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Addition of 0.01 mM HgCl(2) increased the exo-PG2 activity 3.4 times but did not affect exo-PG1. Analysis of the reaction products of (reduced) pentagalacturonate by high-performance anion-exchange chromatography revealed that both enzymes split the substrate from the non-reducing end in a multi-chain attack mode. Exo-PG1 had a broad specificity towards oligogalacturonates with different degrees of polymerisation, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid and Xyl-GalA disaccharide, as identified by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing galacturonic acid and acetylated galacturonic acid, as shown by ESI-ITMS.

Topics: Aspergillus niger; Beta vulgaris; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hexuronic Acids; Hydrogen-Ion Concentration; Oligosaccharides; Pectins; Pisum sativum; Polygalacturonase; Polysaccharides; Substrate Specificity; Temperature

Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells. The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa ( Medicago sativa). However, no significant similarity was found to known PGases. The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family. Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap. A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Daucus carota; DNA, Complementary; DNA, Plant; Escherichia coli; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Genome, Plant; Glycoside Hydrolases; Hexuronic Acids; Immunohistochemistry; In Situ Hybridization; Meristem; Molecular Sequence Data; Pectins; Plant Roots; Saccharomyces cerevisiae; Sequence Analysis, DNA; Sequence Homology, Amino Acid

Diseases of the gastrointestinal system are often related with irritations or pathological changes of mucous membranes. In an ex vivo system based on porcine colonic tissue various neutral and acidic polysaccharides were tested concerning their bioadhesive potential in order to form artificial mucin layers on colon epithelial membranes. Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast highly esterified pectins and neutral polysaccharides were ineffective. Within a structure-activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Polysaccharide layers, located exclusively on the apical membrane surface of colonic tissue, were visualized by fluorescent microscopy. The adhesion of the exogenous galacturonides on the tissue surface was mediated by interaction with the endogenous mucin, for the release of the endogenous mucines with a mucolytic agent resulted in a decreased bioadhesion of exogenous galacturonides. Additionally, mucin-galacturonide synergism was shown by rheological methods. The artificial mucin layers provide protective effects on colonic mucous membranes against toxic agents as shown by incubation of the tissue with TritonX-100.

Topics: Animals; Cell Adhesion; Cell Membrane; Chromatography, High Pressure Liquid; Colon; Intestinal Mucosa; Pectins; Rheology; Swine

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl2, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.

Topics: Botrytis; Calcium Chloride; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Molecular Weight; Pectins; Polygalacturonase

The effects of glucose and of a pectic substrate in the duplication cycle, spore polarization and septation of Aspergillus nidulans were tested in poor and rich media. Growth on poor conditions and on sodium polypectate slowed nuclear duplication and reduced the coupling of polarization to mitosis. Coupling of septation to the third mitosis was also reduced by changing growth conditions. When protein kinase A (PKA) and protein kinase C (PKC) activators were added to the media the results suggested a role for PKA in slowing the duplication cycle, while allowing polarization. Addition of a PKC activator to poor media uncoupled the first septum formation from the third mitosis in a carbon source-regulated manner, suggesting a role for PKC in coordinating cell cycle signals, growth and cytokinesis.

Topics: Aspergillus nidulans; Carbohydrates; Cell Division; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Fungal Structures; Glucose; Mitosis; Pectins; Protein Kinase C; Signal Transduction; Spores, Fungal

The structure and rheological properties of water-soluble polysaccharides from industrialized mango pulp were investigated. Soluble fraction (SF) 2 was heterogeneous on high performance size exclusion chromatography, giving two peaks as determined by multi-angle laser light scattering and refractive index detectors. The presence of starch in SF2 was demonstrated by a positive iodine reaction and by 13C nuclear magnetic resonance (NMR) spectroscopy. The presence of pectic polysaccharides was shown by a calorimetric method, 13C-NMR spectroscopy and carboxyl reduction. The main pectic polysaccharide was polygalacturonic acid; type I rhamnogalacturonan was also detected. Analysis of the rheological properties of SF2 showed a pseudoplastic behavior up to 3 g x l(-1). 'Creep and recovery' tests and analysis performed under a dynamic state revealed a weak gel character for solutions at concentrations of 15, 20 and 30 g x l(-1).

Topics: Iodine; Magnetic Resonance Spectroscopy; Mangifera; Pectins; Polysaccharides; Time Factors

Tanacetan TVF was found to have a branched structure with a backbone of linear alpha-1,4-D-galacturonan. The ramified regions consist of linear alpha-1,2-L-rhamno-alpha-1,4-D-galacturonan as the core. The side chains appear to attach to the 4-position of the L-rhamnopyranose residues. They are present as single beta-galactopyranose residues or a branching beta-1,4-galactopyranan bearing 4,6-substituted beta-D-galactopyranose residues as branched points. In addition, the ramified regions contain side chains of a branched alpha-1,5-arabinofuranan possessing 2,5- and 3,5-substituted alpha-L-arabinofuranose residues as branching points. Some side chains of rhamnogalacturonan appear to be arabinogalactan which contains branched sugar chains of alpha-1,5-arabinofuranan attached to the linear chains of beta-1,4-galactopyranan by 1,3- and 1,6-linkages. The residues of alpha-L-arabinofuranose seem to occupy the terminal positions of the arabinogalactan side chains.

Topics: Asteraceae; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydrolysis; Lithium; Magnetic Resonance Spectroscopy; Methylation; Pectins; Plants; Polysaccharides; Time Factors

Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. To gain insight into the mechanisms that determine specificity in response to a particular stimulus, a biochemical approach has been employed. Photoautotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used as experimental system to characterize MAP kinase activation by different stress-related stimuli. An elicitor preparation of the tomato-specific pathogen Fusarium oxysporum lycopersici was shown to result in the simultaneous induction of four kinase activities that could be separated by ion-exchange chromatography. The simultaneous activation of multiple MAP kinases was further substantiated by distinct pharmacological and immunological properties: a differential sensitivity toward various protein kinase inhibitors and a differential cross-reaction with isoform-specific MAP kinase antibodies. In contrast to the two fungal elicitors chitosan and the F. oxysporum lycopersici preparation, the plant-derived stimuli polygalacturonic acid and salicylic acid were shown to activate distinctly different subsets of MAP kinases. Application of a voltage pulse was introduced as a transient stress-related stimulus that does not persist in the culture. Voltage application activates a distinct set of MAP kinases, resembling those activated by salicylic acid treatment, and generates a refractory state for the salicylic acid response. The inhibitory effect of nifedipine indicates that current application may directly affect voltage-gated calcium channels, thus, providing a tool to study various calcium-dependent pathways.

Topics: Antibodies; Calcium; Calcium Channel Blockers; Cells, Cultured; Chitin; Chitosan; Electric Stimulation; Enzyme Activation; Fusarium; Hydrogen Peroxide; Immunity, Innate; Mitogen-Activated Protein Kinases; Nifedipine; Pectins; Plant Diseases; Precipitin Tests; RNA, Messenger; Salicylic Acid; Solanum lycopersicum

Recent progress in the determination of the intramolecular distribution of methyl-esterified residues in pectic substrates has been made using a fragmentation approach in which endopolygalacturonase is used to digest the polysaccharide and its subsequent (methyl-ester sequence-dependent) digest pattern is determined. This has been facilitated by the separation of partially methyl-esterified enzyme digest fragments using high-performance anion-exchange chromatography at pH 5. Here we demonstrate that capillary electrophoresis can be used as an additional method for separation and quantification of such digest patterns. The technique offers improvements in speed and economy of materials and a straightforward quantification procedure.

Topics: Electrophoresis, Capillary; Esterification; Methylation; Pectins; Polygalacturonase

Arthrobacter simplex ATCC 6946 free and immobilized cells were assayed for their ability to convert 4-androsten-3,17-dione (AD) to 1,4-androstadien-3,17-dione (ADD) in aqueous and liposomal media. Bioconversions were carried out in a 100 ml flask containing 25 ml of AD liposomal or aqueous medium for 3h, and AD concentrations ranging from 0.3 to 1.0 mM were tested. AD/ADD ratios in samples were determined by HPLC. Biotransformation of substrate entrapped in multilamellar vesicles (MLV) was demonstrated to be better than the corresponding free form. In the former case, 2h were necessary to completely bioconvert 1 mM AD. By contrast, 3h were needed to reach 50% bioconversion in (4%) ethanol medium containing 0.63 mM AD. The liposomal medium allows us to perform steroid conversions at high concentrations of AD, reusing immobilized cells in suitable conditions which are non-toxic for microorganisms.

Topics: Androstenedione; Arthrobacter; Liposomes; Pectins; Solutions; Water

The occurrence of pectic polysaccharide epitopes in cells and tissues of the pea testa during late stages of seed development have been examined in relation to anatomy and cell properties. Homogalacturonan, in a highly methyl-esterified form, was present throughout late development in all pea testa cell walls, including the thickened cell walls of the outer macrosclereid layer. Two epitopes, characteristic of the side-chains of the rhamnogalacturonan-I domain of pectic polysaccharides, occurred in restricted and separate cell layers of the pea testa. A (1-->4)-beta-D-galactan epitope was restricted to regions of the outer cell wall of the testa and to inner regions of the macrosclereid layer at 20 DAA and was absent from the osteosclereid and parenchyma cell walls. By 25 DAA the (1-->4)-beta-D-galactan epitope occurred only in the outer epidermal cell walls. A (1-->5)-alpha-L-arabinan epitope was also dependent on the developmental stage of the seed and was found with greatest abundance in the walls of the inner parenchyma cells. Cell separation studies indicated that, although calcium cross-links were involved in the maintenance of the link between the macrosclereid layer and proximal cell layers, most cell-to-cell adhesion in the testa was not due to calcium- or ester-based bonds.

Topics: Antibodies, Monoclonal; Calcium; Cell Adhesion; Cell Wall; Epitope Mapping; Epitopes; Galactans; Glycoside Hydrolases; Pectins; Pisum sativum; Polysaccharides; Seeds

We fed cholesterol-enriched (0.1% w/w) semipurified diets containing 3% of lemon pectin or 3% of the polygalacturonic acid regions fraction (smooth regions fraction) of the lemon pectin to hybrid F1B hamsters for a period of 8 weeks. A control group was fed cellulose and a positive control group was fed psyllium. The feeding of the semipurified diets resulted in an increase of plasma cholesterol levels in all the dietary groups when compared with initial values. The hamsters fed the psyllium, pectin, or the polygalacturonic acid regions fraction had significantly (P < 0.05) lower plasma cholesterol levels than the cellulose fed group throughout the experimental period. Plasma cholesterol levels in the hamsters fed the psyllium, pectin, or polygalacturonic acid regions fraction were not significantly different. Liver cholesterol concentrations were also lower in the hamsters fed the psyllium, pectin, or the polygalacturonic acid regions fraction than in the hamsters fed the cellulose, but this effect reached statistical significance only in the hamsters fed the polygalacturonic acid regions fraction. The results of these studies suggest that the polygalacturonic acid regions of the pectin molecule is responsible for the cholesterol-lowering properties of the pectin.

Topics: Animals; Anticholesteremic Agents; Cellulose; Cholesterol; Cholesterol, Dietary; Cricetinae; Liver; Male; Mesocricetus; Pectins; Psyllium

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

Approaches using immobilized biological materials are very promising for application in different branches of the food industry, especially in the production of fermented beverages. Materials tested by our team for the process of entrapment belong to the family of charged polysaccharides able to form beaded hydrogels by ionotropic gelation (e.g. alginate, pectate, kappa-carrageenan) and synthetic polymers (e.g. polyvinyl alcohol) forming bead- and lens-shaped hydrogels by thermal sol/gel transition. Concentration of a gel, conditions and instrumentation of gelation process, bead and size distribution, porosity, diffusion properties, mechanical, storage and operational stability, and many other parameters were followed and optimized. Our work has been oriented especially to practical applications of immobilized cells. Brewing yeast cells were successfully immobilized by entrapment materials and used in a process of batch and continual production of beer, including primary and secondary fermentation of wort. Other applications include continual production of ethanol by fermentation of different saccharide substrates (molasses, glucose syrup, wheat hydrolysate), mead and non-alcoholic beverages production.

Topics: Beer; Beverages; Cells, Immobilized; Ethanol; Fermentation; Food Microbiology; Food Technology; Hydrogels; Pectins; Saccharomyces cerevisiae; Uronic Acids

Studies of the enzymic digestion of pectic substrates using different polygalacturonase (PG) preparations have revealed evidence for a previously unreported enzyme activity carried out by a contaminating enzyme in one of the preparations. This observed activity involves the demethylation of specific oligogalacturonides, namely 2-methyltrigalacturonic acid and 2,3-dimethyltetragalacturonic acid. However, no large-scale demethylation of highly methylated polymeric substrates is found, demonstrating that the enzyme responsible is not a conventional pectin methylesterase (PME). Furthermore, it has been shown that a commercial sample of fungal PME from Aspergillus niger demethylates all of the oligogalacturonides present as primary products of endo-PG digestion, in contrast with the activity observed here. On the basis of the known methyl ester distribution of the endo-PG-generated fragments and knowledge of which of these oligogalacturonides are demethylated, it is concluded that the observed activity can be explained by the existence of an exo-acting methylesterase that attacks the non-reducing end of the oligogalacturonide molecules.

Topics: Carboxylic Ester Hydrolases; Electrophoresis, Capillary; Methylation; Oligosaccharides; Pectins; Polygalacturonase

Whole bacterial (Pseudomonas putida) and yeast (Saccharomyces cerevisiae) cells were immobilized by entrapment in gel beads of Ca-pectate obtained from sugar beet pulp and gel beads of commercial Ca-alginate (of algal origin). These immobilized-cell particles were tested for Cd2+ removal from dilute aqueous solutions and their mechanical properties were evaluated. Only Ca-pectate gel beads loaded with yeast cells displayed enhanced cadmium-binding efficiency (13.8 mg Cd2+ per g dry matter) as compared to sterile beads (10.7 mg/g). Sterile Ca-pectate gel beads were noticeably more brittle than algal alginate counterparts. The presence of cells reduced the mechanical resistance of both types of beads, however. The practical use of cell-loaded sugar beet Ca-pectate gel as a metal biosorbent is discussed in light of these results.

Topics: Adsorption; Beta vulgaris; Cadmium; Cells, Immobilized; Environmental Pollution; Gels; Humans; Metals; Pectins; Pseudomonas putida; Saccharomyces cerevisiae

Well-characterized pectin samples with a wide range of degrees of esterification (39-74%) were incubated with the solubilized pure alpha and gamma isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the alpha isoform activity at pH 5.6 and to inhibit the gamma isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 +/- 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem. 273, 33150-33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of alpha and gamma isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.

Topics: Amino Acid Sequence; Base Sequence; Carboxylic Ester Hydrolases; DNA Primers; Esterification; Fabaceae; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Molecular Sequence Data; Pectins; Plants, Medicinal; Substrate Specificity

Fusarium moniliforme NCIM 1276 isolated from a tropical mangrove ecosystem produces a single extracellular endo-polygalacturonase with an M(r) of 38 kDa and a carbohydrate content of 4%. It has an alkaline pI of 8.1. The K(m) is 0.12 mg.mL(-1), V(max) is 111.1 micromol.min(-1).mg(-1) and the kcat is 4200 min-1. It has a pH optimum of 4.8. Kinetic and fluorescence data show that tryptophan is involved in binding. An arginine residue at or near the active site may be involved in extended binding of the substrate. A carboxylate and a histidine residue are involved in catalysis. These data are discussed with reference to current literature.

Topics: Amino Acids; Arginine; Binding Sites; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Fusarium; Histidine; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Pectins; Polygalacturonase; Protein Binding; Sequence Analysis, Protein; Spectrometry, Fluorescence; Tryptophan

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Topics: Arginine; Binding Sites; Calcium-Binding Proteins; Cell Wall; Cucurbitaceae; Electrophoresis; Hypocotyl; Models, Molecular; Mutation; Pectins; Peroxidase; Peroxidases; Protein Conformation; Static Electricity

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5 kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2 kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2 kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8 kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH 10 and in the presence of 2 mM Ca2+. Highest enzyme activity was detected at 62 degrees C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167-177], are the first CBM-containing pectinases described to date.

Topics: Amino Acid Sequence; Base Sequence; Carbohydrate Metabolism; Cellulose; Cloning, Molecular; DNA, Bacterial; Molecular Sequence Data; Pectins; Polysaccharide-Lyases; Protein Binding; Pseudomonas; Reproducibility of Results; Sequence Homology, Amino Acid

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.

Topics: Antibodies, Monoclonal; Binding, Competitive; Calcium; Cell Membrane; Cell Wall; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epitopes; Esterification; Immunohistochemistry; Models, Biological; Pectins; Pisum sativum; Plant Proteins; Polygalacturonase; Polysaccharide-Lyases; Protein Structure, Tertiary; Time Factors

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

A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated from sea ice-covered surface water from the Southern Ocean, showed pectinolytic activity on citrus pectin agar. The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplanktis. The supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively. The deduced amino acid sequence of the two ORFs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at 30 degrees C and a pH range of 9 to 10. The Km values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively. Calcium was required for activity on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium.

Topics: Amino Acid Sequence; Antarctic Regions; Cloning, Molecular; Cold Temperature; Enzyme Stability; Gammaproteobacteria; Genes, Bacterial; Geologic Sediments; Hydrogen-Ion Concentration; Molecular Sequence Data; Pectins; Polysaccharide-Lyases; Seawater; Substrate Specificity; Temperature

Selectivity of polyuronide sequestrants (pectate, alginates of various uronide composition) in respect to Sr2+ and Ca2+ ions has been evaluated in terms of thermodynamic affinity. It is suggested that there is no point in the use of pectate as a Sr(2+)-binding agent because at initial stages of reaction it reveals higher affinity to Ca2+ ions in comparison to Sr2+ ions. Contrary to pectate, alginates under similar conditions have higher affinity to Sr2+ ions. It is shown that these ions are bound only by blocks of L-guluronic acid residues in alginate macromolecules. The results obtained lend support to the advisability of the use of alginate preparations with the high content of L-guluronic acid residues for the excretion of Sr2+ ions from human body.

Topics: Alginates; Calcium; Chelating Agents; Humans; Ligands; Macromolecular Substances; Metals, Alkaline Earth; Models, Theoretical; Pectins; Spectrophotometry; Strontium; Thermodynamics; Uronic Acids; Xenobiotics

Cnr (colorless non-ripening) is a pleiotropic tomato (Lycopersicon esculentum) fruit ripening mutant with altered tissue properties including weaker cell-to-cell contacts in the pericarp (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). Whereas the genetic basis of the Cnr mutation is being identified by molecular analyses, here we report the identification of cell biological factors underlying the Cnr texture phenotype. In comparison with wild type, ripe-stage Cnr fruits have stronger, non-swollen cell walls (CW) throughout the pericarp and extensive intercellular space in the inner pericarp. Using electron energy loss spectroscopy imaging of calcium-binding capacity and anti-homogalacturonan (HG) antibody probes (PAM1 and JIM5) we demonstrate that maturation processes involving middle lamella HG are altered in Cnr fruit, resulting in the absence or a low level of HG-/calcium-based cell adhesion. We also demonstrate that the deposition of (1-->5)-alpha-L-arabinan is disrupted in Cnr pericarp CW and that this disruption occurs prior to fruit ripening. The relationship between the disruption of (1-->5)-alpha-L-arabinan deposition in pericarp CW and the Cnr phenotype is discussed.

Topics: Microscopy, Electron, Scanning; Mutation; Pectins; Polysaccharides; Solanum lycopersicum

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.

Topics: Binding Sites; Fusarium; Hexuronic Acids; Kinetics; Models, Chemical; Pectins; Polygalacturonase; Substrate Specificity

A borate-containing pectin was solubilized from sugar beet (Beta vulgaris L. ) cell walls by treatment with 0.5 M imidazole, pH 7. The molecular weight of the pectin was reduced when the borate ester was hydrolyzed by treatment with 1 N HCl. Treatment of the acid-treated pectin with boric acid in the presence of Pb(2+) gave a product whose molecular weight distribution was similar to the imidazole-soluble pectin. The imidazole-soluble pectin was saponified and then digested with endo- and exo-polygalacturonases. These treatments shifted the boron peak at the high molecular weight region to the low molecular weight (10 kDa), which corresponds to rhamnogalacturonan II-borate ester cross-linked dimer (dRG-II-B). The treatment also generated rhamnogalacturonan I (RG-I), dRG-II-B, monomeric rhamnogalacturonan II and galacturonic acid. These results show that imidazole solubilizes a high molecular weight borate-containing pectic complex composed of homogalacturonan-rhamnogalacturonan II and RG-I. Our data suggest that borate esters formed between rhamnogalacturonan II molecules cross-link the macromolecular pectin.

Topics: Borates; Cell Wall; Chenopodiaceae; Chromatography, Gel; Chromatography, High Pressure Liquid; Dimerization; Hydrolysis; Molecular Weight; Monosaccharides; Pectins; Polygalacturonase

Binding isotherms of Pb2+ ions with potassium pectate and potassium alginate with relatively low content of blocks of L-guluronic residues (20%) have been determined. Interactions of Pb2+ ions with polyuronides studied is cooperative. Maximum values of binding constants are an order of magnitude higher than previously determined ones for Ca2+ and Sr2+ ions. Along with ion-coordination ("stoichiometric") interactions, alginate is typified by so-called extra-stoichiometric binding of Pb2+ ions, which presumably proceeds by a coprecipitation mechanism. Limitations of the thermodynamic approach to the selection of sequestrants for human body protection from toxic metal ions are discussed.

Topics: Alginates; Chelating Agents; Humans; Lead; Pectins; Thermodynamics; Uronic Acids

A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35 degrees C in the presence of 5 mM Mn2+ and 0.5% (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous alpha-1,4-linkages.

Topics: Carbohydrate Conformation; Fabaceae; Glucuronosyltransferase; Glycosyltransferases; Hydrogen-Ion Concentration; Kinetics; Membranes; Microsomes; Molecular Weight; Pectins; Plant Proteins; Plants

Pectic polysaccharides are a complex set of macromolecules of the primary cell wall matrix with distinct structural domains. The biosynthesis, organisation and function of these domains within cell wall matrices are poorly understood. An immersion immunofluorescence labelling technique was developed for the in-situ analysis of pectic polysaccharides at the surface of seeds and seedlings of Arabidopsis thaliana (L.) Heynh., and used to investigate the occurrence of pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) epitopes. Seed mucilage appeared to consist of two regions: a highly methyl-esterified HG was a major component throughout the mucilage, while an inner region with relatively low porosity was stabilized by calcium-based HG cross-linking. The small size and transparency of Arabidopsis roots allowed the occurrence of pectic HG and RG-I epitopes at root surfaces to be directly determined on whole-mount preparations. Pectic epitopes were not distributed evenly over root surfaces and were notably absent from lateral root apices and from the surface of root hairs. The use of defined antibody probes in the immersion immunolabelling protocol will be useful for the analysis of the influence of growth conditions and genetic factors on pectic polysaccharides in Arabidopsis.

Topics: Antibodies, Monoclonal; Arabidopsis; Cell Wall; Epitopes; Fluorescent Antibody Technique; Pectins; Plant Epidermis; Plant Roots; Seeds

Alpha-1,4-galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1-->4-linked alpha-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.

Topics: Catalysis; Cell Membrane; Endoplasmic Reticulum; Glucuronosyltransferase; Glycosyltransferases; Golgi Apparatus; Intracellular Membranes; Models, Molecular; Pectins; Pisum sativum; Plant Proteins; Pyrophosphatases; Subcellular Fractions; Sucrose; Uronic Acids

Grape berries (Vitis vinifera L., cv Ugni blanc) were harvested at 12 different weeks of development in 1996 and 1997. Ripening was induced at veraison, the crucial stage of berry softening, and was followed by a rapid accumulation of glucose and fructose and an increase of pH. Total RNAs, crude proteins and cell wall material were isolated from each developmental stage. A partial length cDNA (pme1, accession number AF159122, GenBank) encoding a pectin methyl-esterase (PME, EC 3.1.1.11) was cloned by RT-PCR with degenerate primers. Northern blots revealed that mRNAs coding for PME accumulate from one week before the onset of ripening until complete maturity, indicating that this transcript represents an early marker of veraison and could be involved in berry softening. However, PME activity was detected during all developmental stages. Total activity per berry increased, whereas "specific" activity, on a fresh weight basis, decreased during development. The amount of cell wall material (per berry and per g of berry) followed the same pattern as that of PME activity (total and "specific" respectively), indicating they were tightly correlated and that PME levels varied very little in the cell walls. Nevertheless, the degree of methyl-esterification of insoluble pectins decreased throughout the development from 68% in green stages to less than 20% for the ripe berries, and this observation is consistent with the induction of PME mRNAs during ripening. Relations between transcript expression, PME activity, the DE of insoluble pectic polysaccharides and their involvement in grape berry ripening are discussed.

Topics: Amino Acid Sequence; Carboxylic Ester Hydrolases; Cell Wall; DNA, Complementary; Esterification; Gene Expression Profiling; Molecular Sequence Data; Pectins; RNA, Messenger; Sequence Homology, Amino Acid; Transcription, Genetic; Vitis

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.

Topics: Antibodies, Monoclonal; Calcium; Cell Wall; Galactans; Immunohistochemistry; Microscopy, Electron; Pectins; Solanum tuberosum

Flax fibers have been the subject of many biochemical studies, which revealed that cellulose and pectins are the major constituents of their walls. In contrast, little is known about the location of these polymers within the walls of mature fibers by microscopic methods. This has been technically hampered by the very thick secondary wall of fibers, resulting in inadequate tissue preservation unsuitable for immunogold microscopy. In this study, we adapted the basic chemical fixation, dehydration and infiltration methods to achieve a good preservation of the cell structures of mature fibers and reduced damage to antigens. We were able to apply postembedding immunocytochemical techniques to map the location of various pectic epitopes within the walls of mature fibers. Our immunolabeling data show that homogalacturonans were exclusively found in the middle lamellae and the cell junctions but were not detectable in the secondary wall. In contrast, rhamnogalacturonan I (RG I)-associated epitopes, as well as galactan and arabinan epitopes, were abundantly distributed over the secondary wall of mature fibers.

Topics: Acrylic Resins; Cell Wall; Epitopes; Flax; Immunohistochemistry; Microscopy, Electron; Pectins; Plant Stems; Polysaccharides; Tissue Embedding

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons have a pectin-like structure. The core polysaccharides after treatments with four kinds of hemicellulases and a pectinase contained approximately equal numbers of L-rhamnose and D-galacturonate residues, suggesting the presence of the rhamnogalacturonan (RG) I structure consisting of the diglycosyl repeating unit, -4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-. The lengths of RG chains were calculated as approximately 15, 28, and 100 diglycosyl repeats. The RG components linked to each other by intervention of galacturonan (GN) chains, constituting the backbone of SSPS. All arabinose residues, which constitute 21% of total SSPS sugars, were found to be in side chains from RG regions, and this was also true for galactose residues, which constitute 50% of total sugars. Of arabinose residues, 94% are present as alpha-1,3- or alpha-1,5-arabinans, and 89% of galactose residues were present as beta-1,4-galactans. Galactan chains are modified with arabinose, xylose, fucose, and glucose at the sites close to the RG regions.

Topics: Arabinose; Carbohydrate Conformation; Carbohydrate Sequence; Chromatography, Gel; Chromatography, Ion Exchange; Galactose; Glycine max; Glycoside Hydrolases; Molecular Sequence Data; Pectins; Polygalacturonase; Polysaccharides; Rhamnose

Theophylline pellets were coated with cellulosic (Aquacoat ECD 30, Surelease clear) or acrylic (Eudragit NE30D, RS30D) polymer aqueous dispersions, containing 10% (related to the insoluble polymer content) of pectin HM or calcium pectinate, using a Uni-Glatt fluidized-bed coating apparatus. When commercial pectinolytic enzymes were added to the dissolution media (0.05 M acetate - phosphate buffer, pH 6.0), the release of theophylline from the coated pellets was generally slower than that observed in the media without enzymes. The enzymatic slowing down of the drug release, depending on the type of the aqueous polymer dispersion used, is more important with mixed Eudragit NE/calcium pectinate coated pellets. The results obtained have been examined with regard to the validity of the approach based on the combination of pectins and the insoluble polymer aqueous dispersions intended for specific-delivery of drugs to the colon. The mechanism of the hydrophilic drug release from pellets coated with insoluble polymer aqueous dispersions containing an aqueous gel-forming polymer has been also discussed.

Topics: Absorption; Acrylic Resins; Cellulose; Particle Size; Pectins; Polygalacturonase; Polymers; Polymethacrylic Acids; Solubility; Theophylline; Water

The phytopathogenic fungus Botrytis cinerea produces a set of endopolygalacturonases (endoPGs) which are involved in the enzymatic degradation of pectin in plant cell walls. The endoPG-encoding genes of B. cinerea are differentially expressed when the fungus is grown in liquid culture on different carbon sources. A basic constitutive expression level was observed for two genes, Bcpg1 and Bcpg2, which encode basic isozymes. Galacturonic acid was shown to induce the expression of Bcpg4 and Bcpg6. Low pH of the culture medium resulted in induced expression of the Bcpg3 gene. Expression of the Bcpg5 gene was inducible; however the inducing factors could not be identified. Finally, galacturonic acid-induced expression of the Bcpg4 gene was repressed by the presence of more-favourable carbon sources, such as glucose.

Topics: Arabinose; Botrytis; Carbon; Enzyme Repression; Galactose; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Glucose; Hexuronic Acids; Hydrogen-Ion Concentration; Isoenzymes; Pectins; Polygalacturonase; Rhamnose; RNA, Fungal; Xylose

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

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.

Topics: Aspergillus niger; Carbohydrate Conformation; Carbohydrate Sequence; Hexuronic Acids; Models, Molecular; Molecular Sequence Data; Pectins; Polygalacturonase

The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10-11.

Topics: Carboxylic Ester Hydrolases; Chromatography, Ion Exchange; Esterification; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Pectins; Plant Proteins; Rosales

Polygalacturonic acid, a linear homopolysaccharide, was investigated by capillary electrophoresis (CE) using linear polyacrylamide-coated capillaries and laser-induced fluorescence (LIF) detection. A successful separation of its fluorescently labeled oligomers was achieved through sieving in polyacrylamide entangled matrices. The reaction conditions for the derivatization of polygalacturonic acid were optimized. In studying the interactions between polygalacturonic acid and various metal ions, the end-label, free-solution electrophoretic (ELFSE) technique, developed earlier in our laboratory (Sudor, J., Novotny, M. V., Anal. Chem. 1995, 67, 4205-4209) was found preferable to the sieving method. ELFSE is fast and convenient in that no polymer solutions are needed for the separation. The investigation showed that for the moderately large oligomers, the strongest binding occurred with calcium and cadmium ions, while the smallest interaction was observed with magnesium ions.

Topics: Cadmium; Calcium; Cations, Divalent; Electrophoresis, Capillary; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Oligosaccharides; Pectins; Schiff Bases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

Topics: Alcohol Oxidoreductases; Aldehyde-Ketone Transferases; Candida; Carboxylic Ester Hydrolases; Genes, Fungal; Kinetics; Methanol; Mutagenesis; Pectins; Peroxisomes; Polygalacturonase; Polysaccharide-Lyases; Substrate Specificity

An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.

Topics: Antibodies, Monoclonal; Galactans; Immunoassay; Pectins

Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

Topics: Amino Acid Sequence; Carbohydrate Sequence; Dickeya chrysanthemi; Genomic Library; Genotype; Glucose; Glycerol; Isoenzymes; Kinetics; Molecular Sequence Data; Pectins; Phenotype; Polysaccharide-Lyases; Recombinant Fusion Proteins; Restriction Mapping; Sequence Alignment; Sequence Homology, Amino Acid; Transduction, Genetic

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3-7). Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mukat.mg-1, 36.5 mukat.mg-1 and 415 nkat.mg-1 for endopolygalacturonases I, II and C, respectively. K(m) values were < 0.15 mg.mL-1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes. Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated delta 4,5-unsaturated oligogalacturonates of degree of polymerization 4-8. Whereas endopolygalacturonase I showed a strong preference for generating the delta 4,5-unsaturated dimer, with endopolygalacturonase II the delta 4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the delta 4,5-unsaturated dimer and trimer were observed, although in different ratios.

Topics: Aspergillus niger; Fungal Proteins; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Pectins; Polygalacturonase; Polysaccharide-Lyases; Recombinant Proteins; Substrate Specificity

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.

Topics: Blotting, Southern; Botrytis; Cloning, Molecular; Genes, Bacterial; Genome, Fungal; Glucose; Molecular Sequence Data; Pectins; Phylogeny; Plant Diseases; Polygalacturonase; Sequence Analysis, DNA

A previous study had shown that polyamines adsorb selectively on plant cell walls according to the valence of the polyamine (Messiaen et al. 1997, Plant Physiol. 113: 387-395). In this study, the adsorption of polyamines onto isolated carrot cell walls and onto pure polygalacturonic acid was investigated in the presence of competing mono- and divalent cations (Na+ and Ca2+). Putrescine (Put2+) was unable to remove all the calcium (Ca2+) from cell walls or from polygalacturonic acid. Spermidine (Spd3+) and spermine (Spm4+) adsorbed on all galacturonates and were able to remove Ca2+ completely from both the walls and the pure polygalacturonates. Therefore, Spd3+ and Spm4+, unlike Put2+, prevented polygalacturonic acid from adopting the Ca(2+)-induced supramolecular conformation recognized by the 2F4 anti-pectin monoclonal antibody. We show that the signal transduction cascade otherwise initiated in plant cells by Ca(2+)-bound alpha-1,4-oligogalacturonides was indeed blocked by both Spd3+ and Spm4+, but not by Put2+. The mobilization of cytosolic free Ca2+ and the cytosolic acidification usually observed after treatment with pectic fragments did not occur and the subsequent activation of phenylalanine ammonia-lyase was suppressed. It is hypothesized that the disruption by Spd3+ and Spm4+ of the Ca(2+)-induced supramolecular conformation of pectic fragments was the cause of the inhibition of the pectic signal. We conclude that polyamines can act on plant cell physiology by modulating the transduction of the pectic signal.

Topics: Calcium; Cytosol; Daucus carota; Diamines; Dimerization; Enzyme Activation; Pectins; Phenylalanine Ammonia-Lyase; Polyamines; Signal Transduction; Sodium

Pectin has been investigated for its ability to produce solid calcium pectinate gel (CPG) beads containing bovine serum albumin (BSA). Several factors can influence the properties and release characteristics of the CPG beads. In this study, the effect of calcium concentration, hardening agent and drying condition on the encapsulation and release characteristics of BSA from the matrix gel beads made of calcium pectinate were studied. BSA release studies under conditions mimicking mouth to colon transit have shown that calcium pectinate protects the drug from being released completely in the physiological environment of the upper gastrointestinal tract, and is susceptible to the enzymatic action with consequent drug release. In addition, the release of BSA from CPG beads was strongly affected by calcium concentration and drying condition. However, the release was not particularly affected by the presence of hardening agent at the concentration of 1% or lower. Since the release of BSA as a model protein drug could be controlled by the regulation of the preparation conditions of CPG beads, the CPG beads may be used for a potential oral controlled release system for protein drugs.

Topics: Calcium; Capsules; Chemistry, Pharmaceutical; Excipients; Gels; Glutaral; Microscopy, Electron, Scanning; Pectins; Serum Albumin, Bovine; Temperature

The interaction between D- and L-enantiomers of polylysine and potassium pectate was studied by means of CD, microcalorimetry, and osmometry. Upon binding with pectate, only poly(L-lysine) undergoes a coil to alpha-helix transition, while poly(D-lysine) remains in the disordered state. This suggest that the energetics of the interaction is influenced by stereochemical constraints besides electrostatic forces. Experimental findings from microcalorimetry suggest that a contribution to the overall enthalpy of binding comes from the polysaccharidic moiety. Stoichiometry of the macromolecular complexes studied by osmometry gives a polylysine:pectate ratio of 3:1, in agreement with the respective degree of polymerization of the two polyelectrolytes.

Topics: Calorimetry; Circular Dichroism; Pectins; Polylysine; Protein Structure, Secondary; Solutions; Stereoisomerism; Water

Polygalacturonic acid (PGA) was hydrolyzed by polygalacturonases (PGs) purified from six fungi. The oligogalacturonide products were analyzed by HPAEC-PAD (high performance anion exchange chromatography-pulsed amperimetric detection) to assess their relative amounts and degrees of polymerization. The abilities of the fungal PGs to reduce the viscosity of a solution of PGA were also determined. The potential abilities of four polygalacturonase-inhibiting proteins (PGIPs) from three plant species to inhibit or to modify the hydrolytic activity of the fungal PGs were determined by colorimetric and HPAEC-PAD analyses, respectively. Normalized activities of the different PGs acting upon the same substrate resulted in one of two distinct oligogalacturonide profiles. Viscometric analysis of the effect of PGs on the same substrate also supports two distinct patterns of cleavage. A wide range of susceptibility of the various PGs to inhibition by PGIPs was observed. The four PGs that were inhibited by all PGIPs tested exhibited an endo/exo mode of substrate cleavage, while the three PGs that were resistant to inhibition by one or more of the PGIPs proceed by a classic endo pattern of cleavage.

Topics: Amino Acid Sequence; Chromatography, Ion Exchange; Enzyme Inhibitors; Fungi; Molecular Sequence Data; Oligosaccharides; Pectins; Plant Proteins; Polygalacturonase; Sequence Homology, Amino Acid; Substrate Specificity

The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.

Topics: Carboxylic Ester Hydrolases; Cations, Divalent; Cells, Cultured; Cholic Acids; Dithioerythritol; Enzyme Activation; Enzyme Stability; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Methylation; Methyltransferases; Microsomes; Nicotiana; Oxalates; Pectins; Plants, Toxic; Polygalacturonase; Solubility; Temperature; Water

Modeling simulations have been performed on the four regular glycuronans: alpha-D-(1--->4) polygalacturonic, alpha-L-(1--->4) polyguluronic, beta-D-(1--->4) polymannuronic, and beta-D-(1--->4) polyglucuronic acids. The goal of this study was to characterize the similarities and differences in conformational and configurational behavior as well as in calcium binding in order to progress in the understanding of the physicochemical properties of the parent polysaccharides of industrial interest, namely pectin, alginate and glucuronan. This required the evaluation of the accessible conformational space for the disaccharide subunits of the four homopolymers, using the flexible residue protocol of the MM3 molecular mechanics procedure. The results were used to access the configurational statistics of representative polysaccharide chains, as well as for the determination of the regular polysaccharide helices and their conformational transitions. The surfaces of all regular helices likely to occur for each polyuronide were explored for cation binding using the GRID procedure. Both alpha-D-(1--->4) polygalacturonate and alpha-L-(1--->4) polyguluronate chains exhibit a high specificity for calcium binding, and have well-defined chelation sites. In contrast, beta-D-(1--->4) polymannuronate and beta-D-(1--->4) polyglucuronate chains do not display any stereospecificity for calcium binding. The results gathered from molecular modeling lead to a clear understanding of the different structural features that are displayed by the four ionic polymers.

Topics: Alginates; Calcium; Carbohydrate Conformation; Carbohydrate Sequence; Cations, Divalent; Disaccharides; Glucuronic Acid; Hexuronic Acids; Models, Molecular; Molecular Sequence Data; Pectins; Polysaccharides; Polysaccharides, Bacterial; Uronic Acids

Polygalacturonic acid is a linear carbohydrate polymer of monomeric galacturonic acid. It is commercially available as apple and citrus pectins comprised of a mixture of partially methoxylated and/or amidated polygalacturonic acids with molecular weights ranging from 25,000 to > 100,000 Da. Pectin can be chemically or enzymatically hydrolyzed to yield polygalacturonic acid fractions of diverse average molecular weight ranges and polydispersities for a variety of uses. Pectin and polygalacturonic acid are used extensively as gelling agents and stabilizers by the food industry, and have applications as therapeutic, and diagnostic pharmaceutical agents such as the magnetic resonance imaging agent LumenHance. A simple high-performance size exclusion chromatography (HPSEC) method, employing commonly available non-specialized HPLC instrumentation, is described for use as a rapid molecular weight screening technique to determine the average molecular weight range and polydispersity of polygalacturonic acid intended for use in pharmaceutical formulations. A TosoHaas G3000PWXL HPLC column, 50 mM phosphate buffer (pH approximately 6.9) mobile phase, and refractive index detection were used. A molecular weight calibration curve was linear for polysaccharide standards of 180-100,000 Da with a coefficient of correlation of 0.999. The method was employed to screen commercially available polygalacturonic acid raw materials for average molecular weight data (Mn, Mw, and Mp) and polydispersity (Mw/Mn).

Topics: Chromatography, High Pressure Liquid; Molecular Weight; Pectins; Technology, Pharmaceutical

Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.

Topics: Carbon Isotopes; Cell Wall; Cellulose; Cucumis sativus; Glucans; Hypocotyl; Magnetic Resonance Spectroscopy; Pectins; Plant Proteins; Polymers; Polysaccharides; Protons; Xylans

Two endopolygalacturonases (endo-PGs) secreted at the early stage of cultures of Sclerotinia sclerotiorum grown on polygalacturonate medium, were purified to apparent homogeneity, using ion exchange chromatography. They are glycoproteins of an apparent weight of 42 and 41.5 kDa and a pI of 4.8. The two purified isoforms found in early cultures were not detected in late cultures. Purification of the isoforms secreted at different stages of growth revealed that the increase of polygalacturonase activity during the culture corresponds to a sequential production of enzymes and to the successive replacement of isoforms by new enzymes.

Topics: Ascomycota; Culture Media; Electrophoresis, Polyacrylamide Gel; Isoenzymes; Pectins; Polygalacturonase

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-D-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e-->a) during hydrolysis of alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-D-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

Topics: Aspergillus; Carbohydrate Conformation; Glycoside Hydrolases; Hexuronic Acids; Magnetic Resonance Spectroscopy; Molecular Conformation; Pectins; Rhamnose

Two methods were developed to detect partially methyl-esterified galacturonic acid oligomers, generated by endopolygalacturonase treatment of a 30% methyl-esterified pectin. The enzyme digest was shown, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to contain sodiated galacturonic acid oligomers with a degree of polymerization of 2-12, containing 0-6 methyl esters. Galacturonic acid (monomer) could not be detected because of matrix ions interference in the low mass region. Using high-performance anion-exchange chromatography, with a sodium acetate gradient at pH 5.0 and postcolumn sodium hydroxide addition to allow pulsed amplified detection, a complex elution profile was obtained with the endopolygalacturonase-treated 30% methyl-esterified pectin. All the components eluted before nonesterified tetragalacturonic acid. The partially methyl-esterified oligogalacturonic acids eluted in a discernible series of oligomers with an identical number of nonesterified carboxylic acid groups; the large, more esterified oligomers eluted before small, less esterified oligomers. The methyl esters may hinder the interaction of the neighboring carboxylic acid groups with the anion-exchange resin, thereby giving the components an apparent lower overall negative charge.

Topics: Chromatography, Ion Exchange; Esters; Hydrogen-Ion Concentration; Pectins; Polygalacturonase; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production.

Topics: Aspergillus niger; Biomass; Carboxylic Ester Hydrolases; Cell Membrane; Centrifugation; Chromatography, High Pressure Liquid; Fatty Acids; Fermentation; Glucose; Hexuronic Acids; Pectins; Polygalacturonase

Partially esterified polygalacturonic acid is the main component of pectin in higher plants. The carboxylic groups and their methyl esters markedly affect the ability of the pectin molecules to bind oppositely charged ions and to form gels. In order to make a contribution to the understanding of the mechanisms which regulate the ionic transfer at the soil-root interface and in the apoplast, we report the results of a set of molecular dynamics experiments in which the interactions of four fully deprotonated fragments of polygalacturonic acid, each counting 12 units, 300 water molecules and 48 or 24 Na+ and Ca2+ ions were studied. We observed the formation of Ca2+ bridges between the polygalacturonate chains. The forces driving the aggregation processes are characterized by the formation of strong coulombic interactions between the metal ions and the carboxylate groups. The results are consistent with experiment evidence of the formation of Ca-polygalacturonate organized gels. The Ca-polygalacturonate complex exhibits a lower energy compared to that of Na-polygalacturonate. The ratio of the Na+ and Ca2+ diffusion coefficients agree well with experimental reports.

Topics: Binding Sites; Calcium; Carbohydrate Conformation; Ion Transport; Models, Molecular; Pectins; Plants; Sodium; Thermodynamics

Ethylene can be produced by a variety of developmental and environmental factors such as ripening, the plant hormone auxin, and mechanical wounding via a biosynthetic pathway including AdoMet synthase, ACC synthase, and ACC oxidase steps. ACC synthase and ACC oxidase are known to be encoded by multigene families, and are believed to be differentially expressed in response to various stimuli. In mung bean, ACC synthase is encoded by 7 genes, ACS1, ACS2 ACS3, ACS4, ACS5, ACS6, and ACS7, and ACC oxidase by 2 genes, ACO1 and ACO2. In this study, was have investigated differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls under various conditions by the semiquantitative RT-PCR method. Primers which can specifically bind and amplify each cDNAs of ACS1, ACS2, ACS3, ACS4, ACS5, ACS6, ACS7, and ACO1, and ACO2 were designed and used to monitor the responses to various stimuli. Transcripts of ACO1 and ACO2 were accumulated constitutively in the hypocotyl segments even without andy treatment. After cold treatment on intact plant, transcripts of ACS5, ACS6, and ACS7 were accumulated in the hypocotyl segments. We also found the excision of hypocotyl segments and incubation in a buffer solution, a typical way of chemical treatments to hypocotyl segments, lowered the level of ACO2 transcripts with little change of the level of ACO1 transcripts. In response to incubation with IAA (0.1 mM) of excised hypocotyl segments, transcripts of ACS1, ACS6, and ACS7 were accumulated and the level of ACO2 transcripts was increased. Transcripts of ACS1, ACS2, ACS3, ACS5, ACS6 and ACS7 were induced by incubation with OGA (50 micrograms/ml), while the transcripts of ACS4 were accumulated and the level of ACO2 transcripts was increased by incubation with 1 mM LiCl. Our results strongly suggest that all seven ACC synthase genes and two ACC oxidase genes must be active and each gene is differentially regulated by a different subset of the inducing factors.

Topics: Adjuvants, Immunologic; Amino Acid Oxidoreductases; Cold Temperature; DNA Primers; Enzyme Induction; Fabaceae; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hypocotyl; Indoleacetic Acids; Lithium Chloride; Lyases; Multigene Family; Oligosaccharides; Pectins; Plant Growth Regulators; Plants, Medicinal; Polymerase Chain Reaction; RNA, Plant; Sensitivity and Specificity; Sequence Homology, Amino Acid; Stimulation, Chemical; Transcription, Genetic; Transcriptional Activation; Tromethamine

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42. 9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. DeltahrpZ::nptII or hrpW::OmegaSpr P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.

Topics: Bacterial Outer Membrane Proteins; Chromosome Mapping; Cloning, Molecular; DNA, Bacterial; Genes, Bacterial; Molecular Sequence Data; Mutagenesis; Nicotiana; Pectins; Plants, Toxic; Polysaccharide-Lyases; Pseudomonas; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Solanum lycopersicum

Entrapment of cells within spheres of polysaccharide gel has become the most widely used technique for immobilizing living cells. Polysaccharide pectin, formed gel with calcium ions, was investigated as a precursor of spherical calcium polysaccharide gel beads. The type of pectin sample was found to be important in the formation of the beads. Partially deesterified pectin with a lower degree of esterification provided spherical beads and was chosen for immobilization of the yeast cells, Sacchararomyces cerevisiae, and compared to those with alginate. The effect of storage condition of the beads on the viability of the entrapped cells was also studied. After storage at 4 degreesC or -40 degreesC for 1 month, even lyophilization before storage, the beads with entrapped cells were sufficiently stable when compared to suspension of non-entrapped yeast cells.

Topics: Alginates; Cells, Immobilized; Glucuronic Acid; Hexuronic Acids; Microscopy, Electron, Scanning; Pectins; Polysaccharides; Saccharomyces cerevisiae

Two pectinolytic enzymes were purified from the culture broth of Pseudomonas marginalis pv. marginalis MAFF 03-01173 with total 33% recovery of the initial activity. From the substrate specificities against pectin and polygalacturonic acid, the requirement of calcium ion for the enzymatic activity, and the N-terminal sequences, the enzymes were identified as pectin lyase and pectate lyase. The M,s of pectin lyase and pectate lyase were estimated to be 34,000 and 43,000, respectively, by SDS polyacrylamide gel electrophoresis. Both enzymes showed almost the same pH dependent activity curves with the highest activity at pH 8.3

Topics: Amino Acid Sequence; Bacterial Proteins; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Pectins; Polysaccharide-Lyases; Pseudomonas; Sodium Dodecyl Sulfate

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.

Topics: Chromatography, Ion Exchange; Electric Conductivity; Electrophoresis, Polyacrylamide Gel; Erwinia; Pectins; Plant Diseases; Polygalacturonase; Polymers; Polysaccharide-Lyases; Sodium Dodecyl Sulfate; Solanum tuberosum

An approach to the analysis of isotherms of cooperative binding is developed that allows to calculate approximately affinity profiles, i.e. dependencies of binding constants on binding densities. The comparison of the affinity profiles of the interactions of Ca2+ and Sr2+ ions with sodium pectate as well as sodium alginates with the different content of the blocks of alpha-L-guluronic acid residues (GG-blocks) and blocks of mixed composition showed that (1) pectate has higher affinity to both ions as compared with alginates; (2) the affinity of pectate to Ca2+ ions is comparable to its affinity to Sr2+ ions but in the case of Ca2+ ions the maximum of the affinity profile falls on the substantially lesser value of the binding density in comparison to Sr2+ ions that seems to be unfavorable from the standpoint of the use of this polyuronide in preventive or medicinal nutrition; (3) the affinity of both alginates to Sr2+ ions at relatively low binding densities exceeds their affinity to Ca2+ ions; (4) the affinity of alginates to Sr2+ ions increases with the increase in the content of GG-blocks, whereas the affinity to Ca2+ ions practically does not depend on the composition of the alginate; (5) the maximum binding density of Sr2+ ions to the alginates approximately corresponds to the content of GG-blocks. These results corroborate the practice of the use of alginates rich in residues of alpha-L-guluronic acid for the removal of radioactive strontium from the digestive tract. The binding constants of Pb2+ ions to pectate over a wide range of binding densities are more than an order of magnitude greater than those of alkali-earth metal ions.

Topics: Alginates; Calcium; Chelating Agents; Chemical Phenomena; Chemistry, Physical; Drug Contamination; Food Contamination, Radioactive; Lead; Nutritional Physiological Phenomena; Pectins; Strontium; Thermodynamics; Uronic Acids

A sensitive assay is described for the detection of pectate-depolymerizing enzymes using capillary electrophoresis of a fluorescent end-labeled pectate oligomer. The labeled oligomer is allowed to react with the enzyme either in vitro or in vivo, such as inside the intercellular spaces of a cotton cotyledon, and after an appropriate incubation time the products are analyzed by capillary electrophoresis. The site and mode of action of the pectate-depolymerizing activity can be inferred from the products. Both endo- and exopolygalacturonase activity, and lyase activity, were distinguished. Since only the fluorescent oligomer and products from its labeled reducing end are detected, there is no interference from other compounds; only pectic enzyme activity is detected. By this type of analysis we can show that there is considerable endo- and exo-polygalacturonase activity in the intercellular spaces of cotton cotyledons.

Topics: Cotyledon; Electrophoresis, Capillary; Fluorescence; Glycoside Hydrolases; Gossypium; Hexuronic Acids; Lyases; Naphthalenes; Pectins; Polygalacturonase; Sensitivity and Specificity; Substrate Specificity

Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication.

Topics: Amino Acid Sequence; Base Sequence; Carrier Proteins; Cell Membrane; Cloning, Molecular; DNA, Complementary; Galactans; Hexuronic Acids; Molecular Sequence Data; Pectins; Phosphoproteins; Plant Proteins; Plant Viral Movement Proteins; Plants; Recombinant Proteins; Sequence Homology, Amino Acid; Solanum tuberosum; Viral Proteins

The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.

Topics: Actinomycetales; Amino Acid Sequence; Chromatography, Ion Exchange; Dickeya chrysanthemi; Isoelectric Point; Kinetics; Molecular Sequence Data; Molecular Weight; Pectins; Polysaccharide-Lyases; Sequence Homology, Amino Acid; Species Specificity; Substrate Specificity

We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity of PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by beta-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.

Topics: Allergens; Dose-Response Relationship, Drug; Esterification; Glycoside Hydrolases; Hydrolysis; Pectins; Plant Proteins; Pollen; Polymers; Polysaccharide-Lyases

Structural parameters (e.g. the geometry, partial charges and dipole moment) of the alpha-D-galacturonic residue have been calculated by using semi-empirical quantum mechanical methods on several combinations of either the uncharged or charged forms, GalAH and GalA-, respectively. Three residue types have been explored: (i) the isolated residue, termed GEO1 and GEO1C for GalAH and GalA-, respectively; (ii) the residue with a methyl group attached to the O4 and O1 positions (GEO2 and GEO2C); and (iii) the internal residue in a trimer, e.g. GalAH-GalAH-GalAH or the corresponding fully charged version (GEO3 and GEO3C). The presence of a charged group in the galacturonate residue and the distribution of the excess negative charge on the molecule lead to significant differences in the structural parameters in comparison with those of the uncharged galacturonic residue. These perceptible differences in internal coordinates of GalAH and GalA- residues appear to play a major role in the delimitation of the conformational space that is accessible to the dimers, as clearly seen by inspection of the conformational maps. Although the overall features seem alike, the maps show that the position of the minimum and the shape of the lower energy region significantly change if one or both residues in the dimer are charged. The relevance of these results for the conformational properties of polygalacturonate chains is discussed elsewhere (Ruggiero et al. Int. J. Biol. Macromol. 1995, 17, 213-218).

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Disaccharides; Hexuronic Acids; Models, Molecular; Molecular Sequence Data; Pectins; Trisaccharides

The unperturbed dimensions of poly-alpha-D-galacturonic acid (PGA) as a function of the degree of polymerization (n) and degree of ionization (%GalA-) have been determined by molecular mechanics and Monte Carlo methods. Chain extensions appear to depend substantially on contributions arising from local redistributions of charge on charged and uncharged galacturonic acid residues. Inclusion of methyl-esterified galacturonic acid units results in increased chain extensions. Incorporation of alpha-(1-->2)-L-rhamnose units causes abrupt changes in chain propagation and a reduction in calculated chain extension.

Topics: Carbohydrate Conformation; Glycosides; Monte Carlo Method; Pectins; Structure-Activity Relationship

Pectinase (polygalacturonase) was purified from a commercial pectinase preparation from a mold. Substrate binding of pectinase was measured by centrifugal affinity chromatography using an immobilized substrate, pectic acid. Desorption of pectinase from the affinity matrix with the substrate pectin and pectic acid gave Kd values of 5.3 and 8.5 mg/ml, respectively. Chemical modification of pectinase by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and diethyl pyrocarbonate (DEP) caused a loss of most of the enzyme activity, but the substrate binding ability was not impaired. Thus, the pectinase preparation was digested with lysyl endopeptidase and the resulting peptides were treated with pectic acid-affinity gel. Three peptide fragments, which were recovered from the affinity column and sequenced, were identical to sequences in the second pectinase gene from Aspergillus niger. The first peptide contained 17 amino acids, Asp101-Ser117, and the second and third peptides corresponded to 18 amino acids of Asn152-Asp169. These results indicate that the inactivated pectinase retained substrate binding ability and would function as an acidic polysaccharide recognizing protein.

Topics: Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Enzymes, Immobilized; Molecular Sequence Data; Pectins; Polygalacturonase; Polysaccharides

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

Topics: Amino Acid Sequence; Base Sequence; DNA, Bacterial; Erwinia; Genes, Bacterial; Isoenzymes; Kinetics; Molecular Sequence Data; Molecular Structure; Pectins; Plant Diseases; Polysaccharide-Lyases; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity

Alcohol insoluble solids from apple were extracted in sequence by buffer at 20 degrees C and at 70 degrees C, EDTA/oxalate, and mild alkali, yielding four populations of pectins. These pectins and the insoluble residue were characterized by their sugar composition, degree of esterification (methyl ester and O-acetyl groups), molecular weight distribution, and degradability by the combination of endopolygalacturonase (PG) and pectin esterase (PE) and by rhamnogalacturonase (RGase) after chemical saponification. After PG/PE treatment, the remaining high molecular weight material representing the pectic hairy regions was isolated and characterized. Clear differences were found in the sugar composition of the fractions obtained, while only small variations were observed in the sugar linkage composition. The pectic hairy regions were further degraded by RGase and the digests separated into high molecular weight and oligomeric degradation products. These "RGase oligomers" consisted of between 4 and 9 sugar units with a backbone of alternating rhamnose and galacturonic acid residues, partly substituted with galactose linked to C-4 of the rhamnose moiety. Both the absolute amount of RGase oligosaccharides released as well as the degree of galactose-substitution of the oligomers increased when severer extraction conditions were used. Relatively more RGase oligomers were released from the low molecular weight hairy regions as compared to the high molecular weight fraction. Typical high molecular weight fragments isolated from the RGase digests of various hairy regions included residual segments of the rhamnogalacturonan backbone rich in arabinose and a polymer presumably enriched in xylogalacturonan segments.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Cell Wall; Chromatography, Gel; Chromatography, High Pressure Liquid; Fruit; Glycoside Hydrolases; Molecular Sequence Data; Molecular Structure; Monosaccharides; Pectins

Humoral and cell-mediated immune responses to the capsular polysaccharide (CPS) of M. dispar and polygalacturonic acid (pGaIU--a structurally similar polysaccharide) were investigated in calves experimentally infected with Mycoplasma dispar and in mice immunized with CPS or pGaIU. Sera, tracheobronchial lavage and nasal fluids, collected before and after infection in calves, were checked for the presence of anti-CPS and anti-pGaIU antibodies. The sera from mice injected with CPS or pGaIU were checked for different classes of anti-CPS and anti-pGaIU antibodies. Peripheral blood lymphocytes from calves and splenic lymphocytes from mice were monitored for specific proliferative responses to CPS and pGaIU. At about 2 weeks post-infection, anti-CPS IgM response in serum, anti-CPS and anti-pGaIU IgM and IgA response in lavage fluid and lymphocyte proliferative response was seen in the calves. Mice immunized with CPS and pGaIU gave exclusively IgM responses. No secondary response was seen in mice immunized with CPS in contrast to mice immunized with pGaIU. Antibodies cross-reactive with pGaIU were present in the sera of CPS-immunized mice but antibodies cross-reactive with CPS were not found in pGaIU-immunized mice. No significant blastogenic response was shown by mouse splenocytes to CPS or pGaIU.

Topics: Animals; Antibodies, Bacterial; Bacterial Capsules; Bronchoalveolar Lavage Fluid; Cattle; Cattle Diseases; Enzyme-Linked Immunosorbent Assay; Immunity, Cellular; Immunization; Immunoglobulin A; Immunoglobulin M; Lymphocyte Activation; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Mycoplasma; Nasal Mucosa; Pectins; Pneumonia, Mycoplasma; Polysaccharides, Bacterial

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease.

Topics: Amino Acid Sequence; Calcium Chloride; Cell Division; Chromosome Mapping; Dickeya chrysanthemi; Gene Expression Regulation, Bacterial; Glucuronidase; Hydrogen-Ion Concentration; Isoenzymes; Molecular Sequence Data; Mutagenesis; Pectins; Plants; Polysaccharide-Lyases; Recombinant Fusion Proteins; Sequence Analysis; Sequence Homology, Amino Acid; Transcription, Genetic

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

Linear oligogalacturonic acids (1,4-linked alpha-D-galacturonic acid oligomers), obtained by partial acid hydrolysis of orange polygalacturonides, were studied by negative-ion electrospray ionization mass spectrometry, without prior sample derivatization. After preparative separation using high-resolution anion-exchange chromatography, some fractions enriched in uronic acids were desalted, transferred into a methanol+water solution adjusted to pH 10, and directly submitted to electrospray ionization mass spectrometry in the negative-ion recording mode. Clear molecular mass assignments of the oligomers, covering a degree of polymerization between 4 and 7, were obtained.

Topics: Carbohydrate Sequence; Gas Chromatography-Mass Spectrometry; Molecular Sequence Data; Oligosaccharides; Pectins

1H-NMR and molecular dynamics simulations in vacuo and in water of (1-->4)-alpha-D-galacturono-disaccharide were performed. The results of the molecular dynamics simulations showed that the molecule fluctuates between two conformations characterized by different values of torsion angles around the glycosidic linkage and two different intramolecular hydrogen bonds. When these conformations are extrapolated to a regular polymeric structure, they generate pectic acid compatible with a 2(1)- or a right-handed 3(1)-helix.

Topics: Biopolymers; Carbohydrate Conformation; Carbohydrate Sequence; Disaccharides; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Pectins; Solutions; Thermodynamics

The accumulation of an abnormal, protease-resistant form of the protein PrP (PrP-res) in hosts with scrapie and related transmissible spongiform encephalopathies appears to be important in disease pathogenesis. To gain insight into the mechanism of PrP-res accumulation and the in vivo antiscrapie activity of certain polyanions, we have studied effects of sulfated glycans on PrP metabolism in scrapie-infected neuroblastoma cells. Pentosan polysulfate, like the amyloid-binding dye Congo red, potently inhibited the accumulation of PrP-res in these cells without apparent effects on the metabolism of the normal isoform. The inhibition was due primarily to prevention of new PrP-res accumulation rather than destabilization of preexisting PrP-res. PrP-res accumulation remained depressed in the cultures after removal of the inhibitors. The activities of other sulfated glycans, nonsulfated polyanions, dextran, and DEAE-dextran were compared with those of pentosan polysulfate and Congo red. This comparison provided evidence that the density of sulfation and molecular size are factors influencing anti-PrP-res activity of sulfated glycans. The relative potencies of these compounds corresponded well with their previously determined antiscrapie activities in vivo, suggesting that the prophylactic effects of sulfated polyanions may be due to inhibition of PrP-res accumulation. Since PrP-res amyloid is known to contain sulfated glycosaminoglycans, we reason that these inhibitors may competitively block an interaction between PrP and endogenous glycosaminoglycans that is essential for its accumulation in a protease-resistant, potentially amyloidogenic state.

Topics: Amphotericin B; Animals; Carrageenan; Congo Red; Dextran Sulfate; DNA; Dose-Response Relationship, Drug; Mice; Pectins; Pentosan Sulfuric Polyester; Polyelectrolytes; Polyglutamic Acid; Polymers; Prions; PrPSc Proteins; RNA; Scrapie; Tumor Cells, Cultured

A simple method for the direct measurement of the catalytic properties of immobilized cells in the flow minicalorimeter, the enzyme thermistor (ET), is presented. A Trigonopsis variabilis strain with cephalosporin C-transforming activity was used as the model system. The yeast cells were immobilized either by crosslinking with a homobifunctional reagent or by entrapment in gels. The actual activity of the immobilized cells used in the ET was estimated by means of a stirred-batch reactor measurement in conjunction with HPLC analysis of substrate and products. Similar results were also obtained using D-amino acid oxidase (EC 1.4.3.3) isolated from T. variabilis cells and immobilized by gel entrapment. This calibration procedure was found to be appropriate for all biocatalyst systems used. The thermometric signal was proportional to the amount of biocatalyst immobilized in the ET minicolumn. It was shown that the rate of reaction catalyzed by T. variabilis entrapped in calcium pectate gel was limited by internal diffusion to an extent depending on the cell concentration in the biocatalyst particle. This approach offers a direct method for studying the kinetic properties of immobilized cells.

Topics: Acremonium; Biosensing Techniques; Biotechnology; Calorimetry; Cephalosporins; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Glutaral; Indicators and Reagents; Kinetics; Mathematics; Mitosporic Fungi; Models, Theoretical; Pectins; Polyethyleneimine

Five isozymes (four acidic and one basic) of polygalacturonase were separated by chromatofocusing from the culture filtrate of Botrytis cinerea grown on apple pectin. The isozymes, designated as Polygalacturonase I to V, have isoelectric points of 9.7, 4.9, 4.6, 3.7, and 2.7, respectively, with Polygalacturonase III exhibiting the highest specific activity. Polygalacturonase I appeared to function as an endo-polygalacturonase while the other four isozymes act as exo-polygalacturonases. The pH optima of the isozymes range from pH 4.5 to 5.5 with Polygalacturonase V being less sensitive to higher pH compared with the rest of the isozymes.

Topics: Chromatography, Thin Layer; Culture Media; Fruit; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Isoenzymes; Kinetics; Mitosporic Fungi; Pectins; Polygalacturonase

Although phospholipase C hydrolysis of polyphosphoinositides constitutes one of the major second messenger pathways in animal cells, its participation in signal transduction in higher plants has not been established. To determine whether activation of phosphatidylinositol-directed phospholipase C might be involved in signaling the elicitor-induced oxidative burst in plants, suspension-cultured soybean cells were treated with two stimulants of the H2O2 burst and examined for polyphosphoinositide turnover. Both polygalacturonic acid elicitor and the G protein activator, mastoparan, promoted a transient increase in inositol 1,4,5-trisphosphate (IP3) content that exceeded basal IP3 levels (0.9 +/- 0.4 pmol of IP3/10(6) cells, n = 28) by 2.6- and 7-fold, respectively. In each case, intracellular IP3 content reached a maximum at 1 min post-stimulation and declined to near basal levels during the subsequent 5-10 min. Neomycin sulfate, an inhibitor of polyphosphoinositide hydrolysis, blocked the IP3 transient, and Mas-17, an inactive analogue of mastoparan, induced no change in IP3. Thin layer chromatography of lipid extracts of the soybean cells corroborated the above results by revealing a rapid decrease in phosphatidyl-inositol monophosphate and phosphatidylinositol 4,5-bisphosphate following polygalacturonic acid elicitor and mastoparan (but not Mas-17) stimulation. Since the rise in IP3 preceded H2O2 production and since neomycin sulfate inhibited the appearance of both, we hypothesize that phospholipase C activation might constitute one pathway by which elicitors trigger the soybean oxidative burst.

Topics: Cells, Cultured; Enzyme Activation; Glycine max; Hydrogen Peroxide; Inositol 1,4,5-Trisphosphate; Intercellular Signaling Peptides and Proteins; Neomycin; Pectins; Peptides; Phosphatidylinositols; Respiratory Burst; Signal Transduction; Type C Phospholipases; Wasp Venoms

The growing success of liver transplantation and the shortage of donor livers has turned attention to the possibility of utilizing hepatocytes within artificial liver support systems to allow time for donor livers to become available and to improve the condition of patients with hepatic failure. This study evaluated encapsulated hepatocytes, a technology which might allow the possibility of using xenogenic or human hepatoma cells. Rabbit hepatocytes were encapsulated using the ionic polysaccharides carboxymethylcellulose, chondroitin sulfate A, chitosan, and polygalacturonic acid. Encapsulated cells were maintained in perfusion culture for at least 6 days in heparinized, normal human plasma or in a defined culture medium. Parallel cultures of plated hepatocytes were also conducted. The metabolic capability of the cells was evaluated by following the rates of urea, albumin, and transferrin synthesis and the transformation rate of the drug antipyrine. Protein synthesis and ureogenesis in plasma were depressed from the levels expressed in defined culture medium. Drug detoxification as measured by antipyrine metabolism appeared to be enhanced in plasma. We conclude that encapsulated rabbit hepatocytes retain significant levels of function for at least 6 days of perfusion with human plasma, suggesting the feasibility of this technology as a potential method of short-term liver support.

Topics: Albumins; Animals; Antipyrine; Carboxymethylcellulose Sodium; Cells, Cultured; Chitin; Chitosan; Chondroitin Sulfates; Culture Media; Drug Compounding; Extracorporeal Circulation; Feasibility Studies; Hemostatics; Heparin; Humans; Liver; Liver Failure, Acute; Male; Pectins; Plasma; Rabbits; Time Factors; Transferrin; Urea

The phytopathogenicity of Erwinia chrysanthemi is chiefly supported by the production of pectate lyase isoenzymes, encoded by the pel genes. One of these enzymes, PelA, encoded by the pelA gene, seems to represent only a small part of the total pectate lyase activity, but is required for full bacterial pathogenicity. To study the regulation of pelA gene expression, a pelA::uidA gene fusion was constructed. Expression of this fusion was analysed under various growth conditions and in various genetic backgrounds. Whatever the culture conditions, the pelA gene was weakly expressed. Moreover, pelA expression seems not to be regulated by the pecS gene product, but essentially by the kdgR gene product. In many plant-associated bacteria, genes involved in pathogenicity are induced by certain plant compounds. In this work, we demonstrate that E. chrysanthemi pel genes are induced in the presence of plant extracts, but only in synergy with known pectate lyase inducers (KDG: 2-keto-3-deoxygluconate; DKII: 2,5-diketo-3-deoxygluconate). However, different pel genes did not exhibit the same sensitivity to plant signal molecules. Partial purification of inducing plant compounds suggested that plant extracts contain at least one molecule involved in pectate lyase induction. This compound is thermoresistant, and has a low molecular mass and a very hydrophilic behaviour.

Topics: Carbohydrate Sequence; Dickeya chrysanthemi; Enzyme Induction; Gene Expression Regulation, Bacterial; Glucuronidase; Isoenzymes; Molecular Sequence Data; Pectins; Plant Extracts; Polysaccharide-Lyases; Recombinant Fusion Proteins; Virulence

Calcium pectinate (CaP)--the insoluble salt of pectin--can potentially be used as a colon-specific drug delivery system. The use of CaP as a carrier was based on the assumption that, like pectin, it can be decomposed by specific pectinolytic enzymes in the colon but that it retains its integrity in the physiological environment of the small bowel. The biodegradation of the carrier was characterized by monitoring the percent cumulative release of the insoluble drug indomethacin, incorporated into pectin or CaP matrices. Compressed tablets of pectin and indomethacin were analyzed for degradation in the presence of Pectinex 3XL, a typical pectinolytic enzyme mixture, and in the presence of the human colonic bacterium Bacteroides ovatus. The degradation of CaP-indomethacin tablets was assessed in the presence of Pectinex 3XL and in rat cecal contents. The release of indomethacin was significantly increased (end-time percentage cumulative release vs control) in the presence of Pectinex 3XL (89 +/- 20 vs 16 +/- 2 for CaP tablets), Bacteroides ovatus (12 and 22 vs 5.2 for pectin tablets), and rat cecal contents (61 +/- 16 vs 4.9 +/- 1.1 for CaP tablets). The weight loss of tablet mass was significantly higher (end-time dry weight vs control) in the presence of Pectinex 3XL (0 vs 75 +/- 6% of initial weight for CaP tablets). These findings indicate the potential of CaP, compressed into tablets with insoluble drug, to serve as a specific drug delivery system to the colon.

Topics: Animals; Bacteroides; Cecum; Chromatography, High Pressure Liquid; Colon; Drug Carriers; Drug Delivery Systems; Fermentation; Indomethacin; Pectins; Rats; Tablets

Topics: Carboxylic Ester Hydrolases; Chromatography; Hydrogen-Ion Concentration; Pectins; Polygalacturonase; Reference Standards; Sensitivity and Specificity

Application of heparin, polyadenylate, polyglutamate and polygalacturonate resulted in changes in the electron absorption spectrum of cytochrome c that resembled those after cytochrome c oxidase application at neutral pH. The formed complexes of cytochrome c with polyanions retain the bond of Met-80 with heme iron. Cytochrome c oxidase and the polyanions increased the apparent pKa of alkaline transition of cytochrome c by an order of magnitude.

Topics: Animals; Cattle; Cytochrome c Group; Electron Transport Complex IV; Heparin; Hydrogen-Ion Concentration; Myocardium; Osmolar Concentration; Pectins; Poly A; Polyelectrolytes; Polyglutamic Acid; Polymers; Spectrophotometry

For the first time, reducing values of homologous series of oligosaccharides have been determined by the 2,2'-bicinchoninate assay. The extent of Cu2+ reduction was monitored by spectrophotometric measurement of Cu+/2,2'-bicinchoninate complexes. Conditions of the assay were optimized so that relatively uniform reducing values were obtained with oligosaccharides derived from starch, polygalacturonic acid, and chitin, regardless of degree of polymerization. The uniform values resulted because members of each oligosaccharide series were oxidized to similar levels beyond the aldonic acid stage, while oxidation was limited to the reducing-terminal residue. This conclusion was based on direct measurements of quantitative loss of paramagnetic copper (Cu2+) by electron paramagnetic resonance spectroscopy.

Topics: Chitin; Copper; Electron Spin Resonance Spectroscopy; Mathematics; Oligosaccharides; Oxidation-Reduction; Pectins; Quinolines; Starch

Topics: Biotechnology; Biphenyl Compounds; Carbazoles; Carbohydrate Sequence; Colorimetry; Hexuronic Acids; Indicators and Reagents; Molecular Sequence Data; Oligosaccharides; Pectins

The effect of mucoadhesive polymeric vehicles on the mydriatic efficacy, and on the systemic and ocular absorption of cyclopentolate from eyedrops was studied in albino rabbits. Combining cyclopentolate base to polygalacturonic (CY-PGA) or hyaluronic (CY-HA) acid resulted in an increased mydriatic effect when compared with cyclopentolate hydrochloride (CY-HCl). During the first half an hour, the systemic absorption of cyclopentolate was lower after CY-PGA than after CY-HCl. The ocular penetration of cyclopentolate, based on drug concentrations in aqueous humor 30 minutes after the eyedrop instillation, was increased 3 fold when the polygalacturonate complex was used. CY-PGA, as well as other polymeric salts, might offer a possibility to increase the therapeutic index of cyclopentolate.

Topics: Absorption; Animals; Cyclopentolate; Drug Carriers; Eye; Female; Hyaluronic Acid; Male; Pectins; Rabbits; Random Allocation

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.

Topics: Calibration; Catalysis; Cellulase; Glycoside Hydrolases; Hexuronic Acids; Hydrogen-Ion Concentration; Nitriles; Oxidation-Reduction; Pectins; Polygalacturonase; Polysaccharides; Spectrophotometry, Ultraviolet

Laboratory experiments have been carried out for the removal of heavy metals from hydrogenated vegetable oils using hydrated polyuronides (degree of swelling from 4 to 12.8 ml/g) such as alginic acid, pectic and pectinic acids. The effect of the type of polyuronide, degree of esterification and oil treatment on the degree of demetalization has been studied. It has been shown that with increase in the degree of esterification of the polyuronide the efficiency of demetalization decreases. The second and third treatment of the hydrogenated oil with pectinic acid resulted in a high degree of heavy metal removal. The possibility of efficient demetalization of hydrogenated oils by treatment with water solutions of pectinic acids has also been demonstrated. The degree of metal ion removal increases with decreasing concentration of pectinic acids in the water solution.

Topics: Adsorption; Alginates; Copper; Food Contamination; Glucuronic Acid; Helianthus; Hexuronic Acids; Iron; Metals; Nickel; Pectins; Plant Oils; Sunflower Oil; Zinc

The role of electrostatic interactions in the attachment and fusion at acidic pH of Sindbis virus (SNV) with goose erythrocytes was studied, investigating the effect of several anionic and cationic polyelectrolytes on SNV hemagglutination and hemolysis. In order to establish the target of active drugs, the compounds were incubated either with the virus particles or with the erythrocytes. Dextran sulfate was the only compound able to inhibit the attachment of SNV to the erythrocytes. Fusion of virus with red cells was reduced dose-dependently by the polyanions dextran sulfate, mucin and polygalacturonic acid. On the contrary two polycations, polylysine and polybrene, enhanced viral hemolytic activity. However the effect of polyions is not exclusively related to the electric charge since ineffective molecules were found in both classes of compounds.

Topics: Animals; Chondroitin Sulfates; Dextran Sulfate; Dose-Response Relationship, Drug; Geese; Hemagglutination, Viral; Hemolysis; Heparin; Hexadimethrine Bromide; Histones; Hydrogen-Ion Concentration; Mucins; Pectins; Polylysine; Polymyxin B; Protamines; Sindbis Virus; Vero Cells

Polygalacturonic acid (DPave approximately 20), alpha-1,4-di- and trigalacturonic acids, delta 4,5-alpha-1,4-di- and delta 4,5-alpha-trigalacturonic acids, and several chemically modified derivatives of these oligomers were prepared. Their proteinase inhibitor-inducing activities were determined by supplying solutions of the compounds to young, excised tomato plants through their cut stems. Digalacturonic acid, on a molar basis, was the most active oligomer (ED50 approximately 1.5 micrograms/plant), being about three times more active than the parent oligogalacturonic acid (ED50 approximately 5.5 micrograms/plant). The specific inducing activity of trigalacturonic acid was about half that of digalacturonic acid. Both delta 4,5-di- and delta 4,5-trigalacturonic acids were about half as active as di- and trigalacturonic acids, respectively. Reduction of the hemiacetal (carbonyl) group of the di- and trigalacturonic acids with sodium borohydride completely destroyed proteinase inhibitor inducing activities, indicating that the inducing activity of both acids depends upon an intact hemiacetal at the reducing termini. Reduction of the double bonds of delta 4,5-di- and delta 4,5-trigalacturonic acids by catalytic hydrogenation with H2 (palladium catalyst) produced derivatives with specific inducing activities of approximately one-half that of the parent compounds. Thus, while the reducing termini of oligogalacturonides require an intact hemiacetal for proteinase inhibitor inducing activities, the nonreducing termini of the small oligouronides do not require a C4 hydroxyl nor a C5 proton to be active inducers.

Topics: Carbohydrate Conformation; Hexuronic Acids; Kinetics; Oligosaccharides; Pectins; Plants; Protease Inhibitors; Structure-Activity Relationship

The kinetic study of the de-esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell-shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell-wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell-wall enzymes are involved.

Topics: Cadaverine; Calcium Chloride; Carboxylic Ester Hydrolases; Enzyme Activation; Glycine max; Kinetics; Models, Biological; Pectins; Plants; Polyamines; Putrescine; Sodium Chloride; Spermidine; Spermine

The involvement of Rhizobium enzymes that degrade plant cell wall polymers has long been an unresolved question about the infection process in root nodule symbiosis. Here we report the production of enzymes from Rhizobium leguminosarum bv. trifolii that degrade carboxymethyl cellulose and polypectate model substrates with sensitive methods that reliably detect the enzyme activities: a double-layer plate assay, quantitation of reducing sugars with a bicinchoninate reagent, and activity gel electrophoresis-isoelectric focusing. Both enzyme activities were (i) produced commonly by diverse wild-type strains, (ii) cell bound with at least some of the activity associated with the cell envelope, and (iii) not changed appreciably by growth in the presence of the model substrates or a flavone that activates expression of nodulation (nod) genes on the resident symbiotic plasmid (pSym). Equivalent levels of carboxymethyl cellulase activity were found in wild-type strain ANU843 and its pSym-cured derivative, ANU845, consistent with previous results of Morales et al. (V. Morales, E. Martínez-Molina, and D. Hubbell, Plant Soil 80:407-415, 1984). However, polygalacturonase activity was lower in ANU845 and was not restored to wild-type levels in the recombinant derivative of pSym- ANU845 containing the common and host-specific nod genes within a 14-kb HindIII DNA fragment of pSym from ANU843 cloned on plasmid pRt032. Activity gel electrophoresis resolved three carboxymethyl cellulase isozymes of approximately 102, 56, and 33 kDa in cell extracts from ANU843. Isoelectric focusing activity gels revealed one ANU843 polygalacturonase isozyme with a pI of approximately 7.2. These studies show that R. leguminosarum bv. trifolii produces multiple enzymes that cleave glycosidic bonds in plant cell walls and that are cell bound.

Topics: Carboxymethylcellulose Sodium; Cell Membrane; Cellulase; Fabaceae; Isoenzymes; Pectins; Plants, Medicinal; Plasmids; Polygalacturonase; Rhizobium; Substrate Specificity; Symbiosis

The intrinsic protein fluorescence of the polygalacturonase from Colletotrichium lindemuthianum was exploited in stopped-flow experiments aimed at elucidating the kinetic mechanism for this enzyme. Binding of the polymeric substrate polygalacturonic acid (PGA) essentially produced a triphasic fluorescence profile. There was an initial rapid quench in fluorescence, consistent with the rapid formation of the enzyme-substrate complex, with an equilibrium constant of about 8 x 10(-4)% (w/v) PGA (about 0.27 microM). There then followed a near-constant fluorescence phase, attributable to turnover of the enzyme-substrate complex as a steady-state intermediate. As the concentration of the steady-state intermediate became depleted, towards the end of the reaction, there was a partial return of the fluorescence intensity. This phase is attributed to a final, single turnover of the enzyme at the end of the reaction. The fluorescence intensity does not return to its original level due to product remaining bound at the end of the reaction.

Topics: Kinetics; Mitosporic Fungi; Pectins; Polygalacturonase; Spectrometry, Fluorescence; Substrate Specificity

Arthrobacter simplex ATCC 6946 (viable cells) was immobilized in a calcium polygalacturonate gel. The trapped cells were used for repeated batchwise bioconversion of steroids. Reichstein's compound S and hydrocortisone were dehydrogenated introducing a double bond between C1 and C2 of ring A. The products 1-dehydro S and prednisolone, respectively, were identified by high pressure liquid chromatography. Steroid dehydrogenase activity increased in the system when an artificial electron acceptor, such as menadione (vitamin K3) was present in the reaction mixture. An airlift-type reactor was used to bioconvert up to 90% of substrate in 15 min, under optimal conditions. The gel entrapped cell preparations were used for repeated batch bioconversion during 30 days; 69 batch bioconversions for Reichstein's compound S were performed during 15 days of operation of the reactor. The operational stability of the process and the feasibility of repeated batch bioconversions was shown to be comparable to similar processes.

Topics: Arthrobacter; Cortodoxone; Hydrocortisone; Hydrogen-Ion Concentration; Hydroxysteroid Dehydrogenases; Microspheres; Oxygen; Pectins; Prednisolone; Vitamin K

The carbocyanine dye Stains-all displays spectral colour shifts or metachromasia upon binding to proteins, polysaccharides and other anionic substrates. The conformational status of the binding region of the substrate appears to govern the metachromatic features of the bound Stains-all. We have used this property to derive information about the conformational differences between the two anionic polysaccharides alginate and pectate. The stronger induction of circular dichroism in the 500 nm region of the dye by pectate is indicative of a greater extent of helical order in this polymer in solution than in the alginate or perhaps even hyaluronate chains.

Topics: Alginates; Carbocyanines; Carbohydrate Conformation; Circular Dichroism; Coloring Agents; Pectins; Spectrophotometry; Staining and Labeling

Evidence for the anomeric configurations and attachment sites of 3-deoxy-D-lyxo-2-heptulosaric acid (DHA) and apiosyl residues has been obtained through the characterization of two oligoglycosyl fragments isolated from rhamnogalacturonan II (RG-II). One of the oligoglycosyl fragments, a pentaglycosyl aldonic acid generated by Smith degradation of RG-II, was composed of four D-galactopyranosyluronic acid residues, a DHA residue, and a threonic acid residue (derived from a D-galactopyranosyluronic acid residue). The structural analysis of the pentaglycosyl aldonic acid established the beta-D-configuration for the DHA residue. Furthermore, it established that a previously identified diglycosyl side chain, 5-O-(beta-L-arabinofuranosyl)-DHA was directly attached to O-3 of a D-galactopyranosyluronic acid residue in the backbone of RG-II. The second oligoglycosyl fragment, a peralkylated diglycosyl hex-1-enitol, was generated by hex-5-enose degradation of permethylated and carboxyl-reduced RG-II. The structure of the peralkylated diglycosyl hex-1-enitol, beta-L-Rhap-(1----3')-beta-D-Apif-(1----5)-hex-1-enitol++ +, was determined by a combination of glycosyl-linkage composition analysis and n.m.r. spectroscopy. The n.m.r. data indicated the beta-configuration for the D-apiosyl residue. The isolation and characterization of the diglycosyl hex-1-enitol also established that a previously identified heptaglycosyl side chain was directly attached to O-2 of a D-galactopyranosyluronic acid in the backbone of RG-II.

Topics: Butyrates; Carbohydrate Sequence; Cell Wall; Cells, Cultured; Deoxy Sugars; Glucosides; Mannans; Molecular Sequence Data; Oligosaccharides; Pectins; Pentoses; Sugar Acids; Sugar Alcohols; Trees

The in vitro phosphorylation by [gamma-32P]ATP of a 34-kDa plasma membrane-associated protein (pp34) from tomato and potato is strongly enhanced in the presence of alpha-1,4-D-polygalacturonic acid (PGA) fragments (Farmer, E. E., Pearce, G., and Ryan, C. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1539-1542) that activate the expression of defensive and developmental genes in plant tissues. [gamma-35S]ATP, but not [gamma-35S]GTP, has now been found to strongly label pp34 in the presence of the PGA fragments. PGA-enhanced phosphorylation of pp34 is at one or more threonine residue(s) and therefore is the product of a serine/threonine kinase. alpha-1,4-L-Polyguluronic acid (PGU) enhances thiophosphorylation of pp34, but is less effective than PGA. beta-1,4-D-Polymannuronic acid (PMA) is inactive. In vivo synthesis of proteinase inhibitors in tomato leaves in response to PGA, PGU, and PMA parallels enhancing activities in in vitro phosphorylation assays. The minimum oligogalacturonide lengths that enhance in vitro thiophosphorylation of pp34 are about 14-15 residues, which are near the minimum sizes of uronides required to elicit a variety of localized defensive and developmental responses in plants. The lengths of biologically active galacturonic acid oligomers are of the same length that form strong intermolecular complexes in solution with Ca2+. Uronide-Ca2+ complexes are proposed to be the active molecular species that initiate the signal transduction pathways regulating uronide-responsive genes.

Topics: Adenosine Triphosphate; Autoradiography; Blotting, Western; Calcium; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Oligosaccharides; Pectins; Phosphorylation; Plants; Protease Inhibitors

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

A castor bean cDNA library has been constructed in the expression vector lambda gt11. Screening of 30,000 plaques with antibodies against casbene synthetase yielded six positive clones, from which three purified clones were obtained after sequential screening. The cDNA inserts in these clones ranged in size from 200 to 500 base pairs and were shown to share homologous sequences. One of the clones (pCS4) was examined in hybrid-selected and hybrid-arrested translation experiments and shown to contain a partial sequence of the casbene synthetase coding region. Northern analysis using the pCS4 clone as a probe revealed that the total hybridizable casbene synthetase mRNA amount increased to a maximum at 6 h after treatment of seedlings with pectic fragment elicitor and then decreased steadily with almost the same kinetics as observed previously for the changes in the translatable casbene synthetase mRNA activity under these same conditions. Run-on transcription experiments were performed with nuclei isolated from castor bean seedlings at various time periods after treatment with pectic fragment elicitors. Transcription of the casbene synthetase gene was first detected at 2 h after elicitation, rose to a maximum at 5 h, and fell rapidly thereafter. These results suggest that the accumulation of casbene synthetase, and hence casbene, is governed primarily by the rate of transcription of the casbene synthetase gene during elicitation by pectic fragments.

Topics: Cell Nucleus; Cloning, Molecular; DNA; Gene Expression Regulation; Gene Library; Genes, Plant; Kinetics; Nucleic Acid Hybridization; Oligosaccharides; Pectins; Phosphorus-Oxygen Lyases; Plants; Plants, Toxic; Plasmids; Poly A; Protein Biosynthesis; Ricinus; Ricinus communis; RNA; RNA, Messenger; Transcription, Genetic; Transferases

Vi is a linear homopolymer of 1,4 N-acetyl galactosaminuronic acid. It is present in S. typhi and some other members of Enterobacteriaceae. Vi antigen of S. typhi has been associated with the virulence of the organism and a vaccine based upon this antigen has been found to confer immunity against typhoid. In this paper, we report production and characterization of four hybrid cell clones secreting monoclonal antibodies against Vi capsular polysaccharide. Binding analysis using different derivatives of Vi showed that three monoclonal antibodies reacted with the antigenic determinant constituted by O-acetyl group and one recognised the epitope constituted by N-acetyl and carboxyl groups together. All the antibodies bound to Vi positive strains of S. typhi and did not show any significant reactivity with Vi negative strains of S. typhi, S. paratyphi A, S. paratyphi B and E. coli. Besides their utility in studying the sub-specificity of antibodies produced after vaccination with Vi, these antibodies would be helpful in the diagnosis of typhoid fever.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Binding, Competitive; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Epitopes; Hybrid Cells; Immunoglobulin Isotypes; Mice; Mice, Inbred BALB C; Pectins; Polysaccharides, Bacterial

This paper presents optical and chirooptical data on the interaction of the microscopy-staining agent ruthenium red with carboxylated polysaccharides in dilute aqueous solution. The polysaccharides used are both natural (alginate and pectate) and semisynthetic (C6-oxidized cellulose and C6-oxidized amylose). A preliminary discussion of the molecular structure and conformational features which control the interaction is presented.

Topics: Alginates; Amylose; Cellulose; Chemical Phenomena; Chemistry; Circular Dichroism; Pectins; Polysaccharides; Ruthenium; Ruthenium Red; Spectrum Analysis; Stereoisomerism

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.

Topics: Antibodies, Monoclonal; Bacillus subtilis; Erwinia; Extracellular Matrix; Hydrolysis; Kinetics; Molecular Weight; Pectins; Polysaccharide-Lyases; Substrate Specificity

The thermodynamic properties of the dissociation of natural polyuronic acids in dilute aqueous solution at 25 degrees C are reported. The investigation concerns polygalacturonic acid and alginic acids from different sources, including highly homogeneous mannuronic and gulronic fractions and involved potentiometry, isothermal calorimetry, dilatometry and circular dichroism measurements. The dissociation of polyuronic acids was studied as a function of degree of dissociation, concentration of the polymer and ionic strength. Abnormal behavior upon dissociation was observed only in polyguluronic and polygalacturonic acids and attributed to a pH-induced conformational transition and/or to an interchain disaggregation. Direct molecular weight evidence supports the proposed intramolecular transition of polygalacturonate chain.

Topics: Alginates; Calorimetry; Circular Dichroism; Glucuronic Acid; Hexuronic Acids; Hydrogen-Ion Concentration; Molecular Conformation; Molecular Weight; Pectins; Potentiometry; Protons; Thermodynamics

The ability of Erwinia chrysanthemi to cause soft-rot diseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase E. chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone (pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase (ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6. Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL- ExoPL- mutant of EC16 was constructed that exhibited reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Southern; Calcium; Cloning, Molecular; DNA Mutational Analysis; DNA, Bacterial; Erwinia; Genes, Bacterial; Genomic Library; Molecular Sequence Data; Pectins; Polysaccharide-Lyases; Sodium

1. Conjugates of 1-beta-D-arabinofuranosylcytosine (araC) with polysaccharides containing carboxyl groups, such as polygalacturonic acid (PGA) and carboxymethylated yeast beta-D-glucan (CMG) were prepared. 2. Activation of the polysaccharidic carboxyl group by isobutylchloroformiate and formation of a peptide bond via 4-NH2 group of araC was used for a coupling reaction. 3. Elementary analysis, u.v. and i.r. spectra confirmed the structures of the conjugates. 4. The conjugates were most stable against the hydrolysis under the mild acid conditions. 5. It was also shown that under the physiological conditions trypsin catalyze the conjugate hydrolysis and the catalytic effect is higher than that of chymotrypsine. 6. It is suggested that trypsin or trypsin-like proteases could participate in the hydrolysis of the conjugates in vivo. PGA-araC and CMG-araC showed 1.5- or 2.5-times higher antileukemic activity than both free araC or polysaccharides.

Topics: Animals; Antineoplastic Agents; beta-Glucans; Cytarabine; Female; Glucans; Hydrolysis; Leukemia L1210; Male; Mice; Mice, Inbred DBA; Nitrogen; Pectins; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.

Topics: Alginates; Amino Acids; Aspergillus niger; Blotting, Western; Chemical Phenomena; Chemistry, Physical; Chromatography; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Epichlorohydrin; Glucuronic Acid; Glycoside Hydrolases; Hexuronic Acids; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Macromolecular Substances; Molecular Weight; Pectins; Polygalacturonase

Autoantibodies to DNA (anti-DNAab), found primarily in systemic lupus erythematosus (SLE), cross-react with a variety of antigens. The binding of these antibodies to naturally occurring mucopolysaccharides such as heparan and chondroitin sulfates has led to the suggestion that anti-DNAab have specificity for a polyanionic epitope. In this study, to avoid the use of endogenous immunogens to which humans may have become sensitized, we have used the synthetic polyanions, dextran sulfate (DS), polyvinyl sulfate (PVS) and the semi-synthetic antigen, pectic acid (PA) to evaluate this hypothesis using SLE sera (n = 15) and sera from healthy individuals (controls; n = 15, age and sex matched to SLE group). Inhibition of binding with 125I-DNA was optimal at 1 mg/ml for DS and PVS, and resulted in significant inhibition of binding by both SLE and control sera of native DNA (P less than 0.01, each group); no inhibition was observed with PA, nor was a significant inhibition observed with any antigen on binding of SLE or control sera groups to denatured DNA. We conclude that while on quantitative grounds reaction of anti-DNAab with polyanions may not be clinically relevant, it is clear that, unlike polycarboxylates (e.g. PA), polysulfated polymers, whether aliphatic, as in the case of PVS, or glycosidic, such as DS, react with a subpopulation of anti-DNAab in such a manner as to block significantly the ability of these antibodies to bind DNA.

Topics: Analysis of Variance; Antibodies, Antinuclear; Antigens; Autoimmune Diseases; Dextran Sulfate; Dextrans; DNA; Dose-Response Relationship, Immunologic; Humans; Lupus Erythematosus, Systemic; Molecular Structure; Pectins; Polyelectrolytes; Polymers; Polyvinyls

The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity.

Topics: Aspergillus nidulans; Carbon; Cell Wall; Culture Techniques; Fabaceae; Hydrogen-Ion Concentration; Kinetics; Models, Biological; Mutation; Pectins; Plants, Medicinal; Polygalacturonase; Polysaccharide-Lyases

One of the anti-complementary pectic polysaccharides (AR-2IIa) isolated from the root of Angelica acutiloba Kitagawa gives the "ramified" region (PG-1a,rhamnogalacturonan with neutral side-chains) in addition to oligogalacturonides on digestion with endo-alpha-D-(1--4)-polygalacturonase. When the neutral side-chains in PG-1a were digested with both exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase, approximately 70% of the arabinosyl chains and approximately 30% of the galactosyl chains were released. The resistant product E-PG-1a had the same anti-complementary activity as PG-1a. E-PG-1a gave long (d.p. greater than or equal to 5) and short (d.p. less than or equal to 4) neutral galactosyl chains after degradation of the GalA moiety by base-catalysed beta-elimination in the presence of sodium borodeuteride followed with lithium-mediated degradation. Methylation analysis showed that the long galactosyl chains consisted mainly of terminal, 6-linked and 3,6-disubstituted Gal, and that the short chains were rich in 6-linked Gal. Degradation of the GalA moieties in PG-1a markedly decreased the anti-complementary activity, but the long and short galactosyl chains still expressed approximately 50 and approximately 20%, respectively, of the anti-complementary activity of E-PG-1a.

Topics: Animals; Carbohydrate Sequence; Complement Hemolytic Activity Assay; Complement Inactivator Proteins; Gas Chromatography-Mass Spectrometry; In Vitro Techniques; Methylation; Molecular Sequence Data; Pectins; Plants; Sheep; Structure-Activity Relationship

Topics: Chromatography, Ion Exchange; Gene Expression Regulation; Genes, Plant; Oligosaccharides; Pectins; Plants; Polygalacturonase; Polysaccharide-Lyases; Protease Inhibitors

A mixture of heterotrophic bacteria and collection strains of Escherichia coli and Pseudomonas fluorescens were immobilized in calcium alginate or pectate gels. Comparison of respiratory activity, substrate uptake and biosynthetic capacity of immobilized cells showed that both types of carriers permit a prolonged preservation of metabolic activity but the transfer of substances through the gel is faster in the pectate. Morphological changes include some intracellular structures, partial shrinkage of the plasma membrane of immobilized cells, and transformation of a rod-like cell shape to an oval one.

Topics: Alginates; Bacteria; Escherichia coli; Gels; Glucuronic Acid; Hexuronic Acids; Pectins; Polysaccharides

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40 degrees C. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nonagalacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-alpha-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.

Topics: Animals; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; Glycoside Hydrolases; Gram-Negative Anaerobic Bacteria; Hydrogen-Ion Concentration; Kinetics; Pectins; Polygalacturonase; Polymers; Rumen; Substrate Specificity; Temperature; Viscosity

A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.

Topics: Chromatography, Ion Exchange; Hexuronic Acids; Hydrolysis; Magnetic Resonance Spectroscopy; Pectins; Uronic Acids

Pectins from apple, citrus and sugar beet injected intravenously to ewes markedly stimulated blood prolactin, growth hormone (GH) and cortisol. Pectic acid and polygalacturonic acid exhibited the same property. A preparation of oligogalacturonic acid with a polymerisation degree of 12 to 13 was also active, whereas oligomers with a smaller degree of polymerisation (congruent to 10) were devoid of activity. Pectic acid administered orally to mature virgin rats induced the accumulation of beta-casein in mammary gland. Pectins and some of their derivatives therefore had a lactogenic property and their effect probably resulted from a capacity to trigger lactogenic hormone secretion.

Topics: Administration, Oral; Animals; Caseins; Female; Growth Hormone; Hydrocortisone; Injections, Intravenous; Mammary Glands, Animal; Pectins; Prolactin; Rats; Sheep

A DNA fragment containing a Klebsiella oxytoca gene for polygalacturonate trans-eliminase was cloned into the kanamycin resistance transposon Tn5. This new transposon, designated Tn5-Pga+, had a transposition frequency of 1 X 10(-6). The broad host range plasmid pR751::Tn5-Pga+ was conjugally transferred to a variety of genetic backgrounds. The ability to degrade polygalacturonate was expressed in Aeromonas hydrophila, Alcaligenes eutrophus, Azotomonas insolita, Escherichia coli, Pseudomonas putida and Rhodopseudomonas sphaeroides, but not in Zymomonas mobilis.

Topics: Cloning, Molecular; Conjugation, Genetic; DNA Transposable Elements; Genes, Bacterial; Gram-Negative Bacteria; Klebsiella; Pectins; Plasmids; Polysaccharide-Lyases

Serum amyloid P component (SAP), a normal human plasma glycoprotein, was found in a solid phase ELISA to have Ca2+-dependent binding for keyhole limpet haemocyanin (KLH), pectic acid, trinitrophenylated (TNP) macromolecules, and plastic surfaces. The binding to TNP-KLH was used to develop a sensitive ELISA. The binding of SAP to the ligands mentioned was inhibited by EDTA, KLH, pectic acid, TNP-conjugated macromolecules (bovine serum albumin, polyacrylhydrazide), and p-nitrophenylarsonic acid. Underivatized and DNP-conjugated macromolecules did not inhibit the SAP binding; arsenilic acid, picric acid, and dinitrophenyl were weak inhibitors. SAP bound to TNP-agarose was eluated by either EDTA or p-nitrophenylarsonic acid. Thus, a unique region of SAP is responsible for the polyspecific binding. We suggest that the polyspecific binding of SAP takes place through a Ca2+ bridge: half of the metal coordination sphere is occupied by SAP, with the other half available to interact with metal ligand.

Topics: C-Reactive Protein; Calcium; Chromatography, Gel; Edetic Acid; Enzyme-Linked Immunosorbent Assay; Hemocyanins; Humans; Pectins; Plastics; Protein Binding; Serum Albumin, Bovine; Serum Amyloid P-Component; Surface Properties; Trinitrobenzenes

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.

Topics: Hydrogen-Ion Concentration; Pectins; Polyamines

The genes for two of several pectate lyase isozymes produced by the phytopathogenic enterobacterium Erwinia chrysanthemi 1237 were subcloned and compared by DNA-DNA hybridization, and the encoded proteins were analyzed. The borders of the genes were located on a restriction map by incremental exonuclease III deletions. DNA-DNA hybridization studies revealed a low percentage of mismatch (7 to 17%) between pelB and pelC. No homology was detected between pelC and other regions of the E. chrysanthemi 1237 chromosome, in which three other isozyme genes apparently reside. The pectate lyase isozymes were readily purified by chromatofocusing or granulated-gel bed isoelectric focusing from the periplasmic shock fluids of Escherichia coli subclones. The molecular weights of PLb and PLc were 30,000 and 33,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their isoelectric points were 7.6 and 8.1, respectively, as determined by equilibrium isoelectric focusing in ultrathin polyacrylamide gels. The Km values for PLb and PLc were 0.20 and 0.32 mg/ml, respectively, with polygalacturonate as a substrate. Thin-layer chromatography of reaction products and viscometric assays revealed little difference between the two isozymes. All our data indicate that the genes are duplicates and that the proteins are isofunctional.

Topics: Cloning, Molecular; DNA Restriction Enzymes; DNA, Bacterial; Erwinia; Genes; Genes, Bacterial; Isoelectric Point; Isoenzymes; Kinetics; Molecular Weight; Nucleic Acid Hybridization; Pectins; Polysaccharide-Lyases; Sequence Homology, Nucleic Acid

Effects of various purified dietary fiber components on beta-carotene utilization by the chick were investigated in two experiments (expt.). Eight-day-old Columbian X New Hampshire male (expt. 1) or female (expt. 2) chicks were fed a vitamin A-deficient diet for 1 wk and then fed beta-carotene-supplemented diets containing 0% fiber, 7% arenaceous flour or 7% of a purified fiber source for 4 wk. Results of expt. 1 showed that hemicellulose, lignin and citrus pectin, but not arenaceous flour or polygalacturonic acid, depressed beta-carotene utilization by the chick, as measured by percentage of consumed beta-carotene stored in liver as vitamin A relative to the 0% fiber control. In expt. 2, effects of the methoxyl content of pectin were studied. High and medium methoxyl apple pectin, citrus pectin and polygalacturonic acid reduced storage of vitamin A in liver. Low methoxyl apple pectin had no significant effect on beta-carotene utilization. Thus, several purified forms of dietary fiber significantly reduced beta-carotene utilization by chicks when fed at the 7% supplementary level. Moreover, with pectin, there was an inverse relationship between methoxyl content of pectin and beta-carotene utilization.

Topics: Animals; beta Carotene; Biological Availability; Carotenoids; Chickens; Dietary Fiber; Eating; Female; Male; Mathematics; Molecular Weight; Pectins; Vitamin A Deficiency

Five Bacteroides species that are found in the human colon can utilize polygalacturonic acid (PGA) when they are grown in laboratory media: Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis subsp. a, and Bacteroides sp. strain 3452A (an unnamed DNA-DNA homology group). PGA-degrading enzymes from B. thetaiotaomicron have been isolated and characterized previously. To determine whether a PGA lyase activity in human feces could be attributed to any of these species, we first determined the properties of PGA lyases from the other four Bacteroides species. PGA lyases from all the Bacteroides species were soluble, cell associated, and inducible by PGA. All had similar pH optima (8.4 to 8.8) and similar molecular weights (50,000). All activities were enhanced by calcium. The PGA lyases from the five species differed with respect to isoelectric point: B. thetaiotaomicron (pI 7.5), B. vulgatus (pI 7.7), B. ovatus (pI 5.8, 7.2), B. fragilis subsp. a (pI 6.1), and Bacteroides sp. strain 3452A (pI 7.7). The PGA lyase activity in human feces resembled those of the Bacteroides PGA lyases in that it had a pH optimum of 8.4 to 8.8 and was enhanced by calcium. However, it differed from the Bacteroides PGA lyases both with respect to isoelectric point (pI 4.2 to 4.4) and molecular weight (100,000). On the basis of these findings, it appears that the PGA lyase activity in human feces is not produced by any of the Bacteroides species surveyed in this survey. Moreover, there was no detectable PGA lyase activity in feces that had the same properties as the Bacteroides enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bacteroides; Chromatography, Gel; Colon; Enzyme Induction; Feces; Humans; Isoelectric Focusing; Molecular Weight; Pectins; Polysaccharide-Lyases

A procedure for reducing the Pb content in wines containing high levels of Pb is described. The reduction of Pb by means of pectic acid (PA) depends on the quality grade of the wine. In "Auslese" (wine A) 6250 mg PA/l diminished Pb content in 24 h from 0.88 mg/l to 0.28 mg/l; in "Spätlese" (wine B) from 0.83 mg/l to 0.06 mg/l. Under the same conditions the content of Fe, Zn, Cu decreased in wine A from 8.85 mg/l to 7.05 mg/l, from 4.75 mg/l to 1.25 mg/l, from 0.67 mg/l to 0.57 mg/l and in wine B from 6.70 mg/l to 5.41 mg/l, from 1.17 mg/l to 0.28 mg, 0.46 mg/l to 0.28 mg/l, respectively. PA is removed almost quantitatively by filtration. Sensory properties of treated wines were unchanged with concentrations of PA of 750 mg and 1500 mg/l. A slight effect on taste at 6250 mg PA/l wine cannot be excluded. On account of its high affinity to Pb, PA will probably remove Pb from other liquid foods as well.

Topics: Chemical Phenomena; Chemistry; Lead; Metals; Pectins; Time Factors; Wine

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.

Topics: Aldose-Ketose Isomerases; Bacterial Proteins; Carbohydrate Epimerases; Enzyme Induction; Enzyme Repression; Erwinia; Genes, Bacterial; Gluconates; Mutation; Pectins; Polysaccharide-Lyases; Sugar Alcohol Dehydrogenases

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.

Topics: Amines; Aspergillus niger; Caprolactam; Chromatography, Affinity; Glass; Glycoside Hydrolases; Keratins; Particle Size; Pectins; Polygalacturonase; Polymers; Silica Gel; Silicon Dioxide

When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a Km for PGA of 40 to 70 micrograms/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a delta 4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a Km for PGA of 350 to 400 micrograms/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated.

Topics: Bacteroides; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Glycoside Hydrolases; Pectins; Polygalacturonase; Polysaccharide-Lyases

An intracellular pectinolytic enzyme was isolated from a cell extract of Butyrivibrio fibrisolvens and purified. The optimum pH for enzyme activity was 5.6. The enzyme preferentially degraded de-esterified substrates by hydrolysis of monosaccharide units from the non-reducing end; the only product of degradation was D-galacturonic acid. Values of Km and Vmax for oligo- and polygalacturonates indicated that the best substrate was digalacturonic acid; oligogalacturonates containing either a saturated or a delta 4,5-unsaturated non-reducing end were both degraded. The enzyme was classified as an exo-D-galacturonanase [poly(1,4-alpha-D-galacturonide) galacturonohydrolase (EC 3.2.1.67)].

Topics: Animals; Cattle; Chromatography; Eubacterium; Glycoside Hydrolases; Hydrolysis; Pectins; Rumen

E.s.r. Experiments employing a flow system in conjunction with the TiIII-H2O2 couple show that dextrans react with the hydroxyl radical (HO.) via indiscriminate attack (except that abstraction of hydrogen atoms from carbons which are both linked by glycosidic bonds and included in the pyranose ring may be inhibited, possibly for steric reasons). Acid- and base-catalysed transformations of first-formed radicals have been demonstrated; the suggestion that such reactions can lead to glycosidic cleavage is supported by viscosity studies which confirm the pH-dependence of radical-initiated degradation. For galacturonan and related compounds, e.s.r. results indicate that reaction with HO. proceeds preferentially via abstraction of the hydrogen on the carbon adjacent to the carboxyl group. The crucial step in the subsequent degradation pathway probably involves a pH-independent rearrangement.

Topics: Dextrans; Electron Spin Resonance Spectroscopy; Free Radicals; Glucuronates; Glucuronic Acid; Hexuronic Acids; Hydroxides; Oxidation-Reduction; Pectins; Polysaccharides; Uronic Acids; Viscosity

In our previous paper, we reported that various types of carrageenan, dextran sulfate and fucoidan, which are sulfated homopolysaccharides with high molecular weights, were human T cell mitogens and murine polyclonal B cell activators (PBAs) and that heparin, a sulfated heteropolysaccharide, was a very weak human mitogen and mouse PBA. Here we used cellulose sulfate (Mr 7-9 X 10(3], dextran sulfate with two different low molecular weights (Mr 5 X 10(3) and 8 X 10(3], two different condroitin sulfates (Mr 3.5 X 10(4], polyvinyl sulfate and polygalacturonic acid to investigate mitogenic activities of polysaccharides in detail. The following results were obtained. Low-molecular-weight sulfated homopolysaccharides, dextran sulfate and cellulose sulfate, were very weak or not human T cell mitogens. However, they were better murine PBAs. Sulfated heteropolysaccharides, chondroitin 4-sulfate and chondroitin 6-sulfate, hardly induced mitogenic changes in human T cells and mouse B cells, even though the molecular weight of these substances was more than 1 X 10(4). There were no other polymers examined so far which activated both human T cells and murine B cells. The relationship among molecular size, sulfate groups and lymphocyte activation is discussed in detail.

Topics: Adult; Animals; B-Lymphocytes; Cellulose; Chondroitin Sulfates; Dextran Sulfate; Dextrans; Female; Humans; In Vitro Techniques; Lymphocyte Activation; Male; Mice; Mitogens; Molecular Weight; Pectins; Polysaccharides; Polyvinyls; T-Lymphocytes

Mild acid hydrolysis of a small (Mr = 6 kDa) pectic polysaccharide isolated from tomato leaves, an inducer of the synthesis and accumulation of two proteinase inhibitors in excised tomato plants, yielded a alpha-D-polygalacturonic acid polymer with degree of polymerization = 20 that retained proteinase inhibitor-inducing activity. Enzymic and acid hydrolysis of this polygalacturonan yielded a series of alpha-1,4-D-galacturonic acid oligomers with degrees of polymerization from 2 to 6 which were purified to homogeneity and assayed for proteinase inhibitor-inducing activity in young excised tomato plants. All of the oligomers exhibited activity. The hexagalacturonide possessed the highest activity and the trimer the lowest. The evidence supports a possible role for plant cell wall fragments as systemic messengers that regulate the expression of proteinase inhibitor genes in plant leaves in response to pest attacks.

Topics: Carbohydrates; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Hexuronic Acids; Oligosaccharides; Pectins; Plants; Uronic Acids

Cells of tumorigenic Agrobacterium tumefaciens strain B6 were labeled with [35S]methionine and used to estimate bacterial adsorption to potato tuber discs. After 10 min at pH 7.2, about 5 X 10(5) bacteria adsorb to each 9.0-mm disc. Lengthening the incubation period to 90 min increases adsorption to about 11 X 10(5) bacteria per disc. Bacterial adsorption is reduced under both alkaline and acid pH conditions and increases 2.3-fold if the number of bacterial cells in the inoculum is doubled. Adsorption is increased if citrus pectin, polygalacturonic acid, or demethylated pectin is included in the inoculum; methylated polygalacturonic acid is inactive. The activity of citrus pectin is abolished, however, if the compound is applied to the discs as a pretreatment and then removed prior to inoculation. Lipopolysaccharide preparations from five of six A. tumefaciens strains have no effect on bacterial adsorption. The sixth preparation, from nontumorigenic strain NT-1, reduces adsorption by about 20%.

Topics: Adsorption; Hydrogen-Ion Concentration; Kinetics; Lipopolysaccharides; Pectins; Plant Tumors; Plants; Rhizobium; Time Factors; Vegetables

Penicillium oxalicum produced two isozymes of polygalacturonase (PG) and a pectate lyase (PL). The enzymes were separated and purified following ammonium sulphate precipitation, ion exchange chromatography, ultrogel column chromatography and isoelectric focusing. The first isozyme of polygalacturonase (PGI) was rather unstable hence its properties could not be much assayed. PGII macerated and killed yam tissue in 4 hours but PL was unable to do so. Enzyme assay for the end-products of degradation of sodium polypectate and yam tissue showed that PGI was an exo-enzyme while PGII and PL were endo-enzymes. Endo-polygalacturonase (PGII) appears to play the major role (as the macerating enzyme) in the pathogenesis of yam tissue infected by P. oxalicum.

Topics: Cell Wall; Glycoside Hydrolases; Hydrogen-Ion Concentration; Isoelectric Point; Isoenzymes; Pectins; Penicillium; Plant Diseases; Plants; Polygalacturonase; Polysaccharide-Lyases

The contents of the lower alimentary tract from rats fed a semisynthetic, pectin-supplemented diet showed increased nitrate reductase activity and an increase in the amount of luminal contents in the intestine and cecum. Nitrate reductase activity was associated with the insoluble fraction of the gut contents which was sedimented by centrifugation (5,100 X g,20 min) and was abolished after treating the animals with streptomycin, neomycin, and bacitracin for 7 days. The pectin-dependent increase in cecal size and microbial nitrate reduction were reversed when animals were transferred from a pectin-supplemented onto a control semisynthetic diet. Polygalacturonic acid (pectic acid) was without effect on either cecal size or cecal microbial nitrate reductase activity. The studies demonstrate that pectin influences microbial metabolism in the alimentary tract.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Cecum; Dietary Carbohydrates; Enzyme Induction; Intestine, Small; Male; Nitrate Reductases; Pectins; Rats; Rats, Inbred Strains; Stomach

Pectic enzymes in the supernatants of Erwinia chrysanthemi cultures in late-logarithmic-phase growth on D-galacturonan were resolved into three components: two pectate lyase isozymes and an exo-poly-alpha-D-galacturonosidase previously unreported in this organism. The hydrolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, preparative electrofocusing in Ultrodex gel, and gel filtration through Ultrogel AcA54. The enzyme had a specific activity of 591 mumol/min per mg of protein, a pI of 8.3, a molecular weight of 67,000, a pH optimum of 6.0, and a Km of 0.05 mM for D-galacturonan. Analyses of reaction mixtures by paper chromatography revealed that the enzyme released only digalacturonic acid from D-galacturonan. The action of the hydrolytic enzyme on D-galacturonan labeled at the nonreducing end by partial digestion with pectate lyase revealed that it rapidly released 4,5-unsaturated digalacturonic acid from 4,5-unsaturated pectic polymers. The production of extracellular exo-poly-alpha-D-galacturonosidase was coordinately regulated with pectate lyase production. The action patterns of the two enzymes appeared complementary in the degradation of pectic polymers to disaccharides that stimulated pectic enzyme production and supported bacterial growth.

Topics: Erwinia; Glycoside Hydrolases; Hydrogen-Ion Concentration; Isoenzymes; Molecular Weight; Pectins; Polygalacturonase; Polysaccharide-Lyases

Topics: Calcium; Carbohydrate Conformation; Circular Dichroism; Gels; Pectins

Topics: Calcium; Carbohydrate Conformation; Gels; Pectins; Polygalacturonase

Topics: Methylation; Methyltransferases; Molecular Conformation; Pectins; Plants; S-Adenosylmethionine; Stereoisomerism

Polygalacturonic acid transeliminase (PATE) was produced by all of six Azospirillum strains studied. Characteristics were similar to those of PATE from other bacteria: activity was maximal at pH 8.0 and was stimulated by CaCl2. Polygalacturonic acid was used more readily than pectin as a substrate. Polygalacturonic acid in the medium stimulated PATE production by several but not all strains. In all cases some of the PATE produced in cultures remained bound to cell walls. In one strain, most remained cell wall bound. When nitrogen was supplied as amino acids rather than ammonium salts, the ratio of free to bound enzyme was increased. The strains studied varied considerably to response to nutrient amendments and in maximum PATE activity.

Topics: Bacteria; Calcium; Cell Wall; Hydrogen-Ion Concentration; Pectins; Polysaccharide-Lyases; Species Specificity; Substrate Specificity

Topics: Carbohydrate Conformation; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Pectins; Sodium; Water; X-Ray Diffraction

Topics: Calcium; Carbohydrate Conformation; Gels; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Pectins; X-Ray Diffraction

The depolymerase activity of cell-free extracts of nine species of rumen ciliate protozoa and two mixed protozoal preparations, grown in vivo and in vitro, towards polygalacturonic acid was examined. The highest activity was found with Eremoplastron bovis and Ostracodinium obtusum bilobum while there was none in the spined or spineless forms of Entodinium caudatum and little in Polyplastron multivesticulatum. On the basis of the rapid drop in viscosity, inhibition by EDTA and the production of u.v.-absorbing material, the enzymes from all active species were designated as endopectate lyases (EC4.2.2.2) although some polygalacturonase may be present. Neither pectin nor polygalacturonic acid supported the survival or growth of any of the protozoal species tested.

Topics: Animals; Ciliophora; Hydrogen-Ion Concentration; Pectins; Polygalacturonase; Polysaccharide-Lyases; Rumen; Sheep

Topics: Calcium Chloride; Disaccharides; Edetic Acid; Enzyme Induction; Erwinia; Pectins; Polysaccharide-Lyases; Uronic Acids

Topics: Calcium Chloride; Carbohydrate Metabolism; Chromatography; Clostridium; Dialysis; Glycoside Hydrolases; Pectins; Polysaccharides; Polysaccharides, Bacterial; Renal Dialysis; Research; Uronic Acids

Davis, B. R. (Communicable Disease Center, Atlanta, Ga.), and W. H. Ewing. Lipolytic, pectolytic, and alginolytic activities of Enterobacteriaceae. J. Bacteriol. 88:16-19. 1964.-Of 2,262 cultures of Enterobacteriaceae tested, only cultures of Enterobacter liquefaciens (Aerobacter liquefaciens), Serratia, Proteus vulgaris, and P. mirabilis gave evidence of lipolytic activities. None of the cultures liquefied pectate or alginate. The value of tests for these activities in diagnostic work with Enterobacteriaceae, and in defining the limits of that family, is discussed.

Topics: Alginates; Carbohydrate Metabolism; Classification; Enterobacteriaceae; Glucuronic Acid; Glycerides; Hexuronic Acids; Humans; Lipid Metabolism; Metabolism; Oils; Pectins; Serratia; Toxicology

Topics: Bacteria; Carbohydrate Metabolism; Isomerases; Oxidoreductases; Pectins; Pseudomonas; Uronic Acids

Topics: Bacteria; Carbohydrate Metabolism; Gluconates; Oxidoreductases; Pectins; Pseudomonas; Uronic Acids

Ng, Henry (University of California, Davis) and Reese H. Vaughn. Clostridium rubrum sp. n. and other pectinolytic clostridia from soil. J. Bacteriol. 85:1104-1113. 1963.-Reports in the literature and results of experiments described herein suggest that pectinolytic anaerobes constitute a very heterogeneous group. The cultures isolated in this study all belonged to the genus Clostridium. The following species were identified: C. butyricum, C. fallax, C. multifermentans, and C. indolis. In addition, a species believed to be previously undescribed was named C. rubrum sp. n. The ability to ferment galacturonic acid was found to be adaptive. Some cultures fermented pectin and pectic acid to the same degree, whereas others fermented pectin only partially. The partial fermentation was attributed to the lack of a pectinesterase. On the basis of fermentation balances, it was concluded that the four strains of galacturonic acid fermenters selected for study yielded identical end products in approximately the same proportions. Per mole of galacturonic acid fermented, about 2 moles of CO(2), 1.5 moles of H(2), 1.5 moles of acetic acid, and 0.25 mole of butyric acid were produced.

Topics: Acetic Acid; Chromatography; Clostridium; Clostridium beijerinckii; Fermentation; Manometry; Pectins; Research; Soil; Soil Microbiology; Uronic Acids

Culture filtrates of Erwinia carotovora and Bacillus polymyxa split pectic substances to yield a product that absorbs at 230 mmicro, and which reacts with thiobarbituric acid to form a substance that absorbs at 547 mmicro. This product is believed to be a C-4,5-unsaturated oli-gouronide. The preferred substrate is polygalacturonic acid rather than pectin; the enzyme, provisionally named polygalacturonic-trans-eliminase, is activated by calcium.

Topics: Bacillus; Bacteria; Calcium; Carbohydrate Metabolism; Enzymes; Pectins; Uronic Acids

Nagel, Charles W. (University of California, Davis) and Reese H. Vaughn. Comparison of growth and pectolytic enzyme production by Bacillus polymyxa. J. Bacteriol. 83:1-5. 1962.-Studies were made of pectolytic enzyme production by Bacillus polymyxa during growth. It was found that elaboration of enzyme occurred during logarithmic growth and ceased when the stationary phase was reached. The specific activity of the extra-cellular enzyme remained relatively constant until lysis occurred. The increased specific activity of the intracellular pectolytic enzyme may be explained if one assumes that the rate of secretion of the intracellular enzyme is dependent upon the concentration of the extracellular enzyme. The concentration of the intracellular pectolytic enzyme dropped markedly at the end of the logarithmic growth phase; the enzyme was released into the medium during the stationary growth phase and subsequent lysis of the cells. It was shown that the intra- and extracellular enzymes were similar or identical in that both were calcium-dependent pectic acid transeliminases.

Topics: Bacillus; Calcium; Enzymes; Esterases; Paenibacillus; Pectins; Plasmodiophorida

Topics: Pectins

Topics: Culture Media; Gels; Humans; Pectins

Topics: Animals; Blood Volume; Hemorrhage; Hemostasis; Pectins; Rats

Topics: Coffee; Humans; Pectins; Polysaccharides; Prunus

Topics: Bacillus; Carbohydrate Metabolism; Fermentation; Paenibacillus; Pectins; Plasmodiophorida

Topics: Glycerol; Pectins; Plasma Substitutes; Plasma Volume

Topics: Glycoside Hydrolases; Hydrolysis; Pectins; Polygalacturonase; Saccharomyces cerevisiae

Topics: Glycoside Hydrolases; Hydrolysis; Pectins; Polygalacturonase; Saccharomyces cerevisiae; Uronic Acids

Topics: Beta vulgaris; Pectins; Vegetables

Topics: Clostridium; Fermentation; Humans; Pectins

Topics: Glycoside Hydrolases; Pectins; Polygalacturonase

Topics: Bacteria; Pectins

Topics: Pectins; X-Ray Diffraction

Topics: Flavoring Agents; Food Additives; Fragaria; Malus; Molecular Structure; Pectins

Topics: Pectins; X-Ray Diffraction

Topics: Pectins

Topics: Esters; Glycols; Pectins

Topics: Bacteria; Clostridium; Fermentation; Pectins

Topics: Humans; Pectins; Sodium; X-Ray Diffraction