Hydrolysis of pectins with different degrees and patterns of methylation by the endopolygalacturonase of Fusarium moniliforme.
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The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme. (+info)
Direct interference with rhamnogalacturonan I biosynthesis in Golgi vesicles.
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Pectin is a class of complex cell wall polysaccharides with multiple roles during cell development. Assigning specific functions to particular polysaccharides is in its infancy, in part, because of the limited number of mutants and transformants available with modified pectic polymers in their walls. Pectins are also important polymers with diverse applications in the food and pharmaceutical industries, which would benefit from technology for producing pectins with specific functional properties. In this report, we describe the generation of potato (Solanum tuberosum L. cv Posmo) tuber transformants producing pectic rhamnogalacturonan I (RGI) with a low level of arabinosylation. This was achieved by the expression of a Golgi membrane-anchored endo-alpha-1,5-arabinanase. Sugar composition analysis of RGI isolated from transformed and wild-type tubers showed that the arabinose content was decreased by approximately 70% in transformed cell walls compared with wild type. The modification of the RGI was confirmed by immunolabeling with an antibody recognizing alpha-1,5-arabinan. This is the first time, to our knowledge, that the biosynthesis of a plant cell wall polysaccharide has been manipulated through the action of a glycosyl hydrolase targeted to the Golgi compartment. (+info)
Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion.
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Polygalacturonases (PGs) cleave runs of unesterified GalUA that form homogalacturonan regions along the backbone of pectin. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the wall where two adjacent cells abut and its integrity is important for cell adhesion. Transgenic apple (Malus domestica Borkh. cv Royal Gala) trees were produced that contained additional copies of a fruit-specific apple PG gene under a constitutive promoter. In contrast to previous studies in transgenic tobacco (Nicotiana tabacum) where PG overexpression had no effect on the plant (K.W. Osteryoung, K. Toenjes, B. Hall, V. Winkler, A.B. Bennett [1990] Plant Cell 2: 1239-1248), PG overexpression in transgenic apple led to a range of novel phenotypes. These phenotypes included silvery colored leaves and premature leaf shedding due to reduced cell adhesion in leaf abscission zones. Mature leaves had malformed and malfunctioning stomata that perturbed water relations and contributed to a brittle leaf phenotype. Chemical and ultrastructural analyses were used to relate the phenotypic changes to pectin changes in the leaf cell walls. The modification of apple trees by a single PG gene has offered a new and unexpected perspective on the role of pectin and cell wall adhesion in leaf morphology and stomatal development. (+info)
Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase, a key gene of hemicellulose/pectin formation.
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Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH. (+info)
Polypectate digestion by Yersinia.
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The ability of Yersinia to digest polypectate may be of some value in differentiating Y. enterocolitica and Y. pseudotuberculosis from some of the other fermenting gram-negative bacilli, such as Enterobacter agglomerans, with which they can be confused. Pectolytic activity in Yersinia may also have some teleologic or taxonomic significance about which we do not care to speculate. (+info)
A novel enzyme activity involving the demethylation of specific partially methylated oligogalacturonides.
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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. (+info)
The degree of methylation influences the degradation of pectin in the intestinal tract of rats and in vitro.
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We investigated the degradation, metabolism, fate, and selected effects of pectin in the intestinal tract of rats. Conventional and germfree rats were fed for 3 wk diets containing 6.5% pectin (degree of methylation 34.5, 70.8 and 92.6%, respectively) or pectin-free diets. Pectin passes the small intestine as a macromolecule. The molecular weight distribution of pectins isolated from intestinal contents of germfree rats were unaffected by diet. No or very little galacturonan was found in cecum, colon or feces of most of the conventional rats. In colon contents of some conventional rats, di- and trigalacturonic acid were present. Total anaerobic and Bacteroides counts were greater in groups fed pectin. The concentration of short-chain fatty acids (SCFA) was higher in cecum and feces in all pectin-fed groups. With increasing degree of methylation, the formation rate of SCFA decreased in the cecum of conventional rats. During in vitro fermentation of pectin with fecal flora from rats, unsaturated oligogalacturonic acids appeared as intermediate products. Low-methoxyl pectin was fermented faster than high-methoxyl pectins in vivo and in vitro. Pectin-fed rats had greater ileum, cecum and colon weights. We conclude that structural parameters of pectin influence its microbial degradation in the intestinal tract. (+info)
Pectin synthesis during the wall regeneration of plasmolysed tobacco leaf cells.
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1. Discs of tobacco leaf lamina were floated on media containing D-[U-14C]glucose. Glucose uptake was mainly through the cut edges, and diffusion through the veins and intracellular spaces was slow. 2. Radioactivity was detected in all polysaccharide fractions extracted from the discs, including those associated with the wall. 3. Plasmolysis in sorbitol or KCL decreased the incorporation of radioactive material into all fractions. 4. Incorporation of arabinose into pectins was increased or unaffected by plasmolysis, but the incorporations of other sugars were decreased. Removal of the lower epidermis did not affect this result. 5. Seperate mechanisms for arabinan and polygalacturonorhamman syntheses must exist, and these must differ in their responses to the physiochemical, structural and organizational changes that accompany plasmolysis. (+info)