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Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals.

ABSTRACT: Scission of plant cell wall polysaccharides in vivo has generally been assumed to be enzymic. However, in the presence of l-ascorbate, such polysaccharides are shown to undergo non-enzymic scission under physiologically relevant conditions. Scission of xyloglucan by 1 mM ascorbate had a pH optimum of 4.5, and the maximum scission rate was reached after a 10-25-min delay. Catalase prevented the scission, whereas added H2O2 (0.1-10 mM) increased the scission rate and shortened the delay. Ascorbate caused detectable xyloglucan scission above approx. 5 microM. Dehydroascorbate was much less effective. Added Cu2+ (>0.3 microM) also increased the rate of ascorbate-induced scission; EDTA was inhibitory. The rate of scission in the absence of added metals appeared to be attributable to the traces of Cu (2.8 mg.kg-1) present in the xyloglucan. Ascorbate-induced scission of xyloglucan was inhibited by radical scavengers; their effectiveness was proportional to their rate constants for reaction with hydroxyl radicals (.OH). It is proposed that ascorbate non-enzymically reduces O2 to H2O2, and Cu2+ to Cu+, and that H2O2 and Cu+ react to form .OH, which causes oxidative scission of polysaccharide chains. Evidence is reviewed to suggest that, in the wall of a living plant cell, Cu+ and H2O2 are formed by reactions involving ascorbate and its products, dehydroascorbate and oxalate. Systems may thus be in place to produce apoplastic .OH radicals in vivo. Although .OH radicals are often regarded as detrimental, they are so short-lived that they could act as site-specific oxidants targeted to play a useful role in loosening the cell wall, e.g. during cell expansion, fruit ripening and organ abscission.

SUBMITTER: Fry SC

PROVIDER: S-EPMC1219507 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

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Project description:Background and aimsMany fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals ((•)OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to 'fingerprint' (•)OH-attacked polysaccharides.MethodsWe tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during (•)OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography.Key resultsStrawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA-pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently.ConclusionsGalA-pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents ((•)OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by (•)OH. The evidence shows that (•)OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). (•)OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.

Project description:Symbiotic bacteria inhabiting the human gut have evolved under intense pressure to utilize complex carbohydrates, primarily plant cell wall glycans in our diets. These polysaccharides are not digested by human enzymes, but are processed to absorbable short chain fatty acids by gut bacteria. The Bacteroidetes, one of two dominant bacterial phyla in the adult gut, possess broad glycan-degrading abilities. These species use a series of membrane protein complexes, termed Sus-like systems, for catabolism of many complex carbohydrates. However, the role of these systems in degrading the chemically diverse repertoire of plant cell wall glycans remains unknown. Here we show that two closely related human gut Bacteroides, B. thetaiotaomicron and B. ovatus, are capable of utilizing nearly all of the major plant and host glycans, including rhamnogalacturonan II, a highly complex polymer thought to be recalcitrant to microbial degradation. Transcriptional profiling and gene inactivation experiments revealed the identity and specificity of the polysaccharide utilization loci (PULs) that encode individual Sus-like systems that target various plant polysaccharides. Comparative genomic analysis indicated that B. ovatus possesses several unique PULs that enable degradation of hemicellulosic polysaccharides, a phenotype absent from B. thetaiotaomicron. In contrast, the B. thetaiotaomicron genome has been shaped by increased numbers of PULs involved in metabolism of host mucin O-glycans, a phenotype that is undetectable in B. ovatus. Binding studies of the purified sensor domains of PUL-associated hybrid two-component systems in conjunction with transcriptional analyses demonstrate that complex oligosaccharides provide the regulatory cues that induce PUL activation and that each PUL is highly specific for a defined cell wall polymer. These results provide a view of how these species have diverged into different carbohydrate niches by evolving genes that target unique suites of available polysaccharides, a theme that likely applies to disparate bacteria from the gut and other habitats.

Project description:BackgroundClostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation.ResultsWe cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant.ConclusionIn this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

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Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals.

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