Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif

Hemicellulose polymers were isolated from Argania spinosa leaf cell walls by sequential extractions with alkali. The structure of the two main polymers, xylan and xyloglucan, was investigated by enzyme degradation with specific endoglycosidases followed by analysis of the resulting fragments by high performance anion exchange chromatography (HPAEC) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). The results show that A. spinosa xylan is composed of a beta-(1-->4)-linked-D-xylopyranose backbone substituted with 4-O-methyl-D-glucuronic acid residues. Xyloglucan oligosaccharide subunits were generated by treatment with an endo-(1-->4)-beta-D-glucanase of the xyloglucan-rich hemicellulosic fractions. MALDI-TOF mass spectra and HPAE-PAD chromatography of the pool of endoglucanase-generated xyloglucan oligomers indicated that A. spinosa cell wall contains a XXXG-type xyloglucan. In addition to XXXG, XXFG, XLXG/XXLG, XLFG fragments previously characterised in various plants, a second group of XXXG-type fragments was detected. The primary structure of the major subunit was determined by a combination of sugar analysis, methylation analysis, post-source decay (PSD) fragment analysis of MALDI-TOF MS and 1H NMR spectroscopy. This fragment, termed XUFG, contains a novel beta-D-Xylp-(1-->2)-alpha-D-Xylp side chain linked to C-6 of the second glucose unit from the nonreducing end of the cellotetraose sequence.     

Isolation and characterization of xylans from seed pericarp of Argania spinosa fruit

Hemicellulose-type polysaccharides were isolated from the pericarp of seeds of Argania spinosa (L.) Skeels fruit by sequential alkaline extractions and fractionated by precipitation. Water soluble and water insoluble fractions were obtained, purified and characterized by sugar analysis and 1H and 13C NMR spectroscopy. The water soluble fractions were assumed to be (4-O-methyl-D-glucurono)-D-xylans, with 4-O-methyl-D-glucopyranosyluronic acid groups linked to C-2 of a (1-->4)-beta-D-xylan. The 1H NMR spectrum showed that the water soluble xylans have, on average, one non-reducing terminal residue of 4-O-methyl-D-glucuronic acid for every seven xylose units. The water insoluble fractions consisted of a neutral xylan with linear (1-->4)-beta-D-xylopyranosyl units.     

Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum

Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-β-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall.     

Cell wall polysaccharides from Talaromyces species

The neutral sugars and amino sugars, released by acid hydrolysis of walls and polysaccharidic fractions, of six species of Talaromyces and the infrared spectra have been used to study their interspecific relationships. In whole cell walls neutral sugars ranged from 23 to 39.6% dry weight and were identified as glucose, galactose and mannose. Glucosamine varied from 8 to 19.8% in the samples. Galactosamine (2% or less) was found in T. emersonii and T. rotundus and no galactosamine in the other species. Sequential fractionation of the cell walls with alkali and acid gave several polysaccharidic fractions. The main differences among species were found in the alkali-soluble fraction at 20° (F1). This fraction represented 8 to 33.2% of the whole cell wall and was characterized as an -glucan in T. bacillisporus, T. emersonii, T. luteus and T. rotundus (Group A) and as a -galactofuranosyl containing glucan in T. ohiensis and T. stipitatus (Group B). The alkali-insoluble residue (F4) represented the bulk of the cell wall in all species tested (33.2% to 57.3%) and was characterized as a -glucan/chitin complex. The results may indicate degrees of interspecific relationship in the genus Talaromyces.Abbreviations CWM cell wall material - GLC gas-liquid chromatography - IR infrared - wt weight - CBS Centraal Bureau voor Schimmelcultures (Baarn. The Netherlands) - Ara arabinose - Xyl xylose - Man mannose - Gal galactose - Glc glucose - GlcNH₂ glucosamine - GalNH₂ galactosamine     

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.     

Functional characterisation of Nicotiana tabacum xyloglucan endotransglycosylase (NtXET-1): generation of transgenic tobacco plants and changes in cell wall xyloglucan

To study the function of xyloglucan endotransglycosylase (XET) in vivo we isolated, a tomato (Lycopersicon esculentum Mill.) XET cDNA (GenBank AA824986) from the homologous tobacco (Nicotiana tabacum L.) clone named NtXET-1 (Accession no. D86730). The expression pattern revealed highest levels of NtXET-1 mRNA in organs highly enriched in vascular tissue. The levels of NtXET-1 mRNA decreased in midribs with increasing age of leaves. Increasing leaf age was correlated with an increase in the average molecular weight (MW) of xyloglucan (XG) and a decrease in the relative growth rates of leaves. Transgenic tobacco plants with reduced levels of XET activity were created to further study the biochemical consequences of reduced levels of NtXET-1 expression. In two independent lines, total XET activity could be reduced by 56% and 37%, respectively, in midribs of tobacco plants transformed with an antisense construct. The decreased activity led to an increase in the average MW of XG by at least 20%. These two lines of evidence argue for NtXET-1 being involved in the incorporation of small XG molecules into the cell wall by transglycosylation. Reducing the incorporation of small XG molecules will result in a shift towards a higher average MW. The observed reduction in NtXET-1 expression and increase in the MW of XG in older leaves might be associated with strengthening of cell walls by reduced turnover and hydrolysis of XG. Received: 24 January 2000 / Accepted: 21 July 2000     

Phytochemical analysis and antibacterial activity towards methicillin-resistant Staphylococcus aureus of leaf extracts from Argania spinosa (L.) Skeels

Argania spinosa (L.) Skeels is an endemic Moroccan species belonging to Sapotaceae family. In this work, lipophilic and aqueous extracts were obtained from leaves and subjected to a chemical profiling by MS and LC-MS/MS. Pentacyclic terpenoids were identified and quantified in the lipophilic fraction, while phenolic compounds (mainly belonging to flavonols and flavan-3-ols) were identified in the aqueous fraction. The antibacterial activities of fractions were evaluated in vitro against both reference Gram-positive and -negative bacterial strains and clinical isolates of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA); in addition, the compounds quantified as main components in each extract were assayed against reference strains. A relevant antibacterial activity was observed against reference MSSA and MRSA strains of S. aureus: for the lipophilic fraction, MIC₅₀ values obtained were 177.8 and 170.6 μg/mL for the former and the latter, respectively, while for the aqueous fraction were 215.5 and 233.3 μg/mL. These inhibitory activities could be mainly ascribed to ursolic and oleanolic acids, among pentacyclic terpenoids, and to quercetin concerning phenolic compounds. A remarkable antibacterial activity was also observed against clinical isolates, thus argan leaves can be considered of interest in the chemotherapy of human infections.     

Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.     

Structure of the xyloglucan produced by suspension-cultured tomato cells

The xyloglucan secreted by suspension-cultured tomato (Lycopersicon esculentum) cells was structurally characterized by analysis of the oligosaccharides generated by treating the polysaccharide with a xyloglucan-specific endoglucanase (XEG). These oligosaccharide subunits were chemically reduced to form the corresponding oligoglycosyl alditols, which were isolated by high-performance liquid chromatography (HPLC). Thirteen of the oligoglycosyl alditols were structurally characterized by a combination of matrix-assisted laser-desorption ionization mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Nine of the oligoglycosyl alditols (GXGGol, XXGGol, GSGGol, XSGGol, LXGGol, XTGGol, LSGGol, LLGGol, and LTGGol, [see, Fry, S.C.; York, W.S., et al., Physiologia Plantarum 1993, 89, 1-3, for this nomenclature]) are derived from oligosaccharide subunits that have a cellotetraose backbone. Very small amounts of oligoglycosyl alditols (XGGol, XGGXXGGol, XXGGXGGol, and XGGXSGGol) derived from oligosaccharide subunits that have a cellotriose or celloheptaose backbone were also purified and characterized. The results demonstrate that the xyloglucan secreted by suspension-cultured tomato cells is very complex and is composed predominantly of 'XXGG-type' subunits with a cellotetraose backbone. The rigorous characterization of the oligoglycosyl alditols and assignment of their 1H and 13C NMR spectra constitute a robust data set that can be used as the basis for rapid and accurate structural profiling of xyloglucans produced by Solanaceous plant species and the characterization of enzymes involved in the synthesis, modification, and breakdown of these polysaccharides.     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls

Cell wall material (CWM) was prepared from nine fruit species at two ripening stages (unripe and ripe) and extracted sequentially with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃ and 4 M KOH. Each solubilised fraction and the CWM-residue remaining after 4 M KOH extraction was analysed for non-cellulosic sugar composition. A common pattern of distribution for polyuronide and pectin-associated neutral sugar was observed for all unripe fruit. Most polyuronide was extracted in the CDTA/Na₂CO₃ fractions while 70–93% of the neutral sugar was located on pectic polysaccharides in the 4 M KOH-soluble and CWM-residue fractions. During ripening, most of the galactose was lost from pectic polysaccharides in the CWM-residue. Partial solubilisation of these polysaccharides was achieved by treating the CWM-residue with endopolygalacturonase. The solubilised polysaccharides were separated into two fractions by ion-exchange chromatography. One of these contained polysaccharides with average molecular weights of 400 kDa or larger and consisted of between 70 and 90% arabinogalactan. The galactosyl residues were 80–90% β-1→4 linked, indicating largely unbranched side-chains. The arabinosyl residues were distributed among terminal, 3-, 5-, 2,5-, and 2,3,5-linked residues, indicating a highly ramified structure. The results are discussed with regard to the relationship between pectin solubilisation and galactose loss and their respective contribution to fruit softening. Received: 28 January 1997 / Accepted: 11 March 1997     

Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides

A high-throughput method is described by which Arabidopsis thaliana stems can be screened for variation in cell wall composition after hydrolysis with Driselase or trifluoroacetic acid (TFA). Driselase, a mixture of fungal enzymes, hydrolyses cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides); TFA hydrolyses the matrix polysaccharides, but not cellulose, to monosaccharides. Two different wild-type ecotypes, Columbia and Wassilewskija, showed only minor differences in wall carbohydrate composition. A small number of T-DNA-tagged populations that were screened contained individuals in which the proportion of cellulose, xyloglucan or xylan differed quantitatively from the wild-type. Differences from the wild-type were also observed in the susceptibility of the hemicelluloses to hydrolysis by Driselase, probably reflecting differences in wall architecture.     

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.     

A novel xyloglucan from seeds of Afzelia africana Se. Pers.--extraction, characterization, and conformational properties

This paper is the first multi-scale characterization of the xyloglucan extracted from seeds of the African tree Afzelia africana Se. Pers. It describes the extraction and characterization of this polysaccharide in terms of both primary monosaccharide and oligosaccharide composition. It also includes a study of the seed morphology. Morphological characterization includes optical, transmission, and scanning electron microscopy. The polysaccharide exists in thickened cell walls of the cotyledonary cells, and the extracted xyloglucan is structurally quite similar to those from tamarind seed and detarium. Nevertheless there are some subtle differences in the fine structure, particularly in the oligomeric xyloglucan composition. The chain flexibility of the polysaccharide is also discussed in the light of our recent measurements reported elsewhere [Biomacromolecules2004, 5, 2384-2391].     

Immunogold localization of the cell-wall-matrix polysaccharides rhamnogalacturonan I and xyloglucan during cell expansion and cytokinesis inTrifolium pratense L.; implication for secretory pathways

We have localized two cell-wall-matrix polysaccharides, the main pectic polysaccharide, rhamnogalacturonan I (RG-I), and the hemicellulose, xyloglucan (XG), in root-tip and leaf tissues of red clover (Trifolium pratense L.) using immunoelectron microscopy. Our micrographs show that in both leaf and root tissues RG-I is restricted to the middle lamella, with 80–90% of the label associated with the expanded regions of the middle lamella at the corner junctions between cells. Xyloglucan, however, is nearly exclusively located in the cellulose-microfibril-containing region of the cell wall. Thus, these cell-wall-matrix polysaccharides are present in distinct and complementary regions of the cell wall. Our results further show that during cell expansion both RG-I and XG are present within Golgi cisternae and vesicles, thus confirming that the Golgi apparatus is the main site of synthesis of the non-cellulosic cell-wall polysaccharides. No label is seen over the endoplasmic reticulum, indicating that synthesis of these complex polysaccharides is restricted to the Golgi. The distribution of RG-I and XG in root-tip cells undergoing cell division was also examined, and it was found that while XG is present in the Golgi stacks and cell plate during cytokinesis, RG-I is virtually absent from the forming cell plate.Abbreviations ER endoplasmic reticulum - RG-I rhamnogalacturonan I - XG xyloglucan     

Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus

UV-C irradiation (254 nm) was found to enhance the secretion of some cell-wall-degrading enzymes, especially the following carbohydrases: beta-galactosidase, alpha-L-arabinofuranosidase, polygalacturonase, pectinesterase, cellulase, xylanase, and beta-xylosidase, in the campion callus, contributing thereby to an alteration in the polysaccharide structure. The relative amounts of the galactose and arabinose residues in pectin (silenan) and of arabinose in arabinogalactan of calli irradiated during the exponential phase were shown to decrease during the stationary phase. A decrease in the degree of SV methylesterification was found for the irradiated callus. These alterations were found to persist over a long period of culturing time. Decreasing the relative amounts of the arabinose residues in arabinogalactan and pectin and the galactose residues in silenan corresponded to increasing activity of alpha-L-arabinofuranosidase and beta-galactosidase, respectively, due to treatment with UV-C. UV-C irradiation may be used as a tool for modifying the structural features of the cell-wall polysaccharides, such as the relative amounts of galactose and arabinose residues in the side chains of polysaccharides, with the purpose of obtaining physiologically active polysaccharides with the desired properties and structural features.     

NMR characterization of endogenously O-acetylated oligosaccharides isolated from tomato (Lycopersicon esculentum) xyloglucan

Eight oligosaccharide subunits, generated by endoglucanase treatment of the plant polysaccharide xyloglucan isolated from the culture filtrate of suspension-cultured tomato (Lycopersicon esculentum) cells, were structurally characterized by NMR spectroscopy. These oligosaccharides, which contain up to three endogenous O-acetyl substituents, consist of a cellotetraose core with alpha-D-Xylp residues at O-6 of the two beta-D-Glcp residues at the non-reducing end of the core. Some of the alpha-D-Xylp residues themselves bear either an alpha-L-Arap or a beta-D-Galp residue at O-2. O-Acetyl substituents are located at O-6 of the unbranched (internal) beta-D-Glcp residue, O-6 of the terminal beta-D-Galp residue, and/or at O-5 of the terminal alpha-L-Arap residue. Structural assignments were facilitated by long-range scalar coupling interactions observed in the high-resolution gCOSY spectra of the oligosaccharides. The presence of five-bond scalar coupling constants in the gCOSY spectra provides a direct method of assigning O-acetylation sites, which may prove generally useful in the analysis of O-acylated glycans. Spectral assignment of these endogenously O-acetylated oligosaccharides makes it possible to deduce correlations between their structural features and the chemical shifts of diagnostic resonances in their NMR spectra.