Non-cellulosic polysaccharide distribution during G-layer formation in poplar tension wood fibers: abundance of rhamnogalacturonan I and arabinogalactan proteins but no evidence of xyloglucan.

MAIN CONCLUSION: RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

Tomato GDSL1 is required for cutin deposition in the fruit cuticle.

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

Exploring architecture of xyloglucan cellulose nanocrystal complexes through enzyme susceptibility at different adsorption regimes.

Xyloglucan (XG) is believed to act as a cementing material that contributes to the cross-linking and mechanical properties of the cellulose framework in plant cell walls. XG can adsorb to the cellulose nanocrystal (CNC) surface in vitro in order to simulate this in vivo relationship. The target of our work was to investigate the sorption behavior of tamarind seed XG on CNC extracted from cotton linters at different XG/CNC concentration ratios, that is, different adsorption regimes regarding the XG-CNC complex organization and the enzymatic susceptibility of XG. First, we determined the adsorption isotherm. Second, XG-CNC complexes were enzymatically hydrolyzed using a xyloglucan-specific endoglucanase in order to quantify the different XG fractions involved in binding to CNC and to determine adsorption regimes, that is, presence of loops, tails, and trains. Finally, the architecture of the XG-CNC complex was investigated by transmission electron microscopy imaging of negatively stained XG-CNC suspensions and XG immunolabeled suspensions at different XG/CNC concentration ratios, both before and after xyloglucanase hydrolysis process. This study revealed that an increasing XG/CNC concentration ratio led to a change in the XG binding organization to CNC. At low XG/CNC concentration ratios, almost all XG chains were bound as trains to the CNC surface. In contrast, at increasing XG/CNC concentration ratios, the proportion of loops and tails increases. The organization change induces CNC aggregation to form a cellulose/XG network at low XG/CNC regimes, whereas CNC remains in the form of individual particles at higher XG/CNC regimes. Results are discussed both regarding the biological role of XG in plant cell walls and in the perspective of designing new biobased materials.

Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern.

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.

Brachypodium distachyon grain: characterization of endosperm cell walls.

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ∼52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-ß-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-ß-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.

GDP-D-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato.

The GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-d-mannose to GDP-l-galactose, is generally considered to be a central enzyme of the major ascorbate biosynthesis pathway in higher plants, but experimental evidence for its role in planta is lacking. Using transgenic tomato lines that were RNAi-silenced for GME, we confirmed that GME does indeed play a key role in the regulation of ascorbate biosynthesis in plants. In addition, the transgenic tomato lines exhibited growth defects affecting both cell division and cell expansion. A further remarkable feature of the transgenic plants was their fragility and loss of fruit firmness. Analysis of the cell-wall composition of leaves and developing fruit revealed that the cell-wall monosaccharide content was altered in the transgenic lines, especially those directly linked to GME activity, such as mannose and galactose. In agreement with this, immunocytochemical analyses showed an increase of mannan labelling in stem and fruit walls and of rhamnogalacturonan labelling in the stem alone. The results of MALDI-TOF fingerprinting of mannanase cleavage products of the cell wall suggested synthesis of specific mannan structures with modified degrees of substitution by acetate in the transgenic lines. When considered together, these findings indicate an intimate linkage between ascorbate and non-cellulosic cell-wall polysaccharide biosynthesis in plants, a fact that helps to explain the common factors in seemingly unrelated traits such as fruit firmness and ascorbate content.

Improved colloidal stability of bacterial cellulose nanocrystal suspensions for the elaboration of spin-coated cellulose-based model surfaces.

Well-dispersed suspensions are a prerequisite when preparing smooth model surfaces based on neutral bacterial cellulose nanocrystals (BCNs). However, neutral nanocrystal suspensions present pronounced particle aggregation. Carboxymethyl cellulose (CMC) or xyloglucan (XG) was therefore added to aggregated BCN suspensions. Turbidity measurements, polysaccharide content, and transmission electron microscopy (TEM) analysis revealed that aggregation of BCNs in CMC/BCN and XG/BCN suspensions is dependent on the concentration of CMC and XG in the suspensions. CMC enhances BCN dispersion above the concentration ratio of 0.05. In the case of XG, a better colloidal stability is observed above the concentration ratio of 0.5. Atomic force microscopy (AFM) investigations demonstrated that cellulose-based model surfaces, spin-coated from CMC/BCN or XG/BCN solutions, exhibited more uniform topography and less roughness than the reference BCN model surface.

Fast data preprocessing for chromatographic fingerprints of tomato cell wall polysaccharides using chemometric methods.

The variability in the chemistry of cell wall polysaccharides in pericarp tissue of red-ripe tomato fruit (Solanum lycopersicon Mill.) was characterized by chemical methods and enzymatic degradations coupled to high performance anion exchange chromatography (HPAEC) and mass spectrometry analysis. Large fruited line, Levovil (LEV) carrying introgressed chromosome fragments from a cherry tomato line Cervil (CER) on chromosomes 4 (LC4), 9 (LC9), or on chromosomes 1, 2, 4 and 9 (LCX) and containing quantitative trait loci (QTLs) for texture traits, was studied. In order to differentiate cell wall polysaccharide modifications in the tomato fruit collection by multivariate analysis, chromatograms were corrected for baseline drift and shift of the component elution time using an approach derived from image analysis and mathematical morphology. The baseline was first corrected by using a "moving window" approach while the peak-matching method developed was based upon location of peaks as local maxima within a window of a definite size. The fast chromatographic data preprocessing proposed was a prerequisite for the different chemometric treatments, such as variance and principal component analysis applied herein to the analysis. Applied to the tomato collection, the combined enzymatic degradations and HPAEC analyses revealed that the firm LCX and CER genotypes showed a higher proportion of glucuronoxylans and pectic arabinan side chains while the mealy LC9 genotype demonstrated the highest content of pectic galactan side chains. QTLs on tomato chromosomes 1, 2, 4 and 9 contain important genes controlling glucuronoxylan and pectic neutral side chains biosynthesis and/or metabolism.

Remodeling of pectin and hemicelluloses in tomato pericarp during fruit growth.

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.

Structural characterization of underivatized arabino-xylo-oligosaccharides by negative-ion electrospray mass spectrometry.

Various arabino-xylo-oligosaccharides with known substitution patterns were assessed by negative ESI-Q-TOFMS and ESI-ITMS. The CID spectra of linear xylo-oligosaccharides and of nine isomeric mono- and disubstituted arabino-xylo-oligosaccharides established that structures differing in their substitution pattern can be differentiated by this approach. The negative-ion fragmentation spectra of the deprotonated quasi-molecular ions are mainly characterized by glycosidic cleavage ions from the C-series, which provide sequence informations, and by cross-ring cleavage (0,2)A(i) ions, which provide partial linkage information. When the collision energy increased, the cross-ring cleavage (0,2)A(i) ions underwent consecutive loss of water to produce (0,2)A(i)-18 fragment ions and glycosidic cleavage ions of the B-series are also produced besides the C(i) ions. Contrary to linear xylo-oligosaccharides, C(i) ions, which originate from C-3 monosubstituted xylosyl residues never produce the related cross-ring cleavage (0,2)A(i) ions. Disubstitution at O-2 and O-3 of xylosyl residues appears to enhance the production of the (0,2)A(i) ions compared to monosubstitution. For the differentiation of the mono- and disubstitution patterns of the penultimate xylosyl residue, the relative abundance of the glycosidic cleavage ions at m/z 263 and 299 found on Q-TOF CID spectra plays a relevant role and appears to be more informative than MS(n) spectra obtained on a ion trap instrument.

Induced mutations in tomato SlExp1 alter cell wall metabolism and delay fruit softening.

Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin 1 gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in SlExp1 function. Characterization of two induced mutations, Slexp1-6_W211S, and Slexp1-7_Q213Stop, showed that SlExp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-ß-1,4-d-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1-7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

Sugar beet (Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall.

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after (18)O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (beta-1-->4-linked-dimer), Ara (alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.

Mapping sugar beet pectin acetylation pattern.

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

Negative electrospray ionization mass spectrometry: a method for sequencing and determining linkage position in oligosaccharides from branched hemicelluloses.

Xyloglucans of apple, tomato, bilberry and tamarind were hydrolyzed by commercial endo ß-1-4-D-endoglucanase. The xylo-gluco-oligosaccharides (XylGos) released were separated on CarboPac PA 200 column in less than 15 min, and, after purification, they were structurally characterized by negative electrospray ionization mass spectrometry using a quadrupole time-of-flight (ESI-Q-TOF), a hybrid linear ion trap (LTQ)/Orbitrap and a hybrid quadrupole Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. In order to corroborate the fragmentation routes observed on XylGos, some commercial galacto-manno-oligosaccharides (GalMOs) and glucurono-xylo-oligosaccharides were also studied. The fragmentation pathways of the ionized GalMos were similar to those of XylGos ones. The product ion spectra were mainly characterized by prominent double cleavage (D) ions corresponding to the entire inner side chains. The directed fragmentation from the reducing end to the other end was observed for the main glycosylated backbone but also for the side-chains, allowing their complete sequencing. Relevant cross-ring cleavage ions from (0,2)X(j)-type revealed to be diagnostic of the 1-2-linked- glycosyl units from XylGos together with the 1-2-linked glucuronic acid unit from glucuronoxylans. Resonant activation in the LTQ Orbitrap allowed not only determining the type of all linkages but also the O-acetyl group location on fucosylated side-chains. Moreover, the fragmentation of the different side chains using the MS(n) capabilities of the LTQ/Orbitrap analyzer also allowed differentiating terminal arabinosyl and xylosyl substituents inside S and U side-chains of XylGos, respectively. The CID spectra obtained were very informative for distinction of isomeric structures differing only in their substitution pattern. These features together makes the fragmentation in negative ionization mode a relevant and powerful technique useful to highlight the subtle structural changes generally observed during the development of plant organs such as during fruit ripening and for the screening of cell wall mutants with altered hemicellulose structure.

Evidence for linkage position determination in known feruloylated mono- and disaccharides using electrospray ion trap mass spectrometry.

Various feruloylated arabinose- and galactose-containing mono- and disaccharides with known linkage configurations (2-O-(trans-feruloyl)-L-arabinopyranose, 5-O-(trans-feruloyl)-L-arabinofuranose, O-[2-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->5)-L-arabinofuranose, and O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose) were analyzed by electrospray ionization mass spectrometry using an ion trap or a quadrupole time-of-flight (Q-TOF) mass analyzer. Collision-induced dissociation (CID) experiments using the two mass analyzers generated similar tandem mass spectrometric (MS/MS) fragmentation patterns. However, the ester-bond cleavage ions were more abundant using the Q-TOF mass analyzer. Compared with the positive ion mode, the negative ion mode produces simpler and more useful CID product-ion patterns. For arabinose-containing feruloylated compounds, results obtained with both analyzers show that it is possible to assign the location of the feruloyl group to the O-2 or O-5 of arabinosyl residues. In the characterization of the 2-O-feruloyl and 5-O-feruloyl linkages, the relative abundance of the cross-ring fragment ions at m/z 265 (-60 u or -62 u after 18O-labelling) and at m/z 217 (-108 u or -110 u after 18O-labelling) play a relevant role. For galactose-containing feruloylated compounds, losses of 60, 90 and 120 Da observed in MS3 experiment correspond to the production of 0,2A1, 0,3A1 and (0,2A1-60 Da) cross-ring cleavage ions, respectively, fixing the location of feruloyl group at the O-6 of the galactose residue.

Assignment of acetyl groups to O-2 and/or O-3 of pectic oligogalacturonides using negative electrospray ionization ion trap mass spectrometry.

Partially acetylated and methylated oligogalacturonides produced by enzymatic hydrolysis of sugar beet pectin were analysed by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS). The (18)O labelling of the oligomer reducing end allowed the precise assignment of the fragments resulting from glycosidic bond and cross-ring cleavages. The collisional-induced dissociation of the C(i) and Z(j) fragment ions through sequential MS(n) experiments always displayed (0, 2)A-type cross-ring cleavage ions which were related to C(2)H(4)O(2) losses. These (0, 2)A ions appeared to be highly diagnostic ions allowing the precise location of the acetyl groups to the O-2 and/or O-3 of the acetylated galacturonic acid residues.

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography.

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.

Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls.

After degradation of sugar beet cell walls with Driselase and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1-->5)-linked arabinose units esterified by a central 8-O-4' ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.

Xylanase, beta-glucanase, and other side enzymatic activities have greater effects on the viscosity of several feedstuffs than xylanase and beta-glucanase used alone or in combination.

This study was carried out to evaluate the effects of a pure xylanase, a pure beta-glucanase, a mix of the two pure enzymes, and a commercial enzyme preparation (Quatrazyme HP, Nutri-Tomen Les Ulis, France) on the viscosity exhibited by water-soluble nonstarch polysaccharides of several feedstuffs (Rialto wheat, Sidéral wheat, Isengrain wheat, triticale, rye, barley, oats, corn, wheat bran, rice bran, wheat screenings, soybean meal, rapeseed meal, sunflower meal, and peas). The viscosity depended on the feedstuffs and varieties of the same feedstuff. There was a correlation (R (2) = 0.86) between viscosity of cereals and their arabinoxylan and beta-glucan contents. The correlation was greater (R (2) = 0.99) when the type of cereal was taken into account. The addition of pure xylanase significantly decreased the viscosity of all feedstuffs except sunflower meal (P < or = 0.05). However, pure beta-glucanase was unable significantly to decrease the viscosity of Isengrain wheat, corn, rice bran, wheat screenings, soybean meal, and sunflower meal. There was a greater decrease in viscosity with the combination of xylanase and beta-glucanase than with addition of xylanase or beta-glucanase alone. This synergistic action of xylanase and beta-glucanase was observed only in Rialto wheat, Sidéral wheat, triticale, rye, barley, oats, and peas. Finally, the commercial enzyme preparation produced a greater reduction (P < or = 0.05) in viscosity for all feedstuffs compared to xylanase or beta-glucanase used alone or in combination. The greater effectiveness of the commercial enzyme preparation was due to the presence of side enzymatic activities (arabinofuranosidase, xylosidase, glucosidase, galactosidase, cellulase, and polygalacturonase).

Structural characterisation by both positive- and negative-ion electrospray mass spectrometry of partially methyl-esterified oligogalacturonides purified by semi-preparative high-performance anion-exchange chromatography.

The off-line coupling of high-performance anion-exchange chromatography (HPAEC) to electrospray ionisation/ion trap mass spectrometry (ESI-ITMS) is described. The Dionex carbohydrate membrane desalter (CMD) has been assessed as an on-line chromatographic desalting system to remove the high sodium concentration necessary for the HPAEC separation of partially methyl-esterified oligogalacturonides. The developed HPAEC configuration proved to be suitable for indirect coupling with ESI-ITMS. This paper provides some interesting features of positive- and negative-ion multistage tandem mass spectrometry (MS(n)) analysis of these acidic oligosaccharides. The spectra acquired in both negative- and positive-ion modes show characteristic fragment ions resulting from glycosidic bond and cross-ring cleavages. Some new mass spectrometric fragmentation routes are also described. The positive-ion mode gave more complex spectra but was as informative as the negative-ion mode. ESI-ITMS was revealed to be, as previously reported from direct use on an unseparated enzymatic digest, a powerful sequencing technique for the determination of linkage type and the methyl ester distribution of partially methyl-esterified oligogalacturonides. Moreover, unlike matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS), it gives valuable information on the elution behaviour of these oligomers in relation to their structure, namely the HPAEC co-elution of isomeric structures.