M.M.H. Huisman

A xylogalacturonan epitope is specifically associated with plant cell detachment

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.

The occurrence of internal (1→5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.

Glucuronoarabinoxylans from maize kernel cell walls are more complex than those from sorghum kernel cell walls.

Abstract Water-unextractable solids (WUS) were isolated from maize kernels. They contained 7% of protein, 8% of starch and 57% of non-starch polysaccharides (NSP). These NSP were composed mainly of glucose, xylose, arabinose, and glucuronic acid. Sequential extractions with a saturated Ba(OH) 2 -solution (BE1 extract), and distilled water (BE2 extract) were used to solubilise glucuronoarabinoxylans from maize WUS. Cellulose remained in the insoluble residue. The glycosidic linkage composition of the extracts and their resistance to endo-xylanase treatment indicated that the extracted glucuronoarabinoxylans were highly substituted. In the maize BE1 extract 25% of the xylose was unsubstituted, 38% was monosubstituted and 15% was disubstituted. A new measure for the degree of substitution is defined. The resulting degree of substitution for maize BE1 arabinoxylan (87%) is higher than for sorghum BE1 arabinoxylan (70%). The glucuronoarabinoxylans in maize BE1 can be degraded by a sub-fraction of Ultraflo, a commercial enzyme preparation from Humicola insolens . The digest contains a number of series of oligomers: pentose n , pentose n GlcA, pentose n hexose, and pentose n GlcA 2 .

Enzymatic degradation of cell wall polysaccharides from soybean meal

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.

Changes in cell wall polysaccharides from ripening olive fruits

Abstract Cell wall material was isolated as alcohol insoluble solids (AIS) from olives at three stages of ripeness. AIS contained 38.8 to 45.6% carbohydrates and 21.5 to 24.8% proteins. Glucose, xylose, arabinose and uronic acids were the major constituent sugars. AIS was sequentially extracted with hot buffer (HBSS), chelating agent (ChSS), dilute alkali(DASS), 1 m alkali (1 m ASS), 4 m alkali (4 m ASS), 4 m alkali + borate (A/BO33-), 6 m alkali (6 m ASS) and water (WSS) to leave a cellulose-rich residue (RES). These extracts were characterised by their sugar composition and their molecular weight distribution. The yields, sugar composition, degree of branching and molecular weight distribution of the pectin-rich extracts (HBSS, ChSS, DASS and 6 m ASS) changed during ripening, whereas no significant changes were detected in the other extracts. The degree of branching of the pectins increased with increasing strength of the extractant; the ratio ara:uronic acids increased from 0.46 to 2.59. These extracts were further characterised by their degradability with polygalacturonase (PG) and rhamnogalacturonase (RGase) after chemical saponification. The digests were analysed by high-performance size-exclusion chromatography (HPSEC) and high-performance anion-exchange chromatography (HPAEC). The amount of oligomers released by PG from pectic material in olive fruits of corresponding extracts from various stages of ripeness decreased with increasing ripening stage. Oligomers released by RGase were formed in similar amounts from corresponding extracts from various stages of ripeness as well as from each extract. HBSS and 6 m ASS were fractionated by anion exchange chromatography. Five pools were obtained for each fractionated extract. HBSS was rich in strongly bound, uronic acid-rich polysaccharides, whereas 6 m ASS was rich in unbound, neutral sugar-rich polysaccharides.

Xyloglucan from soybean (Glycine max) meal is composed of XXXG-type building units

Soybean cell wall material was depectinated by extraction with a hot chelating agent and a cold dilute alkali. The hemicelluloses were solubilised from the residue with 1 and 4 M KOH solutions, resulting in 1 M Alkali Soluble Solids (1 MASS) and 4 M Alkali Soluble Solids (4 MASS) fractions. The polysaccharides extracted with 1 M KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucans only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V using both HPAEC and MALDI-TOF MS showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG).

Studies on the structure of a lithium-treated soybean pectin: characteristics of the fragments and determination of the carbohydrate substituents of galacturonic acid

Two galacturonic-acid-containing polysaccharide fractions (ChSS and P) were isolated from soybean meal and subjected to lithium treatment. The fragments obtained were analyzed by using monosaccharide and methylation analyses, and NMR spectroscopy. Lithium degradation of ChSS, followed by sodium borodeuteride reduction, hydrolysis, sodium borohydride reduction, and acetylation afforded alditol acetates, of which the labeled ones reflected residues linked to GalA. As followed from quantifications of the labeled and non-labeled alditols from each constituent monosaccharide by GLC-EIMS, 6 mol% of Ara, 22 mol% of Fuc, 13 mol% of Gal, 53 mol% of Rha, and 57 mol% of Xyl are glycosidically linked to GalA. Analysis of the lithium-treated polymer revealed that it contains arabinogalactan side chains linked to Rha O-4, which consist of a beta-(1 --> 4)-linked galactan substituted with highly branched arabinan chains. On average, an arabinogalactan chain contains up to 29 Gal and 25 Ara residues. Surface plasmon resonance was used to determine conditions for affinity chromatography. Furthermore, this technique confirmed the presence of terminal alpha-Fuc residues in ChSS. Polysaccharide P turned out to be relatively resistant to lithium degradation.

DIFFERENCES IN THE METHYL ESTER DISTRIBUTION OF PECTINS

Both commercial pectin preparations as well as pectins as present in plant cell walls are very complex and heterogeneous molecules. Although the relative amount of the rhamnogalacturonan structural element may very significant for pectins from different origine, most pectins have in common that homogalacturonans forms the major part of the molecule. These homogalacturonan segments partly determine the functional properties of the pectin like its thickening and gelling properties. Especially the level of methyl esters and their distribution over the galacturonan backbone is of major importance. Many effort has been directed to the reveal the pattern of methyl ester distribution.

Pectic Substances from Soybean Cell Walls Distinguish Themselves from other Plant Cell Walls Pectins

In soybean cell wall material, pectic substances are the major non-starch polysaccharide. These pectic substances distinguish themselves from pectic substances of cell wall material from other plants in the absence of homogalacturonan, the presence of fucose residues in the xylogalacturonan, and two uncommon structural features of the pectic arabinogalactan side chains, namely the presence of internal (1,5)-linked arabinofuranose and terminal arabinopyranose. Therefore, these pectic substances are rather resistant to degradation by both established (like polygalacturonase) and novel (like RG-hydrolase) pectic enzymes. The hemicellulosic polysaccharides in the soybean cell wall appeared to be predominantly xyloglucans, composed of XXXG-type building units like most legume xyloglucans.