Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls.

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth.

Turgor-driven plant cell growth depends on wall structure. Two allelic l-fucose-deficient Arabidopsis thaliana mutants (mur1-1 and 1-2) are dwarfed and their rosette leaves do not grow normally. mur1 leaf cell walls contain normal amounts of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II), but only half exists as a borate cross-linked dimer. The altered structure of mur1 RG-II reduces the rate of formation and stability of this cross-link. Exogenous aqueous borate rescues the defect. The reduced cross-linking of RG-II in dwarf mur1 plants indicates that plant growth depends on wall pectic polysaccharide organization.

Detection of expansin proteins and activity during tomato fruit ontogeny.

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.

Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain.

Monomeric rhamnogalacturonan II (mRG-II) was isolated from red wine and the reducing-end galacturonic acid of the backbone converted to L-galactonic acid by treatment with NaBH4. The resulting product (mRG-II'ol) was treated with a cell-free extract from Penicillium daleae, a fungus that has been shown to produce RG-II-fragmenting glycanases. The enzymatically generated products were fractionated by size-exclusion and anion-exchange chromatographies and the quantitatively major oligosaccharide fraction isolated. This fraction contained structurally related oligosaccharides that differed only in the presence or absence of a single Kdo residue. The Kdo residue was removed by acid hydrolysis and the resulting oligosaccharide then characterized by 1- and 2D 1H NMR spectroscopy, ESMS, and by glycosyl-residue and glycosyl-linkage composition analyses. The results of these analyses provide evidence for the presence of at least two structurally related oligosaccharides in the ratio approximately 6:1. The backbone of these oligosaccharides is composed of five (1-->4)-linked alpha-D-GalpA residues and a (1-->3)-linked L-galactonate. The (1-->4)-linked GalpA residue adjacent to the terminal non-reducing GalpA residue of the backbone is substituted at O-2 with an apiosyl-containing side chain. Beta3-L-Araf-(1-->5)-beta-D-DhapA is likely to be linked to O-3 of the GalpA residue at the non-reducing end of the backbone in the quantitatively major oligosaccharide and to O-3 of a (1-->4)-linked GalpA residue in the backbone of the minor oligosaccharide. Furthermore, the results of our studies have shown that the enzymically generated aceryl acid-containing oligosaccharide contains an alpha-linked aceryl acid residue and a beta-linked galactosyl residue. Thus, the anomeric linkages of these residues in RG-II should be revised.

Fungal polygalacturonases exhibit different substrate degradation patterns and differ in their susceptibilities to polygalacturonase-inhibiting proteins.

Polygalacturonic acid (PGA) was hydrolyzed by polygalacturonases (PGs) purified from six fungi. The oligogalacturonide products were analyzed by HPAEC-PAD (high performance anion exchange chromatography-pulsed amperimetric detection) to assess their relative amounts and degrees of polymerization. The abilities of the fungal PGs to reduce the viscosity of a solution of PGA were also determined. The potential abilities of four polygalacturonase-inhibiting proteins (PGIPs) from three plant species to inhibit or to modify the hydrolytic activity of the fungal PGs were determined by colorimetric and HPAEC-PAD analyses, respectively. Normalized activities of the different PGs acting upon the same substrate resulted in one of two distinct oligogalacturonide profiles. Viscometric analysis of the effect of PGs on the same substrate also supports two distinct patterns of cleavage. A wide range of susceptibility of the various PGs to inhibition by PGIPs was observed. The four PGs that were inhibited by all PGIPs tested exhibited an endo/exo mode of substrate cleavage, while the three PGs that were resistant to inhibition by one or more of the PGIPs proceed by a classic endo pattern of cleavage.

Structural characterization of novel L-galactose-containing oligosaccharide subunits of jojoba seed xyloglucans.

Jojoba seed xyloglucan was shown to be a convenient source of biologically active xyloglucan oligosaccharides that contain both L- and D-galactosyl residues [E. Zablackis et al., Science, 272 (1996) 1808-1810]. Oligosaccharides were isolated by liquid chromatography of the mixture of oligosaccharides generated by treating jojoba seed xyloglucan with a beta-(1-->4)-endoglucanase. The purified oligosaccharides were reduced with NaBH4, converting them to oligoglycosyl alditol derivatives that were structurally characterized by a combination of mass spectrometry and 2-dimensional NMR spectroscopy. This analysis established that jojoba xyloglucan oligosaccharides contain the novel side-chain [alpha-L-Gal p-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-], which is structurally homologous to the fucose-containing side-chain [alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-] found in other biologically active xyloglucan oligosaccharides.

Synthesis and characterization of tyramine-derivatized (1-->4)-linked alpha-D-oligogalacturonides.

The reducing end C-1 of (1-->4)-linked alpha-D-oligogalacturonides (oligogalacturonides), with degrees of polymerization (dp) 3 and 13, was coupled to tyramine via reductive amination in the presence of sodium cyanoborohydride. These derivatives were purified in milligram quantities and structurally characterized. Tyramination of trigalacturonic acid proceeded to completion. The yield of apparently homogeneous tyraminated trigalacturonic acid after desalting was 35%. Derivatization of tridecagalacturonide with tyramine was incomplete. The tyraminated tridecagalacturonide was purified to apparent homogeneity using semipreparative high-performance anion-exchange chromatography (HPAEC) with a yield of 30%. The structures of the derivatized oligogalacturonides were established by 1H NMR spectroscopy and electrospray mass spectrometry.

Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. In vitro conditions for the formation and hydrolysis of the dimer.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide present in the walls of growing plant cells. We now report that RG-II, released by endopolygalacturonase treatment of the walls of suspension-cultured sycamore cells and etiolated pea stems, exists mainly as a dimer that is cross-linked by a borate ester. The borate ester is completely hydrolyzed at room temperature within 30 min at pH 1, partially hydrolyzed between pH 2 and 4, and stable above pH 4. The dimer is formed in vitro between pH 2.4 and 6. 2 by treating monomeric RG-II (0.5 mM) with boric acid (1.2 mM); the dimer formed after 24 h at pH 3.4 and 5.0 accounts for approximately 30 and approximately 5%, respectively, of the RG-II. In contrast, the dimer accounts for approximately 80 and approximately 54% of the RG-II when the monomer is treated for 24 h at pH 3.4 and 5.0, respectively, with boric acid and 0.5 m Sr2+, Pb2+, or Ba2+. The amount of dimer formed at pH 3.4 or 5.0 is not increased by addition of 0.5 mM Ca2+, Cd2+, Cu2+, Mg2+, Ni2+, and Zn2+. Steric considerations appear to regulate dimer formation since those divalent cations that enhance dimer formation have an ionic radius >1.1 A. Our data suggest that the borate ester is located on C-2 and C-3 of two of the four 3'-linked apiosyl residues of dimeric RG-II. Our results, taken together with the results of two previous studies (Kobayashi, M., Matoh, T., and Azuma, J.-I. (1996) Plant Physiol. 110, 1017-1020; Ishii, T., and Matsunaga, T. (1996) Carbohydr. Res. 284, 1-9) provide substantial evidence that this plant cell wall pectic polysaccharide is covalently cross-linked.

The structures of arabinoxyloglucans produced by solanaceous plants.

Several structural features, most notably the presence of alpha-L-Araf-(1-->2)-alpha-D-Xylp side chains, distinguish the arabinoxyloglucans (AXGs) produced by solanaceous plants from the xyloglucans produced by other dicotyledonous plants. However, previous studies did not establish the exact order of attachment of the various side chains along the backbone of these AXGs. Therefore, oligosaccharide subunits of the AXGs secreted by suspension-cultured tobacco and tomato cells were generated by treatment of the isolated AXGs with a fungal endo-beta-(1-->4)-D-glucanase (EG). The oligosaccharides were reduced with sodium borohydride to the corresponding oligoglycosyl alditol derivatives and purified by a combination of gel-permeation chromatography, reversed-phase HPLC, and HPAE chromatography. The isolated oligoglycosyl alditols were chemically characterized by NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDITOFMS), fast-atom bombardment mass spectrometry (FABMS), FABMS/MS, and glycosyl-linkage analysis. The results confirmed that the AXGs from these species are composed of a (1-->4)-linked beta-D-Glcp backbone substituted at O-6 with various side chains. Both tobacco and tomato AXG contain alpha-D-Xylp and alpha-L-Araf-(1-->2)-alpha-D-Xylp side chains. However, oligosaccharide fragments of tomato AXG were also shown to contain beta-D-Galp-(1-->2)-alpha-D-Xylp and beta-Araf-(1-->3)-alpha-L-Araf-(1-->2)-alpha-D-Xylp side chains that are not present in the tobacco AXG. This is the first report of beta-Araf residues in a xyloglucan. The primary structures of 20 oligosaccharides generated by EG-treatment of tobacco AXG were determined. The generation of such a large number of oligosaccharides is due in part to the presence of O-acetyl substituents at O-6 of many of the backbone beta-D-Glcp residues of tobacco AXG. The presence of either an O-acetyl or a glycosidic substituent at O-6 of a beta-D-Glc p residue in the AXG backbone protects the glycosidic bond of this residue from cleavage by the EG. Removal of the O-acetyl substituents prior to EG-treatment of the AXG-results in oligosacharide fragments that are smaller than those produced by EG-treatment of the O-acetylated AXG. Therefore, analysis of the complex mixture of oligosaccharides obtained by EG treatment of native tobacco AXGs provides information regarding the distribution of AXG side chains that would be lost if the AXG is de-O-acetylated prior to EG-treatment. Furthermore, the large library of oligosaccharide fragments generated by this approach revealed additional correlations between the structural features of AXGs and diagnosis chemical shift effects in their 1H NMR spectra.

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots.

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner.

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.

Improved protocol for the formation of N-(p-nitrobenzyloxy)aminoalditol derivatives of oligosaccharides.

An improved procedure has been developed for the rapid derivatization of oligosaccharides with UV-detectable p-nitrobenzylhydroxylamine (PNB). The improved conditions used result in quantitative derivatization of neutral oligosaccharides. Sialylated oligosaccharides can also be quantitatively PNB-derivatized without detectable desialylation. Of the oligosaccharides tested, only the derivatization of oligogalactosyluronic acids was incomplete (yield approximately 70%). PNB-derivatization of tamarind seed xyloglucan oligosaccharides results in products with improved chromatographic properties during HPAEC. These PNB derivatives were also subjected to hydrophilic interaction chromatography (HILIC) and analyzed by on-line LC-MS. On-line LC-MS is readily usable with HILIC, as this chromatographic technique does not require salt-containing solvents. Approximately 10 pmol of a PNB-derivatized oligosaccharide can be identified and quantitated utilizing this method.

Characterization of an endo-beta-1,4-glucanase gene induced by auxin in elongating pea epicotyls.

A gene (EGL1) encoding an endo-beta-1,4-D-glucanase (EGase, EC 3.2.1.4) of pea (Pisum sativum) has been cloned and characterized. EGL1 encodes a 486-amino acid polypeptide, including a 24-mer putative signal peptide. The mature protein has a calculated molecular mass of 51.3 kD and an isoelectric point of 9.1. This pea EGase shares significant similarity with EGases from other plant species, but it appears to be distinct from the EGases associated with abscission and fruit ripening. Although EGL1 transcripts are detected in all parts of pea plants, they are relatively abundant in flowers and young pods undergoing rapid growth and most abundant in elongating epicotyls of etiolated seedlings. When epicotyl segments (6 mm long, 4 mm from the apical hook) are incubated in a 5 microM solution of the synthetic auxin analog 2,4-dichlorophenoxyacetic acid, the concentration of EGL1 mRNA increases about 10-fold when the segments elongate most rapidly.

The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase.

Rhamnogalacturonan I (RG-I), a major pectic component of the primary walls of plant cells, is believed to play an important role in determining both the structure and functions of the walls. A more detailed structural description of RG-I is likely to lead to a greater understanding of the biological roles of this polysaccharide. Two enzymes secreted by Aspergillus aculeatus that have been cloned and expressed in a fungal system (Kofod et al., J. Biol. Chem., 269, 29182-29189, 1994) cleave the RG-I backbone in an endo fashion and should assist in the further structural characterization of this polysaccharide. We found that both of the available preparations of the cloned enzymes were contaminated with exoglycanases, reducing their utility in structurally characterizing RG-I. We purified the enzymes to apparent homogeneity by ion-exchange chromatography and then used the purified enzymes to generate backbone oligosaccharide fragments from partially debranched sycamore RG-I. The backbone oligosaccharides, which were separated from larger pieces of partially debranched RG-I by gel-permeation chromatography, have been structurally characterized by 1H-NMR spectroscopy, electrospray MS, GC-MS, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and UV spectroscopy. The results of these analyses establish that rhamnogalacturonase A (RGase A) is an endohydrolase that cleaves the -4)-alpha-D-GalpA-(1-2)-alpha-L-Rhap glycosidic linkage. However, the purported rhamnogalacturonase B (RGase B) is, in fact, an endolyase that cleaves the -2)-alpha-L-Rhap-(1-4)-alpha-D-GalpA glycosidic linkage, thereby generating oligosaccharides terminating at the non-reducing end with a hex-4-enopyranosyluronic acid residue.

Metabolism of xyloglucan generates xylose-deficient oligosaccharide subunits of this polysaccharide in etiolated peas.

Oligosaccharide subunits of xyloglucan were isolated from the stems and roots of etiolated pea plants and structurally characterized. The two most abundant subunits of pea xyloglucan are the well-known nonasaccharide, XXFG, and heptasaccharide, XXXG. In addition, significant amounts of oligosaccharides that have not previously been reported to be subunits of pea xyloglucan were detected, including a decasaccharide, XLFG, two octasaccharides, XLXG and XXLG, a pentasaccharide, XXG, and a trisaccharide, XG. Several novel oligosaccharide subunits, including the octasaccharide, GXFG, and the hexasaccharide, GXXG, were also found. Xyloglucan oligosaccharides generated by treatment of intact pea stem cell walls were compared to oligosaccharides generated by endoglucanase treatment of xyloglucan polysaccharides obtained by subsequent alkali extraction of the same cell walls. The results suggest that the xyloglucan in etiolated pea stems is distributed between at least two domains, one of which is distinguished by its enzyme accessibility. We further hypothesize that the chemical modification of a xyloglucan during cell-wall maturation depends on its physical environment (i.e., the domain in which it resides). For example, only the endoglucanase-released material, representing the enzyme-accessible xyloglucan domain, contains significant amounts of the two unusual oligosaccharide subunits, GXXG and GXFG, both of which have a nonreducing terminal glucosyl residue. This structure may be generated during cell-wall maturation by the sequential action of an endolytic enzyme (such as xyloglucan endotransglycosylase or endoglucanase) and an alpha-xylosidase.

Characterization of a monoclonal antibody that recognizes an arabinosylated (1-->6)-beta-D-galactan epitope in plant complex carbohydrates.

Monoclonal antibody CCRC-M7 is representative of a group of antibodies with similar binding specificity that were generated using the plant cell-wall pectic polysaccharide, rhamnogalacturonan I, as immunogen. The epitope recognized by CCRC-M7 is present in several plant polysaccharides and membrane glycoproteins. Selective enzymatic or chemical removal of arabinosyl residues from rhamnogalacturonan I reduced, but did not abolish, the ability of CCRC-M7 to bind to the polysaccharide. In contrast, enzymatic removal of both arabinosyl and galactosyl residues from rhamnogalacturonan I completely abolished binding of CCRC-M7 to the resulting polysaccharide. Competitive ELISAs using chemically defined oligosaccharides to compete for the CCRC-M7 binding site showed that oligosaccharides containing (1-->6)-linked beta-D-galactosyl residues were the best competitors among those tested, with the tri-, penta-, and hexa-saccharides being equally effective. The combined results from indirect and competitive ELISAs suggest that the minimal epitope recognized by CCRC-M7 encompasses a (1-->6)-linked beta-galactan containing at least three galactosyl residues with at least one arabinosyl residue attached.

Structural characterization of the pectic polysaccharide, rhamnogalacturonan-II.

An octasaccharide was released from sycamore cell wall rhamnogalacturonan-II (RG-II) by selective acid hydrolysis of the glycosidic linkages of apiosyl residues and purified to homogeneity by gel-permeation and high-performance anion-exchange chromatographies. The octasaccharide 1 contains a terminal nonreducing beta-L-arabinofuranosyl residue linked to position 2 of the alpha-L-rhamnopyranosyl residue of the aceric acid-containing heptasaccharide 2 that had been previously isolated from RG-II [M.W. Spellman et al. Carbohydr. Res., 122 (1983) 131-153]. Heptasaccharide 2 and octasaccharide 1 were found to be mono- or di-O-acetylated. The O-acetyl groups were located, by ESMSMS, on the terminal nonreducing 2-O-methyl-alpha-L-fucosyl residue and/or on the 2-linked beta-L-aceryl acid residue. Octasaccharide 1 and heptasaccharide 2 have the following structures: [structure: see text]

Purification, cloning and characterization of two xylanases from Magnaporthe grisea, the rice blast fungus.

Magnaporthe grisea, the fungal pathogen that causes rice blast disease, secretes two endo-beta-1,4-D-xylanases (E. C. 3.2.1.8) when grown on rice cell walls as the only carbon source. One of the xylanases, XYN33, is a 33-kD protein on sodium dodecyl sulfate-polyacrylamide gel and accounts for approximately 70% of the endoxylanase activity in the culture filtrate. The second xylanase, XYN22, is a 22-kD protein and accounts for approximately 30% of the xylanase activity. The two proteins were purified, cloned, and sequenced. XYN33 and XYN22 are both basic proteins with calculated isoelectric points of 9.95 and 9.71, respectively. The amino acid sequences of XYN33 and XYN22 are not homologous, but they are similar, respectively, to family F and family G xylanases from other microorganisms. The genes encoding XYN33 and XYN22, designated XYN33 and XYN22, are single-copy in the haploid genome of M. grisea and are expressed when M. grisea is grown on rice cell walls or on oatspelt xylan, but not when grown on sucrose.

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.

Eleven newly characterized xyloglucan oligoglycosyl alditols: the specific effects of sidechain structure and location on 1H NMR chemical shifts.

Eleven previously uncharacterized oligosaccharides, each containing from seventeen to twenty glycosyl residues, were isolated from the xyloglucan produced by suspension-cultured Acer pseudo-platanus cells and characterized by 1H NMR spectroscopy, fast-atom bombardment mass spectrometry, and matrix-assisted laser-desorption mass spectrometry. The complex mixture of xyloglucan oligosaccharides released by endo-(1-->4)-beta-glucanase (Trichoderma reesei) treatment of cell walls was similar to that released by digestion of the soluble xyloglucan present in the culture medium. The oligosaccharides were converted to oligoglycosyl alditols by borohydride reduction and purified by a combination of gel-permeation (Bio-Gel P-2) chromatography, normal-phase HPLC, reversed-phase HPLC, and high-performance anion-exchange (HPAE) chromatography. Eleven new oligoglycosyl alditols (along with several others that had been previously characterized) were isolated and characterized, allowing additional correlations between xyloglucan structure and specific chemical shift effects in the 1H NMR spectra to be determined. The correlations between structural and spectral features deduced in this study will facilitate the structural determination of a wide range of xyloglucans and their subunit oligosaccharides.