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Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-L-methionine to O-4 of α-D-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co(2+) for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.

Glucuronoxylans with a backbone of 1,4-linked β-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked β-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.

Cdc42, a member of the Rho-family small GTP-binding proteins, is a pivotal signaling switch that cycles between active GTP-bound and inactive GDP-bound forms, controlling actin cytoskeleton organization and cell polarity. In this report, we show that MgCdc42, a Cdc42 ortholog in Magnaporthe grisea, is required for its plant penetration. Consequently, the deletion mutants show dramatically decreased virulence to rice due to the arrest of penetration and infectious growth, which may be attributed to the defect of turgor and superoxide generation during the appressorial development in Mgcdc42 deletion mutants. In addition, the mutants also exhibit pleotropic defects including gherkin-shaped conidia, delayed germination as well as decreased sporulation. Furthermore, dominant negative mutation leads to a similar phenotype to that of the deletion mutants, lending further support to the conclusion that MgCdc42 is required for the penetration and virulence of M. grisea.

Xyloglucan is a well-characterized hemicellulosic polysaccharide that is present in the cell walls of all seed-bearing plants. The cell walls of avascular and seedless vascular plants are also believed to contain xyloglucan. However, these xyloglucans have not been structurally characterized. This lack of information is an impediment to understanding changes in xyloglucan structure that occurred during land plant evolution. In this study, xyloglucans were isolated from the walls of avascular (liverworts, mosses, and hornworts) and seedless vascular plants (club and spike mosses and ferns and fern allies). Each xyloglucan was fragmented with a xyloglucan-specific endo-glucanase and the resulting oligosaccharides then structurally characterized using NMR spectroscopy, MALDI-TOF and electrospray mass spectrometry, and glycosyl-linkage and glycosyl residue composition analyses. Our data show that xyloglucan is present in the cell walls of all major divisions of land plants and that these xyloglucans have several common structural motifs. However, these polysaccharides are not identical because specific plant groups synthesize xyloglucans with unique structural motifs. For example, the moss Physcomitrella patens and the liverwort Marchantia polymorpha synthesize XXGGG- and XXGG-type xyloglucans, respectively, with sidechains that contain a beta-D-galactosyluronic acid and a branched xylosyl residue. By contrast, hornworts synthesize XXXG-type xyloglucans that are structurally homologous to the xyloglucans synthesized by many seed-bearing and seedless vascular plants. Our results increase our understanding of the evolution, diversity, and function of structural motifs in land-plant xyloglucans and provide support to the proposal that hornworts are sisters to the vascular plants.

Xylan is the second most abundant polysaccharide in dicot wood, and thus elucidation of the xylan biosynthetic pathway is required to understand the mechanisms controlling wood formation. Genetic and chemical studies in Arabidopsis have implicated three genes, FRAGILE FIBER8 (FRA8), IRREGULAR XYLEM8 (IRX8) and IRREGULAR XYLEM9 (IRX9), in the biosynthesis of glucuronoxylan (GX), but the biochemical functions of the encoded proteins are not known. In this study, we determined the effect of the fra8, irx8 and irx9 mutations on the activities of xylan xylosyltransferase (XylT) and glucuronyltransferase (GlcAT). We show that microsomes isolated from the stems of wild-type Arabidopsis exhibit XylT and GlcAT activities in the presence of exogenous 1,4-linked beta-d-xylooligomers. Xylooligomers ranging in size from two to six can be used as acceptors by XylT to form xylooligosaccharides with up to 12 xylosyl residues. We provide evidence that the irx9 mutation results in a substantial reduction in XylT activity but has no discernible effect on GlcAT activity. In contrast, neither XylT nor GlcAT activity is affected by fra8 and irx8 mutations. Our results provide biochemical evidence that the irx9 mutation results in a deficiency in xylan XylT activity, thus leading to a defect in the elongation of the xylan backbone.

Mutations of Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9 were previously shown to cause a collapsed xylem phenotype and decreases in xylose and cellulose in cell walls. In this study, we characterized IRX8 and IRX9 and performed chemical and structural analyses of glucuronoxylan (GX) from irx8 and irx9 plants. IRX8 and IRX9 are expressed specifically in cells undergoing secondary wall thickening, and their encoded proteins are targeted to the Golgi, where GX is synthesized. 1H-NMR spectroscopy showed that the reducing end of Arabidopsis GX contains the glycosyl sequence 4-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-GalpA-(1-->4)-D-Xylp, which was previously identified in birch (Betula verrucosa) and spruce (Picea abies) GX. This indicates that the reducing end structure of GXs is evolutionarily conserved in woody and herbaceous plants. This sequence is more abundant in irx9 GX than in the wild type, whereas irx8 and fragile fiber8 (fra8) plants are nearly devoid of it. The number of GX chains increased and the GX chain length decreased in irx9 plants. Conversely, the number of GX chains decreased and the chain length heterodispersity increased in irx8 and fra8 plants. Our results suggest that IRX9 is required for normal GX elongation and indicate roles for IRX8 and FRA8 in the synthesis of the glycosyl sequence at the GX reducing end.

Secondary walls in wood are the most abundant biomass produced by plants. Understanding how plants make wood is not only of interest in basic plant biology but also has important implications for tree biotechnology. Three recent papers report exciting findings regarding a group of novel glycosyltransferases (GTs) involved in secondary wall synthesis. Because little is known about genes involved in the synthesis of wood polysaccharides other than cellulose, the identification of these GTs is a breakthrough in the molecular dissection of wood formation.

Magnaporthe grisea, a destructive ascomycetous pathogen of rice, secretes cell wall-degrading enzymes into a culture medium containing purified rice cell walls as the sole carbon source. From M. grisea grown under the culture conditions described here, we have identified an expressed sequenced tag, XYL-6, a gene that is also expressed in M. grisea-infected rice leaves 24 h postinoculation with conidia. This gene encodes a protein about 65% similar to endo-beta-1,4-D-glycanases within glycoside hydrolase family GH10. A M. grisea knockout mutant for XYL-6 was created, and it was shown to be as virulent as the parent strain in infecting the rice host. The proteins secreted by the parent strain and by the xyl-6Delta mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-beta-1,4-D-glucanase or endo-beta-1,4-D-xylanase activities. Two protein-containing peaks with endo-beta-1,4-D-xylanase activity secreted by the parent strain are not detectable in the column eluant of the proteins secreted by the mutant. The two endoxylanases (XYL-6alpha and XYL-6beta) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6alpha is a fragment of XYL-6beta and that XYL-6beta is identical to the deduced protein sequence encoded by the XYL-6 gene. Finally, XYL-6 was introduced into Pichia pastoris for heterologous expression, which resulted in the purification of a fusion protein, XYL-6H, from the Pichia pastoris culture filtrate. XYL-6H is active in cleaving arabinoxylan. These experiments unequivocally established that the XYL-6 gene encodes a secreted endo-beta-1,4-D-xylanase.

Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation.

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.

Arabidopsis thaliana mur1 is a dwarf mutant with altered cell-wall properties, in which l-fucose is partially replaced by l-galactose in the xyloglucan and glycoproteins. We found that the mur1 mutation also affects the primary structure of the pectic polysaccharide rhamnogalacturonan II (RG-II). In mur1 RG-II a non-reducing terminal 2- O-methyl l-galactosyl residue and a 3,4-linked l-galactosyl residue replace the non-reducing terminal 2- O-methyl l-fucosyl residue and the 3,4-linked l-fucosyl residue, respectively, that are present in wild-type RG-II. Furthermore, we found that a terminal non-reducing l-galactosyl residue, rather than the previously reported d-galactosyl residue, is present on the 2- O-methyl xylose-containing side chain of RG-II in both wild type and mur1 plants. Approximately 95% of the RG-II from wild type and mur1 plants is solubilized as a high-molecular-weight (>100 kDa) complex, by treating walls with aqueous potassium phosphate. The molecular mass of RG-II in this complex was reduced to 5-10 kDa by treatment with endopolygalacturonase, providing additional evidence that RG-II is covalently linked to homogalacturonan. The results of this study provide additional information on the structure of RG-II and the role of this pectic polysaccharide in the plant cell wall.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that was first identified in 1978 as a quantitatively minor component of suspension-cultured sycamore cell walls. Subsequent studies have shown that RG-II is present in the primary walls of angiosperms, gymnosperms, lycophytes, and pteridophytes and that its glycosyl sequence is conserved in all vascular plants examined to date. This is remarkable because RG-II is composed of at least 12 different glycosyl residues linked together by more than 20 different glycosidic linkages. However, only a few of the genes and proteins required for RG-II biosynthesis have been identified. The demonstration that RG-II exists in primary walls as a dimer that is covalently cross-linked by a borate diester was a major advance in our understanding of the structure and function of this pectic polysaccharide. Dimer formation results in the cross-linking of the two homogalacturonan chains upon which the RG-II molecules are constructed and is required for the formation of a three-dimensional pectic network in muro. This network contributes to the mechanical properties of the primary wall and is required for normal plant growth and development. Indeed, changes in wall properties that result from decreased borate cross-linking of pectin may lead to many of the symptoms associated with boron deficiency in plants.

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.

A basic, 51 kDa protein was purified from suspension-cultured tomato and shown to inhibit the hydrolytic activity of a xyloglucan-specific endoglucanase (XEG) from the fungus Aspergillus aculeatus. The tomato (Lycopersicon esculentum) protein, termed XEG inhibitor protein (XEGIP), inhibits XEG activity by forming a 1 : 1 protein:protein complex with a Ki approximately 0.5 nm. To our knowledge, XEGIP is the first reported proteinaceous inhibitor of any endo-beta-1,4-glucanase, including the cellulases. The cDNA encoding XEGIP was cloned and sequenced. Database analysis revealed homology with carrot extracellular dermal glycoprotein (EDGP), which has a putative role in plant defense. XEGIP also has sequence similarity to ESTs from a broad range of plant species, suggesting that XEGIP-like genes are widely distributed in the plant kingdom. Although Southern analysis detected only a single XEGIP gene in tomato, at least five other XEGIP-like tomato sequences have been identified. Similar small families of XEGIP-like sequences are present in other plants, including Arabidopsis. XEGIP also has some sequence similarity to two previously characterized proteins, basic globulin 7S protein from soybean and conglutin gamma from lupin. Several amino acids in the XEGIP sequence, notably 8 of the 12 cysteines, are generally conserved in all the XEGIP-like proteins we have encountered, suggesting a fundamental structural similarity. Northern analysis revealed that XEGIP is widely expressed in tomato vegetative tissues and is present in expanding and maturing fruit, but is downregulated during ripening.

The monoclonal antibody, CCRC-M1, which recognizes a fucose (Fuc)-containing epitope found principally in the cell wall polysaccharide xyloglucan, was used to determine the distribution of this epitope throughout the mur1 mutant of Arabidopsis. Immunofluorescent labeling of whole seedlings revealed that mur1 root hairs are stained heavily by CCRC-M1, whereas the body of the root remains unstained or only lightly stained. Immunogold labeling showed that CCRC-M1 labeling within the mur1 root is specific to particular cell walls and cell types. CCRC-M1 labels all cell walls at the apex of primary roots 2 d and older and the apices of mature lateral roots, but does not bind to cell walls in lateral root initials. Labeling with CCRC-M1 decreases in mur1 root cells that are undergoing rapid elongation growth such that, in the mature portions of primary and lateral roots, only the walls of pericycle cells and the outer walls of epidermal cells are labeled. Growth of the mutant on Fuc-containing media restores wild-type labeling, where all cell walls are labeled by the CCRC-M1 antibody. No labeling was observed in mur1 hypocotyls, shoots, or leaves; stipules are labeled. CCRC-M1 does label pollen grains within anthers and pollen tube walls. These results suggest the Fuc destined for incorporation into xyloglucan is synthesized using one or the other or both isoforms of GDP-D-mannose 4,6-dehydratase, depending on the cell type and/or developmental state of the cell.

A 2-O-methylfucosyl-containing heptasaccharide was released from red wine rhamnogalacturonan II (RG-II) by acid hydrolysis of the glycosidic linkage of the aceryl acid residue (AceA) and purified to homogeneity by size-exclusion and high-performance anion-exchange chromatographies. The primary structure of the heptasaccharide was determined by glycosyl-residue and glycosyl-linkage composition analyses, ESIMS, and by 1H and 13C NMR spectroscopy. The NMR data indicated that the pyranose ring of the 2,3-linked L-arabinosyl residue is conformationally flexible. The L-Arap residue was confirmed to be alpha-linked by NMR analysis of a tetraglycosyl-glycerol fragment, [alpha-L-Arap-(1-->4)-beta-D-Galp-(1-->2)-alpha-L-AcefA-(1-->3)-beta-L-Rhap-(1-->3)-Gro], generated by Smith degradation of RG-II. Our data together with the results of a previous study,(1) establish that the 2-O-Me Fuc-containing nonasaccharide side chain of wine RG-II has the structure (Api [triple bond] apiose): [see structure]. Data are presented to show that in Arabidopsis RG-II the predominant 2-O-MeFuc-containing side chain is a mono-O-acetylated heptasaccharide that lacks the non-reducing terminal beta-L-Araf and the alpha-L-Rhap residue attached to the O-3 of Arap, both of which are present on the wine nonasaccharide.

A characteristic plant response to microbial attack is the production of endo-beta-1,3-glucanases, which are thought to play an important role in plant defense, either directly, through the degradation of beta-1,3/1,6-glucans in the pathogen cell wall, or indirectly, by releasing oligosaccharide elicitors that induce additional plant defenses. We report the sequencing and characterization of a class of proteins, termed glucanase inhibitor proteins (GIPs), that are secreted by the oomycete Phytophthora sojae, a pathogen of soybean, and that specifically inhibit the endoglucanase activity of their plant host. GIPs are homologous with the trypsin class of Ser proteases but are proteolytically nonfunctional because one or more residues of the essential catalytic triad is absent. However, specific structural features are conserved that are characteristic of protein-protein interactions, suggesting a mechanism of action that has not been described previously in plant pathogen studies. We also report the identification of two soybean endoglucanases: EGaseA, which acts as a high-affinity ligand for GIP1; and EGaseB, with which GIP1 does not show any association. In vitro, GIP1 inhibits the EGaseA-mediated release of elicitor-active glucan oligosaccharides from P. sojae cell walls. Furthermore, GIPs and soybean endoglucanases interact in vivo during pathogenesis in soybean roots. GIPs represent a novel counterdefensive weapon used by plant pathogens to suppress a plant defense response and potentially function as important pathogenicity determinants.

An 85-kDa beta-glucosidase/xylosidase (BGX1) was purified from the axenically grown phytopathogenic oomycete, Phytophthora infestans. The bgx1 gene encodes a predicted 61-kDa protein product which, upon removal of a 21 amino acid leader peptide, accumulates in the apoplastic space. Extensive N-mannosylation accounts for part of the observed molecular mass difference. BGX1 belongs to family 30 of the glycoside hydrolases and is the first such oomycete enzyme deposited in public databases. The bgx1 gene was found in various Phytophthora species, but is apparently absent in species of the related genus, Pythium. Despite significant sequence similarity to human and murine lysosomal glucosylceramidases, BGX1 demonstrated neither glucocerebroside nor galactocerebroside-hydrolyzing activity. The native enzyme exhibited glucohydrolytic activity towards 4-methylumbelliferyl (4-MU) beta-D-glucopyranoside and, to lesser extent, towards 4-MU-D-xylopyranoside, but not towards 4-MU-beta-D-glucopyranoside. BGX1 did not hydrolyze carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting high substrate specificity and/or specific cofactor requirements for enzymatic activity.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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 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.

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.

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.

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.

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.

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]

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.

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 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.

A combination of commercially available preparations of Aspergillus niger beta-D-galactosidase, endo-alpha-L-arabinanase, alpha-L-arabinosidase, and endo-beta-D-galactanase has been used to generate oligoglycosyl fragments of the backbone of rhamnogalacturonan I (RG-I) that had been isolated from the walls of suspension-cultured sycamore cells. The backbone-cleaving enzyme, which is present in the beta-D-galactosidase preparation, only fragments the RG-I backbone when many of the neutral oligoglycosyl side chains have been removed by the other exo- and endo- glycanases. The oligosaccharides released from the backbone were separated from the partially fragmented RG-I and then purified, as their oligoglycosyl aldonic acids, by HPAEC-PAD. Those backbone fragments with degrees of polymerization (dp's) between 2 and 11 were characterized using one- and two-dimensional 1H NMR spectroscopy, electrospray mass spectrometry, and glycosyl-residue and glycosyl-linkage composition analyses. Two series of oligoglycosyl fragments were identified. The quantitatively predominant series has the structure alpha-D-GalpA-(1 --> 2)- alpha-L-Rhap-[ --> 4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap-(1 --> ]n-4-D-GalpA, and the quantitatively minor series has the structure alpha-L-Rhap-[ --> 4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap-(1 --> ]n-4-D- GalpA (n = 1-5). Thus, the enzyme preparations contain an alpha-L-rhamnosidase in addition to the endo- rhamnogalacturonase. The products of the endo-rhamnogalacturonase provide additional evidence that the backbone of RG-I is composed of the diglycosyl repeating unit: --> 4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap- (1 -->. The endo-rhamnogalacturonase from the A. niger beta-D-galactosidase preparation and the endo- rhamnogalacturonase secreted by Aspergillus aculeatus [H.A. Schols et al. Carbohydr. Res., 206 (1990) 117-129] have the same substrate specificities and generate similar oligoglycosyl fragments.

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.

Monoclonal antibodies (McAbs) generated against rhamnogalacturonan I (RG-I) purified from suspension-cultured sycamore maple (Acer pseudoplatanus) cells fall into three recognition groups. Four McAbs (group I) recognize an epitope that appears to be immunodominant and is present on RG-I from maize and sycamore maple, pectin and polygalacturonic acid from citrus, gum tragacanth, and membrane glycoproteins from suspension-cultured cells of maize, tobacco, parsley, bean, and sycamore maple. A second set of McAbs (group II) recognizes an epitope present in sycamore maple RG-I but does not bind to any of the other polysaccharides or glycoproteins recognized by group I. Lastly, one McAb, CCRC-M1 (group III), binds to RG-I and more strongly to xyloglucan (XG) from sycamore maple but not to maize RG-I, citrus polygalacturonic acid, or to the plant membrane glycoproteins recognized by group I. The epitope to which CCRC-M1 binds has been examined in detail. Ligand competition assays using a series of oligosaccharides derived from or related to sycamore maple XG demonstrated that a terminal alpha-(1-->2)-linked fucosyl residue constitutes an essential part of the epitope recognized by CCRC-M1. Oligosaccharides containing this structural motif compete with intact sycamore maple XG for binding to the antibody, whereas structurally related oligosaccharides, which do not contain terminal fucosyl residues or in which the terminal fucosyl residue is linked alpha-(1-->3) to the adjacent glycosyl residue, do not compete for the antibody binding site. The ligand binding assays also indicate that CCRC-M1 binds to a conformationally dependent structure of the polysaccharide. Other results of this study establish that some of the carbohydrate epitopes of the plant extracellular matrix are shared among different macromolecules.

Rhamnogalacturonan I (RG-I), a pectic polysaccharide isolated from the walls of suspension-cultured sycamore cells, was shown by glycosyl-residue composition analysis to contain D-glucosyluronic acid (GlcpA) residues (1 mol%) and 4-O-methyl-D-glucosyluronic acid (4-O-Me-GlcpA) residues (0.5 mol%). These monosaccharides were shown, by glycosyl-linkage analysis, to be present in RG-I as terminal nonreducing residues. The glycosyl sequences containing GlcpA and 4-O-Me-GlcpA were determined by structurally characterizing the acidic oligosaccharides released by partial acid hydrolysis of RG-I. Six acidic oligosaccharides were purified by semipreparative high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and characterized by glycosyl-residue and glycosyl-linkage composition analyses, GLC-CIMS, GLC-EIMS, electrospray MS (ESMS), and 1H NMR spectroscopy. We propose that three of the acidic oligosaccharides characterized, 4-O-Me-beta-D-GlcpA-(1-->6)-D-Gal, beta-D-GlcpA-(1-->6)-D-Gal, and beta-D-GlcpA-(1-->4)-D-Gal, originate from the galactosyl-containing side chains of RG-I. The three other acidic oligosaccharides characterized, alpha-D-GalpA-(1-->2)-L-Rha, alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA+ ++-(1-->2)-alpha-L-Rha, and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA+ ++-(1-->2)-alpha-L- Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rha, were generated by partial hydrolysis of the RG-I backbone. No evidence was obtained for the presence of galactosyluronic acid in the side chains of RG-I.(ABSTRACT TRUNCATED AT 250 WORDS)

The borohydride-reduced forms (oligoglycosyl alditols) of two isomeric octasaccharides (Glc4Xyl3Gal) that are released from xyloglucans of various plant species upon treatment with a fungal endo-(1-->4)-beta-glucanase were isolated and structurally characterized. A mixture of oligosaccharides that is released from tamarind seed xyloglucan by the endo-(1-->4)-beta- glucanase was digested with a commercially available beta-galactosidase (Aspergillus niger). The beta-galactosidase selectively hydrolyzed the galactosyl residue of one of the two isomeric octasaccharides present in the mixture. A homogeneous preparation of the beta-galactosidase-resistant octasaccharide was prepared by high-resolution gel-permeation chromatography of the enzyme-digestion products. Spectroscopic characterization of the oligoglycosyl alditol prepared by reduction of this octasaccharide confirmed the previously proposed structure that had been based on analysis of the mixture of isomeric octasaccharides. The availability of large amounts of the pure, reduced octasaccharide and of a pure, reduced pentasaccharide (Glc3Xyl2) made it possible to completely assign their 1H and 13C NMR spectra. In addition, the borohydride-reduced form of the beta-D-galactosidase-susceptible octasaccharide isomer was purified by high pH anion-exchange chromatography of the endo-(1-->4)-beta-glucanase-released octasaccharides from rape-seed xyloglucan (no beta-galactosidase treatment), and its 1H and 13C NMR spectra were assigned. Additional correlations between specific structural features of xyloglucan oligoglycosyl alditols and the positions of specific resonances in their NMR spectra were deduced and added to the extensive list that we have compiled. The effects of recording the NMR spectra of the xyloglucan oligoglycosyl alditols in the presence of borate salts, which could lead to incorrect structural assignments, are also described.

A Phytophthora megasperma f.sp. glycinea cell wall glucan preparation was previously shown to protect tobacco plants against viral infection. Eleven plant defense-related genes were assayed for elevated mRNA accumulation levels in response to glucan treatment of tobacco plants. The expression of only one of these genes, a glycine-rich protein (GRP) gene, was induced by glucan application. Elevated GRP gene mRNA levels could be detected within 15 min of glucan treatment and reached maximum levels at 4 h post-treatment followed by a slow decline to 8 h. The maximum induction of the GRP gene was approximately ninefold above H2O-treated control plants. Northern blot analysis showed that a single mRNA species of 1.4 kb was responding to the glucan treatment. GRP genes occur in tobacco as members of a multigene family, but only one specific GRP gene was induced by the glucan treatment. A genomic copy of this responding GRP gene was cloned and sequenced. This tobacco GRP gene is homologous to the petunia ptGRP1 gene and the French bean GRP1.8-gene, but is not closely related to the French bean GRP1.0 gene. GRP gene expression has previously been associated with disease resistance in plants, but it remains to be determined whether beta-glucan activation of the tobacco GRP gene results in the observed resistance to virus.

Rhamnogalacturonan I (RG-I) has been isolated from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus), and additional structural features of the polysaccharide were elucidated. Treatment of RG-I with a purified endo-(1-->5)-alpha-L-arabinanase released a series of arabinose-containing oligosaccharides with degrees of polymerization (dp's) between 2 and 20. These oligosaccharides were shown, by glycosyl-linkage composition analysis, to contain terminal, 5-, and (3-->5)-linked Araf residues. These results provide evidence that a branched arabinan is attached to the backbone of RG-I. RG-I was freed of 95% of its arabinosyl residues by treating the polysaccharide with a combination of endo-(1-->5)-alpha-L-arabinanase and alpha-L-arabinosidase. No galacturonic acid was released by these enzymes, which is evidence that the arabinosyl-containing portions of the side chains do not contain galactosyluronic acid residues. The galactose-containing portions of the side chains of RG-I were not fragmented by an endo-(1-->4)-beta-D-galactanase. However, approximately 85% of the galactose and small amounts of galacturonic acid were released by digestion of arabinose-depleted RG-I with a combination of endo- and exo-beta-D-galactanases. The galacturonic acid may have been released by small amounts of an exo-alpha-galactosyluronidase contaminating the galactanases. Treatment of RG-I with this mixture of endo- and exo-glycanases resulted in a relatively size-homogeneous, almost side chain-free backbone composed of the O-acetylated diglycosyl repeating unit -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap. A combination of 1H NMR spectroscopy and periodate oxidation established that the backbone repeating unit contained a single O-acetyl substituent on C-2 or C-3 of each galactosyluronic acid residue.

The structural features required for xyloglucan oligosaccharides to inhibit 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-beta-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8') was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific alpha-xylosidase from pea seedlings. XG8' was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth.

The complete primary structures of seven oligosaccharide subunits of the xyloglucan secreted by suspension-cultured Acer pseudoplatanus cells were determined. The oligosaccharides, ranging in size from 17 to 20 glycosyl residues, were generated by treatment of the xyloglucan with an endo-beta-(1----4)-glucanase. The oligosaccharide components of a fraction obtained by Bio-Gel P-2 chromatography of enzyme-treated xyloglucan were further purified by normal-phase h.p.l.c. and then converted to the corresponding oligoglycosyl alditols by reduction with NaBH4. The oligoglycosyl alditols, after purification to near homogeneity by reversed-phase h.p.l.c., were structurally characterized by 1H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry (f.a.b.-m.s.), and analysis of their glycosyl-residue and glycosyl-linkage compositions. Novel structural elements of xyloglucans were observed in this study, including beta-D-xylopyranosyl and alpha-L-arabinofuranosyl-(1----3)-beta-D-xylopyranosyl sidechains. The results also extend our list of correlations between 1H-n.m.r. resonances and specific structural features of xyloglucans and thus enhance our ability to determine the structures of xyloglucans from various sources.

The extracellular anionic polysaccharide produced by the bacterium Agrobacterium radiobacter (ATCC 53271) contains D-galactose, D-glucose, and pyruvic acid in the molar ratio 2:15:2. Analysis of the methylated polysaccharide indicated the presence of terminal, non-reducing glucosyl, 3-, 4-, 6-, 2,4-, and 4,6-linked glucosyl residues, 3-linked 4,6-O-[(S)-1-carboxyethylidene]glucosyl residues, and 3-linked galactosyl residues. Partial acid hydrolysis of the methylated polysaccharide, followed by reduction with NaB2H4 and then O-ethylation, gave a mixture of alkylated oligoglycosyl alditols that were separated by reversed-phase h.p.l.c. and analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. Smith degradation of the polysaccharide gave three diglycosyl alditols that were separated by semi-preparative, high-pH anion-exchange chromatography, and were analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The polymer obtained by NaBH4 reduction of the periodate-oxidized polysaccharide was methylated, and the noncyclic acetals were hydrolyzed with aq. 90% formic acid to generate a mixture of partially O-methylated mono- and di-glycosyl alditols. The partially O-methylated oligoglycosyl alditols were O-ethylated. The resulting alkylated oligoglycosyl alditols were separated by reverse-phase h.p.l.c. and then characterized by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The results from the studies described here provide strong evidence that the acidic polysaccharide secreted by A. radiobacter (ATCC 53271) has a heptadecasaccharide repeating unit.