Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain.

Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes.

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.

Differential recognition of plant cell walls by microbial xylan-specific carbohydrate-binding modules.

Glycoside hydrolases that degrade plant cell walls have complex molecular architectures in which one or more catalytic modules are appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs promote binding to polysaccharides and potentiate enzymic hydrolysis. Although there are diverse sequence-based families of xylan-binding CBMs, these modules, in general, recognize both decorated and unsubstituted forms of the target polysaccharide, and thus the evolutionary rationale for this diversity is unclear. Using immunohistochemistry to interrogate the specificity of six xylan-binding CBMs for their target polysaccharides in cell walls has revealed considerable differences in the recognition of plant materials between these protein modules. Family 2b and 15 CBMs bind to xylan in secondary cell walls in a range of dicotyledon species, whereas family 4, 6, and 22 CBMs display a more limited capability to bind to secondary cell walls. A family 35 CBM, which displays more restricted ligand specificity against purified xylans than the other five protein modules, reveals a highly distinctive binding pattern to plant material including the recognition of primary cell walls of certain dicotyledons, a feature shared with CBM15. Differences in the specificity of the CBMs toward walls of wheat grain and maize coleoptiles were also evident. The variation in CBM specificity for ligands located in plant cell walls provides a biological rationale for the repertoire of structurally distinct xylan-binding CBMs present in nature, and points to the utility of these modules in probing the molecular architecture of cell walls.

Monoclonal antibodies to plant cell wall xylans and arabinoxylans.

Two rat monoclonal antibodies have been generated to plant cell wall (1-->4)-beta-D-xylans using a penta-1,4-xylanoside-containing neoglycoprotein as an immunogen. The monoclonal antibodies, designated LM10 and LM11, have different specificities to xylans in relation to the substitution of the xylan backbone as indicated by immunodot assays and competitive-inhibition ELISAs. LM10 is specific to unsubstituted or low-substituted xylans, whereas LM11 binds to wheat arabinoxylan in addition to unsubstituted xylans. Immunocytochemical analyses indicated the presence of both epitopes in secondary cell walls of xylem but differences in occurrence in other cell types.

Novel cell wall architecture of isoxaben-habituated Arabidopsis suspension-cultured cells: global transcript profiling and cellular analysis.

The herbicide isoxaben is a highly specific and potent inhibitor of cellulose synthesis in plants. Nevertheless, suspension-cultured cells can be habituated to grow in high concentrations of isoxaben, and apparently compensate for the disruption of cellulose synthesis by the modulation of other cell wall components. We have habituated Arabidopsis cells to isoxaben and characterized the cellular and genetic consequences. Near whole-genome transcript profiling implicated novel genes in cell wall assembly and extended our understanding of the activity of known cell wall-related genes including glycosyltransferases involved in cellulose and pectin biosynthesis. Habituation does not appear to be mediated by stress response processes, nor by functional redundancy within the cellulose synthase (AtCesA) family. Uniquely, amongst the cellulose synthase superfamily, AtCslD5 was highly upregulated and may play a role in the biosynthesis of the novel walls of habituated cells. In silico analysis of differentially expressed genes with unknown functions identified a putative glycosyltransferase and collagen-like putative cell wall protein.

Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth.

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

A monoclonal antibody to feruloylated-(1-->4)-beta-D-galactan.

We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(1-->4)-beta-D-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno-assays and immunofluorescence microscopy indicated that LM9 binding is specific to samples and cell walls obtained from species belonging to this family. In a series of competitive-inhibition enzyme-linked immunosorbent assays with potential oligosaccharide haptens, the most effective inhibitor was O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose (Gal2F). LM9 is therefore a useful antibody probe for the analysis of phenolic substitution of cell wall pectic polymers and of cell wall structure in the Amaranthaceae including sugar beet (Beta vulgaris L.) and spinach (Spinacia oleracea L.).

Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers.

Novel molecular probes have been developed for the analysis and detection of polysaccharides in plant cell walls using carbohydrate-binding modules (CBMs) derived from modular glycoside hydrolases belonging to families 2a, 6, and 29. Recombinant forms of these proteins containing his-tags, in conjunction with anti-his-tag detection, provide a flexible system that utilizes CBMs as molecular probes in a range of applications. Assays for the rapid analysis of the binding of CBMs to polysaccharides and oligosaccharides using nitrocellulose-based CBM macroarrays and microtiter plate-based CBM capture and competitive-inhibition assays are described. We also demonstrate the use of CBMs with his-tags for the localization of their target ligands in planta. The generation of molecular probes from other families of CBMs will dramatically increase the repertoire of molecular probes available to determine the developmental and functional aspects of plant cell walls.

A xylogalacturonan epitope is specifically associated with plant cell detachment.

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

Cell wall pectic (1-->4)-beta-d-galactan marks the acceleration of cell elongation in the Arabidopsis seedling root meristem.

Here we demonstrate that the pectic rhamnogalacturonan-I-associated LM5 (1-->4)-beta-d-galactan epitope occurs in a restricted manner at the root surface of intact Arabidopsis seedlings. The root surface occurrence of (1-->4)-beta-d-galactan marks the transition zone at or near the onset of rapid cell elongation and the epitope is similarly restricted in occurrence in epidermal, cortical and endodermal cell walls. The extent of surface (1-->4)-beta-d-galactan occurrence is reduced in response to genetic mutations (stp-1, ctr-1) and hormone applications that reduce root cell elongation. In contrast, the application of the arabinogalactan-protein (AGP) binding beta-glucosyl Yariv reagent (betaGlcY) that disrupts cell elongation results in the persistence of (1-->4)-beta-d-galactan at the root surface and in epidermal, cortical and endodermal cell walls. This latter observation indicates that modulation of pectic (1-->4)-beta-d-galactan may be an event downstream of AGP function during cell expansion in the Arabidopsis seedling root.

Regulation of pectic polysaccharide domains in relation to cell development and cell properties in the pea testa.

The occurrence of pectic polysaccharide epitopes in cells and tissues of the pea testa during late stages of seed development have been examined in relation to anatomy and cell properties. Homogalacturonan, in a highly methyl-esterified form, was present throughout late development in all pea testa cell walls, including the thickened cell walls of the outer macrosclereid layer. Two epitopes, characteristic of the side-chains of the rhamnogalacturonan-I domain of pectic polysaccharides, occurred in restricted and separate cell layers of the pea testa. A (1-->4)-beta-D-galactan epitope was restricted to regions of the outer cell wall of the testa and to inner regions of the macrosclereid layer at 20 DAA and was absent from the osteosclereid and parenchyma cell walls. By 25 DAA the (1-->4)-beta-D-galactan epitope occurred only in the outer epidermal cell walls. A (1-->5)-alpha-L-arabinan epitope was also dependent on the developmental stage of the seed and was found with greatest abundance in the walls of the inner parenchyma cells. Cell separation studies indicated that, although calcium cross-links were involved in the maintenance of the link between the macrosclereid layer and proximal cell layers, most cell-to-cell adhesion in the testa was not due to calcium- or ester-based bonds.