Stefan Eberhard

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

Pectin and xylan are generally considered as separate cell wall glycan networks distinct from cell wall proteins. This work describes a cell wall proteoglycan with pectin and arabinoxylan covalently attached to an arabinogalactan protein, identifying a cross-linked matrix polysaccharide wall protein architecture with implications for wall structure, function, and synthesis. Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.

Pectic Cell Wall Fragments Regulate Tobacco Thin-Cell-Layer Explant Morphogenesis.

Pectic fragments of cell wall polysaccharides, released from the walls of suspension-cultured sycamore cells by treatment with endopolygalacturonase, were tested for morphogenesis-regulating activity in a modified tobacco thin-cell-layer explant (TCL) bioassay (D. Mohnen, S. Eberhard, V. Marfa, N. Doubrava, P. Toubart, D. J. Gollin, T.A. Gruber, W. Nuri, P. Albersheim, and A. Darvill, manuscript submitted). The pectic fragments inhibited the formation of roots on TCLs grown on a root-inducing medium containing 15 micromolar indole-3-butyric acid and 0.5 micromolar kinetin. Addition of the pectic fragments to a root-inducing medium containing 7 micromolar indole-3-butyric acid and 0.15 micromolar kinetin caused roots to form on the basal end of TCLs. TCLs cultured on this medium in the absence of added pectic fragments formed roots along their entire length. The pectic fragments induced polar tissue enlargement and the formation of flowers on TCLs cultured on transition medium. The flower-inducing activity was stable to heat treatment and proteolytic digestion. Pectic fragments isolated from the walls of suspension-cultured tobacco cells were as effective as those from the walls of sycamore cells in inducing de novo flower formation in the TCLs. These results support the hypothesis that oligosaccharins from plant cell walls regulate morphogenesis.

Moss and liverwort xyloglucans contain galacturonic acid and are structurally distinct from the xyloglucans synthesized by hornworts and vascular plants.

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.

Mutations in Multiple XXT Genes of Arabidopsis Reveal the Complexity of Xyloglucan Biosynthesis

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxt1 xxt5 and xxt2 xxt5) and a triple mutant (xxt1 xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxt1 xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls.

Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells

Abstract. Mutation of the Arabidopsis thaliana (L.) Heynh. gene MUR1, which encodes an isoform of GDP-D-mannose-4,6-dehydratase, affects the biosynthetic conversion of GDP-mannose to GDP-fucose. Cell walls in the aerial tissues of mur1 plants are almost devoid of α-L-fucosyl residues, which are partially replaced by closely related α-L-galactosyl residues. A line of suspension-cultured A. thaliana cells was generated from leaves of mur1 plants and the structure of the xyloglucan in the walls of these cells was structurally characterized. Xyloglucan fractions were prepared from the walls of both wild-type (WT) and mur1 cells by sequential extraction with a xyloglucan-specific endoglucanase (XEG) and aqueous KOH. Structural analysis of these fractions revealed that xyloglucan produced by cultured mur1 cells is similar, but not identical to that isolated from leaves of mur1 plants. As previously reported for mur1 leaves, the xyloglucan from cultured mur1 cells contains less than 5% of the fucose present in the xyloglucan from WT cells. Fucosylation of the xyloglucan is substantially restored when mur1 cells are grown in medium supplemented with L-fucose. Xyloglucan isolated from leaves contains more oligosaccharide subunits in which the central sidechain is terminated with a β-D-galactosyl residue than does xyloglucan prepared from cultured cells. This was observed for both mur1 and WT plants, indicating that this correlation is independent of the mur1 mutation and that it is possible to distinguish changes due to genetic mutation from those due to the physiological state of the cells in culture. Suspension-cultured cells thus provide a convenient source of genetically altered cell wall material, facilitating the biochemical characterization of mutations that affect cell wall structure.

Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides.

Abstract— Application of Aloe barbadensis poly/oligosaccharides to UV‐irradiated skin prevents photosuppression of delayed‐type hypersensitivity (DTH) responses in mice. We tested the hypothesis that these carbohydrates belong to a family of biologically active, plant‐derived polysaccharides that can regulate responses to injury in animal tissues. C3H mice were exposed to 5 kJ/m2 UVB from unfiltered FS40 sunlamps and treated with between 1 pg and 10 μg tamarind xyloglucans or control polysaccharides methylcellulose or dextran in saline. The mice were sensitized 3 days later with Candida albicans. Tamarind xylogiucans and purified Aloe poly/oligosaccharides prevented suppression of DTH responses in vivo and reduced the amount of interleukin (IL)‐IO observed in UV‐irradiated murine epidermis. Tamarind xyloglucans were immunoprotective at low picogram doses. In contrast, the control polysaccharides methylcellulose and dextran had no effect on immune suppression or cutaneous IL10 at any dose. Tamarind xyloglucans and Aloe poly/oligosaccharides also prevented suppression of immune responses to alloantigen in mice exposed to 30 kJ/m2 UVB radiation. To assess the effect of the carbohydrates on keratinocytes, murine Pam212 cells were exposed to 300 J/m2 UVB radiation and treated for 1 h with tamarind xyloglucans or Aloe poly/oligosaccharides. Treatment of keratinocytes with immunoprotective carbohydrates reduced IL‐10 production by approximately 50% compared with the cells treated with UV radiation alone and completely blocked suppressive activity of the culture supernatants in vivo. The tamarind xyloglucans also blocked UV‐activated phosphorylation of SAPK/JNK protein but had no effect on p38 phosphorylation. These results indicate that animals, like plants, may use carbohydrates to regulate responses to environmental stimuli.

Loss of Arabidopsis GAUT12/IRX8 causes anther indehiscence and leads to reduced G lignin associated with altered matrix polysaccharide deposition

GAlactUronosylTransferase12 (GAUT12)/IRregular Xylem8 (IRX8) is a putative glycosyltransferase involved in Arabidopsis secondary cell wall biosynthesis. Previous work showed that Arabidopsis irregular xylem8 (irx8) mutants have collapsed xylem due to a reduction in xylan and a lesser reduction in a subfraction of homogalacturonan (HG). We now show that male sterility in the irx8 mutant is due to indehiscent anthers caused by reduced deposition of xylan and lignin in the endothecium cell layer. The reduced lignin content was demonstrated by histochemical lignin staining and pyrolysis Molecular Beam Mass Spectrometry (pyMBMS) and is associated with reduced lignin biosynthesis in irx8 stems. Examination of sequential chemical extracts of stem walls using 2D 13C-1H Heteronuclear Single-Quantum Correlation (HSQC) NMR spectroscopy and antibody-based glycome profiling revealed a reduction in G lignin in the 1 M KOH extract and a concomitant loss of xylan, arabinogalactan and pectin epitopes in the ammonium oxalate, sodium carbonate, and 1 M KOH extracts from the irx8 walls compared with wild-type walls. Immunolabeling of stem sections using the monoclonal antibody CCRC-M138 reactive against an unsubstituted xylopentaose epitope revealed a bi-lamellate pattern in wild-type fiber cells and a collapsed bi-layer in irx8 cells, suggesting that at least in fiber cells, GAUT12 participates in the synthesis of a specific layer or type of xylan or helps to provide an architecture framework required for the native xylan deposition pattern. The results support the hypothesis that GAUT12 functions in the synthesis of a structure required for xylan and lignin deposition during secondary cell wall formation.

Oligosaccharins - Plant Regulatory Molecules

The discoveries that complex carbohydrates are tissue-specific cell-surface antigens and the receptors for hormones, toxins, bacteria, and viruses have created considerable interest in complex carbohydrates. Furthermore, the complex carbohydrate portions of some glycoprotein hormones are required for their biological activity, and the complex carbohydrates of some glycoprotein enzymes keep the enzymes stable, while the carbohydrates of some glycoproteins direct the glycoproteins to their proper locations. Moreover, the immune response to glycoproteins is often directed to their carbohydrate side chains, and the carbohydrate chains have been shown to affect the activity and residence time of glycoprotein pharmaceuticals. Still another discovery that has generated considerable interest in complex carbohydrate research is that these molecules perform regulatory functions in plants and animals, often at the level of controlling gene expression.

β-1,3-Glucans are components of brown seaweed (Phaeophyceae) cell walls.

LAMP is a cell wall-directed monoclonal antibody (mAb) that recognizes a β-(1,3)-glucan epitope. It has primarily been used in the immunolocalization of callose in vascular plant cell wall research. It was generated against a brown seaweed storage polysaccharide, laminarin, although it has not often been applied in algal research. We conducted in vitro (glycome profiling of cell wall extracts) and in situ (immunolabeling of sections) studies on the brown seaweeds Fucus vesiculosus (Fucales) and Laminaria digitata (Laminariales). Although glycome profiling did not give a positive signal with the LAMP mAb, this antibody clearly detected the presence of the β-(1,3)-glucan in situ, showing that this epitope is a constituent of these brown algal cell walls. In F. vesiculosus, the β-(1,3)-glucan epitope was present throughout the cell walls in all thallus parts; in L. digitata, the epitope was restricted to the sieve plates of the conductive elements. The sieve plate walls also stained with aniline blue, a fluorochrome used as a probe for callose. Enzymatic digestion with an endo-β-(1,3)-glucanase removed the ability of the LAMP mAb to label the cell walls. Thus, β-(1,3)-glucans are structural polysaccharides of F. vesiculosus cell walls and are integral components of the sieve plates in these brown seaweeds, reminiscent of plant callose.

Covalent Cross-Linking of Primary Cell Wall Pectic Polysaccharides is Required for Normal Plant Growth

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that is present in the primary walls of all higher plant cells. Recent research has revealed much about the structure and function of RG-II. RG-II exists in the wall predominantly as a dimer that is cross-linked by a 1:2 borate diol ester. The formation of the RG-II dimer in muro is proposed to generate a covalently cross-linked pectic network that contributes to the physical and biochemical properties of the wall. We have investigated the function of this pectic network using a dwarf Arabidopsis mutant (mur1) that synthesizes RG-II with an altered glycosyl residue composition and a dwarf Arabidopsis mutant (bor1) that is defective in root-to-shoot translocation of boron. The walls of these mutants contain reduced amounts of the RG-II dimer. The amounts of borate cross-linked RG-II in the walls and morphology of mur1 and bor1 plants sprayed with boric acid are comparable to wild-type plants. Our study demonstrates a major role for borate cross-linking of primary wall pectic polysaccharides in plant growth.