Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

<p>Abstract</p> <p>Background</p> <p>Molecular probes are required to detect cell wall polymers <it>in-situ </it>to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.</p> <p>Results</p> <p>Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types.</p> <p>Conclusion</p> <p>These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.</p

Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans of plant cell walls.

The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls. Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.This work was supported by the European Union Seventh Framework Programme (FP7 2007-2013) under the WallTraC project (Grant Agreement number 263916). (This article reflects the authors’ views only and the European Union is not liable for any use that may be made of the information contained herein). The work was also supported by the United Kingdom Biotechnology and Biological Research Council (BBSRC, Grant BB/K017489/1). JX acknowledges support from the Chinese Scholarship Council, TAT from a BBSRC studentship and MGR from the Danish Strategic Research Council and The Danish Council for Independent Research, Technology and Production Sciences as part of the GlycAct project (FI 10-093465). We acknowledge kind gifts of enzymes from Harry Gilbert and oligosaccharides from Sanna Koutaniemi. We thank Theodora Tryfona for mass spectrometry analysis.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00425-015-2375-

Comparative glycan profiling of Ceratopteris richardii 'C-Fern' gametophytes and sporophytes links cell-wall composition to functional specialization

Background and Aims: Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii 'C-Fern', a widely used model system for ferns. Methods: Microarrays of sequential diamino-cyclohexane-tetraacetic acid(CDTA) and NaOH extractions of gametophytes, spores and different organs of 'C-Fern' sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue-and cell-specific distribution of glycan epitopes. Key Results: While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. Conclusions: The differences and similarities in glycan cell-wall composition between 'C-Fern' gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte

Polyploidy Affects Plant Growth and Alters Cell Wall Composition

Polyploidization has played a key role in plant breeding and crop improvement. Although its potential to increase biomass yield is well described, the effect of polyploidization on biomass composition has largely remained unexplored. Here, we generated a series of Arabidopsis (Arabidopsis thaliana) plants with different somatic ploidy levels (2n, 4n, 6n, and 8n) and performed rigorous phenotypic characterization. Kinematic analysis showed that polyploids developed slower compared to diploids; however, tetra- and hexaploids, but not octaploids, generated larger rosettes due to delayed flowering. In addition, morphometric analysis of leaves showed that polyploidy affected epidermal pavement cells, with increased cell size and reduced cell number per leaf blade with incrementing ploidy. However, the inflorescence stem dry weight was highest in tetraploids. Cell wall characterization revealed that the basic somatic ploidy level negatively correlated with lignin and cellulose content, and positively correlated with matrix polysaccharide content (i.e. hemicellulose and pectin) in the stem tissue. In addition, higher ploidy plants displayed altered sugar composition. Such effects were linked to the delayed development of polyploids. Moreover, the changes in polyploid cell wall composition promoted saccharification yield. The results of this study indicate that induction of polyploidy is a promising breeding strategy to further tailor crops for biomass production.status: publishe

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls-1

Nitrocellulose sheets and the relative binding of LM15 determined using an enzyme-linked secondary antibody detection system. Binding intensities were quantified and the horizontal scale indicates relative binding. The result shown is representative of at least three separate glycan profiling assays.<p><b>Copyright information:</b></p><p>Taken from "Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls"</p><p>http://www.biomedcentral.com/1471-2229/8/60</p><p>BMC Plant Biology 2008;8():60-60.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409341.</p><p></p

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls-11

Absorbance units. The result shown is representative of at least three separate experiments. LM15 was used at a 100-fold dilution and hapten oligosaccharides tested at five-fold dilutions from 1 mg/ml.<p><b>Copyright information:</b></p><p>Taken from "Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls"</p><p>http://www.biomedcentral.com/1471-2229/8/60</p><p>BMC Plant Biology 2008;8():60-60.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409341.</p><p></p

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls-3

Tained with Calcofluor White showing extent of cell walls. c. LM15 binding to cell walls of nasturtium cotyledon parenchyma cells. d. Micrograph c combined with Calcofluor White fluorescence. e. CCRCM1 binding to cell walls of nasturtium cotyledon parenchyma cells. f. Micrograph e combined with Calcofluor White fluorescence. Arrowheads in a and b indicate inner cell wall. Arrowheads in c and d indicate inner edge of abundant LM15 immunolabelling. Arrowheads in e and f indicate inner cell wall and region of CCRCM1 immunolabelling. Scale = 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls"</p><p>http://www.biomedcentral.com/1471-2229/8/60</p><p>BMC Plant Biology 2008;8():60-60.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409341.</p><p></p

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls-4

Showing all cell walls. c. LM15 binding to an equivalent section. The antibody binds most strongly to distal region of protoxylem (arrowheads). d. An equivalent section to c that had been pre-treated with pectate lyase to remove pectic HG. LM15 binds strongly to epidermal and parenchyma cell walls. e. Section immunolabelled with pectic HG probe JIM5. f. Equivalent section to e pre-treated with pectate lyase indicates that the JIM5 epitope had been abolished. Arrows indicate inner edge of pith parenchyma. Dotted shaft arrows indicate junction between non-sclerifed parenchyma cell walls with sclerified parenchyma/fibre cell walls. cp = cortical parenchyma, pp = pith parenchyma, ep = epidermis, vb = vascular bundle, pf = phloem fibre bundle. Scale = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls"</p><p>http://www.biomedcentral.com/1471-2229/8/60</p><p>BMC Plant Biology 2008;8():60-60.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409341.</p><p></p