Background: Carbohydrate binding modules (CBMs) contribute to the enzymatic degradation of complex polysaccharide structures. Results: New CBMs display specificity for decorated glucans through an extensive hydrophobic platform that interacts with both backbone and side chain structures. Conclusion: CBMs that bind to complexβ-glucans exploit different components of these ligands as specificity determinants. Significance: CBMs can utilize the side chains of decorated glucans as specificity determinants.© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
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Cell wall hemicellulosic polysaccharides are structurally complex and diverse. Knowledge about the synthesis of cell wall hemicelluloses and their biological roles is limited. Quantitative trait loci (QTL) mapping is a helpful tool for the dissection of complex phenotypes for gene identification. In this study, we exploited the natural variation in cell wall monosaccharide levels between a common wild rice, Yuanj, and an elite indica cultivar, Teqing, and performed QTL mapping with their introgression lines (ILs). Chemical analyses conducted on the culms of Yuanj and Teqing showed that the major alterations are found in glucose and xylose levels, which are correlated with specific hemicellulosic polymers. Glycosidic linkage examination revealed that, in Yuanj, an increase in glucose content results from a higher level of mixed linkageβ-glucan (MLG), whereas a reduction in xylose content reflects a low level of xylan backbone and a varied arabinoxylan (AX) structure. Seventeen QTLs for monosaccharides have been identified through composition analysis of the culm residues of 95 core ILs. Four major QTLs affecting xylose and glucose levels are responsible for 19 and 21% of the phenotypic variance, respectively. This study provides a unique resource for the genetic dissection of rice cell wall formation and remodeling in the vegetative organs.
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The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3;1,4)-β-d-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.
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The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15),β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region oftracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.
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Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise,in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.
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Carbohydrate-binding modules (CBMs) are a set of tools that can be used as molecular probes for studying plant cell walls and cellulose-based substrates. CBMs from enzymes of bacterial and fungal origin present a range of recognition capabilities for crystalline and amorphous cellulose. Here cellulose-directed CBMs have been used to visualize and quantify crystallinity changes in cellulose II-based polymers following NaOH treatment. Cellulose II polymers used were in the form of lyocell fibers, which are derived from eucalyptus wood pulp. The supramolecular structure, morphology, and existence of‘skin-core’ model in the fiber were examined using CBM-labeling techniques. Changes in cellulose crystallinity showed maxima at 3.33 mol dm−3 NaOH (under treatment conditions of 49 N m−1 at 25 ◦C) and 4.48 mol dm−3 NaOH (under treatment conditions of 147 N m−1 at 40 ◦C); CBM methodswere also suitable for quantifying changes within amorphous regions. Quantification of crystallinity changes using CBM labeling techniques was achieved in combination with image analysis, which was shown to reflect the same crystallinity changes as measured using ATR-FTIR methods. It was demonstrated that CBM-labeling techniques were able to validate the proposed ‘skin-core’ model of lyocell fibers, comprising a semi-permeable fiber skin and a porous core.
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Cellulose is generally found in the context of complex plant cell wall materials and mostly in association with other glycans. Cellulose-directed carbohydrate-binding modules (CBMs) can be readily adapted to a range of methods for the in situ imaging of cellulose structures within plant cell walls or other cellulose-based materials. Protocols for the preparation and selection of plant materials, their fixation and processing for preparation of sections for CBM labeling, and fluorescence imaging procedures are described. Approaches to direct methods in which CBMs are directly coupled to fluorophores and indirect methods in which staged incubations with secondary reagents are used for the fluorescence imaging of CBM binding to materials are discussed and presented.© 2012 Elsevier Inc.
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Arabinogalactan proteins (AGP) are a diverse family of proteoglycans associated with the cell surfaces of plants. AGP have been implicated in a wide variety of plant cell processes, including signaling in symbioses. This study investigates the existence of putative AGP in free-living cyanobacterial cultures of the nitrogen-fixing, filamentous cyanobacteria Nostoc punctiforme and Nostoc sp. strain LBG1 and at the symbiotic interface in the symbioses between Nostoc spp. and two host plants, the angiosperm Gunnera manicata (in which the cyanobacterium is intracellular) and the liverwort Blasia pusilla (in which the cyanobacterium is extracellular). Enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence analyses demonstrated that three AGP glycan epitopes (recognized by monoclonal antibodies LM14, MAC207, and LM2) are present in free-living Nostoc cyanobacterial species. The same three AGP glycan epitopes are present at the Gunnera-Nostoc symbiotic interface and the LM2 epitope is detected during the establishment of the Blasia-Nostoc symbiosis. Bioinformatic analysis of the N. punctiforme genome identified five putative AGP core proteins that are representative of AGP classes found in plants. These results suggest a possible involvement of AGP in cyanobacterial-plant symbioses and are also suggestive of a cyanobacterial origin of AGP.
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• Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. • Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. • Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. • This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.
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In some species, a crucial role has been demonstrated for the seed endosperm during germination. The endosperm has been shown to integrate environmental cues with hormonal networks that underpin dormancy and seed germination, a process that involves the action of cell wall remodeling enzymes (CWREs). Here, we examine the cell wall architectures of the endosperms of two related Brassicaceae, Arabidopsis (Arabidopsis thaliana) and the close relative Lepidium (Lepidium sativum), and that of the Solanaceous species, tobacco (Nicotiana tabacum). The Brassicaceae species have a similar cell wall architecture that is rich in pectic homogalacturonan, arabinan, and xyloglucan. Distinctive features of the tobacco endosperm that are absent in the Brassicaceae representatives are major tissue asymmetries in cell wall structural components that reflect the future site of radicle emergence and abundant heteromannan. Cell wall architecture of the micropylar endosperm of tobacco seeds has structural components similar to those seen in Arabidopsis and Lepidium endosperms. In situ and biomechanical analyses were used to study changes in endosperms during seed germination and suggest a role for mannan degradation in tobacco. In the case of the Brassicaceae representatives, the structurally homogeneous cell walls of the endosperm can be acted on by spatially regulated CWRE expression. Genetic manipulations of cell wall components present in the Arabidopsis seed endosperm demonstrate the impact of cell wall architectural changes on germination kinetics.
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Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth, are major repositories for photosynthetically accumulated carbon, and, in addition, impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose, hemicelluloses, and pectic polysaccharides. During the evolution of land plants, polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.
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Plant cell walls are diverse composites of complex polysaccharides. Molecular probes such as monoclonal antibodies (MABs) and carbohydrate-binding modules (CBMs) are important tools to detect and dissect cell wall structures in plant materials. We provide an account of methods that can be used to detect cell wall polysaccharide structures (epitopes) in plant materials and also describe treatments that can provide information on the masking of sets of polysaccharides that may prevent detection. These masking -phenomena may indicate potential interactions between sets of cell wall polysaccharides, and methods to uncover them are an important aspect of cell wall immunocytochemistry.
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Glycosyltransferases of the Cellulose Synthase Like D (CSLD) subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/ csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were co-expressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant.
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A range of molecular probes for cell wall polysaccharides has been used to explore the structure and location of water-extracted pectic polysaccharides occurring in fractions isolated from ginseng roots. The LM19 homogalacturonan (HG) epitope was abundant in an HG fraction and analysis of LM19 binding to a rhamnogalacturonan-I (RG-I) rich-fraction indicated that the LM19 epitope is sensitive to acetylation. A specific RG-I epitope (LM16), four arabinogalactan-protein (AGP) epitopes (LM2, LM14, JIM16, MAC207) and an extensin epitope (JIM20) were found to be abundant and co-located in several isolated polysaccharide fractions including an arabinogalactan fraction and two RG-I fractions. Detection of the RG-I, AGP and extensin epitopes identified in isolated polysaccharide fractions in sections of ginseng roots indicated that they were most abundant in secretory cavities found in the cortical regions of ginseng roots. In addition, the immunocytochemical study indicated that polysaccharide epitope masking is a widespread phenomenon in the primary cell walls of ginseng roots.
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Cell wall appositions (CWAs), formed by the deposition of extra wall material at the contact site with microbial organisms, are an integral part of the response of plants to microbial challenge. Detailed histological studies of CWAs in fern roots do not exist. Using light and electron microscopy we examined the (ultra)structure of CWAs in the outer layers of roots of Asplenium species. All cell walls studded with CWAs were impregnated with yellow-brown pigments. CWAs had different shapes, ranging from warts to elongated branched structures, as observed with scanning and transmission electron microscopy. Ultrastructural study further showed that infecting fungi grow intramurally and that they are immobilized by CWAs when attempting to penetrate intracellularly. Immunolabelling experiments using monoclonal antibodies indicated pectic homogalacturonan, xyloglucan, mannan and cellulose in the CWAs, but tests for lignins and callose were negative. We conclude that these appositions are defense-related structures made of a non-lignified polysaccharide matrix on which phenolic compounds are deposited in order to create a barrier protecting the root against infections.
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Cotton fiber cellulose is highly crystalline and oriented; when native cellulose (cellulose I) is treated with certain alkali concentrations, intermolecular hydrogen bonds are broken and Na-cellulose I is formed. At higher alkali concentrations Na-cellulose II forms, wherein intermolecular and intramolecular hydrogen bonds are broken, ultimately resulting in cellulose II polymers. Crystallinity changes in cotton fibers were observed and assigned using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and X-ray diffraction (XRD) subsequent to sodium hydroxide treatment and compared with an in situ protein-binding methodology using cellulose-directed carbohydrate-binding modules (CBMs). Crystallinity changes observed using CBM probes for crystalline cellulose (CBM2a, CBM3a) and amorphous cellulose (CBM4-1, CBM17) displayed close agreement with changes in crystallinity observed with ATR-FTIR techniques, but it is notable that crystallinity changes observed with CBMs are observed at lower NaOH concentrations (2.0 mol dm(-3)), indicating these probes may be more sensitive in detecting crystallinity changes than those calculated using FTIR indices. It was observed that the concentration of NaOH at which crystallinity changes occur as analyzed using the CBM labeling techniques are also lower than those observed using X-ray diffraction techniques. Analysis of crystallinity changes in cellulose using CBMs offers a new and advantageous method of qualitative and quantitative assessment of changes to the structure of cellulose that occur with sodium hydroxide treatment.
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We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.
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beta-1-4-Galactan has been detected in Sitka spruce [Picea sitchensis (Bong.) Carriere] by an immunofluorescence procedure. The anti-beta-1-4-galactan monoclonal antibody LM5 was found to bind to compression wood (CW) tissue as identified by cell morphology features. In cross-sections, year rings with very fine bands of galactan-containing tissue were found. Cells in these bands expressed no morphological CW features. This finding was interpreted to imply that beta-1-4-galactan synthesis is one of the first physiological reactions of CW formation. In cases where the CW character was pronounced, galactan was exclusively located in the outer cell-wall layers and was absent from the majority of the inner S2 layer.
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Pectin is a high value functional food ingredient widely used as a gelling agent and stabilizer. It is also an abundant, ubiquitous and multifunctional component of the cell walls of all land plants. Food scientists and plant scientists therefore share a common goal to better understand the structure and functionalities of pectic polymers at the molecular level. The basic properties of pectin have been known for nearly 200 years, but recently there has been tremendous progress in our understanding of the very complex fine structure of pectic polymers and pectinolytic enzymes. This has been made possible by synergies between plant and food research and by the application of a range of state-of-the-art techniques including enzymatic fingerprinting, mass spectrometry, NMR, molecular modelling, and monoclonal antibodies. With this increased knowledge comes the prospect of novel applications. Producers are beginning to develop a new generation of sophisticated designer pectins with specific functionalities. Moreover, the ability to manipulate pectin in planta would have a major impact on fruit and vegetable quality and processing, as well as on pectin production.
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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.
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Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.
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Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1(phage)) with specificity for the un-esterified regions of the homogalacturonan backbone of pectic polymers. Although phage particles are essential during mAb selection and amplification, their large size results in phage mAbs being poor probes for immunocytochemistry. In order to overcome this and to extend the utility of the PAM 1 mAb, we describe here the production of a phage-free, soluble scFv version of the PAM1 mAb (PAM1(scFv)). Using the new PAM1(scFv) probe, the occurrence of the HG epitope recognized can now be localized with high resolution within micro-domains of plant cell walls.
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The moss, Physcomitrella patens, is an established model system for the study of genetics, physiology and development. Its relatively simple developmental plan and genetic tractability make it ideal for the study of cell walls and in particular for analyses of the mechanisms governing the development and expansion of tip-growing cells.
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(.) Xylans are known to be major cellulose-linking polysaccharides in secondary cell walls in higher plants. (.) We used two monoclonal antibodies (LM10 and LM11) for a comparative immunocytochemical analysis of tissue and cell distribution of xylans in a number of taxa representative of all major tracheophyte and bryophyte lineages. (.) The results show that xylans containing the epitopes recognized by LM10 and LM11 are ubiquitous components of secondary cell walls in vascular and mechanical tissues in all present-living tracheophytes. In contrast, among the three bryophyte lineages, LM11 binding was detected in specific cell-wall layers in pseudoelaters and spores in the sporophyte of hornworts, while no binding was observed with either antibody in the gametophyte or sporophyte of liverworts and mosses. (.) The ubiquitous occurrence of xylans containing LM10 and LM11 epitopes in tracheophytes suggests that the appearance of these polysaccharides has been a pivotal event for the evolution of highly efficient vascular and mechanical tissues. LM11 binding in the sporophyte of hornworts, indicating the presence of relatively highly substituted xylans ( possibly arabinoxylans), separates these from the other bryophytes and is consistent with recent molecular data indicating a sister relationship of the hornworts with tracheophytes.
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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 immunoclot 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.
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Cell biological, structural, and genetic approaches have demonstrated the presence of arabinogalactan proteins (AGPs) in the moss Physcomitrella patens and provided evidence for their function in cell expansion and specifically in the extension of apical tip-growing cells. Inhibitor studies indicated that apical cell expansion in P. patens is blocked by synthetic AGP binding beta-glucosyl Yariv reagent (beta GlcYR). The anti-(1 ->5)-alpha-L-arabinan monoclonal antibody LM6 binds to some AGPs in P. patens, to all plasma membranes, and to the cell wall surface at the most apical region of growing protonemal filaments. Moreover, LM6 labeling of cell walls at the tips of apical cells of P. patens was abolished in the presence of beta GlcYR, suggesting that the localized movement of AGPs from the plasma membrane to the cell wall is a component of the mechanism of tip growth. Biochemical and bioinformatic analyses were used to identify seven P. patens ESTs encoding putative AGP core proteins from homology with Arabidopsis thaliana, Brassica napus, and Oryza sativa sequences and from peptide fragments isolated from beta GlcYR-precipitated AGPs. Gene knockout by homologous recombination of one of these genes, P. patens AGP1, encoding a classical AGP core protein, resulted in reduced cell lengths in protonemal filaments, indicating a role for AGP1 in apical cell expansion in P. patens.
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Pectins are a highly complex family of cell wall polysaccharides comprised of homogatacturonan (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.
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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.).
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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.
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With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.
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Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.
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