RESUMEN
Root exudates and rhizosheaths of attached soil are important features of growing roots. To elucidate factors involved in rhizosheath formation, wild-type (WT) barley (Hordeum vulgare L. cv. Pallas) and a root hairless mutant, bald root barley (brb), were investigated with a combination of physiological, biochemical, and immunochemical assays. When grown in soil, WT barley roots bound â¼5-fold more soil than brb per unit root length. High molecular weight (HMW) polysaccharide exudates of brb roots had less soil-binding capacity than those of WT root exudates. Carbohydrate and glycan monoclonal antibody analyses of HMW polysaccharide exudates indicated differing glycan profiles. Relative to WT plants, root exudates of brb had reduced signals for arabinogalactan-protein (AGP), extensin, and heteroxylan epitopes. In contrast, the root exudate of 2-week-old brb plants contained â¼25-fold more detectable xyloglucan epitope relative to WT. Root system immunoprints confirmed the higher levels of release of the xyloglucan epitope from brb root apices and root axes relative to WT. Epitope detection with anion-exchange chromatography indicated that the increased detection of xyloglucan in brb exudates was due to enhanced abundance of a neutral polymer. Conversely, brb root exudates contained decreased amounts of an acidic polymer, with soil-binding properties, containing the xyloglucan epitope and glycoprotein and heteroxylan epitopes relative to WT. We, therefore, propose that, in addition to physically structuring soil particles, root hairs facilitate rhizosheath formation by releasing a soil-binding polysaccharide complex.
Asunto(s)
Hordeum , Anticuerpos Monoclonales/metabolismo , Carbohidratos , Epítopos/metabolismo , Exudados y Transudados , Hordeum/genética , Hordeum/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Polímeros/metabolismo , Polisacáridos/metabolismo , Suelo/químicaRESUMEN
Plant and algal 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 these materials. We provide an account of methods that can be used to detect cell wall polysaccharide structures (epitopes) in plant and marine algal materials and also describe treatments that can provide information on the masking 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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Organismos Acuáticos/química , Arabidopsis/química , Pared Celular/química , Polisacáridos/análisis , Pared Celular/ultraestructura , Laminaria/química , Proteínas Recombinantes/metabolismo , Resinas de Plantas/química , Fijación del Tejido , Ceras/químicaRESUMEN
Rhizosheaths function in plant-soil interactions, and are proposed to form due to a mix of soil particle entanglement in root hairs and the action of adhesive root exudates. The soil-binding factors released into rhizospheres to form rhizosheaths have not been characterised. Analysis of the high-molecular-weight (HMW) root exudates of both wheat and maize plants indicate the presence of complex, highly branched polysaccharide components with a wide range of galactosyl, glucosyl and mannosyl linkages that do not directly reflect cereal root cell wall polysaccharide structures. Periodate oxidation indicates that it is the carbohydrate components of the HMW exudates that have soil-binding properties. The root exudates contain xyloglucan (LM25), heteroxylan (LM11/LM27) and arabinogalactan-protein (LM2) epitopes, and sandwich-ELISA evidence indicates that, in wheat particularly, these can be interlinked in multi-polysaccharide complexes. Using wheat as a model, exudate-binding monoclonal antibodies have enabled the tracking of polysaccharide release along root axes of young seedlings, and their presence at root hair surfaces and in rhizosheaths. The observations indicate that specific root exudate polysaccharides, distinct from cell wall polysaccharides, are adhesive factors secreted by root axes, and that they contribute to the formation and stabilisation of cereal rhizosheaths.
Asunto(s)
Raíces de Plantas/metabolismo , Polisacáridos/metabolismo , Suelo , Triticum/metabolismo , Zea mays/metabolismo , Glucanos/metabolismo , Xilanos/metabolismoRESUMEN
Because they suck phloem sap and act as vectors for phytopathogenic viruses, aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has been described as a defensive element for plants during infections with phytopathogens. However, its role during aphid infestation remains unexplored. Using immunofluorescence assays and biochemical approaches, the HG methylesterification status and associated modifying enzymes during the early stage of Arabidopsis (Arabidopsis thaliana) infestation with the green peach aphid (Myzus persicae) were analyzed. Additionally, the influence of pectin methylesterase (PME) activity on aphid settling and feeding behavior was evaluated by free choice assays and the Electrical Penetration Graph technique, respectively. Our results revealed that HG status and HG-modifying enzymes are significantly altered during the early stage of the plant-aphid interaction. Aphid infestation induced a significant increase in total PME activity and methanol emissions, concomitant with a decrease in the degree of HG methylesterification. Conversely, inhibition of PME activity led to a significant decrease in the settling and feeding preference of aphids. Furthermore, we demonstrate that the PME inhibitor AtPMEI13 has a defensive role during aphid infestation, since pmei13 mutants are significantly more susceptible to M. persicae in terms of settling preference, phloem access, and phloem sap drainage.
Asunto(s)
Áfidos/patogenicidad , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/parasitología , Pectinas/metabolismo , Animales , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and ß-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to ß-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.
Asunto(s)
Sistemas CRISPR-Cas , Enzimas/genética , Frutas/fisiología , Pectinas/metabolismo , Solanum lycopersicum/fisiología , Pared Celular/química , Pared Celular/metabolismo , Enzimas/metabolismo , Esterificación , Galactanos/genética , Galactanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Solanum lycopersicum/genética , Mutación , Pectinas/genética , Pectinas/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Antibody-based approaches have been used to study cell wall architecture and modifications during the ripening process of two important fleshy fruit crops: tomato and strawberry. Cell wall polymers in both unripe and ripe fruits have been sequentially solubilized and fractions analyzed with sets of monoclonal antibodies focusing on the pectic polysaccharides. We demonstrate the specific detection of the LM26 branched galactan epitope, associated with rhamnogalacturonan-I, in cell walls of ripe strawberry fruit. Analytical approaches confirm that the LM26 epitope is linked to sets of rhamnogalacturonan-I and homogalacturonan molecules. The cellulase-degradation of cellulose-rich residues that releases cell wall polymers intimately linked with cellulose microfibrils has been used to explore aspects of branched galactan occurrence and galactan metabolism. In situ analyses of ripe strawberry fruits indicate that the LM26 epitope is present in all primary cell walls and also particularly abundant in vascular tissues. The significance of the occurrence of branched galactan structures in the side chains of rhamnogalacturonan-I pectins in the context of ripening strawberry fruit is discussed.
Asunto(s)
Epítopos/química , Fragaria/metabolismo , Frutas/metabolismo , Galactanos/metabolismo , Solanum lycopersicum/metabolismo , Celulosa/metabolismo , Fragaria/genética , Frutas/genética , Galactanos/genética , Solanum lycopersicum/genética , Pectinas/metabolismoRESUMEN
A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.
Asunto(s)
Arabidopsis/metabolismo , Beta vulgaris/metabolismo , Galactanos/metabolismo , Poaceae/metabolismo , Anticuerpos Monoclonales , Arabidopsis/citología , Beta vulgaris/citología , Pared Celular/metabolismo , Epítopos , Galactanos/química , Galactanos/inmunología , Fenómenos Mecánicos , Análisis por Micromatrices , Microscopía de Fuerza Atómica , Floema/citología , Floema/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Tallos de la Planta/citología , Tallos de la Planta/metabolismo , Poaceae/citologíaRESUMEN
Soil is a crucial component of the biosphere and is a major sink for organic carbon. Plant roots are known to release a wide range of carbon-based compounds into soils, including polysaccharides, but the functions of these are not known in detail. Using a monoclonal antibody to plant cell wall xyloglucan, we show that this polysaccharide is secreted by a wide range of angiosperm roots, and relatively abundantly by grasses. It is also released from the rhizoids of liverworts, the earliest diverging lineage of land plants. Using analysis of water-stable aggregate size, dry dispersion particle analysis and scanning electron microscopy, we show that xyloglucan is effective in increasing soil particle aggregation, a key factor in the formation and function of healthy soils. To study the possible roles of xyloglucan in the formation of soils, we analysed the xyloglucan contents of mineral soils of known age exposed upon the retreat of glaciers. These glacial forefield soils had significantly higher xyloglucan contents than detected in a UK grassland soil. We propose that xyloglucan released from plant rhizoids/roots is an effective soil particle aggregator and may, in this role, have been important in the initial colonization of land.
Asunto(s)
Glucanos/metabolismo , Plantas/metabolismo , Suelo/química , Xilanos/metabolismo , Álcalis/química , Carbono/análisis , Glucanos/ultraestructura , Compuestos Orgánicos/análisis , Xilanos/ultraestructuraRESUMEN
Plant cell wall glycans are important polymers that are crucial to plant development and serve as an important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily on monoclonal antibodies (mAbs) for localization and visualization of glycans, using e.g. immunofluorescent microscopy. Here, we describe the detailed epitope mapping of the mAb LM5 that is shown to bind to a minimum of three sugar residues at the non-reducing end of linear beta-1,4-linked galactan. The study uses de novo synthetic analogues of galactans combined with carbohydrate microarray and competitive inhibition ELISA for analysis of antibody-carbohydrate interactions.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Pared Celular/química , Epítopos/química , Galactanos/química , Galactosa/química , Oligosacáridos/metabolismo , Pectinas/química , Anticuerpos Monoclonales/química , Oligosacáridos/químicaRESUMEN
Zygotes from Fucus species have been used extensively to study cell polarization and rhizoid outgrowth, and in this model system cell wall deposition aligns with the establishment of polarity. Monoclonal antibodies are essential tools for the in situ analysis of cell wall glycans, and here we report the characteristics of six monoclonal antibodies to alginates (BAM6-BAM11). The use of these, in conjunction with monoclonal antibodies to brown algal sulfated fucans, has enabled the study of the developmental dynamics of the Fucus zygote cell walls. Young zygotes are spherical and all alginate epitopes are deposited uniformly following cellulose deposition. At germination, sulfated fucans are secreted in the growing rhizoid wall. The redistribution of cell wall epitopes was investigated during treatments that cause reorientation of the growth axis (change in light direction) or disrupt rhizoid development (arabinogalactan-protein-reactive Yariv reagent). Alginate modeling was drastically impaired in the latter, and both treatments cause a redistribution of highly sulfated fucan epitopes. The dynamics of cell wall glycans in this system have been visualized in situ for the first time, leading to an enhanced understanding of the early developmental mechanisms of Fucus species. These sets of monoclonal antibodies significantly extend the available molecular tools for brown algal cell wall studies.
Asunto(s)
Pared Celular/metabolismo , Fucus/metabolismo , Semillas/metabolismo , Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Fucus/crecimiento & desarrollo , Germinación/fisiología , Semillas/crecimiento & desarrolloRESUMEN
MAIN CONCLUSION: 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.
Asunto(s)
Anticuerpos Monoclonales/análisis , Pared Celular/metabolismo , Células Vegetales/metabolismo , Polisacáridos/metabolismo , Anticuerpos Monoclonales/metabolismo , Pectinas/metabolismo , Xilanos/metabolismoAsunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Polisacáridos/metabolismo , Proteínas de Arabidopsis/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación ProteicaRESUMEN
BACKGROUND: While it is kno3wn that complex tissues with specialized functions emerged during land plant evolution, it is not clear how cell wall polymers and their structural variants are associated with specific tissues or cell types. Moreover, due to the economic importance of many flowering plants, ferns have been largely neglected in cell wall comparative studies. RESULTS: To explore fern cell wall diversity sets of monoclonal antibodies directed to matrix glycans of angiosperm cell walls have been used in glycan microarray and in situ analyses with 76 fern species and four species of lycophytes. All major matrix glycans were present as indicated by epitope detection with some variations in abundance. Pectic HG epitopes were of low abundance in lycophytes and the CCRC-M1 fucosylated xyloglucan epitope was largely absent from the Aspleniaceae. The LM15 XXXG epitope was detected widely across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection in ground tissues. Mannan epitopes were found to be associated with the development of mechanical tissues. We provided the first evidence for the presence of MLG in leptosporangiate ferns. CONCLUSIONS: The data sets indicate that cell wall diversity in land plants is multifaceted and that matrix glycan epitopes display complex spatio-temporal and phylogenetic distribution patterns that are likely to relate to the evolution of land plant body plans.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Pared Celular/metabolismo , Helechos/clasificación , Helechos/metabolismo , Especificidad de Órganos , Filogenia , Polisacáridos/metabolismo , Epítopos/metabolismo , Helechos/citología , Técnica del Anticuerpo Fluorescente Indirecta , Galactanos/metabolismo , Glucanos , Mananos/metabolismo , Análisis por Micromatrices , Pectinas/metabolismo , Floema/metabolismo , Extractos Vegetales/metabolismo , Polisacárido Liasas/metabolismo , XilanosRESUMEN
Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.
Asunto(s)
Phaeophyceae/química , Polisacáridos/aislamiento & purificación , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Pared Celular/metabolismo , Cromatografía por Intercambio Iónico , Mapeo Epitopo , Masculino , Phaeophyceae/clasificación , Polisacáridos/química , Ratas , Ratas Wistar , SalinidadRESUMEN
Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.
Asunto(s)
Pared Celular/metabolismo , Grano Comestible/metabolismo , Oryza/metabolismo , Polisacáridos/metabolismo , Triticum/metabolismo , Grano Comestible/crecimiento & desarrollo , Endospermo/metabolismo , Técnica del Anticuerpo Fluorescente , Oryza/crecimiento & desarrollo , Triticum/crecimiento & desarrolloRESUMEN
Little is known of the dynamics of plant cell wall matrix polysaccharides in response to the impact of mechanical stress on plant organs. The capacity of the imposition of a mechanical stress (periodic brushing) to reduce the height of the inflorescence stem of Arabidopsis thaliana seedlings has been used to study the role of pectic arabinans in the mechanical properties and stress responsiveness of a plant organ. The arabinan-deficient-1 (arad1) mutation that affects arabinan structures in epidermal cell walls of inflorescence stems is demonstrated to reduce the impact on inflorescence stem heights caused by mechanical stress. The arabinan-deficient-2 (arad2) mutation, that does not have detectable impact on arabinan structures, is also shown to reduce the impact on stem heights caused by mechanical stress. The LM13 linear arabinan epitope is specifically detected in epidermal cell walls of the younger, flexible regions of inflorescence stems and increases in abundance at the base of inflorescence stems in response to an imposed mechanical stress. The strain (percentage deformation) of stem epidermal cells in the double mutant arad1 × arad2 is lower in unbrushed plants than in wild-type plants, but rises to wild-type levels in response to brushing. The study demonstrates the complexity of arabinan structures within plant cell walls and also that their contribution to cell wall mechanical properties is a factor influencing responsiveness to mechanical stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Pared Celular/química , Pectinas/metabolismo , Pentosiltransferasa/metabolismo , Polisacáridos/metabolismo , Anticuerpos Monoclonales , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Fenómenos Biomecánicos , Epítopos , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Inflorescencia/química , Inflorescencia/citología , Inflorescencia/genética , Inflorescencia/fisiología , Mutación , Especificidad de Órganos , Pentosiltransferasa/genética , Epidermis de la Planta/química , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Epidermis de la Planta/fisiología , Tallos de la Planta/química , Tallos de la Planta/citología , Tallos de la Planta/genética , Tallos de la Planta/fisiología , Plantas Modificadas Genéticamente , Polisacáridos/inmunología , Plantones/química , Plantones/citología , Plantones/genética , Plantones/fisiología , Estrés MecánicoRESUMEN
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.
Asunto(s)
Pared Celular , Análisis por Micromatrices , Plantas , Polisacáridos , Pared Celular/química , Pared Celular/metabolismo , Análisis por Micromatrices/instrumentación , Análisis por Micromatrices/métodos , Plantas/química , Plantas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Pared Celular/química , Plantas/química , Polisacáridos/análisis , Inmunohistoquímica , Microscopía Fluorescente , MicrotomíaRESUMEN
Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/microbiología , Botrytis/fisiología , Pared Celular/metabolismo , Inmunidad Innata/inmunología , Mutación/genética , Enfermedades de las Plantas/inmunología , Acetilación , Adaptación Fisiológica , Alelos , Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , ADN Bacteriano/genética , Epítopos/inmunología , Proteínas Fúngicas/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Mutagénesis Insercional/genética , Proteínas Mutantes/aislamiento & purificación , Pectinas/metabolismo , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Xilanos/metabolismoRESUMEN
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.