RESUMO
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.
Assuntos
Hordeum , Anticorpos Monoclonais/metabolismo , Carboidratos , Epitopos/metabolismo , Exsudatos e Transudatos , Hordeum/genética , Hordeum/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Polímeros/metabolismo , Polissacarídeos/metabolismo , Solo/químicaRESUMO
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.
Assuntos
Afídeos/patogenicidade , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Pectinas/metabolismo , Animais , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de PlantasRESUMO
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.
Assuntos
Raízes de Plantas/metabolismo , Polissacarídeos/metabolismo , Solo , Triticum/metabolismo , Zea mays/metabolismo , Glucanos/metabolismo , Xilanos/metabolismoRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Enzimas/genética , Frutas/fisiologia , Pectinas/metabolismo , Solanum lycopersicum/fisiologia , Parede Celular/química , Parede Celular/metabolismo , Enzimas/metabolismo , Esterificação , Galactanos/genética , Galactanos/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Solanum lycopersicum/genética , Mutação , Pectinas/genética , Pectinas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Beta vulgaris/metabolismo , Galactanos/metabolismo , Poaceae/metabolismo , Anticorpos Monoclonais , Arabidopsis/citologia , Beta vulgaris/citologia , Parede Celular/metabolismo , Epitopos , Galactanos/química , Galactanos/imunologia , Fenômenos Mecânicos , Análise em Microsséries , Microscopia de Força Atômica , Floema/citologia , Floema/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/metabolismo , Poaceae/citologiaRESUMO
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.
Assuntos
Glucanos/metabolismo , Plantas/metabolismo , Solo/química , Xilanos/metabolismo , Álcalis/química , Carbono/análise , Glucanos/ultraestrutura , Compostos Orgânicos/análise , Xilanos/ultraestruturaRESUMO
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.
Assuntos
Epitopos/química , Fragaria/metabolismo , Frutas/metabolismo , Galactanos/metabolismo , Solanum lycopersicum/metabolismo , Celulose/metabolismo , Fragaria/genética , Frutas/genética , Galactanos/genética , Solanum lycopersicum/genética , Pectinas/metabolismoRESUMO
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.
Assuntos
Parede Celular/metabolismo , Fucus/metabolismo , Sementes/metabolismo , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Fucus/crescimento & desenvolvimento , Germinação/fisiologia , Sementes/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Anticorpos Monoclonais/metabolismo , Parede Celular/metabolismo , Gleiquênias/classificação , Gleiquênias/metabolismo , Especificidade de Órgãos , Filogenia , Polissacarídeos/metabolismo , Epitopos/metabolismo , Gleiquênias/citologia , Técnica Indireta de Fluorescência para Anticorpo , Galactanos/metabolismo , Glucanos , Mananas/metabolismo , Análise em Microsséries , Pectinas/metabolismo , Floema/metabolismo , Extratos Vegetais/metabolismo , Polissacarídeo-Liases/metabolismo , XilanosRESUMO
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.
Assuntos
Parede Celular/metabolismo , Grão Comestível/metabolismo , Oryza/metabolismo , Polissacarídeos/metabolismo , Triticum/metabolismo , Grão Comestível/crescimento & desenvolvimento , Endosperma/metabolismo , Imunofluorescência , Oryza/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Anticorpos Monoclonais/análise , Parede Celular/metabolismo , Células Vegetais/metabolismo , Polissacarídeos/metabolismo , Anticorpos Monoclonais/metabolismo , Pectinas/metabolismo , Xilanos/metabolismoRESUMO
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.
Assuntos
Parede Celular , Análise em Microsséries , Plantas , Polissacarídeos , Parede Celular/química , Parede Celular/metabolismo , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Plantas/química , Plantas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Parede Celular/química , Pectinas/metabolismo , Pentosiltransferases/metabolismo , Polissacarídeos/metabolismo , Anticorpos Monoclonais , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Fenômenos Biomecânicos , Epitopos , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inflorescência/química , Inflorescência/citologia , Inflorescência/genética , Inflorescência/fisiologia , Mutação , Especificidade de Órgãos , Pentosiltransferases/genética , Epiderme Vegetal/química , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Epiderme Vegetal/fisiologia , Caules de Planta/química , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/fisiologia , Plantas Geneticamente Modificadas , Polissacarídeos/imunologia , Plântula/química , Plântula/citologia , Plântula/genética , Plântula/fisiologia , Estresse MecânicoRESUMO
Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.
Assuntos
Parede Celular/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Celulose/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/metabolismo , Pisum sativum/metabolismo , Pectinas/metabolismo , Polissacarídeo-Liases/metabolismo , Polissacarídeos/metabolismo , Nicotiana/metabolismo , Triticum/metabolismo , Xilanos/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/microbiologia , Botrytis/fisiologia , Parede Celular/metabolismo , Imunidade Inata/imunologia , Mutação/genética , Doenças das Plantas/imunologia , Acetilação , Adaptação Fisiológica , Alelos , Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , DNA Bacteriano/genética , Epitopos/imunologia , Proteínas Fúngicas/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Mutagênese Insercional/genética , Proteínas Mutantes/isolamento & purificação , Pectinas/metabolismo , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Transporte Proteico , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Xilanos/metabolismoRESUMO
Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ácido Fólico/metabolismo , Peptídeo Sintases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Homeostase , Isoenzimas/genética , Isoenzimas/metabolismo , Família Multigênica , Ácido Pantotênico , Pectinas/metabolismo , Peptídeo Sintases/genética , Fenótipo , Sementes/enzimologia , SacaroseRESUMO
How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-ß-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.
Assuntos
Parede Celular/metabolismo , Mananas/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Esterificação , Técnica Direta de Fluorescência para Anticorpo , Magnoliopsida , Masculino , Pectinas/metabolismo , Pinus , Ratos , Ratos Wistar , beta-Manosidase/metabolismoAssuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Polissacarídeos/metabolismo , Proteínas de Arabidopsis/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação ProteicaRESUMO
Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of beta-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.
Assuntos
Anticorpos Monoclonais/metabolismo , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Beta vulgaris/metabolismo , Parede Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/metabolismo , Glicosídeo Hidrolases/metabolismo , Microscopia de Fluorescência , Polissacarídeos/imunologia , RatosRESUMO
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.