RESUMEN
Plant cell walls display a considerable degree of diversity in their compositions and molecular architectures. In some cases the functional significance of a particular cell wall type appears to be easy to discern: secondary cells walls are often reinforced with lignin that provides durability; the thin cell walls of pollen tubes have particular compositions that enable their tip growth; lupin seed cell walls are characteristically thickened with galactan used as a storage polysaccharide. However, more frequently the evolutionary mechanisms and selection pressures that underpin cell wall diversity and evolution are unclear. For diverse green plants (chlorophytes and streptophytes) the rapidly increasing availability of transcriptome and genome data sets, the development of methods for cell wall analyses which require less material for analysis, and expansion of molecular probe sets, are providing new insights into the diversity and occurrence of cell wall polysaccharides and associated biosynthetic genes. Such research is important for refining our understanding of some of the fundamental processes that enabled plants to colonize land and to subsequently radiate so comprehensively. The study of cell wall structural diversity is also an important aspect of the industrial utilization of global polysaccharide bio-resources.
RESUMEN
BACKGROUND AND AIMS: The anatomy of Equisetum stems is characterized by the occurrence of vallecular and carinal canals. Previous studies on the carinal canals in several Equisetum species suggest that they convey water from one node to another. METHODS: Cell wall composition and ultrastructure have been studied using immunocytochemistry and electron microscopy, respectively. Serial sectioning and X-ray computed tomography were employed to examine the internode-node-internode transition of Equisetum ramosissimum. KEY RESULTS: The distribution of the LM1 and JIM20 extensin epitopes is restricted to the lining of carinal canals. The monoclonal antibodies JIM5 and LM19 directed against homogalacturonan with a low degree of methyl esterification and the CBM3a probe recognizing crystalline cellulose also bound to this lining. The xyloglucan epitopes recognized by LM15 and CCRC-M1 were only detected in this lining after pectate lyase treatment. The carinal canals, connecting consecutive rings of nodal xylem, are formed by the disruption and dissolution of protoxylem elements during elongation of the internodes. Their inner surface appears smooth compared with that of vallecular canals. CONCLUSIONS: The carinal canals in E. ramosissimum have a distinctive lining containing pectic homogalacturonan, cellulose, xyloglucan and extensin. These canals might function as water-conducting channels which would be especially important during the elongation of the internodes when protoxylem is disrupted and the metaxylem is not yet differentiated. How the molecularly distinct lining relates to the proposed water-conducting function of the carinal canals requires further study. Efforts to elucidate the spatial and temporal distribution of cell wall polymers in a taxonomically broad range of plants will probably provide more insight into the structural-functional relationships of individual cell wall components or of specific configurations of cell wall polymers.
Asunto(s)
Acuaporinas/metabolismo , Pared Celular/química , Pared Celular/ultraestructura , Equisetum/metabolismo , Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Inmunoquímica/métodos , Microscopía Electrónica/métodos , Reguladores del Crecimiento de las Plantas/metabolismo , Fenómenos Fisiológicos de las Plantas , España , Tomografía Computarizada por Rayos X/métodosRESUMEN
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.
Asunto(s)
Pared Celular/química , Pared Celular/ultraestructura , Helechos/química , Helechos/ultraestructura , Interacciones Huésped-Patógeno , Raíces de Plantas/química , Raíces de Plantas/ultraestructura , Helechos/microbiología , Técnica del Anticuerpo Fluorescente , Hongos/patogenicidad , Microscopía , Raíces de Plantas/microbiología , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND AND AIMS: Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae. METHODS: Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed. KEY RESULTS: The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100 % bootstrap value and composed of mainly epiphytic species. CONCLUSIONS: This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species.
Asunto(s)
Evolución Biológica , Helechos/citología , Helechos/genética , Raíces de Plantas/citología , Bélgica , Pared Celular/ultraestructura , Fluorescencia , Lignina/análisis , Microscopía Confocal , Filogenia , Proteínas de Plantas/genética , Raíces de Plantas/genética , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
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.
Asunto(s)
Pectinas/metabolismo , Pared Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Conformación Molecular , Pectinas/química , Plantas/genética , Plantas/metabolismo , Poligalacturonasa/genética , Poligalacturonasa/metabolismoRESUMEN
Pectic polysaccharides are a complex set of macromolecules of the primary cell wall matrix with distinct structural domains. The biosynthesis, organisation and function of these domains within cell wall matrices are poorly understood. An immersion immunofluorescence labelling technique was developed for the in-situ analysis of pectic polysaccharides at the surface of seeds and seedlings of Arabidopsis thaliana (L.) Heynh., and used to investigate the occurrence of pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) epitopes. Seed mucilage appeared to consist of two regions: a highly methyl-esterified HG was a major component throughout the mucilage, while an inner region with relatively low porosity was stabilized by calcium-based HG cross-linking. The small size and transparency of Arabidopsis roots allowed the occurrence of pectic HG and RG-I epitopes at root surfaces to be directly determined on whole-mount preparations. Pectic epitopes were not distributed evenly over root surfaces and were notably absent from lateral root apices and from the surface of root hairs. The use of defined antibody probes in the immersion immunolabelling protocol will be useful for the analysis of the influence of growth conditions and genetic factors on pectic polysaccharides in Arabidopsis.
Asunto(s)
Arabidopsis/química , Pectinas/análisis , Anticuerpos Monoclonales/farmacología , Arabidopsis/citología , Arabidopsis/metabolismo , Pared Celular/química , Epítopos/análisis , Técnica del Anticuerpo Fluorescente , Pectinas/química , Epidermis de la Planta/química , Epidermis de la Planta/citología , Raíces de Plantas/química , Raíces de Plantas/citología , Semillas/química , Semillas/citologíaRESUMEN
Cnr (colorless non-ripening) is a pleiotropic tomato (Lycopersicon esculentum) fruit ripening mutant with altered tissue properties including weaker cell-to-cell contacts in the pericarp (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). Whereas the genetic basis of the Cnr mutation is being identified by molecular analyses, here we report the identification of cell biological factors underlying the Cnr texture phenotype. In comparison with wild type, ripe-stage Cnr fruits have stronger, non-swollen cell walls (CW) throughout the pericarp and extensive intercellular space in the inner pericarp. Using electron energy loss spectroscopy imaging of calcium-binding capacity and anti-homogalacturonan (HG) antibody probes (PAM1 and JIM5) we demonstrate that maturation processes involving middle lamella HG are altered in Cnr fruit, resulting in the absence or a low level of HG-/calcium-based cell adhesion. We also demonstrate that the deposition of (1-->5)-alpha-L-arabinan is disrupted in Cnr pericarp CW and that this disruption occurs prior to fruit ripening. The relationship between the disruption of (1-->5)-alpha-L-arabinan deposition in pericarp CW and the Cnr phenotype is discussed.
Asunto(s)
Pectinas/metabolismo , Polisacáridos/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/ultraestructura , Microscopía Electrónica de Rastreo , MutaciónRESUMEN
Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.
Asunto(s)
Pared Celular/química , Pectinas/química , Pisum sativum/química , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Calcio/metabolismo , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Epítopos , Esterificación , Inmunohistoquímica , Modelos Biológicos , Proteínas de Plantas/metabolismo , Poligalacturonasa/metabolismo , Polisacárido Liasas/metabolismo , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Copper-containing amine oxidase (CuAO) has been proposed to play a role in H2O2 production in plant cell walls during cell development and in response to pathogen attack. We have compared the localisation of CuAO in pea (Pisum sativum L.), lentil (Lens culinaris M.) and chick pea (Cicer arietinum L.) grown under different light conditions, using both immuno- and histochemical techniques. The enzyme was detected by indirect immunofluorescence in the cell walls of parenchyma tissues of etiolated pea and lentil plants and was particularly abundant at intercellular spaces. Upon de-etiolation, CuAO largely disappeared from cortical cell walls except in the region of intercellular spaces. In the apical internode of light-grown seedlings, CuAO occurred mainly in cortical cell walls and, to some extent, in cell walls of xylem vessels. In both the elongation zone and mature regions of roots, CuAO was restricted to cortical cell walls and some cell junctions close to the meristem. Extensin epitopes co-localised to intercellular spaces of the cortex in de-etiolated pea, indicating that CuAO may have a role in cell wall strengthening at intercellular spaces. In chick pea, the localisation of the enzyme varied between different cultivars that have differing susceptibility to the fungus Ascochyta rabiei. In a susceptible cultivar Calia, immunogold labelling localised CuAO to cell walls of the cortex, as in lentil and pea, while in a resistant cultivar Sultano, it was most abundant in xylem vessels and, in light-grown plants, in the epidermis. These expression patterns are discussed with regard to the possible functions of amine oxidase in cell growth, cell differentiation and pathogen resistance.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Pared Celular/enzimología , Fabaceae/enzimología , Peróxido de Hidrógeno/metabolismo , Estructuras de las Plantas/enzimología , Amina Oxidasa (conteniendo Cobre)/inmunología , Amina Oxidasa (conteniendo Cobre)/efectos de la radiación , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/fisiología , División Celular/fisiología , Pared Celular/efectos de la radiación , Cicer/citología , Cicer/enzimología , Cicer/crecimiento & desarrollo , Epítopos , Fabaceae/citología , Fabaceae/crecimiento & desarrollo , Glicoproteínas/metabolismo , Inmunidad Innata , Inmunohistoquímica , Lens (Planta)/citología , Lens (Planta)/enzimología , Lens (Planta)/crecimiento & desarrollo , Luz , Pisum sativum/citología , Pisum sativum/enzimología , Pisum sativum/crecimiento & desarrollo , Enfermedades de las Plantas , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/citología , Estructuras de las Plantas/efectos de la radiación , Especificidad de la EspecieRESUMEN
The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.
Asunto(s)
Anticuerpos Monoclonales , Epítopos/análisis , Oligosacáridos/análisis , Pectinas/química , Pectinas/inmunología , Polisacáridos/análisis , Técnicas Químicas Combinatorias , Ensayo de Inmunoadsorción Enzimática/métodos , Hibridomas , Oligosacáridos/inmunología , Pectinas/análisis , Biblioteca de Péptidos , Polisacáridos/inmunologíaRESUMEN
Modifications in cell wall pectic polysaccharides are thought to influence cell-cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo- galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1-->4)-beta-D-galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26-30 days after anthesis), whereas homogalacturonan and pectic (1-->5)-alpha-L-arabinan are present in cotyledon cell walls throughout development. (1-->4)-beta-galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1-->4)-beta-galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1-->4)-beta-galactan appearance, indicated that the cotyledons with the galactan-rich cell wall layer were twice as firm as those with no detectable (1-->4)-beta-galactan.
Asunto(s)
Pared Celular/metabolismo , Cotiledón/metabolismo , Galactanos/metabolismo , Pisum sativum/metabolismo , Anticuerpos Monoclonales , Pared Celular/química , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Cotiledón/química , Cotiledón/crecimiento & desarrollo , Epítopos , Galactanos/biosíntesis , Galactanos/química , Inmunohistoquímica , Pisum sativum/química , Pisum sativum/crecimiento & desarrollo , Pectinas/química , Factores de TiempoRESUMEN
Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhydroxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. leguminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.
Asunto(s)
Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Pisum sativum/metabolismo , Rhizobium leguminosarum/metabolismo , Simbiosis/fisiología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Anticuerpos Monoclonales/farmacología , Técnica del Anticuerpo Fluorescente , Peróxido de Hidrógeno/farmacología , Hibridación in Situ , Pisum sativum/microbiología , Peroxidasa/metabolismo , Putrescina/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhizobium leguminosarum/patogenicidadRESUMEN
Scanning electron microscopic examination of intact tomato (Lycopersicon esculentum) pericarp and isolated pericarp cell walls revealed pit fields and associated radiating ridges on the inner face of cell walls. In regions of the cell wall away from pit fields, equivalent ridges occurred in parallel arrays. Treatment of isolated cell walls with a calcium chelator resulted in the loss of these ridges, indicating that they contain homogalacturonan-rich pectic polysaccharides. Immunolabeling procedures confirmed that pit fields and associated radiating ridges contained homogalacturonan. Epitopes of the side chains of pectic polysaccharides were not located in the same regions as homogalacturonan and were spatially regulated in relation to pit fields. A (1-->4)-beta-galactan epitope was absent from cell walls in regions of pit fields. A (1-->5)-alpha-arabinan epitope occurred most abundantly at the inner face of cell walls in regions surrounding the pit fields.
Asunto(s)
Pectinas/metabolismo , Polisacáridos/metabolismo , Solanum lycopersicum/metabolismo , Anticuerpos Monoclonales , Pared Celular/metabolismo , Pared Celular/ultraestructura , Epítopos/química , Solanum lycopersicum/inmunología , Solanum lycopersicum/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Inmunoelectrónica , Pectinas/química , Pectinas/inmunología , Polisacáridos/química , Polisacáridos/inmunologíaRESUMEN
Homogalacturonan (HG) is a multi-functional pectic polysaccharide of primary cell walls involved in calcium cross-linking and gel formation, and the regulation of ionic status and porosity of the cell wall matrix, and is a source of oligosaccharins functioning in development and defence. Phase display monoclonal antibodies with specificity for de-esterified stretches ('blocks') of pectic HG have been isolated from a naive phage display library without the need for immunization of animals or conjugation of an oligosaccharide to protein. These antibodies, designated PAM1 and PAM2, bind specifically to de-esterified and un-substituted HG. Assays with a series of pectins de-esterified by the action of plant or fungal pectin methyl esterases indicated that the antibodies were specific to de-esterified blocks resulting from the blockwise action of plant pectin methyl esterases. Analysis of antibody binding to a series of oligogalacturonides indicated that optimal binding required in the region of 30 de-esterified GalA residues. The recognition of such a large epitope by these antibodies allows the HG block architecture of primary cell walls to be identified and localized for the first time. Furthermore, we have demonstrated that monoclonal antibodies with high specificity and avidity to cell wall epitopes can be generated using a 'single pot' phage display approach.
Asunto(s)
Anticuerpos Monoclonales , Pared Celular/inmunología , Pectinas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Arabidopsis/química , Arabidopsis/inmunología , Bacteriófagos , Secuencia de Bases , Calcio , Pared Celular/química , Cartilla de ADN/genética , Epítopos/química , Inmunización , Datos de Secuencia Molecular , Pectinas/química , Homología de Secuencia de AminoácidoRESUMEN
We have studied the spatial distributions of arabinogalactan-protein (AGP) epitopes on the surface of maize embryogenic calli and roots using monoclonal antibodies JIM4 and MAC207. For this purpose, a new immunogold-scanning electron microscopy (SEM) method was employed which is based on liquid substitution of samples with glycerol. Using this method, we were able to show that the AGP epitopes are distributed along callus and root surfaces and they decorate filamentous structures. In callus cells, the JIM4 epitope was specifically enriched in the outer extracellular layers covering compact clusters of embryogenic meristematic callus cells. In roots, the MAC207 epitope was abundant on the root epidermal surface corresponding to the outer root pellicle, but was only occasionally found on the mucilage layer covering the root cap cells. Silver-enhanced gold particles, indicating AGP epitopes, were often linearly arranged suggesting that AGPs associate with filamentous structures both on the surface of embryogenic calli and root epidermal cells. These results indicate that AGPs are components of the outer extracellular layers and networks that cover the surface of roots and cells undergoing somatic embryogenesis.
Asunto(s)
Mucoproteínas/análisis , Proteínas de Plantas/análisis , Plantas/química , Epítopos , Técnica del Anticuerpo Fluorescente , Glicerol , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Mucoproteínas/inmunologíaRESUMEN
An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.
Asunto(s)
Inmunoensayo/métodos , Pectinas/análisis , Anticuerpos Monoclonales , Galactanos/análisisRESUMEN
A neoglycoprotein (a heptasaccharide of (1-->5)-alpha-L-linked-arabinosyl residues linked to bovine serum albumin) has been used to generate a rat monoclonal antibody specific to a linear chain of (1-->5)-alpha-L-arabinan which is a structural feature of the side chains of pectins. The antibody, designated LM6, detected 100 ng of debranched sugar beet arabinan in an immunodot binding assay and 1 microgram of commercial citrus pectin in a similar assay. Hapten inhibition studies indicated that the antibody recognized 5-6 Ara residues and 50% inhibition of antibody binding in a competitive inhibition ELISA was achieved with ca. 2ng (21 nM) of (1-->5)-alpha-L-Arabinohexaose. The antibody will be useful for the localization of arabinans in plant tissue and will have uses in the analyses of pectin structure. We report here on the localization of the arabinan epitope in lemon fruits using tissue printing.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Polisacáridos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Bovinos , Chenopodiaceae/química , Citrus/química , Citrus/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Glicoproteínas/inmunología , Masculino , Pectinas/química , Pectinas/inmunología , Polisacáridos/química , Ratas , Ratas WistarRESUMEN
To develop antibody probes for the neutral side chains of pectins, antisera were generated to a pectic galactan isolated from tomato (Lycopersicon esculentum) pericarp cell walls and to a (1[->]4)-[beta]-galactotetraose-bovine serum albumin neoglycoprotein. The use of these two antisera in immunochemical assays and immunolocalization studies indicated that they had very similar specificities. A monoclonal antibody (LM5) was isolated and characterized subsequent to immunization with the neoglycoprotein. Hapten inhibition studies revealed that the antibody specifically recognized more than three contiguous units of (1[->]4)-[beta]-galactosyl residues. The antigalactan antibody was used to immunolocalize the galactan side chains of pectin in tomato fruit pericarp and tomato petiole cell walls. Although the LM5 epitope occurs in most cell walls of the tomato fruit, it was absent from both the locular gel and the epidermal and subepidermal cells. Furthermore, in contrast to other anti-pectin antibodies, LM5 did not label the cell wall thickenings of tomato petiole collenchyma.
RESUMEN
This review covers the generation and use of antibodies to defined components of plant and algal cell walls and how these have contributed to our understanding of the spatial and developmental regulation of cell walls. Particular emphasis is placed upon the generation and characterization of monoclonal antibodies to matrix polysaccharides, extensins, and arabinogalactan-proteins of higher plants, and algal polysaccharides and glycoproteins. Immunolocalization studies are discussed in relation to the identification of molecular domains within cell walls, cell adhesion, cell differentiation, and the establishment of plant interactions with other organisms.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Células Vegetales , Proteínas de Plantas/inmunología , Polisacáridos/inmunología , Pared Celular/inmunología , Epítopos Inmunodominantes/inmunología , Desarrollo de la Planta , Proteínas de Plantas/análisis , Plantas/inmunología , Polisacáridos/análisisRESUMEN
Seedlings of Arabidopsis thaliana were germinated and grown in medium containing beta-glucosyl Yariv reagent (beta GlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of beta GlcY on the seedlings was to reduce the overall growth of both the root and the shoot. beta GlcY only accumulated in the root tissues and the reduced growth of the shoot appeared to be an indirect effect of impaired root growth. Reduced root growth was a consequence of a reduction in cell elongation during the postproliferation phase of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. beta GlcY penetrated roots as far as the endodermis and it is suggested that the interaction of beta GlcY with AGPs in the load-bearing cell layers inhibited root elongation. When beta GlcY was added to carrot suspension-cultured cells that had been induced to elongate rather than proliferate, cell elongation was inhibited. The AGP-unreactive alpha-galactosyl Yariv reagent (alpha GalY) had no biological activity in either of these systems.