Valerie Vreeland

MINIREVIEW—POLYPHENOLS AND OXIDASES IN SUBSTRATUM ADHESION BY MARINE ALGAE AND MUSSELS

Minireviews do not have abstracts.

Analysis of Plant-Substratum Adhesives

“Adhesives are social substances. They unite materials, creating a whole that is greater than the sum of its parts” (Skeist and Miron 1990). The principles of industrial adhesives are equally applicable to plant adhesives. Adhesive bonding is studied with a multidisciplinary approach, including aspects of surface chemistry, rheology, wetting, mechanical properties and fracture mechanics (Pocius 1986). The biological dimension provides additional levels of complexity in plant adhesive formation.

Pectate distribution and esterification in Dubautia leaves and soybean nodules, studied with a fluorescent hybridization probe

Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.

Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga, Fucus

Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immunoglobulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function.

DIFFERENTIAL LOCALIZATION OF CARRAGEENAN GELLING SEQUENCES IN KAPPAPHYCUS ALVAREZII VAR. TAMBALANG (RHODOPHYTA) WITH FITC-CONJUGATED CARRAGEENAN OLIGOSACCHARIDES1

The ability of kappa (κ) and iota (ι) carrageenans to form gels is dependent upon the regular repeat of disaccharide units along the carbohydrate chain. Short, chemically‐ and enzymatically‐purified fragments of κ and ι carrageenan were conjugated to fluorescein and used as specific hybridization probes for localization of κ and ι carrageenan gelling sequences within the cells walls and intercellular matrices of Kappaphycus alvarezii (Doty) Doty. The probes label cell walls and intercellular matrices under ionic conditions appropriate for gelation of κ and ι carrageenans. The distribution of κ and ι carrageenans in the matrix and cell walls of K. alvarezii was determined with respect to cell type (epidermis, cortex, medulla, and central axis) and age. The κ‐probe labels the cell walls of all cell types except epidermal in both young and old tissues. In contrast, the ι‐probe labels the cell walls of the epidermis in both young and old tissue and the cell walls of the thylles only in old tissue. Both probes label intercellular matrix material; however, ι‐probe labelling is very much weaker than κ‐probe labelling. The results indicate that FITC‐conjugated carrageenan oligosaccharides are useful tools that provide information on gelling subunit distribution.

MONOCLONAL ANTIBODIES AS MOLECULAR MARKERS FOR THE INTRACELLULAR AND CELL WALL DISTRIBUTION OF CARRAGEENAN EPITOPES IN KAPPAPHYCUS (RHODOPHYTA) DURING TISSUE DEVELOPMENT1

Carrageenan, the major cell wall carbohydrate of certain red algae, is variable in structure and gelling properties. Sequence types include gelling (kappa and iota) and nongelling (lambda) types in addition to precursors, often in hybrid molecules containing more than one precursor and/or sequence type. Molecular markers to subunits were needed to study carrageenan synthesis, cell wall organization, and the relationship between structure and function. Monoclonal antibodies were produced to carrageenan, and their specificities were determined by competitive enzyme immunoassay. Antibodies were identified with specificities related to kappa, iota, and lambda carrageenan. The patterns of immunofluorescence localization on Kappaphycus alvarezii = Eucheuma alvarezii var. tambalang (Doty) sections were distinctive for each antibody. The antibody to a kappa‐related epitope labeled mature tissue strongly; antibodies to an iota‐related epitope and a lambda‐related epitope labeled weakly, consistent with the kappa‐enriched carrageenan produced by this alga. Kappa‐related epitopes were distributed throughout the wall and matrix, whereas iota‐related epitopes were concentrated in the middle lamella. Lambda‐related epitopes were localized primarily at the plant cuticle where kappa and iota antigens were lacking. An antibody appeared to be specific for a precursor of the gelling subunits because it showed maximal wall and intracellular labeling at the youngest developmental stage. All antibodies labeled intracellular inclusions in the transition zone between the epidermis and medulla during the development of medullary cells from the peripheral meristem in young branches. The results demonstrate the intracellular synthesis of epitopes related to all major carrageenan subunits and their differential extracellular distribution.

Assay-dependent specificity of a monoclonal antibody with alginate

Abstract Monoclonal antibody 2-8.7, prepared to carbohydrates extracted from Fucus , labelled cell-wall antigen with greater intensity after calcium alginate cross-links were disrupted. The suspected alginate specificity of this antibody was confirmed by modified enzyme-immunoassay methods. Incubation conditions were found in which antibody 2-8.7 was specific for the polyguluronate sequences of alginate. Immunofluorescence labelling by antibody 2-8.7 on Fucus germling sections confirmed specificity changes related to the antigen-antibody incubation conditions.

Identification of associating carbohydrate sequences with labelled oligosaccharides : Localization of alginate-gelling subunits in cells walls of a brown alga.

The gelling subunit of alginate, the major cell-wall polysaccharide of brown algae, was used as a molecular marker for identification of this cell-wall carbohydrate subunit at the cellular level. Short polyguluronate chains were conjugated to fluorescein and used as a probe to identify the gelling regions of alginate in tissue sections from a brown alga. The specificity of the probe for gelling subunits was demonstrated by lack of cell-wall labelling in the absence of calcium, correlation between divalent-cation binding affinities of polyguluronate with labelling intensity, and lack of labelling by fluorescein-conjugated nongelling subunits. The probe labelling-pattern also differed from sulfated fucan distribution. Extracellular matrix and cell walls were labelled on sections of vegetative blade, stipe and reproductive frond of Fucus gardneri Silva. Probe labelling was rapid, being virtually complete within 5 min. Probe labelling in seawater differed markedly from labelling at lower ionic strength and is interpreted as reflecting alginategelling properties in natural conditions. High-and low-affinity binding sites are discussed in terms of gelling-subunit length and steric availability. Fluorescein-conjugated polygalacturonate, which also forms calcium dimers, labelled extracellular alginate by formation of mixed polygalacturonate-polyguluronate dimers. Binding by the alginate hybridization probe differs from nucleic-acid hybridization in divalent-cation bridging and the lack of both a conformational transition and polymer polarity.

Application of quantitative immunoelectrophoretic analysis to the assay of the carbohydrate antigens, alginate and fucan

Abstract Quantitative immunoelectrophoretic analysis was applied to two acidic carbohydrate antigens, the major extracellular carbohydrates from brown seaweeds. These are the alginates (linear polyuronides) and the fucans (a family of branched heteropolysaccharides). They are representative of many of the types of physicochemical and immunochemical variability encountered both within and among preparations of carbohydrate antigens. Immunizing preparations of each carbohydrate contained several antigenic components. Rocket immunoelectrophoresis, inhibition of rocket immunoelectrophoresis and thin-layer gel filtration combined with crossed-line immunoelectrophoresis were employed to compare carbohydrate samples prepared in different ways and from different sources. Samples of each carbohydrate varied in immunoprecipitability, and inhibition of rocket immunoelectrophoresis was used to quantitate individual antigenic components in heterologous samples. Factors influencing the precipitability of alginates and fucans are discussed in comparison with hapten systems and other carbohydrate antigens. Possible sources of multiple antigenic components in a purified carbohydrate preparation are also discussed.