Biosynthesis of lectin in roots of germinating and adult cereal plants

Root tips of wheat, rye, barley and rice seedlings contain lectins which are identical to the respective embryo lectins with respect to their molecular weight, sugar-specificity and serological properties. Using in vivo labelling techniques, it could be demonstrated that lectin is synthesized de novo in these tissues. The presence of lectin mRNA in seedlings was confirmed by in-vitro synthesis of lectin in root-tip extracts. Lectin synthesis occurs both in primary and first adventitious roots and is confined to the apical part (2mm) of the root. As seedling development proceeds, lectin synthesis in root tips gradually decreases. Adventitious roots of adult (five to six months old) wheat, rye and barley, but not rice, plants also contain lectins which are indistinguisable from the embryo lectins by the above-mentioned criteria. These lectins are synthesized in vivo in isolated root tips (5 mm) with labelled cysteine and in vitro in cell-free extracts prepared from root tips. Synthesis of lectin in roots of adult plants is also confined to the apical (2 mm) tip of the roots. At the molecular level, root lectin synthesis is very similar to that in embryos. All root lectins are synthesized as 23 000-M_r precursors which are post-translationally converted into the mature 18 000-M_r polypeptides. The observation that seedling roots and adventitious roots of six-month-old plants actively synthesize lectins strongly indicates that lectin genes are expressed in these tissues. In addition, since the root lectins are indistinguishable from the embryo lectins, we postulate that the same lectin genes are expressed.Abbreviations ABA abscisic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Immunocytochemistry in plants with colloidal gold conjugates

Summary The chitin-binding lectin wheat germ agglutinin (WGA) is found at the periphery of wheat embryos, and a similar lectin is present at the root tips of older plants (Mishkind et al. 1982). Although a ferritin-conjugated secondary antibody is adequate for localizing WGA in embryos, native electron-opaque particles make the electron microscope identification of added label equivocal in other wheat tissues. As reported here, however, unambiguous ultrastructural localization of WGA-like lectin in adult wheat roots can be obtained with rabbit anti-WGA followed by colloidal gold-labeled goat anti-rabbit (GAR) IgG. Colloidal gold (CG) was prepared by the reduction of gold chloride with citrate, ascorbate or phosphorous. GAR IgG, prepared from serum by antigen affinity chromatograhy, was adsorbed to the gold particles to produce a stabilized suspension of GAR-CG. Localization was performed on 8–12 M frozen sections of tissue fixed in 4% paraformaldehyde, 0.3% glutaraldehyde, and 0.75% acrolein in phosphate-buffered saline containing 1M sucrose. Localization with GAR-CG was first compared to that ascertained in embryos using other probes and was then extended to the roots of adult plants. An advantage of the GARCG method is that it permits the visualization of antigen at both the light and electron microscope levels in the same section. At the light level, the anti-WGA-GAR-CG complex appears as a red stain that is localized in specific tissues of embryos and in the caps and outer layers of adult roots. Sections in which lectin was detected at the light microscope level were embedded in plastic and sectioned for subcellular examination. Electron dense gold particles indicative of WGA are found at the periphery of protein bodies in wheat embryos and in vacuoles of the roots of adult plants. Sections incubated with control IgG lack reaction product.     

Control of high affinity lectin binding to an integral membrane glycoprotein in lipid bilayers

High affinity binding of wheat germ agglutinin to glycophorin is demonstrated to be potently affected by non-specific interaction of the receptor with other protein and oligosaccharide structures present at the membrane surface. It is suggested that this may represent a significant general mechanism of receptor control.     

Multiple Hormonal Control of the Lectin Content in Roots of Wheat Seedlings

The effects of 0.2 M GA, 4.4 M benzyladenine (BA), and 5.7 M IAA on the contents of wheat germ agglutinin (WGA) and ABA in the roots of four-day-old wheat (Triticum aestivumL.) seedlings were studied. All phytohormones tested almost doubled the level of WGA. BA and IAA evidently stimulated the WGA accumulation by inducing ABA accumulation, whereas GA affected the level of the WGA in an ABA-independent way. The authors conclude that phytohormones control the WGA level via several pathways.     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Labelling of regenerating retinal pigment epithelium by colloidal iron oxide and ferritin conjugated to wheat germ agglutinin

Summary In order to determine if there are biochemical changes in plasma-membrane oligosaccharides of regenerating retinal pigment epithelium, the binding of colloidal iron oxide at low pH and ferritin-conjugated wheat germ agglutinin — probes of sialic acid and N-acetylglucosamine on the cell surface — was examined electron-microscopically. An animal model of retinal pigment epithelium regeneration — rabbits with sodium iodate induced retinopathy — was used. In this model, large expanses of regenerating pigment epithelium are present for comparison with zones of spared pgiment epithelium in the same animals. In thin sections examined by transmission electron microscopy, ferritin-conjugated wheat germ agglutinin appeared to bind more intensely to the exposed plasma membrane of regenerating retinal pigment epithelium than to spared pigment epithelium, or that of normal rabbits. Morphometry verified this. Colloidal iron oxide intensely labelled the plasma membranes of regenerating, spared, and normal pigment epithelium, and was visibly reduced after exposure of tissue to neuraminidase. The observations indicate that the plasma membrane of regenerating retinal pigment epithelium bears sialic acid and N-acetylglucosamine residues as in normal retinal pigment epithelium. However, the amount of plasma membrane bearing exposed N-acetylglucosamine increases during regeneration.     

Studies on chloroplast division in young leaf tissues of some higher plants

Summary The process of chloroplast division in young leaves of four species (bean, spinach, wheat, and maize) was investigated by light and electron microscopy. Two types of division, i.e., by fission, and by partition were observed.Chloroplast division by fission prevailed in the plant species examined, as shown by the relative abundance of dumbbell-shaped plastids, the characteristic stage in this type of division. Electron dense material, most commonly in the shape of a ring structure in the isthmus of the dividing plastid, was nearly always present in wheat and maize. Similar, but less distinct structures were usually observed in the neck region of constricted bean and spinach chloroplasts.Chloroplast division by partition was found in young leaf tissues of bean and spinach, but was not observed in wheat and maize. The main indication of this type of division is a centripetal invagination of the inner limiting membrane of the plastid envelope which progressively divides the chloroplast stroma into two, nearly equal, parts. Specific membraneous structures resembling myelin figures were usually found close to a dividing chloroplast and may participate in chloroplastokinesis.     

Lectin Analysis of Surface Saccharides in Two Trichomonas vaginalis Strains Differing in Pathogenicity*

SYNOPSIS. Surface saccharides in 2 Trichomonas vaginalis strains, the moderately pathogenic, JH34A, and the mild, JH162A, were analyzed with the aid of plant lectins. Concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and lectin from the garden pea (GPA) were employed in agglutination tests and in treatment of ultrathin sections for electron microscopy according to the horseradish peroxidase-3,3′-diaminobenzidine method. With Con A and WGA, small quantitative differences were noted between the 2 strains in the results of agglutination and in the reaction-product deposits observed by electron microscopy. Distribution of the binding sites for the 2 lectins was also somewhat different in the JH34A and JH162A trichomonads. In general, the reactions with the more pathogenic strain were slightly stronger. Although the reactions with SBA and CBA lectins were weaker than those with Con A or WGA, they provided the means for qualitative differentiation between the 2 trichomonad strains. SBA alone agglutinated the JH34A strain and formed demonstrable deposits on the cell surfaces. On the other hand, only CBA reacted with JH162A flagellates. The garden pea lectin failed to bind to the surface of either strain. On the basis of results obtained with the control preparations incubated in the presence of specific inhibitors, it was concluded that both strains had α-methyl-D-mannoside and/or α-methyl-D-mannoside-like as well as N-acetyl-D-glucosamine residues on their surfaces. In addition, JH34A strain had D-lactose-containing residues while JH162A trichomonads had residues with D-galactose. Neither strain appeared to possess residues containing N-acetyl-D-galactosamine.     

Sequence variability in three wheat germ agglutinin isolectins: Products of multiple genes in polyploid wheat

Summary Three highly homologous wheat germ isolectins (95–97%) are distinct gene products in hexaploid wheat. The amino acid sequences of two of these [wheat germ agglutinin 1 (WGA1) and 2 (WGA2)] are compared with sequence date derived from a complementary DNA (cDNA) clone for the third isolection (WGA3). This comparison includes three corrections to earlier amino acid sequences data of both WGA1 and WGA2 at positions 109 (from Ser to Phe), 134 (from Gly to Lys), and 150 (from Gly to Trp). These reassignments are based on new results from crystal structure refinement and amino acid sequence data of WGA1, as well as the recently determined nucleotide sequence of WGA3. In addition, the C-terminal residue of WGA1 has been revised to Gly 171 and now differs from WGA2 (Ala 171). Four other positions, Asn9, Ala53, Gly119, and Ser 123, at which WGA1 and WGA2 are identical but differ from the DNA sequence of WGA3, were also reinvestigated by amino acid sequencing techniques and confirmed.Variability among the three isolectins is observed at a total of 10 sequence positions: 9, 53, 56, 59, 66, 93, 109, 119, 123, and 171. Pairwise comparisons indicate that WGA3 deviates to a much larger extent from WGA1 (at eight positions) and from WGA2 (at seven positions) than the latter from one another (at five positions). Eight of the 10 mutations are equally distributed between domians B and C, the two intrior and more highly conserved of the four WGA domains (A, B, C, D). Correlation of the variable residues with the three-dimensional structure indicates that all except the two previously described B-domain residues, 56 and 59 (Wright and Olafsdottir 1986), are easily accommodated at the dimer surface.WGA3 displays a higher degree of inter-domain similarity than found in WGA1 and WGA2. Of the seven variable positions that are located in the domain core (residues 3–31), five are in perfect agreement with our earlier predicted domain ancestor sequence. This suggests that of the three isolectins WGA3 is most closely related to the common ancestral molecule.     

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank

This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

Developmental characterization of the wheat germ agglutinin binding proteins, wst31 and wst34, enriched in prestalk and stalk cells of Dictyostelium discoideum

Abstract. The onset of prestalk differentiation of Dictyostelium discoideum has been thought to be triggered by differentiation inducing factor (DIF), which is secreted by differentiating cells. We characterized the cell-type specific proteins, wst31 (prestalk and stalk specific) and wst34 (stalk specific), using the mutant HM44 which is defective in DIF-production, and examined the effects of DIF and cAMP on the formation of the proteins. In the mutant HM44, wst34 was formed only in the presence of exogenous DIF as reported for other prestalk/stalk markers (e.g. pDd63 and acid phosphatase-2), which indicates the DIF-requirement for this protein. By contrast, the accumulation of wst31 in this mutant occurred in the presence of cAMP regardless of the presence of exogenous DIF. Thus, we propose a new and distinct state (or stage) in prestalk differentiation, where the expression of wst31 occurs but not that of pDd63 or acid phosphatase-2.     

Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin-agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70+/-14 nM for CB and 12+/-3 mM for glucose binding to GLUT1, are similar to those reported earlier.     

Prespore-to-stalk conversion involves the production of a pathway-specific glycoprotein, wst25, in the cellular slime mould Dictyostelium discoideum

We have previously identified a stalk-specific wheat germ agglutinin (WGA)-binding protein, wst34, in the cellular slime mould Dictyostelium discoideum [Biochem. Cell Biol. 68 (1990) 699]. Here, we found another stalk-specific WGA-binding protein, wst25, which was detected with two antisera that recognize wst34. Using the two marker proteins, we then analyzed and compared the pathways of prestalk-to-stalk maturation and prespore-to-stalk conversion in vitro and in vivo. Prestalk cells isolated from normally formed slugs can be converted to stalk cells (designated StI) in vitro with 8-bromo-cAMP (Br-cAMP), whereas prespore cells isolated from slugs can be converted to fully vacuolated stalk cells (designated StII) in vitro with Br-cAMP and DIF-1. During the process of prespore-to-stalk conversion, prespore-specific mRNAs, D19 and 2H3, disappeared rapidly, while prestalk-specific mRNAs, ecmA and ecmB, appeared at 2h of incubation and increased thereafter. Most importantly, however, the StII cells thus formed were biochemically different from the StI cells originated from prestalk cells; that is, StI cells expressed wst34 but not wst25, while StII cells expressed wst25 but not wst34. When prespore cells isolated from slugs were allowed to develop on a substratum, they differentiated into spores and stalk cells and formed fruiting bodies, and the stalk cells formed from prespore cells in vivo expressed wst25 but not wst34. The present results indicate that there are two types of stalk cells, StI (prestalk-origin) and StII (prespore-origin), and that wst34 and wst25 are the specific markers for StI and StII, respectively.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Juxtaposition of the generative cell and vegetative nucleus in the mature pollen grain of amaryllis (Hippeastrum vitatum)

Summary Computer-generated, three-dimensional reconstructions from serial ultrathin sections were used to investigate the spatial organization and extent of association between the generative cell and vegetative nucleus within the mature pollen grain of amaryllis. In all cases examined, the highly lobed vegetative nucleus was found in close proximity and positioned laterally to the elongated, oval shaped generative cell. Numerous projections of the vegetative nucleus come to within 53 nm of the inner vegetative cell plasma membrane which surrounds the generative cell. These areas of close association may continue transversely around the generative cell for a distance of up to 4 m. Although an association exists between the generative cell and vegetative nucleus of the mature pollen grain, it is apparent that several changes must take place after pollination in order to achieve the high amount of close contact that occurs between the vegetative nucleus and the numerous terminal cell extensions of the leading sperm in the pollen tube of amaryllis (Mogensen 1986). Thus, this study demonstrates that the spatial organization among components of the male germ unit in the mature pollen grain does not necessarily reflect relationships that ultimately exist among these components within the pollen tube.     

Wheat Germ Agglutinin Inhibits the Effects of Nerve Growth Factor on the Phosphorylation of Proteins in PC12h Cells

Treatment of PC12h cells in tissue culture with nerve growth factor (NGF) led to an increased incorporation of [32P]orthophosphoric acid into specific proteins. The increased phosphorylation of 60,000-dalton and 20,000-dalton proteins in the 0.2% Triton X-100 detergent-soluble fraction, of 35,000-dalton protein in the 0.2% Triton X-100 detergent-insoluble fraction, and of slow migrating protein (SMP) in the nonhistone nuclear fraction was observed upon NGF treatment. On the other hand, wheat germ agglutinin (WGA) treatment of PC12h cells induced a slightly decreased phosphorylation of these NGF-responsive proteins. Incubation of cell-free extracts from PC12h cells with [gamma-32P]ATP led to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with NGF, the labeling of the 100,000-dalton protein was substantially and selectively reduced. In contrast, treatment of PC12h cells with WGA led to an increased phosphorylation of the 100,000-dalton protein in cell-free extracts. Thus, NGF and WGA showed opposite effects on the phosphorylation of specific proteins in both intact cells and cell-free extracts. In addition, it was also observed in both systems that pre- and posttreatment of PC12h cells with WGA abolished the effects of NGF on the phosphorylation and produced a phosphorylation pattern similar to that from PC12h cells treated only with WGA. In parent PC12 cells, it has been reported that the treatment of cells with WGA inhibits NGF binding to its receptors and converts the rapidly dissociating receptors to slowly dissociating receptors. Thus, WGA in conjunction with NGF, results in the practical disappearance of rapidly dissociating receptors on cells.(ABSTRACT TRUNCATED AT 250 WORDS)