Immunocytochemical localization of wheat germ agglutinin in wheat

Immunocytological techniques were developed to localize the plant lectin, wheat germ agglutinin (WGA), in the tissues and cells of wheat plants. In a previous study we demonstrated with a radioimmunoassay that the lectin is present in wheat embryos and adult plants both in the roots and at the base of the stem. We have now found, using rhodamine, peroxidase, and ferritin-labeled secondary antibodies, that WGA is located in cells and tissues that establish direct contact with the soil during germination and growth of the plant In the embryo, WGA is found in the surface layer of the radicle, the first adventitious roots, the coleoptile, and the scutellum. Although found throughout the coleorhiza and epiblast, it is at its highest levels within the cells at the surface of these organs. In adult plants, WGA is located only in the caps and tips of adventitious roots. Reaction product for WGA was not visualized in embryonic or adult leaves or in other tissues of adult plants. At the subcellular level, WGA is located at the periphery of protein bodies, within electron-translucent regions of the cytoplasm, and at the cell wall-protoplast interface. Since WGA is found at potential infection sites and is known to have fungicidal properties, it may function in the defense against fungal pathogens.     

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     

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     

Wheat lectin as a factor in plant-microbial communication and a stress response protein

Wheat lectin (wheat germ agglutinin, WGA), a representative of a broad group of cereal lectins, is excreted by plant roots into the surrounding medium and interacts with both pathogenic microflora and growth-stimulating rhizobacteria. WGA was found to serve as a molecular signal for the rhizobacterium Azospirillum brasilense, which forms endophytic and associative symbioses with wheat plants. The bacterial response to the lectin was pleiotropic: WGA at concentrations from 10^?10 to 10^?6 M exerted a dose-dependent effect on a range of processes in the bacterium that are important for the establishment and functioning of symbiosis. Plants with different WGA content differed in their responses to severe nitrogen starvation and to seed treatment with Azospirillum.     

Stress-induced accumulation of wheat germ agglutinin and abscisic Acid in roots of wheat seedlings

Wheat germ agglutinin (WGA) levels in roots of 2-day-old wheat seedlings increased up to three-fold when stressed by air-drying. Similar results were obtained when seedling roots were incubated either in 0.5 molar mannitol or 180 grams per liter polyethylene glycol 6000, with a peak level of WGA after 5 hours of stress. Longer periods of osmotic treatment resulted in a gradual decline of WGA in the roots. Since excised wheat roots incorporate more [³⁵S]cysteine into WGA under stress conditions, the observed increase of lectin levels is due to de novo synthesis. Measurement of abscisic acid (ABA) levels in roots of control and stressed seedlings indicated a 10-fold increase upon air-drying. Similarly, a five- and seven-fold increase of ABA content of seedling roots was found after 2 hours of osmotic stress by polyethylene glycol 6000 and mannitol, respectively. Finally, the stress-induced increase of WGA in wheat roots could be inhibited by growing seedlings in the presence of fluridone, an inhibitor of ABA synthesis. These results indicate that roots of water-stressed wheat seedlings (a) contain more WGA as a result of an increased de novo synthesis of this lectin, and (b) exhibit higher ABA levels. The stress-induced increase of lectin accumulation seems to be under control of ABA.     

Wheat lectin as a factor in plant-microbial communication and a stress response protein

Wheat lectin (wheat germ agglutinin, WGA), a representative of a broad group of cereal lectins, is excreted by plant roots into the surrounding medium and interacts with both pathogenic microflora and growth-stimulating rhizobacteria. WGA was found to serve as a molecular signal for the rhizobacterium Azospirillum brasilense, which forms endophytic and associative symbioses with wheat plants. The bacterial response to the lectin was pleiotropic: WGA at concentrations from 10(-10) to 10(-6) M exerted a dose-dependent effect on a range of processes in the bacterium that are important for the establishment and functioning of symbiosis. Plants with different WGA content differed in their responses to severe nitrogen starvation and to seed treatment with Azospirillum.     

Retrograde tracing of neural pathways with a protein gold complex. II. Electron microscopic demonstration of projections and collaterals

This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGA*HRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGA*HRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.     

Abscisic Acid Control of Lectin Accumulation in Wheat Seedlings and Callus Cultures : Effects of Exogenous ABA and Fluridone

Wheat germ agglutinin is found in wheat embryos and a similar lectin is present in the roots of older plants. We report here that 10 micromolar abscisic acid (ABA) produces an average two to three-fold enhancement in the amount of lectin in the shoot base and the terminal portion of the root system of hydroponically grown wheat seedlings. Although ABA stunts seedling growth, a similar growth inhibition produced by ancymidol is not accompanied by elevated lectin levels. To further clarify the role of ABA, wheat callus cultures were employed. Callus derived from immature embryos was grown on growth medium containing various combinations of ABA and 2,4-dichlorophenoxyacetic acid. Those grown in the presence of 10 micromolar ABA exhibit the largest increases in lectin compared to material grown on other regimes. The involvement of ABA in lectin accumulation was further probed with fluridone, an inhibitor of carotenoid synthesis which has also been linked to depressed levels of endogenous ABA. Wheat seedlings grown in the presence of 1 or 10 milligrams per liter fluridone have few or no carotenoids, and wheat germ agglutinin levels in the shoot base and roots are lower compared to controls. The greatest effect (a 39% reduction in the shoot base) is produced at an herbicide concentration of 10 milligrams per liter. Exogenous 10 micromolar ABA greatly stimulates lectin accumulation in the presence of fluridone, but the levels are not as high as those produced by ABA alone. These results indicate that lectin synthesis is under ABA control in both wheat embryos and adult plants.     

The Role of Wheat Germ Agglutinin in Plant–Bacteria Interactions: A Hypothesis and the Evidence in Its Support

Wheat plants are known to develop the associative symbiosis with the rhizobacterium Azospirillum brasilense.We studied the interaction of a lectin, wheat germ agglutinin (WGA), which is also found in wheat roots, with A. brasilense, strain sp245. When added to the azospirillum culture to the final concentration of 10^–8to 10^–9M, WGA enhanced IAA production, dinitrogen fixation, and ammonium excretion by bacterial cells. WGA also promoted the synthesis of proteins, both new and those already present in bacterial cells. The hypothesis that WGA is a signal molecule rerouting the bacterial metabolism in the direction favorable for the growth and development of the host plant has been put forward. It is suggested that signal properties of WGA are the basis for one of the functions of this lectin and essential for the effective associative symbiosis.     

Wheat germ agglutinin in wheat seedling roots: induction by elicitors and fungi

Summary Treatment of wheat (Triticum aestivum L.) seedlings with elicitors originating from either plant or fungal cell walls induces about a 2-fold increase of wheat germ agglutinin (WGA) in the roots. While the WGA content in roots of healthy plants normally decreases as a function of germination time, a transient accumulation of WGA could be observed in plants challenged with different fungi, including Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa. Peak levels in challenged roots were 2 to 5 times as high as in control plants. Most of this induced WGA could be released from the roots by soaking them in a solution of the hapten N-acetylglucosamine. On the basis of the results obtained it is postulated that WGA may be involved in the defence of wheat against fungal attack.     

Lectin levels in tissues of cultured immature wheat embryos

Levels of wheat germ agglutinin have been determined by radioimmunoassay in tissues of immature wheat embryos cultured under different conditions in order to determine the suitability of the lectin as a marker for somatic embryogenesis. Embryos cultured on media favouring continued embryo development accumulated lectin in a similar manner to zygotic embryos in planta unless precocious germination occurred. Embryos cultured on media containing 2,4-D produced callus, and some of this developed somatic embryos. Both embryogenic and non-embryogenic callus contained WGA, that in non-embryogenic callus possibly arising from developmentally arrested root primordia.Abbreviations ABA abscisic acid - dpa days post anthesis - PBS phosphate buffered saline, (10 mM KH₂PO₄ K₂HPO₄, 145 mM NaCl, pH 7.4) - RIA radioimmunoassay - WGA wheat germ agglutinin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Timing, localization, and control of wheat germ agglutinin synthesis in developing wheat embryos

The lectin, wheat germ agglutinin (WGA), is synthesized de novo by developing wheat (Triticum aestivum, L.) embryos but is not synthesized or localized in developing endosperm as shown by radioimmunoassay. Young embryos removed from the grain and cultured on a defined medium germinate precociously and concomitantly cease WGA synthesis. In vitro precocious germination of young embryos is reversibly inhibited by low levels (1–100 μM) of the plant growth substance abscisic acid (ABA). Embryos inhibited from germinating by this growth regulator not only continue synthesizing WGA, but do so at an accelerated rate when compared with embryos left associated with the grain.     

Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D.Hyphal tips and septae were strongly labelled by short pulses of [³H] N-acetyl-D-glucosamine ([³H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA).The dynamics of wall formation was followed by pulse and chase as well as by pulse and wash treatments in which the colony was shortly exposed to [³H]GlcNAc and then freed from the radioactive chitin precursor.Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis.FITC-WGA binding, [³H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - GlcNAc N-acetyl-D-glucosamine     

Distribution and changes of glycoconjugates in rat colonic mucosa during development

Summary A study was made of the modifications of glycoconjugates in rat colonic mucosa during development. Sections of the caecum, and proximal and distal portions of the colon from Sprague Dawley rats at different stages of development (embryos, fetuses, suckling, weaning and adult rats) were examined. The sections were incubated with a battery of eight fluoresceinated lectins: DBA, SBA, WGA, LFA, PNA, GS-I, UEA-I and Con A. Some sections were treated with neuraminidase, and others were submitted to sequential saponification-neuraminidase treatment prior to incubation with the lectin (WGA, PNA or LFA). The intensity of the fluorescence was evaluated and graded from absent (–) to very positive (4+). Gradual and progressive changes were seen in colonic glycoconjugates during development. These changes revealed a unique developmental pattern for each lectin, which was independent for each cellular compartment (goblet cells, luminal surface and supranuclear region). Local and regional differences, observed between the different colonic sections, were already present from early stages of development. Moreover, our study showed that for several glycoconjugates, the differentiation process in colonic mucosa began in the distal region and continued through to the proximal region, the former being the first to reach the adult pattern. In the caecum, some lectins maintained a fetal pattern throughout all the periods of development up to the adult stage.     

Wheat germ agglutinin participates in regulation of cell division in apical root meristem of wheat seedlings

A study was made of cell division activity and hormonal status in roots of 4 day old wheat seedling treated with wheat germ agglutinin (WGA). The revealed stimulating effect of WGA on mitotic index (MI) and cell area in root extension zone was specific for this lectin, because gliadin, taken as a control protein, caused no changes in growth parameters. Phytolectins (phytohemagglutinin and concanavalin A) possessing properties of mitogens rendered no such essential influence on cell growth of wheat. Immunoassay has shown that WGA-treatment leads to accumulation of auxins and cytokinins in roots. This suggest participation of WGA in regulation of MI of meristem cells in roots of seedlings during their interaction with phytohormones.     

Abscisic acid promotes lectin biosynthesis in developing and germinating rice embryos

Immature rice (Oryza sativa, L) embryos isolated about 12 days post anthesis are fully able to develop into young seedlings when cultured in vitro. Concomitantly, they rapidly loose their lectin synthesis activity. Abscisic acid added to the nutrient medium prevents precocious germination of the immature embryos and simultaneously strongly promotes lectin biosynthesis activity. Similarly, abscisic acid keeps mature embryos grown in a nutrient medium in a dormant state and maintains their lectin synthesis activity, whereas control embryos rapidly germinate but also quickly loose their lectin synthesis activity. It appears, therefore, that rice lectin is typically synthesized in embryos which are kept in a dornant state.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - WGA wheat germ agglutinin - DPA days post anthesis     

Wheat germ agglutinin regulates cell division in wheat seedling roots

The mitogenic activity of wheat germ agglutinin (WGA) has been studied in roots of 4-day-old wheat seedlings. WGA had a more pronounced stimulating effect on cell division than the known mitogens concanavalin A and phytohemagglutinin whereas gliadin had no effect. Treatment of wheat seedling roots with exogenous WGA led to the accumulation of indoleacetic acid and cytokinins, hormones that play an important role in the activation of plant cell growth. The data on the combined effect of 24-epibrassinolide and WGA on cell division and accumulation of phytohormones in seedling roots support a possible link between the endogenous WGA level and hormonal regulation of cell division in the root meristem of wheat plants.     

Distribution and changes of glycoconjugates in rat colonic mucosa during development. A histochemical study using lectins

A study was made of the modifications of glycoconjugates in rat colonic mucosa during development. Sections of the caecum, and proximal and distal portions of the colon from Sprague Dawley rats at different stages of development (embryos, fetuses, suckling, weaning and adult rats) were examined. The sections were incubated with a battery of eight fluoresceinated lectins: DBA, SBA, WGA, LFA, PNA, GS-I, UEA-I and Con A. Some sections were treated with neuraminidase, and others were submitted to sequential saponification-neuraminidase treatment prior to incubation with the lectin (WGA, PNA or LFA). The intensity of the fluorescence was evaluated and graded from absent (-) to very positive (4+). Gradual and progressive changes were seen in colonic glycoconjugates during development. These changes revealed a unique developmental pattern for each lectin, which was independent for each cellular compartment (goblet cells, luminal surface and supranuclear region). Local and regional differences, observed between the different colonic sections, were already present from early stages of development. Moreover, our study showed that for several glycoconjugates, the differentiation process in colonic mucosa began in the distal region and continued through to the proximal region, the former being the first to reach the adult pattern. In the caecum, some lectins maintained a fetal pattern throughout all the periods of development up to the adult stage.     

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.Abbreviations IgG immunoglobulin G - M_r relative molecular weight - PBS phosphate-buffered saline - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis