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.     

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.     

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.     

Role of abscisic acid in regulation of wheat seedling growth under salinity stress

Growth of wheat seedlings (Triticum aestivum L. cv. Mehran-89), in hydroponic culture, was affected by abscisic acid (ABA). Using salinity stress and exogenous ABA application (10-6 M) to enhance endogenous ABA level, the growth of roots was more suppressed than the growth of shoots. On the other hand, norflurazon, which inhibits ABA biosynthesis, reduced only the growth of shoots. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

Fusarium sp. growth inhibition by wheat germ agglutinin.

The antifungal role of wheat germ agglutinin (WGA) isolated from a Romanian dihaploid variety of wheat against two pathogenic fungal species of Fusarium, F. graminearum and F. oxysporum, is demonstrated. WGA was prepared from unprocessed wheat germs by a new purification procedure using chitin and fetuin-Sepharose as affinity chromatography supports. SDS-PAGE and chitinase assay showed that the WGA preparation migrated as a single protein band and was devoid of any contaminating enzyme chitinase, well known for its antifungal effects. Based on its affinity for N-acetylglucosamine residues, WGA binding to the chitin-containing walls of the fungi was detected by fluorescence microscopy using WGA coupled with fluorescein isothiocyanate (FITC). In vitro testing of WGA action on early developmental stages of both fungal strains resulted in various modifications of the germ tubes, visualised by light microscopy: swelling, vacuolation of the cellular content and lysis of cell walls. Viability tests performed on potato tuber slices showed that the microbial infection was prevented from spreading by pretreatment of the fungal suspension with WGA.     

Distribution of wheat germ agglutinin in young wheat plants

A liquid phase, competition-binding radioimmunoassay for wheat germ agglutinin, with a detection limit of 10 nanograms, was developed in order to determine the distribution of this lectin in young wheat plants. Affinity columns for wheat germ agglutinin removed all antigenically detectable activity from crude extracts of wheat tissue; thus, the antigenic cross-reactivity detected by the assay possesses sugar-binding specificity similar to the wheat germ-derived lectin. The amount of lectin per dry grain is approximately 1 microgram, all associated with the embryo. At 34 days of growth, the level of lectin per plant was reduced by about 50%, with approximately one-third in the roots and two-thirds in the shoot. The data also indicate that actively growing regions of the plant (the bases of the leaves and rapidly growing adventitious roots) contain the highest levels of lectin. Half of the lectin associated with the roots could be solubilized by washing intact roots in buffer containing oligomers of N-acetylglucosamine, whereas the remainder is liberated only upon homogenization of the tissue.     

Subunit structure of wheat germ agglutinin

Cells isolated by enzymic digestion of embryonic tendon were incubated under N₂ so that they synthesized and accumulated the unhydroxylated form of procollagen which is known as protocollagen and which is largely comprised of pro-α chains linked by interchain disulfide bonds. The cells were then exposed to O₂ so that the intracellular protocollagen was hydroxylated and secreted as procollagen. When the hydroxylation was allowed to proceed at 31° or 34°, the procollagen secreted into the medium was triple-helical but its hydroxyproline content was less than two-thirds and its hydroxylysine content was less than half the control. Even when the hydroxylation was allowed to occur at 37°, the procollagen secreted by the cells was under-hydroxylated by about 15% in terms of its hydroxyproline content and about 45% in terms of its hydroxylysine content. The results may have consequences for collagen synthesis by tendons and similar tissues $\text{in vivo}$, since temporary anoxia in such tissues may well lead to the synthesis of a less stable procollagen or to fibers of decreased tensile strength.     

Modification of nerve growth factor receptor properties by wheat germ agglutinin

PC12 is a nerve growth factor (NGF) responsive cell line which exhibits two classes of NGF receptors distinguishable by different kinetic rate constants, sensitivity to trypsin and resistance to Triton detergent solubilization. Whereas incubation of PC12 cells with wheat germ agglutinin (WGA) prior to addition of 125I-NGF inhibits binding of NGF to both classes of receptors, treatment with WGA subsequent to incubation with NGF does not inhibit NGF binding but causes the class of NGF receptors which exhibit rapid or "Fast" dissociation kinetics prior to lectin treatment to be converted to the form which exhibits "Slow" dissociation kinetics. This WGA-mediated receptor conversion is lectin specific, blocked by N-acetyl-D-glucosamine, occurs at similar rates at 4 and 37 degrees C, and is not impaired by a metabolic poison. NGF receptors converted by WGA, like pre-existing Slow receptors, are resistant to trypsinization and remain associated to Triton X-100 extracted "cytoskeletons." Very similar results were obtained for NGF receptors on a human melanoma cell line A875. These results suggest that Fast and Slow receptors are two interconvertible forms of a single protein, rather than distinct proteins. The significance of the generality of these properties for NGF receptors from diverse species and cell types is discussed.     

Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress

Worldwide, dryland salinity is a major limitation to crop production. Breeding for salinity tolerance could be an effective way of improving yield and yield stability on saline-sodic soils of dryland agriculture. However, this requires a good understanding of inheritance of this quantitative trait. In the present study, a doubled-haploid bread wheat population (Berkut/Krichauff) was grown in supported hydroponics to identify quantitative trait loci (QTL) associated with salinity tolerance traits commonly reported in the literature (leaf symptoms, tiller number, seedling biomass, chlorophyll content, and shoot Na⁺ and K⁺ concentrations), understand the relationships amongst these traits, and determine their genetic value for marker-assisted selection. There was considerable segregation within the population for all traits measured. With a genetic map of 527 SSR-, DArT- and gene-based markers, a total of 40 QTL were detected for all seven traits. For the first time in a cereal species, a QTL interval for Na⁺ exclusion (wPt-3114-wmc170) was associated with an increase (10%) in seedling biomass. Of the five QTL identified for Na⁺ exclusion, two were co-located with seedling biomass (2A and 6A). The 2A QTL appears to coincide with the previously reported Na⁺ exclusion locus in durum wheat that hosts one active HKT1;4 (Nax1) and one inactive HKT1;4 gene. Using these sequences as template for primer design enabled mapping of at least three HKT1;4 genes onto chromosome 2AL in bread wheat, suggesting that bread wheat carries more HKT1;4 gene family members than durum wheat. However, the combined effects of all Na⁺ exclusion loci only accounted for 18% of the variation in seedling biomass under salinity stress indicating that there were other mechanisms of salinity tolerance operative at the seedling stage in this population. Na⁺ and K⁺ accumulation appear under separate genetic control. The molecular markers wmc170 (2A) and cfd080 (6A) are expected to facilitate breeding for salinity tolerance in bread wheat, the latter being associated with seedling vigour.     

Effects of wheat germ agglutinin on membrane transport

(1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD50 ranged from 1 to 5 microgram wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids alpha-aminoisobutyric acid and cycloleucine and inhibited the uptake of thymidine. In contrast, 2-deoxy-D-glucose uptake was not altered and colchicine uptake was enhanced. (2) The inhibition of alpha-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent Km. Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of alpha-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseolus vulgaris E-phytohemagglutinin and L-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 microgram/ml. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems.     

Folding and homodimerization of wheat germ agglutinin

Wheat germ agglutinin (WGA) is emblematic of proteins that specialize in the recognition of carbohydrates. It was the first lectin reported to have a capacity for discriminating between normal and malignant cells. Since then, it has become a preferred model for basic research and is frequently considered in the development of biomedical and biotechnological applications. However, the molecular basis for the structural stability of this homodimeric lectin remains largely unknown, a situation that limits the rational manipulation and modification of its function. In this work we performed a thermodynamic characterization of WGA folding and self-association processes as a function of pH and temperature by using differential scanning and isothermal dilution calorimetry. WGA is monomeric at pH 2, and one of its four hevein-like domains is unfolded at room temperature. Under such conditions, the agglutinin exhibits a fully reversible thermal unfolding that consists of three two-state transitions. At higher pH values, the protein forms weak, nonobligate dimers. This behavior contrasts with that observed for the other plant lectins studied thus far, which form strong, obligate oligomers, indicating a distinctly different molecular basis for WGA function. For dimer formation, the four domains must be properly folded. Nevertheless, depending on the solution conditions, self-association may be coupled with folding of the labile domain. Therefore, dimerization may proceed as a rigid-body-like association or a folding-by-binding event. This hybrid behavior is not seen in other plant lectins. The emerging molecular picture for the WGA assembly highlights the need for a reexamination of existing ligand-binding data in the literature.     

Isolation and characterization of plant growth-promoting rhizobacteria from wheat roots by wheat germ agglutinin labeled with fluorescein isothiocyanate

Thirty-two isolates were obtained from wheat rhizosphere by wheat germ agglutinin (WGA) labeled with fluorescein isothiocyanate (FITC). Most isolates were able to produce indole acetic acid (65.6%) and siderophores (59.3%), as well as exhibited phosphate solubilization (96.8%). Fourteen isolates displayed three plant growth-promoting traits. Among these strains, two phosphate-dissolving ones, WS29 and WS31, were evaluated for their beneficial effects on the early growth of wheat (Triticum aestivum Wan33). Strain WS29 and WS31 significantly promoted the development of lateral roots by 34.9% and 27.6%, as well as increased the root dry weight by 25.0% and 25.6%, respectively, compared to those of the control. Based on 16S rRNA gene sequence comparisons and phylogenetic positions, both isolates were determined to belong to the genus Bacillus. The proportion of isolates showing the properties of plant growth-promoting rhizobacteria (PGPR) was higher than in previous reports. The efficiency of the isolation of PGPR strains was also greatly increased by WGA labeled with FITC. The present study indicated that WGA could be used as an effective tool for isolating PGPR strains with high affinity to host plants from wheat roots. The proposed approach could facilitate research on biofertilizers or biocontrol agents.     

Effect of wheat germ agglutinin on T lymphocyte activation

Wheat germ agglutinin (WGA) is low mitogenic or nonmitogenic for human T lymphocytes and inhibits phytohemagglutinin (PHA)-induced mitotic response of the lymphocytes. In this study, the effect of WGA was analyzed in terms of interleukin 2 (IL2) production, expression of IL2 receptor, and IL2 responsiveness of the T lymphocytes. WGA as well as PHA could induce IL2 mRNA and IL2 production and also elevate cytoplasmic free Ca2+ concentration. The IL2 production was reduced by inhibitors of calmodulin and protein kinase C. The IL2 receptor (Tac) expression was induced at about 20% of the lymphocytes by WGA and the expression induced by PHA was not blocked by the addition of WGA. The lymphocytes precultured with WGA for 3 days could proliferate by the addition of IL2 after removal of WGA. The IL2-dependent proliferation of PHA-blasts was blocked by the addition of WGA. These results indicate that WGA inhibits T lymphocyte proliferation by inhibiting the responsiveness of the lymphocytes to IL2 but not by interfering with IL2 production and IL2 receptor expression.     

Activity and specificity of covalently immobolized wheat germ agglutinin toward cell surfaces

Wheat germ agglutinin protein, which is able to agglutinate tumor cells better than normal cells, was covalently bound to polyacrylamide gel beads. The specific binding activity of the protein was preserved on these beads and was expressed heterogeneously by the binding of mouse leukemia cells (L1210) to the protein coupled gels. The selective activity of the immobilized protein was maximal when the number of sites available to covalently couple the protein was lowest. The application of this observation to the general field of covalent immobilization of proteins and enzymes may be of considerable utility.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

Tissue zinc distribution in maize seedling roots and its action on growth

Zinc (Zn) distribution over tissues and organs of maize (Zea mays L.) seedlings and its action on root growth, cell division, and cell elongation were studied. Two-day-old seedlings were incubated in the 0.25-strength Hoagland solution containing 2 or 475 μM Zn(NO₃)₂. Zn toxicity was assessed after the inhibition of primary root increment during the first and second days of incubation. The content of Zn was determined by atomic absorption spectrometry in the apical (the first centimeter from the root tip) and basal (the third centimeter from the kernel) root parts. Zn distribution in various tissues was studied by histochemical methods, using a metallochromic indicator zincon and fluorescent indicator Zinpyr-1 and light and confocal scanning fluorescent light microscopy, respectively. To evaluate Zn effects on growth processes, the average length of the meristem; the length of fully elongated cells; the number of meristematic cells in the cortex row; and duration of the cell cycle were measured. When the Zn concentration in the solution was high, the Zn content per weight unit was higher in the basal root part due to its accumulation in lateral root primordial. Zn was also accumulated in both the meristem apoplast and cell protoplasts. In the basal and middle root parts, Zn was detected essentially in all tissues predominantly in the apoplast. Zn inhibited both cell division and elongation. Under Zn influence, the size of the meristem and the number of meristematic cells decreased, which was determined by an increase in the cell cycle duration. The length of the fully elongated cells was also reduced. A comparison of Zn distribution and growth-suppressing activity with other heavy metals studied earlier allows a conclusion that toxic action of heavy metals is mainly determined by physical and chemical properties of their ions and specific patterns of their transport and distribution. As a result, two basic processes determining root growth, e.g., cell division and elongation, could be affected differently.