Detection of lectins in nodulated peanut and soybean plants

The direct double-antibody enzymelinked immunosorbent assay system was used in the detection and measurement of seed lectins from peanut (Arachis hypogaea L.) and soybean (Glycine max L.) plants (PSL and SBL, respectively) that had been inoculated with their respective rhizobia. Concentrations of PSL dropped to undetectable levels in peanut roots at 9 d and stems and leaves at 27 d after planting; SBL could no longer be detected in soybean roots at 9 d and in stems and leaves at 12 d. A lectin antigenically similar to PSL was first detected in root nodules of peanuts at 21 d reaching a maximum of 8 g/g at 29 d then decreasing to 2.5 g/g at 60 d. There was no evidence of a corresponding lectin in soybean nodules.Sugar haemagglutination inhibition tests with neuraminidase-treated human blood cells established that PSL and the peanut nodule lectin were both galactose/lactose-specific. Further tests with rabbit blood cells demonstrated a second mannosespecific lectin in peanut nodule extracts that was not detected in root extracts of four-week-old inoculated plants or six-week-old uninoculated plants, although six-week-old root extracts from inoculated plants showed weak lectin activity. The root extracts from both nodulated and uninoculated plants contained another peanut lectin that agglutinated rabbit but not human blood cells. Haemagglutination by this lectin was, however, not inhibited by simple sugars but a glycoprotein, asialothyroglobulin, was effective in this respect.Abbreviations DAS double antibody sandwich - ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - PSL peanut seed lectin - SBL soybean lectin     

Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.

The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.     

Localization of mannose- and galactose-binding lectins in an effective peanut nodule

Summary Mannose/glucose- and galactose-binding lectins (ML and GL respectively, were located by immunogold labelling in tissues of a peanut (Arachis hypogaea) nodule induced by an effectiveBradyrhizobium sp. strain. Light and electron microscopic examination of silver-enhanced semithin and ultrathin sections, respectively, revealed that both lectins were widely distributed throughout the cortex and bacteroidal zones although ML was more abundant. The lectins were predominantly in the vacuoles of cortical cells but GL was absent from, or at low concentration in, a two-cell-thick layer of cortical cells surrounding the bacteroidal region. Only ML was detected in cells of the vascular bundle endodermis and in central vascular bundle cells; neither lectin was found in pericycle cells. Bacteroidal cells contained abundant ML in the nuclei and cytoplasm surrounding bacteroids while GL was mainly located in the central vacuoles of these cells. Neither lectin was associated with bacteroid surfaces, peribacteroid membranes, plant cell walls or cell organelles and membranes. The above observations indicate that the nodule lectins are not symbiotic cell recognition determinants and suggest that they have protein storage functions.Abbreviations BSA bovine serum albumin - GL galactose-binding lectin - ML mannose-binding lectin - PBS phosphate-buffered saline - PBST phosphate-buffered saline plus Tween     

Developmental changes in the bark lectin of Sophora japonica L.

Lectin is the major protein in the phloem tissue of S. japonica. By immunohistochemistry using anti-seed lectin antibody it was demonstrated that the lectin was localized in the ray and the axial parenchyma. Neither lectin nor other cross-reactive materials were observed in the cambium, sieve tubes and companion cells. The distribution and localization changed in relation to tissue development. Lectin content in the bark changed during the year, the average in summer being about 50% of that in winter. The distribution of lectin in the bark in winter was similar from the innermost (youngest) to the outermost (oldest) region. In contrast, in summer the innermost region hardly contained any lectin, and the outermost region contained less lectin than the middle. Lectin localization in tissues and cells differed also depending on tissue age. In new tissue, produced in the current year, lectip was absent in summer, was located in the cytoplasmic layer between cell wall and vacuole in autumn, and sequestered in the vacuoles in winter. On the other hand, lectin in old tissue (formed in the previous year) was located throughout the year mainly within the vacuoles, with only very small contents in the cytoplasmic layer in autumn. Within the outermost (oldest) region, in which the lectin content was low in summer, the cells which bordered the outer bark never contained any lectin in summer. The intracellular localization in autumn in new tissue, determined by immunogold electron microscopy, was in the lumen of the endoplasmic reticulum and vesicles, with gold particles hardly present in the cytoplasm. From these findings we conclude that lectin is synthesized on the endoplasmic reticulum and most vigorously in the new tissue in autumn, and that it is mainly consumed in the outermost bark regions, where dilatation occurs and-or where cork cambium is differentiated.Abbreviations ELISA enzyme-linked immunosorbent assay - ER endoplasmic reticulum - kDa kilodalton Retired. Anatomical terms in this paper are used according to Multilingual glossary of terms used in wood anatomy edited by the Committee on Nomenclature, International Association of Wood Anatomists; reprints may be obtained from the Office of the Secretary-Treasurer, Universitätsstrasse 2, CH-8092 Zürich 6, Switzerland.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Transcription of ENOD8 in Medicago truncatula nodules directs ENOD8 esterase to developing and mature symbiosomes

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.     

与玉米混作改善花生铁营养对其根瘤形态结构及豆血红蛋白含量的影响

通过土培方法研究了与玉米混作对花生根瘤形态结构及固氮功能的影响。结果表明,玉米与花生混作能够明显地改善花生铁营养、提高根瘤豆血红蛋白的含量。同时,单作花生根瘤细胞液泡化程度较高,正在发育的根瘤细胞内类菌体数量明显地比混作的花生低。成熟根瘤细胞类菌体周膜外空间(细胞壁以内、周膜外的空间)体积变大。说明单作花生固氮酶活性较低的原因是缺铁抑制了豆血红蛋白的合成和改变了根瘤形态结构以及类菌体的超微结构。     

Effect of mannose-binding lectin from peanut and pea on the stem borer Chilo partellus

A mannose-binding lectin found in vegetative tissues of peanut, Arachis hypogaea, was compared with mannose-binding lectin from pea, Pisum sativum, for toxic effects on larvae of the stem borer Chilo partellus (Swinhoe). After 10 days, the mortality of larvae fed on artificial diet containing 0.5% (m/m) peanut lectin was 46.2%. The mortality of larvae fed on 1.0% peanut lectin was similar (48.1%) but insects were significantly smaller than those of the 0.5% treatment. Larvae of both lectin treatments stopped feeding within three days. Larval size and mortality was not significantly reduced by 0.1% peanut lectin and 1% heat-treated lectin did not show toxic effects. The mannose-binding lectin from pea was not toxic to C. partellus at concentrations up to 1%. Peanut lectin bound to the apical membranes of columnar epithelial cells in the mid-gut of C. partellus. This suggests that peanut lectin has an antinutritive action and that it may protect vegetative tissues of peanut against insect pests.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Monospecific antibodies against synthetic T-antigen. Characteristics of their specificity and use in the identification of T-antigenic determinants on the cell surface

Monospecific antibodies directed to a Thomsen-Friedenreich antigen (T-antigen) were obtained using artificial antigen. T-antigen immunodominant alpha-disaccharide Galbeta (1----3) GalNAc alpha 1-(T alpha) and its beta-anomer Gal beta (1----3) GalNAc beta 1-(T beta) were bound to bovine serum albumin (BSA) and cytochrome C (CCC) through a spacer (sp = -O(CH2)3NHCO (CH2)4CO-) by the azide method to give neoglycoproteins T alpha-sp-BSA, T alpha-sp-CCC and T beta-sp-BSA. Anti-T alpha antiserum was obtained by immunization of rabbits with T alpha-sp-BSA and then purified by sequential affinity chromatography on BSA-Sepharose and T alpha-sp-BSA-Sepharose to yield monospecific anti-T IgG antibodies. As elucidated by ELISA method, binding T alpha-sp-BSA to the antibodies was inhibited by T alpha-sp-CCC, T alpha-sp-OEt, asialofetuin, T alpha-OBzl, the activity of the inhibitors decreasing in the above order. Methyl beta-galactopyranoside, benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside, disaccharide Gal beta (1----3) GalNAc and H-sp-BSA were inactive. The inhibitory analysis suggests that both disaccharide moiety T alpha- and a definite part of the spacer are important for the binding and that T alpha-OCH2 seems to be the minimal recognized structure. In immunoprecipitation tests the antibodies react with T alpha-sp-BSA but not with T beta-sp-BSA, whereas peanut (Arachis hypogaea) lectin (PNL) precipitated both T alpha- and T beta-sp-BSA. These data suggest the significance of the alpha-galactosaminide bond for the antibody recognition. Desialylated human erithrocites (natural T-antigen) were effectively agglutinated with the antibodies. Murine cortical thymocytes (obtained by agglutination-sedimentation method using PNL) were agglutinated with the antibodies only partially (67%), while these cells as well as the cells unaffected by the antibodies were completely agglutinated with PNL. These results indicate to different contents of glycoproteins (T alpha) and glycolipids (T beta) oligosaccharide determinants on the surface of cortical thymocytes species.     

Terminal galactosyl residues of cell-surface glycoconjugates exposed on both human and murine immature T- and B-cells

Summary Exposure of terminal galactosyl residues on cell-surface molecules as detected by their ability to bind peanut lectin (PNL) is found to be characteristic for immature cortical human and murine thymic lymphocytes. While in prenatal mice PNL staining is found to be uniformly distributed among all thymic lymphocytes, in adult thymi a cortico-medullary gradient is detectable concerning the PNL-binding capacity of thymic cortical lymphocytes, a phenomenon that appears to be correlated to their maturational degree.In secondary lymphatic tissue, i.e. lymph nodes and spleen of man, mouse and rat, strongly labeled cells are found exclusively in germinal centers. Ultrastructurally, these cells could be identified as centrocytes and centroblasts. These observations suggest that exposed galactosyl moieties of cell-surface glycoconjugates are expressed by undifferentiated lymphocytes of both T-and B-cell lineage.Furthermore, it could be shown that PNL-binding properties of immature cells are not restricted to lymphatic tissue but can also be demonstrated on various embryonic cells of non-lymphatic origin in distinct developmental stages. Thus, they might have a fundamental significance in the course of maturation processes.Supported by the Deutsche Forschungsgemeinschaft     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Immunogold localization of callose and other cell wall components in pea nodule transfer cells

Summary Transfer cells are located adjacent to xylem and phloem elements in pea nodule vascular tissues. The composition of the labyrinthine wall intrusions was investigated by immunogold labeling using specific antibody probes. Callose antigen was found at the base of newly formed cell wall intrusions and also in adjacent plasmodesmata. Sections through developed labyrinthine intrusions revealed that wall ingrowths had an internal structure with small domains of callose suggesting the presence of channels or vents. Xyloglucan and pectin antigens were uniformly distributed within the wall, but the distribution of extensin antigens was variable, with different antigens being detected in different regions of the wall ingrowth. A lectinlike glycoprotein, PsNLEC-1, was localized in intercellular spaces associated with nodule transfer cells. Previously, expression of this component was observed in other types of cells showing complex involution of the plasma membrane, namely root cortical cells harboring arbuscular mycorrhizae and nodule cells harboring nitrogen-fixing rhizobia.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Endoplasmic Reticulum Subcompartments in a Plant Parasitic Fungus and in Baker's Yeast: Differential Distribution of Lumenal Proteins

Bachem, U., and Mendgen, K. 1995. ER subcompartments in a plant parasitic fungus and in baker's yeast: Differential distribution of lumenal proteins. Experimental Mycology 19, 137-152. His-Asp-Glu-Leu (HDEL)-bearing proteins were quantified in different endoplasmic reticulum (ER) subcompartments of Saccharomyces cerevisiae and the plant parasite Uromyces viciae-fabae by immuno-electron microscopy (immuno-EM). In both fungi, the immunogold labeling of these proteins within the ER was three times greater than within the nuclear envelope. In U. viciae-fabae, the ER in germinating uredospores differed from the ER in fungal structures produced within the plant, e.g., haustoria. In haustoria, the cisternal ER differentiated large tubular-vesicular complexes (TVC). TVC contained higher levels of HDEL-bearing proteins than ordinary ER cisternae. ELISA readings also indicated an increased concentration of these proteins in isolated haustoria compared to germinating uredospores. In S. cerevisiae, the ER was differentiated into cortical and internal regions. Immuno-EM revealed that labeling of the binding protein (BiP) was lower in the ER of the cell cortex. Heat shock increased BiP signals, but the relative distribution within the ER did not change. Our results suggest that ER subcompartments can be differentiated by immunogold labeling of proteins with a retention signal. In special cases, such as in the parasitic phase of rust fungi, these proteins accumulate to higher levels in ER subcompartments, probably as a response to plant-induced stress.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

玉米/花生混作改善花生铁营养对花生根瘤碳氮代谢及固氮的影响

研究了石灰性土壤上玉米 (Zea mays L.) /花生 (Arachishypogaea L.)混作改善花生铁营养对花生光合速率、光合产物的运输、花生各部位糖类含量、固氮酶活性以及根瘤内碳氮代谢及其有关酶活性的影响。结果表明 ,玉米 /花生混作改善花生铁营养能够明显增强固氮酶活性 ,进而增加了间作花生根瘤氨基酸的含量 ,这主要是由于玉米 /花生混作改善花生铁营养促进了花生光合作用 ,提高光合产物数量 ,增加光合产物由地上部向地下部的运输 ,但是处理间花生根瘤蔗糖和可溶性糖含量变化不大 ,单作花生根瘤还积累较多淀粉 ,说明不是光合产物的供应导致了花生固氮活性的差异。玉米 /花生混作对花生根瘤碳水化合物代谢水平影响较大 ,混作花生根瘤异柠檬酸脱氢酶 (IDH)、苹果酸脱氢酶 (MDH)、琥珀酸脱氢酶活性明显高于单作 ,而磷酸烯醇丙酮酸羧激 (PEPCK)活性低于单作花生 ,表明混作花生根瘤内三羧酸循环代谢水平较高 ,形成类菌体直接吸收利用的能量物质苹果酸和琥珀酸多 ,能够满足类菌体的固氮需求 ,因此 ,玉米 /花生混作改善花生铁营养增强根瘤碳水化合物代谢水平是提高花生固氮作用的重要原因之一     

Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells

A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels.