Development and Distribution of Dolichos biflorus Lectin as Measured by Radioimmunoassay

A radioimmunoassay, capable of detecting the Dolichos biflorus lectin at concentrations as low as 400 ng/ml, was developed and used to follow the distribution of this lectin in the plant during its life cycle.

The lectin was first detected in the seeds of the plant 27 days after flowering and rapidly attained the high level of lectin present in the mature seed. The lectin content of the plant is highest in the seeds and cotyledons and decreases as the storage materials of the cotyledons decrease.

A low but measurable amount of material that reacts with antibodies to the seed lectin was detected in the leaves, stems, and pods of the plant. This material gives a precipitin band of only partial identity to the seed lectin when tested in immunodiffusion against antiserum to the seed lectin.

No lectin was detected by the radioimmunoassay in the roots of the plant at any stage of development.

     

Subcellular Localizations of Two Dolichos biflorus Lectins

The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRM]) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.

Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCl, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied [¹²⁵I]CRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.

The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions.

     

Production of a Lectin in Tissue Cultures of Dolichos biflorus

Callus cultures have been produced from the epicotyl and leaves, hypocotyl, and roots of germinating Dolichos biflorus seeds. These cultures were initiated on media containing 2,4-dichlorophenoxyacetic acid and kinetin, transferred to media with increased amounts of these hormones, and then maintained on hormone-free media. Extracts of these cultures were examined by radioimmunoassays specific for the lectin from the seeds of this plant and for a lectin that is present only in the stems and leaves of the intact plant. Although the seed lectin was not detected in any cultures, the stem and leaf lectin was produced in those cultures grown on the hormone free media. Lectin isolated from these cultures had subunits identical in electrophoretic mobilities to the subunits from the lectin isolated from intact stems and leaves. Levels of this lectin decreased when the cells were transferred back to media containing hormones and increased again upon transfer to the hormone-free media. The absence of exogenous hormones and the production of lectin were also correlated with the rapid growth and greening of the cells. Immunofluorescence and immunocytochemical studies on sections of cultured cells indicated that the stem and leaf lectin is associated with the cytoplasm as well as the cell wall as has been found in previous studies on the subcellular localization of this lectin in the intact plant.     

Establishment of the fungal entomopathogen Beauveria bassiana as a season long endophyte in jute (Corchorus olitorius) and its rapid detection using SCAR marker

An experiment was conducted to introduce the entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) as an endophyte in jute (Corchorus olitorius), a bast fibre crop through seed treatment. Colonization of root, leaf, stem, capsule, and seed were assessed through plating on selective medium and PCR based detection using B. bassiana specific SCAR markers. Endophytic colonization was detected in all the plants grown from treated seeds, but all the plant parts were not colonized. Colonization was detected in leaves, stems, and green capsules but not in roots and seeds. The endophytic colonization was influenced by both plant part and sampling period. Colonization was greater in leaves (55.87%) compared to stems (12.53%) and capsules (42.44%). The percent colonization was higher in case of 60?days old plants (43.34%) than in 30?days (23.89%) and 120?days (35.39%) old plants. As B. bassiana has already been reported to be pathogenic on jute pests, namely semilooper (Anomis sabulifera) and bihar hairy caterpillar (Spilosoma obliqua), its season long endophytic colonization within jute plant suggests a novel approach of biological control of these pests through seed treatment with the entomopathogen.     

A Novel Calcium Oxalate Crystal Growth Inhibitory Protein from the Seeds of Dolichos biflorus (L.)

Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate, using phytotherapy is being sought. Dolichos biflorus seeds, which are used as dietary food in India, possess antilithiatic properties. In the present study, a novel dimeric antilithiatic protein (98 kDa) from its seeds was purified based on its ability to inhibit calcium oxalate crystallization in vitro. Amino acid analysis of Dolichos biflorus antilithiatic protein showed abundant acidic amino acids. The mascot search engine presented sequence similarity with a calcium binding protein, calnexin of Pisum sativum from the m/z data obtained by MALDI TOF mass spectrometer. Above results demonstrate the anticalcifying/antilithiatic nature of a novel protein from the seeds of Dolichos biflorus and thus open new vistas for using plant proteins as therapeutic agents to treat urolithiasis.     

Allelopathic potential of Bothriochloa laguroides var. laguroides (DC.) Herter (Poaceae: Andropogoneae)

Bothriochloa laguroides var. laguroides (DC.) Herter, a native bluestem of America, has been shown to produce many biologically active compounds. The allelopathic potential of aqueous extracts from roots, stems and leaves was examined. Lettuce seeds (Lactuca sativa) and maize (Zea mays), the common allelopathy bioassay systems, as well as seeds from two native species, wintergreen paspalum (Paspalum guenoarum) and lovegrass (Eragrostis curvula), were germinated in the presence of aqueous extracts. Percent seed germination, root and shoot elongation were measured. After 4 and 7 days root, stem and leaf extracts caused inhibition of root and shoot elongation in all four species tested. Aqueous extracts were generally less inhibitory to seed germination. Aqueous extracts from different parts of B. laguroides var. laguroides show therefore allelopathic effects inhibiting, in particular, growth of competing plants.     

Electron microscopy of human erythrocytes agglutinated by lectin from Codium fragile ssp. tomentosoides and pseudo-haemagglutinin from Ascophyllum nodosum

Human erythrocytes were exposed to extracts of Codium fragile ssp. tomentosoides, Ascophyllum nodosum, Dolichos biflorus seeds and a solution of purified polyphenols from Ascophyllum nodosum. In each case the erythrocytes appeared to agglutinate. Agglutinates were examined by scanning electron microscopy. Photomicrographs of the erythrocytes exposed to lectin-containing extracts of C. fragile and D. biflorus showed the typical appearance of agglutinated cells. Photomicrographs of the erythrocytes exposed to the crude extract and purified polyphenols from A. nodosum were similar to each other, but quite different in appearance to erythrocytes agglutinated by the lectins from C. fragile and D. biflorus. This evidence adds further support to the contention that lectin presence recorded in brown algal extracts is largely due to ‘pseudoagglutination’ produced by polyphenols contained in these extracts.     

Isolation and characterization of a lectin from the roots of Dolichos biflorus

A lectin has been isolated from the roots of 7-day-old Dolichos biflorus plants and has been compared with the D. biflorus seed lectin. The root lectin differs from the seed lectin in molecular weight, subunit stoichiometry, amino acid composition, amino terminal amino acid sequence, and isoelectric focusing pattern. However, the root lectin has in common with the seed lectin a specificity for N-acetyl-D-galactosamine, and upon denaturation the root lectin will react weakly with antiserum made to denatured seed lectin. Distribution studies of this lectin in germinating seedlings show that the highest levels of lectin are found in 1-day-old roots. Upon dissection and analysis of 7-day-old roots, the highest levels of the lectin are in the uppermost segment. In addition, isoforms of this lectin also exist in the stems and leaves of the plant.     

An isoflavone diglycoside from the seeds of Dolichos biflorus

-5-Hydroxy-7,3′,4′-trimethoxy-8-methylisoflavone 5-neohesperidoside has been identified from the seeds of Dolichos biflorus.     

The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum

The seeds of Spartium junceum contained a large quantity of lectin-like protein that did not appear to be either a hemagglutinin or active lectin. The cross-reactive material (CRM), like most legume seed lectins, was a tetrameric glycoprotein of about 130,000 M_r. The singlesized subunits of about 33,000 M_r were not covalently associated. The amino acid composition was typical of legume lectins and was rich in hydroxy-amino acids and poor in sulfur-containing amino acids. The Spartium CRM contained about 3.5% covalently associated carbohydrate, most likely of the high-mannose type, since the CRM was precipitated by concanavalin A. The CRM was localized by electron-microscopic immunocytochemistry and found to be exclusively in protein-filled vacuoles (protein bodies). Because this protein was so similar immunologically, structurally, and in its physiology, to classic legume seed lectins, it is most likely a lectin homolog. Similar seed lectin CRMs appear to be both common and widespread in the Leguminosae.     

Distribution of Lectins in the Jumbo Virginia and Spanish Varieties of the Peanut, Arachis hypogaea L

Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs.     

Detection and characterization of a lectin from non-seed tissue ofPhaseolus vulgaris

The distribution of lectin in various tissues ofPhaseolus vulgaris L. (cv. red) has been investigated using a sensitive solid-phase enzyme immunoassay. Roots, leaves and stems from 3- to 4-week-old plants were screened for their lectin content; low levels could be detected in all organs, with a relative distribution of 37% in roots, 20% in leaves and 43% in stems. The lectin from stemsleaves and roots was then isolated from 5- to 6-week-old plants using extraction, salt fractionation and affinity chromatography on immobilized porcine thyroglobulin. A comparative study of the seed lectin and the lectin isolated from 5- to 6-week-old plants was made using hemagglutination, inhibition of hemagglutination, immunodiffusion, polyacrylamide and agarose electrophoresis. The results showed that lectin isolated from the different tissues was immunologically identical and exhibited the same subunit structure and similar isolectin composition as the seed lectin.Abbreviations EDTA ethylenediaminetetraacetic acid - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Lectins and the soybean-Rhizobium symbiosis: I. Immunological investigations of soybean lines,the seeds of which have been reported to lack the 120 000 dalton soybean lectin

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunofiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lactin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography.Phosphate buffered saline extracts of roots, hypocotyls, stems, and leaves of 3–66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 μg soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were modulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.     

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     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number (($\text{0.26±0.03) · 10}{}^6\text{sites/cell}$) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites of Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by $\text{N-}\text{acetylgalactosamine}$, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

Role of Lectins in Plant-Microorganism Interactions: II. Distribution of Soybean Lectin in Tissues of Glycine max (L.) Merr

Three different assay procedures have been used to quantitate the levels of soybean (Glycine max [L.] Merr.) lectin in various tissues of soybean plants. The assays used were a standard hemagglutination assay, a radioimmunoassay, and an isotope dilution assay. Most of the lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was present in all of the tissues of young seedlings, but decreased as the plants matured and was not detectable in plants older than 2 to 3 weeks. Soybean lectin isolated from seeds of several soybean varieties were identical when compared by several methods.     

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically

Plant seed lectins play a defense role against plant-eating animals. Here, GalNAc-specific Vicia villosa B₄ lectin was found to inhibit hydrolysis of UDP-GalNAc by animal nucleotide pyrophosphatases, which are suggested to regulate local levels of nucleotide sugars in cells. Inhibition was marked at low concentrations of UDP-GalNAc, and was reversed largely by the addition of GalNAc to the reaction mixture. In contrast, lectin inhibited enzymatic hydrolysis of other nucleotide sugars, such as UDP-Gal and UDP-GlcNAc, only to a small extent, and GalNAc did not affect such an inhibition. The binding constant of the lectin for UDP-GalNAc was as high as 2.8×10⁵ M^?1 at 4°C, whereas that for GalNAcα-1-phosphate was 1.3×10⁵ M^?1. These findings indicate that lectin inhibition of pyrophosphatase activity toward low concentrations of UDP-GalNAc arises mainly from competition between lectin and enzyme molecules for UDP-GalNAc. This type of inhibition was also observed to a lesser extent with GalNAc-specific Wistaria floribunda lectin, but not apparently with GalNAc-specific soybean or Dolichos biflorus lectin. Thus, V. villosa B₄ lectin shows unique binding specificity for UDP-GalNAc and has the capacity to modulate UDP-GalNAc metabolism in animal cells.     

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.     

Lectin receptor-like kinase LecRK-VIII.2 is a missing link in MAPK signaling-mediated yield control

The energy allocation for vegetative and reproductive growth is regulated by developmental signals and environmental cues, which subsequently affects seed output. However, the molecular mechanism underlying how plants coordinate yield-related traits to control yield in changing source–sink relationships remains largely unknown. Here, we discovered the lectin receptor-like kinase LecRK-VIII.2 as a specific receptor-like kinase that coordinates silique number, seed size, and seed number to determine seed yield in Arabidopsis (Arabidopsis thaliana). The lecrk-VIII.2 mutants develop smaller seeds, but more siliques and seeds, leading to increased yield. In contrast, the plants overexpressing LecRK-VIII.2 form bigger seeds, but less siliques and seeds, which results in similar yield to that of wild-type plants. Interestingly, LecRK-VIII.2 promotes the growth of the rosette, root, and stem by coordinating the source–sink relationship. Additionally, LecRK-VIII.2 positively regulates cell expansion and proliferation in the seed coat, and maternally controls seed size. The genetic and biochemical analyses demonstrated that LecRK-VIII.2 acts upstream of the mitogen-activated protein kinase (MAPK) gene MPK6 to regulate silique number, seed size, and seed number. Collectively, these findings uncover LecRK-VIII.2 as an upstream component of the MAPK signaling pathway to control yield-related traits and suggest its potential for crop improvement aimed at developing plants with stable yield, a robust root system, and improved lodging resistance.

A lectin receptor-like kinase regulates yield-related traits and coordinates the source–sink relationship in Arabidopsis.