Identification of Albizia lebbeck seed coat chitin-binding vicilins (7S globulins) with high toxicity to the larvae of the bruchid Callosobruchus maculatus

Seed coat is a specialized maternal tissue that interfaces the embryo and the external environment during embryogenesis, dormancy and germination. In addition, it is the first defensive barrier against penetration by pathogens and herbivores. Here we show that Albizia lebbeck seed coat dramatically compromises the oviposition, eclosion and development of the bruchid Callosobruchus maculatus. Dietary supplementation of bruchid larvae with A. lebbeck seed coat flour causes severe weight loss and reduces survival. By means of protein purification, mass spectrometry and bioinformatic analyses, we show that chitin-binding vicilins are the main source of A. lebbeck tegumental toxicity to C. maculatus. At concentrations as low as 0.1 percent, A. lebbeck vicilins reduce larval mass from 8.1 ± 1.7 (mass of control larvae) to 1.8 ± 0.5 mg, which corresponds to a decrease of 78 percent. Seed coat toxicity constitutes an efficient defense mechanism, hindering insect predation and preventing embryo damage. We hypothesize that A. lebbeck vicilins are good candidates for the genetic transformation of crop legumes to enhance resistance to bruchid predation.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15 percent SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/æg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

The leaves of green plants as well as a cyanobacterium, a red alga, and fungi contain insulin-like antigens

We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae

Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus)

The presence of phaseolin (a vicilin-like 7S storage globulin) peptides in the seed coat of the legume Phaseolus lunatus L. (lima bean) was demonstrated by N-terminal amino acid sequencing. Utilizing an artificial seed system assay we showed that phaseolin, isolated from both cotyledon and testa tissues of P. lunatus, is detrimental to the nonhost bruchid Callosobruchus maculatus (F) (cowpea weevil) with ED50 of 1.7 and 3.5 percent, respectively. The level of phaseolin in the seed coat (16.7 percent) was found to be sufficient to deter larval development of this bruchid. The expression of a C. maculatus-detrimental protein in the testa of nonhost seeds suggests that the protein may have played a significant role in the evolutionary adaptation of bruchids to legume seeds

Alfa-amylase inhibitors of legume seeds and their involvement in the resistance to bruchid beetles

The presence of inhibitors of alfa-amylases from several sources (bacterial, plant, insect and mammallian) was investigated in seeds of several food legumes. No inhibitor of any of the tested enzymes was found in Phaseolus lunatus (Lima bean) seeds while the presence of inhibitors of insect (bruchid), porcine pancreas and human saliva alfa-amylases was confirmed in the seeds of P. vulgaris (common bean). Glycine max (soybean) seeds showed alfa-als for all the tested enzymes except for the porcine pancreatic amylase. Although we have found low levels of alfa-als in both bruchid-susceptible and resistant cowpea (Vigna unguiculata) seeds their presence does not correlate with the resistance shown by the seeds of some cultivars to the cowpea weevil Callosobruchus maculatus. The results reported here suggest that alfa-Als are not involved in the resistance of seeds of some cowpea lines to C. maculatus and that variant vicilins, the 7 S storage proteins involved in this resistance, do not show any inhibitory towards bruchi alfa-amylases

Anti-inflammatory and analgesic activity of a water-soluble fraction from shark cartilage

The anti-inflammatory and analgesic activities of a water-soluble fraction (WSF), extracted with 0.1 M ammonium bicarbonate, pH 8.0, from shark cartilage were studied in several experimental mod-els. Orally administered WSF (1O mg/kg) caused 25.7 and 23.6 per cent inhibition of the paw edema produced in female Wistar rats (200-250 g) by carrageenan and dextran, respectively, after 3 h, as compared to controls. WSF administered orally had no effect on acetic acid-induced writhings in male Swiss mice (25-30 g) at the dose of 0.01 mg/kg, but caused 52.8 and 61.4 per cent inhibition at the doses of 0.1 and 0.5 mg/kg, respectively, compared to controls (No. of writhings/20 min, means ñ SEM: treated groups = 18.6 ñ 2.5, N = 12 and 15.2 ñ 1.4, N = 12, respectively; controls = 39.3 ñ 1.3, N = 77). In the formalin test (male Swiss mice, 25-30 g), orally administered WSF (0.5 and 1 mg/kg) caused 12.0 and 46.6 per cent inhibition of licking time, respectively, only in the 2nd phase (infiammatory) of the test (licking time, means ñ SEM: treated group = 18.3 ñ 4.4 sec, N = 7 and 11.1 ñ 3.4 sec, N = 13; controls = 20.8 ñ 2.4 sec, N = 44). The results suggest that a molecule of a protein nature in shark cartilage is probably responsible for the effects observed.

Chitin-binding proteins from cowpea (Vigna unguiculata) seeds

Vicilins (7S storage proteins) from cowpea (Vigna unguiculata) and other legume seeds were shown to bind to chitin, to regenerated chitin (fully acetylated chitin) and to chitosan (deacetylated chitin). Adsorbed vicilins were desorbed from these matrices by acetic and hydrochloric acids and by highly polymerized soluble chitosan. Proteins such as the lectin of common bean (PHA), soybean trypsin inhibitor (Kunitz), a beta-1,3-glucanase from cowpea seeds, bovine pancreatic alpha-chymotrypsin, chicken ovalbumin, serum albumin and rabbit-gamma- globulin did not bind. The present result is the first description of vicilin binding to chitin but other proteins, such as wheat germ agglutinin (WGA), a lectin that contains the so called "chitin-binding domain", and a chitinase isolated from cowpea seeds, which are involved in the defense mechanisms of plants against insects and fungi, were also shown to bind to chitin as previously reported. The binding of vicilins to chitin is probably effected not through a "chitin-binding domain" because they do not share this sequence with the defense-related proteins cited above. We propose that this association of vicilins with chitin may be related to the effect of variant vicilins on the development of Callosobruchus maculatus (bruchid) in resistant cowpea seeds.

Purification and properties of storage proteins (vicilins) from cowpea (Vigna unguiculata) seeds which are susceptible or resistant to the bruchid beethe Callosobruchus maculatus

Vicilins (7S storage proteins) from seeds of cowpea (Vigna unguiculata) cultivars which are susceptible or resistant to the bruchil veetle C. maculatus were purified by size-exclusion and ion-exchange chromatography. The vicilins were partially characterized by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions, by Western blotting and by amino acid analysis. The variant vicilins from C. maculatus-resistant seeds do not differ appreciably from vicilins from susceptible seeds by these criteria except that they are more strongly bound to DEAE-Sepharose, suggesting differences in charge between the various molecules

The resistance of seeds of cowpea (Vigna unguiculata) to the cowpea weevil (Callosobruchus maculatus)

Cowpea (Vigna unguiculata) seeds are heavily damaged during storage by the bruchid Callosobruchus maculatus. Seeds of some Nigerian varieties showed a strong resistance to this bruchid. By utilizing biochemical and entomological techniques we were able to rule out the paticipation of proteolytic enzyme (trypsin, chimotrypsin, subtilisin and papain) inhibitors, lectins, and tannins in the resistance mechanisms. Fractionation of the seed meal of a resistant variety suggests that the factor(s) responsible for the effect is (are) concentrate in the globulin fraction.

Acidic proteinases from resting cotyledons of cowpea (Vigna unguiculata)

Copwpea (Vigna unguiculata) seed cotyledons contain acidic hemoglobinase activity that can be inhibited by the aspartic acid proteinase specific inhibitor pepstatin. Two of these proteinases were isolated by affinity and ion exchange chromatography. Their relative molecular weights were 69,000 and 45,000 as calculated by gel filtration chromatography