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%, 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%. 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.

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% 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.

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/µg 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.

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 pre-treated 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.

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.

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.

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%, respectively. The level of phaseolin in the seed coat (16.7%) 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.

Frações protéicas obtidas de sementes de feijão-de-porco (Canavalia ensiformis), bem como o aminoácido canavanina presente nestas sementes, foram avaliadas em sua capacidade de inibir o crescimento dos fungos fitopatogênicos Macrophomina phaseolina, Colletotrichum gloesporioides, Sclerotium rolfsii e Fusarium oxysporum. Constatamos que a maior parte das proteínas examinadas e também canavanina não apresentaram nenhum efeito sobre o crescimento desses fungos. Por outro lado, a proteína tóxica canatoxina mostrou-se efetiva, a uma concentração de 2%, na inibição do crescimento de M. phaseolina, C. gloesporioides e S. rolfsii.

Protein fractions obtained from seeds of the jack bean (Canavalia ensiformis) as well as the amino acid canavanine, present in these seeds, were tested for their capacity to inhibit the growth of the phytopathogenic fungi Macrophomina phaseolina, Colletotrichum gloesporioides, Sclerotium rolfsii and Fusarium oxysporum. We found that most of the proteins examined and also canavanine did not have any effect on the growth of these fungi. On the other hand the toxic protein canatoxin was found to be effective, at a concentration of 2%, in the inhibition of the growth of M. phaseolina, C. gloesporioides and S. rolfsii.

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.