Vicilin storage proteins (7S globulins) isolated from Vigna
unguiculata (cowpea) seeds were shown
to interfere with the germination of spores or conidia of the fungi
Fusarium solani, Fusarium
oxysporum, Colletotrichum musae, Phytophtora
capsici, Neurospora
crassa, and
Ustilago maydis
sporidia. Cowpea vicilins have been shown to bind
to fungal structures, possibly chitin-containing
structures of the cell wall, and can be desorbed by strong acid.
The results presented in this paper
are in agreement with data previously obtained on the chitin-binding
properties of cowpea vicilins
and the effect they exert on the development and survival of the
storage pest insect Callosobruchus
maculatus (Coleoptera: Bruchidae).
Keywords: 7S storage globulins; cowpea seeds; Vigna unguiculata;
chitin-binding proteins; fungi;
spore germination
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.
Correspondence
A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines.
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 C 4 -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.
Plants exude a variety of substances through their roots, germinating seeds and aerial parts. Some of these released compounds seem to have an inhibitory effect against pathogens. The aim of this work was to investigate and identify antifungal proteins present in exudates from imbibed cowpea seeds (Vigna unguiculata (L.) Walp). The obtained exudation was analyzed in regard to specific protein activities by enzymatic or immunological assays for plant defense proteins, from 4 h to 48 h of seed imbibition. Our results show that cowpea seeds exudates present several defense related proteins characterized as b-1,3-glucanases, cystatins, vicilins and lipid transfer proteins (LTPs), as well as a storage vacuole membrane a-TIP protein, since the very first hours of imbibition. These exudates also have an ''in vitro'' inhibitory effect on the growth of the fungus Fusarium oxysporum f. sp. phaseoli. Our results suggest that seed exudates should promote seed protection from soil pathogens.
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