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

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 insulin in plants is not accepted by the scientific community in general. In this review we discuss this paradigm and retrieve information that strongly suggests that insulin is indeed found in plants. We present results, which indicate that a protein molecule with the same amino acid sequence as bovine insulin is expressed in leguminous plants. Additionally, we provide evidence that proteins associated with insulin signalling pathways in vertebrates are also found in association with insulin-like molecules in plants.

We have investigated the survival of Callosobruchus maculatus larvae when reared on resistant IT81D 1045 Vigna unguiculata seeds, whose resistance has been associated with variant forms of vicilins. Here, we present data which show that larvae of C. maculatus feeding on embryonic axis of resistant cowpea reach a mass of around 28 times higher than those feeding on cotyledonary tissues. Additionally, incorporation of 5-10% of embryonic axis flour on artificial seeds made of resistant flour restores toxicity of seeds to the bruchid. Vicilins purified from both susceptible and resistant embryonic axis had no deleterious effects either on insect development or on insect survival until a level of 4% of incorporation. In contrast, vicilins from resistant cotyledons show an LD 50 (50% lethal dose) and WD 50 (half weight dose) of 2%. Total vicilin contents of embryonic axes were around two times lower than in cotyledonary tissues, while proteolytic activities of all four proteinase classes were always higher in the former tissues. By 2D-PAGE we visualized eight protein spots, which seem to be exclusively found on resistant cotyledons.