Deleterious effects of Schinus terebinthifolius Raddi seed flour on cowpea weevil, Callosobruchus maculatus (F.), larval development.

Schinus terebinthifolius, Raddi, has been extensively studied due to its anti-inflammatory and antibiotic properties. S. terebinthifolius was also toxic to some insects, however little has been explored about the nature of its insecticide compounds or the toxicity of this plant to insect species. In this work, we investigate the toxicity of S. terebinthifolius seed flour against the insect C. maculatus. S. terebinthifolius seed flour interfered with the post hatch development of the C. maculatus larvae, decreasing larval survival, mass and length. Using DEAE-cellulose chromatography, five protein fractions were isolated, a non-retained fraction (NRF) and four retained fractions, eluted with 0.25, 0.5, 0.7 and 1.0 M NaCl. Proteins with varying molecular masses were observed in all fractions. The majority protein bands were identified by mass spectrometry analysis and among the main identified proteins are 11S globulins (such glycinin), lipoxygenase, chitinases, 7S globulins (vicilins, canavalin and ß conglycinin), annexin, catalase and sucrose binding protein. All DEAE-protein fractions were toxic to the insect, interfering with the post hatch larval development and survival. Decreases greater than 90% were observed in the larval mass and length at 20 days after oviposition (DAO) for larvae raised on diet containing 0.5% of some fractions. Alterations in the level of proteins, glucose and in the activity of the enzymes lipases and cysteine proteases were also detected in these larvae. Our results show that seeds of S. terebinthifolius have an arsenal of toxic proteins with potential for the control of the insect C. maculatus.

Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

PvD1 defensin, a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis.

Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD1 from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD1 could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD1 defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD1 defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.

Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves' ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain.

MAIN CONCLUSION: Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues.

In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings.

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH4)2SO4 precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (Km=9.86mM, Vmax=24.66 EU [DeltaAmin(-1)]) and catechol (Km=3.44mM, Vmax=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.

Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests.

Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.

The seed coat of Phaseolus vulgaris interferes with the development of the cowpea weevil [Callosobruchus maculatus (F. ) (Coleoptera: Bruchidae)]

We have confirmed here that the seeds of the common bean (Phaseolus vulgaris, L.) do not support development of the bruchid Callosobruchus maculatus (F.), a pest of cowpea [Vigna unguiculata (L.) Walp] seeds. Analysis of the testa (seed coat) of the bean suggested that neither thickness nor the levels of compounds such as tannic acid, tannins, or HCN are important for the resistance. On the other hand, we have found that phaseolin (vicilin-like 7S storage globulin), detected in the testa by Western blotting and N-terminal amino acid sequencing, is detrimental to the development of C. maculatus. As for the case of other previously studied legume seeds (Canavalia ensiformis and Phaseolus lunatus) we suggest that the presence of vicilin-like proteins in the testa of P. vulgaris may have had a significant role in the evolutionary adaptation of bruchids to the seeds of leguminous plants.

Temporal and tissue localization of a cowpea (Vigna unguiculata) cystatin.

A cowpea (Vigna unguiculata L. Walpers cv. Pitiúba) cystatin was analysed to determine its localization during development and germination of cowpea seeds, using western blotting with a specific antiserum. The pattern of immunoreactive proteins changed during development, with the major reactive bands present in dried seeds being mobilized after a 62-h period of imbibition. Immunohistochemical analysis revealed that cowpea cystatin is distributed in both embryonic axes and cotyledons with the highest level being present in the outer cell walls of the adaxial surface of the cotyledons.