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%, 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.

Chitin-binding proteins; Seed coat; Callosobruchus maculatus; Insect bruchid

Authorship

A.J. Souza

Universidade Estadual do Norte Fluminense, Centro de Biociências e Biotecnologia , Laboratório de Química e Função de Proteínas e Peptídeos, Campos dos Goytacazes, Rio de Janeiro, BrazilUniversidade Estadual do Norte FluminenseBrazilCampos dos Goytacazes, Rio de Janeiro, BrazilUniversidade Estadual do Norte Fluminense, Centro de Biociências e Biotecnologia , Laboratório de Química e Função de Proteínas e Peptídeos, Campos dos Goytacazes, Rio de Janeiro, Brazil

A.T.S. Ferreira

Fundação Oswaldo Cruz, Instituto Oswaldo Cruz , Departamento de Fisiologia e Farmacodinâmica, Rio de Janeiro, Rio de Janeiro, BrazilFundação Oswaldo CruzBrazilRio de Janeiro, Rio de Janeiro, BrazilFundação Oswaldo Cruz, Instituto Oswaldo Cruz , Departamento de Fisiologia e Farmacodinâmica, Rio de Janeiro, Rio de Janeiro, Brazil

J. Perales

D.G. Beghini

K.V.S. Fernandes

J. Xavier-Filho

T.M. Venancio

A.E.A. Oliveira

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (5)
Tables (1)

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Figure 1.
Toxicity of Albizia lebbeck seed coat flour to Callosobruchus maculatus larval development and survival at 20 days after oviposition. The seed coat protein was incorporated into artificial seeds at different relative concentrations (0.1, 0.25, 0.5, and 1.0%). Experiments were carried out in triplicate and the data shown are the means ± SD of these results. *P < 0.05 compared to control (t-test).

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Figure 2.
CM-Sepharose ion exchange chromatography (2.0 x 21.0 cm) of Albizia lebbeck seed coat proteins previously precipitated with ammonium sulfate (0-90% saturation) and dissolved in sodium acetate buffer, pH 5.0. The column was equilibrated and chromatography was developed with 0.1 M sodium acetate buffer, pH 5.0. The non-retained fraction was eluted with 0.1 M sodium acetate buffer, pH 5.0 (CI fraction) and adsorbed proteins were desorbed with 0.25 and 0.5 M NaCl (CII and CIII fractions, respectively).

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Figure 3.
Toxicity of flour and of the CI fraction prepared from Albizia lebbeck seed coat to Callosobruchus maculatus larvae. A and B, Larval mortality. B, a-d: larval mortality in artificial seeds containing the CI fraction (arrows). a: the larva died inside the egg; b: perforation of artificial seed; c and d: dead larvae found inside the artificial seed. e-j: development of larvae on natural A. lebbeck and Vigna unguiculata seeds. e: larva inside the egg on the 5th day after oviposition; f: inferior surface of the egg on the 5th day after oviposition; g: larva inside the egg on the 16th day after oviposition; h: A. lebbeck seed coat partially perforated on the 16th day after oviposition; i: hole across the host seed coat from V. unguiculata on the 6th day; j: hole on the V. unguiculata cotyledon surface on the 6th day after oviposition. The bar inside Panel a indicates magnification for Panels a-d. The bar inside Panel e indicates magnification for Panels e-g. The bar inside Panels h/i indicates magnification for Panels h-j. C, Larval mass at 20 days after oviposition. The seed coat fraction was incorporated into artificial seeds at different concentrations (0.1, 0.25, 0.5, and 1.0%). Experiments were done in triplicate and the data shown are the means ± SD of these results. *P < 0.05 compared to control (t-test).

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Figure 4.
A, Sephacryl S-100 gel filtration chromatography of the CI fraction of Albizia lebbeck seed coat previously obtained by CM-Sepharose chromatography resulting in three fractions (SI, SII, and SIII). The column was equilibrated and developed with 50 mM potassium phosphate buffer, pH 7.8. B, SDS-polyacrylamide gel electrophoresis of the SI fraction (106 to 116 mL).

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Figure 5.
Chitin-binding affinity of Albizia lebbeck seed coat SI protein fraction and quantification of vicilin-like proteins in the non-retained and adsorbed chitin fractions (desorbed with 0.1 M HCl).

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Table 1.
Mass spectrometry analysis of the peptides from the fraction of Albizia lebbeck seed coat obtained by Sephacryl S-100 gel filtration chromatography (SI fraction).

Figure 1. Toxicity of Albizia lebbeck seed coat flour to Callosobruchus maculatus larval development and survival at 20 days after oviposition. The seed coat protein was incorporated into artificial seeds at different relative concentrations (0.1, 0.25, 0.5, and 1.0%). Experiments were carried out in triplicate and the data shown are the means ± SD of these results. *P < 0.05 compared to control (t-test).

Figure 2. CM-Sepharose ion exchange chromatography (2.0 x 21.0 cm) of Albizia lebbeck seed coat proteins previously precipitated with ammonium sulfate (0-90% saturation) and dissolved in sodium acetate buffer, pH 5.0. The column was equilibrated and chromatography was developed with 0.1 M sodium acetate buffer, pH 5.0. The non-retained fraction was eluted with 0.1 M sodium acetate buffer, pH 5.0 (CI fraction) and adsorbed proteins were desorbed with 0.25 and 0.5 M NaCl (CII and CIII fractions, respectively).

Figure 3. Toxicity of flour and of the CI fraction prepared from Albizia lebbeck seed coat to Callosobruchus maculatus larvae. A and B, Larval mortality. B, a-d: larval mortality in artificial seeds containing the CI fraction (arrows). a: the larva died inside the egg; b: perforation of artificial seed; c and d: dead larvae found inside the artificial seed. e-j: development of larvae on natural A. lebbeck and Vigna unguiculata seeds. e: larva inside the egg on the 5th day after oviposition; f: inferior surface of the egg on the 5th day after oviposition; g: larva inside the egg on the 16th day after oviposition; h: A. lebbeck seed coat partially perforated on the 16th day after oviposition; i: hole across the host seed coat from V. unguiculata on the 6th day; j: hole on the V. unguiculata cotyledon surface on the 6th day after oviposition. The bar inside Panel a indicates magnification for Panels a-d. The bar inside Panel e indicates magnification for Panels e-g. The bar inside Panels h/i indicates magnification for Panels *h-j. C, Larval mass at 20 days after oviposition. The seed coat fraction was incorporated into artificial seeds at different concentrations (0.1, 0.25, 0.5, and 1.0%). Experiments were done in triplicate and the data shown are the means ± SD of these results. P < 0.05 compared to control (t-test).

Figure 4. A, Sephacryl S-100 gel filtration chromatography of the CI fraction of Albizia lebbeck seed coat previously obtained by CM-Sepharose chromatography resulting in three fractions (SI, SII, and SIII). The column was equilibrated and developed with 50 mM potassium phosphate buffer, pH 7.8. B, SDS-polyacrylamide gel electrophoresis of the SI fraction (106 to 116 mL).

Figure 5. Chitin-binding affinity of Albizia lebbeck seed coat SI protein fraction and quantification of vicilin-like proteins in the non-retained and adsorbed chitin fractions (desorbed with 0.1 M HCl).

Table 1. Mass spectrometry analysis of the peptides from the fraction of Albizia lebbeck seed coat obtained by Sephacryl S-100 gel filtration chromatography (SI fraction).

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

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