Anais da Academia Brasileira de Ciências ISSN: 0001-3765 [email protected] Academia Brasileira de Ciências Brasil Silva, Luciana B.; Sales, Maurício P.; Oliveira, Antônia E. A.; Machado, Olga L. T.; Fernandes, Kátia V. S.; Xavier-Filho, José The seed coat of Phaseolus vulgaris interferes with the development of the cowpea weevil [Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)] Anais da Academia Brasileira de Ciências, vol. 76, núm. 1, março, 2004, pp. 57-65 Academia Brasileira de Ciências Rio de Janeiro, Brasil Available in: http://www.redalyc.org/articulo.oa?id=32776106 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Anais da Academia Brasileira de Ciências (2004) 76(1): 57-65 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc The seed coat of Phaseolus vulgaris interferes with the development of the cowpea weevil [Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)] LUCIANA B. SILVA1, MAURÍCIO P. SALES2, ANTÔNIA E.A. OLIVEIRA1, OLGA L.T. MACHADO1, KÁTIA V.S. FERNANDES1 and JOSÉ XAVIER-FILHO1 1Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, 28015-620 Campos dos Goytacazes, RJ, Brasil 2Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN, Brasil Manuscript received on August 21, 2003; accepted for publication on October 1, 2003; contributed by José Xavier-Filho* ABSTRACT We have confirmed here that the seeds of the common bean (Phaseolus vulgaris, L.) do not support develop- ment 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 stud- ied 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. Key words: common bean, phaseolin (vicilin), testa, resistance, cowpea weevil. INTRODUCTION tect them against attack by pests, which presum- ably have co-evolved with the species (Sales et al. The common bean (P. vulgaris L.) is a widely 2000). Some of these pests like the weevils Acan- cultivated, high-protein containing, legume origi- thoscelides obtectus Say and Zabrotes subfasciatus nated in the New World which has been domes- Boh. (Coleoptera: Bruchidae) attack the common ticated both in Meso America and South America bean seeds both in the field and in storage causing (Smartt 1990). The common bean seeds are en- severe losses to farmers (Schoonhoven et al. 1983). dowed with an array of low and high molecular Other bruchids like C. maculatus F., a pest of the weight substances that protect them against attack seeds of V. unguiculata (L.) Walp. (cowpea), do by many herbivores (Smartt 1990). Many proteins not attack the seeds of P. vulgaris (Applebaum et like lectins, α-amylase inhibitors, arcelins, trypsin al. 1970). The reason why C. maculatus does not inhibitors and others, while protecting the seeds develop in the seeds of the common bean may be ex- against insects and herbivores in general do not pro- plained by their mutual isolation since the bruchid is *Member of Academia Brasileira de Ciências an Old World species while P. vulgaris has its origin Correspondence to: José Xavier-Filho E-mail: [email protected] in the New World. An Acad Bras Cienc (2004) 76 (1) 58 LUCIANA B. SILVA ET AL. This discrimination may be explained by dif- discarded (40 days). Three replicates were run. For ferent mechanisms one of them being possibly the each replicate one seed was chosen for counting the presence of proteins in the testa, which may cause holes made in the testa by larvae. a delay in the development or entail death of young The effects of the various seed and testa meals larvae. We have recently suggested that the seeds of and fractions thereof on C. maculatus development the legumes P. , and C. ensiformis which are of New were assessed through an artificial seed assay World origin and related to P. vulgaris are resistant (Macedo et al. 1993). Briefly, artificial seeds (400 to attack by C. maculatus due to the presence of vi- mg) were made of finely ground cowpea seed meal cilins (7S storage globulins), in the testa (Oliveira et (EPACE-10) mixed with the materials of interest. al. 1999, Moraes et al. 2000). For testa meals we used concentrations of 1.0, 2.0, 4.0, 8.0 and 16% and for globulin fractions these MATERIALS AND METHODS were 1.0, 2.0, 4.0 and 8.0%. Infestation of artificial seeds was accomplished Seeds. Common bean [P. vulgaris, WAF-9 (white by placing 2-day old female insects in glass vials testa) and ‘‘preto’’ (black testa) genotypes] seeds containing two seeds for 24 hours at 28˚C, 60% were provided by Empresa de Pesquisa Agropecuá- relative humidity. Excess laid eggs were removed ria do Rio de Janeiro (Campos dos Goytacazes, RJ, from the seeds leaving 2-3 eggs per seed. After 20 Brazil). Seeds of the C. maculatus-susceptible cow- days (at 28˚C, 60% relative humidity) infested seeds pea (V. unguiculata) cultivars EPACE-10 (brown were opened and the weight and number of larvae testa) and ‘‘fradinho’’ (white testa) were obtained were counted. Dose-response curves were drawn from Centro de Ciências Agrárias, Universidade Fe- and the effective doses for 50% response (ED50,%) deral do Ceará (Fortaleza, Brazil) and in local mar- and lethal-doses (LD50, %) were calculated. Con- kets. C. maculatus-resistant cowpea seeds (IT81D- trol seeds were made with the meal of cowpea seeds 1045, brown testa) were obtained from IITA (In- without additions. All the experiments were run in ternational Institute of Tropical Agriculture, Ibadan, triplicate and the data shown are the average of these. Nigeria), Ibadan, Nigeria trough Centro Nacional de Pesquisa do Arroz e Feijão-Embrapa, Goiânia, Seed protein isolation. Seeds were carefully dis- Brazil. C. maculatus (cowpea weevil) were pro- sected in order to separate embryonic tissues from vided by J.H. Ribeiro dos Santos from Centro de the testa. The finely ground meals of testa-free Ciências Agrárias, Universidade Federal do Ceará seeds and testa of P. vulgaris (WAF-9) were ex- (Fortaleza, Brazil) and were maintained in culture tracted (1:10, meal to buffer ratio) for3hat5˚C, (28-30˚C and 65-75% relative humidity). We em- with 50 mM sodium tetra borate buffer, pH 8.0; the ployed an antibody against cowpea vicilins raised resulting slurry was centrifuged at 10000 × g for in white rabbits previously prepared by us and an 30 min at 4˚C. The crude extracts obtained were antibody against P. vulgaris phaseolin kindly pro- fractionated by ammonium sulfate and the proteins vided by Dr. Maarten Chrispeels (Dept. of Biology, precipitating between 70 and 90% saturation were University of California, San Diego, USA). collected, dialyzed against water and freeze dried C. maculatus behavior. Development of C. macu- for further analysis. latus on P. vulgaris seeds was observed by placing Finely ground testa tissues from the several 2-day old females in glass vials containing ten seeds seeds utilized were extracted (1 g in 20 ml) with at 28˚C, 60% relative humidity. After 2 days insects a solution, which was 7 M urea and 0.1% acetic were discarded and the numbers of laid and hatched acid for 16 h under continuous stirring, at 5˚C. The eggs were counted. Seeds were observed daily for suspensions were centrifuged at 10000 × g for 30 any emergence of adults until the experiment was min at 4˚C. The supernatants were dialyzed against An Acad Bras Cienc (2004) 76 (1) BEAN SEED COAT PROTEINS INTERFERE WITH BRUCHID DEVELOPMENT 59 water and the samples containing testa proteins were aration on a reverse-phase C18 column (4.6mm× recovered by freeze-drying. 2.5mm) under isocratic conditions, according to the Embryo vicilins (or phaseolin) were purified by manufacturer’s instructions. The sequences were chromatography in a Sephacryl S-400 column (60× compared to amino acid data banks. The se- 2.5 cm) employing 0.1 M Tris-HCl, 0.25 M NaCl, quences selected were submitted to automatic align- pH 8.0 as equilibration and elution buffer (Macedo ment, which was performed by using the NCBI- et al. 1995). BLAST search system. Chemical analysis. Protein was determined by the dye binding method of Bradford (1976) or alterna- RESULTS tively by the absorbance at 280 nm. An ELISA assay employing an antibody produced against P. vulgaris The thickness of the testa of the seeds of P. vul- phaseolin was used to quantify phaseolin in testa and garis employed in this study (WAF-9 and ‘‘preto’’ . ± . embryo tissues. genotypes) was 0 13 0 03 mm and of seeds of The presence of phenols in testa and embryo V. unguiculata (EPACE-10 and IT81D-1045 geno- . ± . ± . tissues and their quantitation were determined by a types) was 0 11 0 03 mm and 0 12 0 02 mm, procedure described by Chiang and Jackay (1988) respectively. Several components of the testa were that is based on the formation of color complexes measured in these and other seeds.