Identification, biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation
Introduction
Common bean (Phaseolus vulgaris L.) is a legume of significant socioeconomic and nutritional importance and is extensively cultivated worldwide (Messina, 2014). This plant species has biochemical and structural defense mechanisms against plant pathogens, and this capacity was developed during evolution. Structural defense mechanisms limit pathogen infections and involve adaptations related to the increased thickness of the wax layer, cuticles, stomata, lenticels, trichomes, and hairs (Glas et al., 2012; Malinovsky et al., 2014). Antimicrobial peptides (AMPs) serve as a biochemical defense mechanism and are a key component in plant innate immunity (Egorov and Odintsova, 2012; Campos et al., 2018).
AMPs are essential in the defense against pathogens and are widely found across plant and animal species (Silva et al., 2011; Egorov and Odintsova, 2012; Campos et al., 2018). AMPs usually have a low molecular weight (<10 kDa), 10 to 100 amino acid residues, and unique physicochemical properties, including amphipathicity, hydrophobicity, and cationicity (Ponnappan et al., 2015; Budagavi et al., 2017).
In plants, AMPs protect against several adverse conditions, including pathogen attack, and a broad spectrum of activity against fungi, Gram-positive and Gram-negative bacteria, enveloped viruses, and parasites (Nguyen et al., 2011; Scorciapino et al., 2017). AMPs have been isolated from different plant species and different plant organs, including flowers, leaves, fruits, tubers, roots, and especially seeds (Pelegrini et al., 2008; Carvalho and Gomes, 2009; Nawrot et al., 2014). Many defense proteins have been identified in legume seeds, especially in the genus Phaseolus, including defensins (Games et al., 2008; Mello et al., 2014), lipid-transfer proteins (LTPs), proteinase inhibitors and α-amylase inhibitors, among others (Moreno et al., 1990; Ye et al., 2001; Carlini and Grossi-de-Sá, 2002).
The seed coat is the first protective barrier against penetration of insects, including insects of the Chrysomelidae family, such as Callosobruchus maculatus (F.) (Souza et al., 2011; De Sá et al., 2014; De Sá et al., 2018). Previous studies reported that the toxicity of the seed coat, such as Phaseolus lunatus, Phaseolus vulgaris, Canavalia ensiformis, Vigna unguiculata, Glycine max, Albizia lebbeck and others impaired the development of C. maculatus (Moraes et al., 2000; Silva et al., 2004, 2016; Souza et al., 2011; Cruz et al., 2016; Silva et al., 2018; De Sá et al., 2018). De Sá et al. (2014) showed that the number of C. maculatus eggs laid on P. vulgaris seed coats decreased about 48%. A high mortality (80%) was observed in larvae fed with P. vulgaris seed coat flour and the surviving larvae showed reductions of approximately 50% in body mass (De Sá et al., 2014). These results indicate the presence toxic/repellant phytochemicals in P. vulgaris seed coat.
In the past few years, AMPs have stimulated the interest in developing genetically engineered plant cultivars resistant to pathogens to minimize the excessive use of agrochemicals in agriculture (Holaskova et al., 2015). The objective of this study is to identify AMPs in common bean genotypes by biochemical and enzymatic analyses and assess their biological roles in seed defense.
Section snippets
Seeds
Nineteen common bean genotypes from the Germplasm Bank of the Darcy Ribeiro State University of the North of Rio de Janeiro (Universidade Estadual do Norte Fluminense Darcy Ribeiro – UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil (2239, 2240, 2251, 2252, 2253, 2256, 2260, 2263, 2264, 2265, 2266, 2267, 2268, 2271, 2274, 2275, 2286, 2287, and 2288) were collected on farms from two regions of the state of Rio de Janeiro (North and Lakes).
The seeds of V. unguiculata (L.) (cv. Fradinho)
Electrophoretic profile of proteins
The electrophoretic profiles of the proteins of the crude extracts of seeds of the 19 common bean genotypes obtained after protein extraction are shown in Fig. 1. Thirteen lanes corresponding to 13 UENF genotypes (2239, 2240, 2251, 2253, 2263, 2264, 2265, 2266, 2267, 2268, 2286, 2287, and 2288) contained one 26.6 kDa protein band. Fifteen lanes corresponding to 15 UENF genotypes (2239, 2240, 2251, 2253, 2256, 2260, 2263, 2264, 2265, 2266, 2267, 2271, 2275, 2286, and 2287) contained protein
Discussion
We initially investigated the presence of defense proteins, including LTPs and defensins, from the crude extracts of seeds of common beans. LTPs were detected in the extracts of nine genotypes (Fig. 2) whereas defensins were detected in the extracts of all genotypes (Fig. 3). Previous characterization studies of plant defensin in seeds of P. vulgaris and V. unguiculata (Wong et al., 2006; Games et al., 2008; Mello et al., 2011), showed that these peptides were able to inhibit α-amylase enzyme
CRediT authorship contribution statement
Elisângela Knoblauch Viega de Andrade: Formal analysis, Investigation, Validation, Data curation, Writing - original draft. Rosana Rodrigues: Formal analysis, Validation, Data curation, Writing - original draft, Supervision, Project administration, Funding acquisition. Gabriela da Costa Vieira Bard: Investigation, Formal analysis, Validation, Data curation, Writing - original draft. Lídia da Silva Pereira: Investigation. Kayan Eudorico Ventury Baptista: Investigation, Formal analysis,
Declaration of competing interest
The authors do not have potential conflict of interest to disclose.
Acknowledgments
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. This work was performed at the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF). We acknowledge the financial support from the Brazilian agencies CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico and FAPERJ - Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro.
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