Identification, biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation

Highlights

• Peptides from antimicrobial families were identified in all genotypes analyzed.

• Extracts were able to inhibit the C. maculatus gut amylase activity.

• Extracts were able to inhibit the human salivary amylase activity.

• Natural seeds from all genotypes did not inhibit insect oviposition.

• Larvae did not survive after feeding on natural seeds from all genotypes.

Abstract

Common bean is a legume of significant socioeconomic importance and is cultivated worldwide. This crop is affected by several pests and diseases, which cause considerable economic losses and reduce yield. In recent years, several studies have demonstrated the role of proteins and peptides with activity against a wide range of insects and pathogens. The objective of this work was to identify defense proteins, such as antimicrobial peptides, protease and amylases inhibitors in common bean genotypes and evaluate the relationship of these proteins with Phaseolus vulgaris seed resistance to Callosobruchus maculatus infestation. Nineteen common bean genotypes were subjected to protein extraction, pH 5.4, and precipitation with ammonium sulfate at 70% saturation. The obtained extracts were separated by tricine gel electrophoresis. Experiments were carried out with natural seeds of common beans and artificial seeds (Vigna unguiculata seeds covered with seed coats of common beans) to evaluate the rate of oviposition and development of the insect species Callosobruchus maculatus. Lipid-transfer proteins were identified in nine genotypes whereas defensins were present in all genotypes. The inhibitory activity of α-amylases and trypsin and fungal development were determined in crude extracts (50 μg mL⁻¹). The results also indicated that the extracts from all bean genotypes inhibited the activity of human salivary α-amylase and C. maculatus larval α-amylase. Except for the extracts of four genotypes, all other extracts inhibited trypsin activity. None of the extracts from the evaluated bean genotypes inhibited the growth of tested fungi. Natural seeds from all genotypes did not inhibit insect oviposition, however, the larvae did not survive after feeding on these seeds. Artificial seeds containing seed coat flour of all genotypes inhibited the oviposition of C. maculatus, indicating that the seed coat was also repellent to insect.

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.