Elsevier

Computers in Biology and Medicine

Volume 56, 1 January 2015, Pages 67-81
Computers in Biology and Medicine

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea)

https://doi.org/10.1016/j.compbiomed.2014.10.019Get rights and content

Highlights

  • Genes for a significant number of proteases and protease inhibitors have been identified in chickpea.

  • Most of them have close orthologs in Medicago truncatula and Glycine max.

  • Although codon preference was seen for the catalytic residues but the relation is complex.

  • A differential pattern of gene expression was observed in multiple plant tissues.

  • The docking and molecular dynamics simulation showed chickpea protease-inhibitor molecular recognition pattern.

Abstract

Background

Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now.

Methods

Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand.

Results

In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein–ligand and inter and intramolecular protein–protein hydrogen bonds.

Discussion

The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea.

Introduction

Plants, being sessile in nature, are continuously exposed to a broad range of environmental stress conditions that adversely affect their growth, development, and productivity. Plants encounter both biotic and abiotic stress conditions during their complete life cycle. In biotic stress, plants face the threat of infection by pathogens (including bacteria, fungi, viruses and nematodes) and attack by herbivore pests [1]. Abiotic stress includes exposure of plants to drought, salinity, heat, cold, chilling, freezing, nutrient deprivation, high light intensity, ozone (O3) and anaerobic stresses [2]. Therefore, in order to withstand such situations, plants have developed diverse mechanisms to detect such environmental changes and respond in such a manner to reduce damage while conserving crucial resources for growth and reproduction. A stress response is initiated when plants perceive stress at the cellular level. Stress recognition activates signal transduction pathways that transmit information within the individual cell and throughout the plant [3]. Further it leads to alterations in the gene expression pattern, which modify growth and development and even influence reproductive capabilities. Various studies have shown the devastating effect of such adverse environmental conditions on the productivity of crops.

According to a report by Food and Agriculture Organization (FAO) 2008 chickpea is one of the ancient and second most widely grown crops in the world [4]. It is the primary source of human dietary nitrogen, rich in proteins, carbohydrates, fibres, vitamins, minerals, sans any cholesterol content [5]. Various studies have shown that consuming chickpea reduces blood cholesterol level [6]. Chickpea crop loss caused by abiotic stress exceeds those due to biotic stress. Major crop damage is caused due to drought, salinity, and cold. The following diseases are also commonly seen in chickpea affecting its productivity severely: Ascochyta blight caused by Ascochyta rabiei (Pass) Labr.; Botrytis Grey Mould (BGM) caused by Botrytis cinerea Pers; Fusarium wilt caused by Fusarium oxysporum f. sp. ciceri; Dry root rot caused by Macrophomina phaseolina; Collar rot caused by Sclerotium rolfsii; and Phytophthora root rot caused by Phytophthora medicaginis. Another major concern is the attack by insect pests that mainly includes Pod borer caused by Helicoverpa armigera Hubner; leaf miner caused by Liriomyza ciceriana Rondani; and seed beetle caused by Callosobruchus spp. in major production areas [7].

Plants are well equipped with a large proteolytic machinery which hydrolyze the non-functional proteins of the cells and make the resultant amino acids available for recycling. Along with that, proteases also take part in several biological processes like recognition of pathogens and pests and the induction of effective defence responses, programmed cell death, countering water deficit etc. [8], [9], [10]. Although proteases play an indispensable role in the maintenance and survival of the organisms they can be harmful if over expressed. Apart from the synthesis of these enzymes as inactive pre-proteins, their activity is also controlled by interaction with protease inhibitors (PIs) that leads to the formation of less active or fully inactive complexes [8]. Plant protease inhibitors (PPIs) are generally small proteins commonly present in the storage tissues like seeds, tubers, and aerial parts of the plants. One of the important roles of PPIs in plant defence mechanism is in triggering response to attack by insects or due to pathogenesis and wounding. They react by inhibiting the proteases present in the insect gut or that secreted by the microorganism which result in reduced amount of amino acids available for their growth and development [11].

Previously Yan et al. reported the genome-wide analysis of regulatory proteases in Taenia solium genome [12]. However, in our study, an in silico search of both proteases and PIs together in chickpea genome was conducted. A significant number of members belonging to four protease families (aspartate protease, cysteine protease, serine protease, and metalloprotease) and PIs (cysteine and serine protease inhibitors) were identified in the chickpea proteome (1.28% of the total chickpea genome encodes proteases out of which 0.8% are with catalytic residues). Phylogenetic analysis of each class of proteases showed clustering that matched with their functional diversification and domain diversity. Orthologs were identified in other sequenced genomes also, the maximum number of orthologs was found in the genome of Glycine max. Chickpea proteases and PI genes were further characterized for their chromosomal location, domain classification, gene architecture, gene duplication events, analysis of codon composition and gene expression. The binding studies of the PIs/ proteases with their cognate target proteases/ inhibitors were carried out to get more insight about the mode of binding and affinity. Fig. 1 perspicuously depicts the workflow followed in the research represented here. These findings will help to select candidate stress-related genes in chickpea, experimentally characterize their function and manipulate them to enhance the stress tolerance capacity of chickpea crop.

Section snippets

Gene identification

A draft genome of chickpea was retrieved from legume information system (LIS) database (http://cicar.comparative-legumes.org/). Estimated genome size of Cicer arietinum is around 740 Mb which consisted of 28,269 gene models and 7163 scaffolds covering 544.73 Mb (over 70% of the estimated genome size) [13]. The proteases (aspartate protease, cysteine protease, serine protease, and metalloprotease) and protein protease inhibitors (cysteine protease inhibitor and serine protease inhibitors) of

Aspartate protease (AP)

A hidden Markov model profile (PF00026) search using HMMER (hmmsearch) resulted in the identification of a total of 89 CaAPs (C. arietinum APs), out of which 66 sequences had the two complete Asp-Thr/Ser-Gly motifs (Fig. S1). The remaining sequences had either an incomplete ASP domain or present in the data only a single motif. Out of these 66 sequences, genomic locations of 56 sequences were known and named from CaAP1–CaAP56 based on their order on the chromosomes. The remaining 8 sequences

Conclusion

Developing disease resistant and stress tolerant varieties of plants is an important objective of breeding crops. Therefore, to help developing stress tolerant and pathogen resistant varieties, we have studied proteases and PIs in chickpea genome. A large fraction of chickpea genome encodes proteases and PI genes which provide resistance against various environmental stress conditions. A differential pattern of gene expression was observed in more than one plant tissue under study. The

Summary

Being sessile, plants are often exposed to a wide range of detrimental environmental conditions which adversely affect their growth and productivity. However, they have developed distinct mechanisms to perceive environmental changes and respond to such complex stress conditions, thus minimize damage and conserve the valuable resources required for the growth and reproduction. Proteases and protease inhibitors are one of the important classes of enzymes which play an important role during

Conflict of interest statement

The authors have no conflict of interest.

Acknowledgements

RS thanks Council of Scientific and Industrial Research (CSIR), India for Senior Research Fellowship. This work is carried out under CSIR-National Chemical Laboratory Centre of Excellence in Scientific Computation (CoE-SC) project.

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