Unveiling the Potential of OsRuvB DNA Helicases in Enhancing Salinity Stress Tolerance in Chickpea

Research Article

Austin J Plant Bio. 2023; 9(1): 1037.

Unveiling the Potential of OsRuvB DNA Helicases in Enhancing Salinity Stress Tolerance in Chickpea

Deepak Kumar1; Nita Lakra1*; Parul Sharma2; Annu Luhach1; Abbu Zaid3,4

1Department of Molecular Biology, Biotechnology and Bioinformatics CCS Haryana Agricultural University, India

2Department of Botany and Plant Physiology, CCS Haryana Agricultural University, India

3Plant Physiology and Biochemistry Section, Department of Botany, Aligarh Muslim University, Aligarh, India

4Department of Botany, GGM Science College, Cluster University Jammu, India

*Corresponding author: Nita Lakra Department of Molecular Biology, Biotechnology and Bioinformatics CCS Haryana Agricultural University, India. Email: nitahaubotany2019@gmail.com

Received: August 24, 2023 Accepted: October 09, 2023 Published: October 16, 2023

Abstract

Chickpea (Cicer arietinum L.) is a self-pollinated true diploid (2n=2x=16) cool season leguminous crop that ranks second among food grain legumes after soybean. It grows under a wide range of climatic conditions and is highly sensitive to salt stress. In the present study, transgenic chickpea plants (var. HC-1) carrying OsRuvB gene were screened for salt stress. Putative transformants were screened at an early stage through PCR amplification using gene specific primers and a transformation frequency of 36.2% was observed. Physio-biochemical analysis of selected T2 transgenic plants subjected to 100 mM salt stress showed that transgenic plants were able to maintain higher chlorophyll content, relative water content, cell viability, proline content, Na+/K+ content, catalase and peroxidase activity compared to the wild type plants. Whereas electrolytic leakage and lipid peroxidation were relatively less as compared to the wild type plants under 100 mM stress. Among all transgenic lines, line 8 performed well with respect to all the parameters studied and can be taken further for the development of transgenic chickpea plants for salt stress tolerance.

Keywords: Chickpea; Transgenic; OsRuvB; Physio-biochemical; Salt stress

Introduction

Chickpea (Cicer arietinum L.) is a self-pollinated true diploid (2n=2x=16) leguminous crop with a 738 Mbp genome that ranks second among food grain legumes in the world after soybean. It is grown in a wide range of environments in over 50 countries in subtropical and temperate regions of the world [1].

Although chickpea is grown in over 50 countries, 90% of the area under chickpea cultivation is in the developing countries, with southern and South-East Asian countries accounting for >79% of the global production [2]. Globally chickpea harvested area has been expanded from 9.63 million ha in 1980 to 12.65 million ha in 2016. Global chickpea production has also increased from 4.85 million tons in 1980 to 12.09 million tons in 2016. During 2017-18, a total of 25.23 million tons pulses were produced from 29.99 million ha. Out of total pulses production, a total of 112.29 lakh tons chickpea was produced from 105.61 lakh ha area which accounted for 35. 21 per cent and 44.50 per cent of area and production of total pulses, respectively [3]. The global growth rate of pulse production over the last decade has been 2.61%. In India, Madhya Pradesh is the highest chickpea producing state with a share of 41% of the national production. The other major chickpea producing states are Rajasthan, Maharashtra, Andhra Pradesh, Telangana, Uttar Pradesh, and Karnataka, which cover 95% the area under chickpea cultivation (State wise share to total production and area of chickpea in India 2015–2016). Cultivars grown in India are either native (desi) types or Mediterranean (Kabuli) types. The growth trends of area and production of pulses in Haryana found declining from 1970-71 to 2016-17. In 2017-2018 the production of chickpea went down from 36.4 thousand tons from 32 thousand ha of area [4].

Chickpea seeds consist of 19.3% protein, 64.6% carbohydrate, and vitamins [5]. Although chickpea has a high yield potential (4000 kg/ha), actual yields are quite low due to biotic and abiotic stresses [6]. High salinity is one of the major abiotic stress factors that reduce plant growth ultimately hindering crop productivity [7]. At least 20% of all irrigated lands are salt affected, with some estimates being as high as 50% [8]. Salinity is a soil condition characterized by a high concentration of soluble salts. Soils are classified as saline when ions concentration is such that osmotic pressure produced by ions are equivalent to that generated by 40 mM NaCl (i.e., 0.2 MPa) or higher [9]. Abiotic stresses affect various morphological, physiological, and biochemical processes, though all plants in a timely and well-coordinated response such that tolerant genotypes which are well adapted adaptation and survive under stress [10]. Excess of soluble salts in the soil leads to osmotic stress, resulting inion imbalances and toxicity, resulting in retarted plant growth [11,12]. Under stress conditions, Reactive Oxygen Species (ROS) are commonly generated and stored which cause oxidative damage to biomolecules such as lipids and proteins, resulting in cell death later in the process [13]. One of the approaches to overcome the consequence of salt stress is increasing salt tolerance in crop plants. Salinity is a complex trait which is associated with various cellular mechanisms [14].

Plant breeders have achieved some success in producing salt-tolerant lines/cultivars of some crops through conventional breeding; however, the main issue that conventional plant breeders have faced is the low magnitude of genetically based variation in the gene pools of most crop species. However, because of reproductive barriers, transferring salt-tolerant genes from wild relatives to domesticated crops is not easy [15] and thus very few examples of this approach being used effectively could be found in the literature [16,17]. The overall approach is time-consuming and labor-intensive; undesirable genes are frequently transferred alongside desirable ones; and reproductive barriers limit transfer of favorable alleles from inter-specific and inter-generic sources. Because of these factors, genetic engineering has emerged as an alternative strategy to conventional breeding for crop quality and impend yield potential in most crops. Nonetheless, plant biologists have focused heavily on genes that encode ion transport proteins, compatible organic solutes, antioxidants, heat-shock, and late embryogenesis abundant proteins as well as transcription factors for gene regulation to improve salt tolerance traits in various crops through genetic engineering [15]. Plants respond to stress by changing gene/transcript/protein expression levels at the molecular level. Overexpression of several stress-induced genes, including helicases, provides salinity stress tolerance in crop plants [18]. DNA helicases act as molecular proteins in a variety of cellular mechanisms and are required for nearly all DNA metabolic activities, including pre-mRNA splicing [19]. RuvB DNA helicases are capable of imparting salinity tolerance in Arabidopsis, rice, and pigeon pea [20]. RuvB is a member of AAA+ (ATPases Associated with diverse cellular Activities) superfamily, and part of SF6 superfamily which belongs to helicase class. Most of the helicases belong to DEAD-box protein superfamily. They are involved in regulation of cellular machinery such as DNA repair recombination, replication, transcription, translation initiation, ribosome biogenesis. So, they play a crucial role in stabilization of growth during stress conditions in plants. There are reports that helicases are up-regulated in response to abiotic stress in plants and help in survival under stressed conditions. Some of the examples of helicases which are activated under abiotic stresses are PDH45, PDH47, STRS1, STRS2, MCM6, p68 etc. [21-24]. The role of helicases has been established in various cellular functions such as replication, transcription, translation, gene regulation, DNA damage repair, chromatin remodeling and stress tolerance [25-28]. RuvB is a SF-6 type DNA helicase associated with diverse cellular activities such as protein folding, proteolysis, cytoskeleton regulation, and transcriptional control [29-32]. RuvB is well characterized in Escherichia coli [33], but now there are reports on characterization of RuvB in rice [34].

In the present study, Haryana chana 1 (HC-1) variety has been used to for the over expression of OsRuvB. HC-1 is a highly cultivated variety of chickpea used for commercial cultivation. India is the largest producer, consumer, and importer of pulse crops [35]. Due to the consumers and Government price support policies which are predominantly in favor of cereal crops it causes global production still lags behind [36]. Through given RuvB orthologous function in stress response, role in cellular functional reprogramming biotic stress we investigated its role in abiotic stress tolerance. To our knowledge, we were able to generate T2 chickpea heterologous expressing OsRuvB.

Materials and Methods

Materials

Plant Materials: In the present study chickpea variety, HC-1 and OsRuvB transgenic lines were used for studying morpho-physiological and biochemical responses of OsRuvB against salt stress. Seeds of chickpea were procured from Molecular Biology Biotechnology and Bioinformatics department, CCS HAU, Hisar.

OsRuvB, Plasmid and Agrobacterium tumefaciens strain: Agrobacterium tumefaciens strain: LBA4404 containing pCAMBIA1301 harboring OsRuvB gene was previously used for genetic transformation of HC-1 chickpea variety (Patent no. 252590) (Figure 1) [37]. The strain was a gift from Tuteja, N.K. ICGEB, Delhi.