SA and AM symbiosis modulate antioxidant defense mechanisms and asada pathway in chickpea genotypes under salt stress

Highlights

• SA-seed priming enhanced mycorrhizal RC by decreasing endogenous SA level.

• Salt-induced enhanced antioxidants were insufficient to control ROS-build up.

• Seed priming escalated basic antioxidants and reduce oxidative stress.

• AM up-regulated Asada cycle more promisingly than SA.

• +SA+AM proved most effective in imparting salt tolerance in chickpea.

Abstract

Salt stress disturbs redox homeostasis by perturbing equilibrium between generation and removal of reactive oxygen species (ROS), which alters the normal metabolism of plants through membrane damage, lipid peroxidation and denaturation of proteins. Salicylic acid (SA) seed priming and arbuscular mycorrhizal (AM) fungi impart salt tolerance in legumes by maintaining redox balance. The present investigation focused on the relative and combined applications of SA and Rhizoglomus intraradices in scavenging ROS in Cicer arietinum L. (chickpea) genotypes (salt tolerant-PBG 5, relatively sensitive-BG 256) subjected to salt stress. Despite the enhanced antioxidant mechanisms under salt stress, ROS (superoxide, O₂− and hydrogen peroxide, H₂O₂) accumulation increased significantly and induced lipid peroxidation and lipoxygenase (LOX) activities, which disrupted membrane stability, more in BG 256 than PBG 5. Salt stress also caused redox imbalance by lowering ascorbate/dehydroascorbate (ASA/DHA) and reduced glutathione/oxidized glutathione (GSH/GSSG) ratios, indicating that redox-homeostasis was crucial for salt-tolerance. Exogenous SA was more promising in reducing ROS-generation and lipid-peroxidation, which provided higher membrane stability as compared to AM inoculation. Although, the enzymatic antioxidants were more active in SA treated plants, yet, AM inoculation outperformed in increasing reformative enzyme activities of Foyer-Halliwell-Asada cycle, which resulted in higher plant biomass in a genotype-dependent manner. SA increased AM root colonization and provided functional complementarity to R. intraradices and thereby strengthening antioxidant defense mechanisms through their cumulative contribution. The study suggested the use of +SA+AM as an eco-friendly tool in imparting salt tolerance in chickpea genotypes subjected to long-term salinity.

Introduction

Cicer arietinum L. (chickpea) is the second most cultivated leguminous crop with 13.1 million tons per annum (mt annum⁻¹) production, commonly grown on an estimated 13.5 million hectares (mha) of land in tropical, subtropical, temperate and semi-arid areas, worldwide. India is amongst the chief producers of chickpea, having 8.63 mha area under cultivation, 7.85 mt annum⁻¹ production with an average yield of 900 kg per hectare (kg ha⁻¹) (CIME, 2010; FAOSTAT, 2015; Muehlbauer and Sarker, 2017). It constitutes 20–30% protein, 40–59% carbohydrate, 3% fibre, 3–6% oil, 4% ash, and is a good source of absorbable ions like Ca, P, Mg, Fe, K and essential B vitamins (Ibrikci et al., 2003). Along with dietary constituents, it is an affluent source of antioxidants (Segev et al., 2010) and compared to other pulse crops, anti-nutritious components are nearly absent (Williams and Singh, 1987). It is cultivated in those areas which are heavily affected by salinity. Despite its economic importance, it is highly prone to salt stress (Flowers et al., 2010), with considerable deviation reported in chickpea germplasm for salt tolerance (Turner et al., 2013).

Salt stress is a detrimental condition of plants, which is produced due to high accumulation of dissolved ions in soil water, out of which Na⁺ and Cl⁻ are the predominant ones. Salt stress induces water deficiency and reduction of chloroplast stromal volume, which lead to overproduction of reactive oxygen species (ROS) such as singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), superoxide (O₂⁻) and hydroxyl (OH) radicals, which seriously disrupt normal metabolism by causing denaturation of proteins, membrane damage and lipid peroxidation (Csiszar et al., 2018). Moreover, the membrane damage increases due to high activity of Lipoxygenase (LOX), which leads to formation of linolenic hydroperoxides along with ¹O₂ as a by-product. To decrease these ROS, plants accelerate its enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPOX) and enzymes of the ascorbate-glutathione (ASA-GSH) cycle: ascorbate peroxidase (APOX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) (Foyer and Noctor, 2011). SOD catalyzes the dismutation of O₂⁻ to H₂O₂ and O_2, hence, deactivates the metal-catalyzed Haber-Weiss-type reaction and reduces the generation of toxic hydroxyl (OH) radicals from O₂⁻ (Haber and Weiss, 1932). CAT, GPOX and APOX detoxify H₂O₂ by degrading it into O₂ and H₂O in peroxisomes (Lucini et al., 2015). In addition, APOX simultaneously oxidizes ASA to dehydroascorbate (DHA), which destabilizes the redox equilibrium. During oxidation of ASA, first and foremost monodehydroascorbate (MDHA) is generated, which is in itself an efficient electron acceptor. It either re-reduces to ASA by MDHAR using NAD(P)H directly and electrons eliminated from the photosynthetic electron transport chain. In case, this reduction does not occur rapidly, MDHA disproportionates to ASA and DHA in the second step, and the latter is then recycled to ASA by DHAR (E.C. 7.8.5.1) (Arora et al., 2002). Glutathione protects thiol groups on enzymes by acting as disulfide reductant and also participates in DHAR-catalyzed regeneration of ASA by scavenging ¹O₂ as well as OH radicals. In such reactions, reduced glutathione (GSH) is oxidized to glutathione disulfide (GSSG), which perturbs the redox equilibrium and for balancing such condition, GR gets activated and regenerate GSH by using NADPH (Garg and Singla, 2015). However, under salt stress, ROS generation overpowers its removal and thereby leads to oxidative stress in plants. In the recent years, researchers have focused on the roles of salicylic acid (SA) and arbuscular mycorrhizal (AM) fungi in combating salt-induced oxidative stress.

SA is reflected as a persuasive phytohormone, as it regulates diverse metabolisms in plants (Hayat et al., 2010). Recently, exogenous SA has been reported to impart salt tolerance in Cicer arietinum L. by positively regulating seed germination (Boukraa et al., 2013), photosynthetic response (Asadi et al., 2013), relative membrane permeability, nutrient uptake, ion homeostasis etc. (Garg and Bharti, 2018). A few reports indicate that the oxidative stress produced in plants under stressed conditions could be ameliorated by the exogenous supply of SA (Janda et al., 2007; Hayat et al., 2010). Pre-soaking the seeds of Cicer arietinum L. in SA solution has been reported to provide membrane stability by decreasing lipid peroxidation (Asadi et al., 2013) and triggers the activities of some antioxidant enzymes namely, SOD, CAT and APOX (Boukraa et al., 2013). The varied influence of SA on protective enzyme activities could be associated with H₂O₂ metabolism (Janda et al., 2003). In addition to SA, AM fungi, widely found in salt affected soils (Yamato et al., 2008; Wilde et al., 2009) can also assist legumes to cope with salt stress conditions (Pandey and Garg, 2017; Garg and Bharti, 2018). Several authors have demonstrated the beneficial effects of AM fungi in enhancing the biomass accumulation of salt stressed legumes such as Cicer arietinum (Garg and Singla, 2015; Garg and Bhandari, 2016), Cajanus cajan (Garg and Manchanda, 2009; Garg and Chandel, 2015; Pandey and Garg, 2017), Phaseolus vulgaris (Aroca et al., 2007), Pisum sativum (Barnawal et al., 2014), Vicia faba (Abeer et al., 2014) etc. via strengthening the antioxidant enzyme activities and reducing ROS build-up.

Although, some literature is available on the influence of SA/AM treatments in combating oxidative stress in various legumes, but, as per our knowledge, no research has been cited, till date, on the relative and combined impacts of SA and AM inoculation in strengthening antioxidant defense mechanisms in Cicer arietinum L. exposed to salinity. Hence, unfolding the role of signal molecule-SA in combination with AM inoculation is a vital step that might strengthen antioxidant defense mechanisms and impart salt tolerance in plants. The present investigation was, therefore, carried out to deliver a more thorough perception into the influence of SA seed priming/+AM inoculation in modulating antioxidant defense machinery that imparts salt tolerance in chickpea genotypes. By keeping this aim into consideration, the study was fragmented into three objectives i.e. to evaluate (1) the influence of salt stress on ROS build-up and their scavenging enzymes along with reformative enzymes of Foyer-Halliwell-Asada pathway (2) the relative roles of SA seed priming and AM inoculation on endogenous SA levels and their impact on root colonization by Rhizoglomus intraradices in salt stressed Cicer arietinum L. genotypes (3) response of two substantially salt tolerant genotypes towards individual as well as combined application of SA seed priming and AM inoculation in establishing redox homeostasis under salt stress.