Elsevier

Journal of Plant Physiology

Volume 181, 1 June 2015, Pages 20-29
Journal of Plant Physiology

Hydrogen sulfide alleviates toxic effects of arsenate in pea seedlings through up-regulation of the ascorbate–glutathione cycle: Possible involvement of nitric oxide

https://doi.org/10.1016/j.jplph.2015.03.015Get rights and content

Abstract

In plants, hydrogen sulfide (H2S) is an emerging novel signaling molecule that is involved in growth regulation and abiotic stress responses. However, little is known about its role in the regulation of arsenate (AsV) toxicity. Therefore, hydroponic experiments were conducted to investigate whether sodium hydrosulfide (NaHS; a source of H2S) is involved in the regulation of AsV toxicity in pea seedlings. Results showed that AsV caused decreases in growth, photosynthesis (measured as chlorophyll fluorescence) and nitrogen content, which was accompanied by the accumulation of As. AsV treatment also reduced the activities of cysteine desulfhydrase and nitrate reductase, and contents of H2S and nitric oxide (NO). However, addition of NaHS ameliorated AsV toxicity in pea seedlings, which coincided with the increased contents of H2S and NO. The cysteine level was higher under AsV treatment in comparison to all other treatments (As-free; NaHS; AsV + NaHS). The content of reactive oxygen species (ROS) and damage to lipids, proteins and membranes increased by AsV while NaHS alleviated these effects. Enzymes of the ascorbate–glutathione cycle (AsA–GSH cycle) showed inhibition of their activities following AsV treatment while their activities were increased by application of NaHS. The redox status of ascorbate and glutathione was disturbed by AsV as indicated by a steep decline in their reduced/oxidized ratios. However, simultaneous NaHS application restored the redox status of the ascorbate and glutathione pools. The results of this study demonstrated that H2S and NO might both be involved in reducing the accumulation of As and triggering up-regulation of the AsA–GSH cycle to counterbalance ROS-mediated damage to macromolecules. Furthermore, the results suggest a crucial role of H2S in plant priming, and in particular for pea seedlings in mitigating AsV stress.

Introduction

Arsenate (AsV), being a dominant As species in aerobic soils and due to its chemical similarity with inorganic phosphate, enters the plant cell through the phosphate transport systems (Zhao et al., 2009, LeBlanc et al., 2013, Singh et al., 2015). The co-transport of phosphate or AsV and protons through H2PO4/H+ symporters requires at least 2H+ for each H2PO4 or H2AsO4 transported (Ullrich-Eberius et al., 1989). Once inside the plant cell, AsV is reduced efficiently to arsenite (AsIII), thus suggesting that most plants have a high capacity for the reduction of AsV (Zhao et al., 2009). Consequently, AsIII may be rendered non-toxic by forming a complex with phytochelatins or may be transported to the vacuoles (Cobbett, 2000, Indriolo et al., 2010). However, when plants are not able to adjust As levels inside the cell, As toxicity occurs. In plants, adverse effects of As exposure are duration, dosage and species-dependent. Adverse impacts of AsV may occur at morphological, physiological, biochemical, proteomic, transcriptomic and genomic levels (Wojas et al., 2010, Requejo and Tena, 2012). Inhibition of seed germination and a decline in growth are common responses of AsV toxicity (Requejo and Tena, 2012). Reduction in photosynthetic pigments and, consequently, photosynthesis has also been reported in various photosynthetic organisms under AsV stress (Wang et al., 2012, Singh et al., 2013, Srivastava et al., 2013). Studies demonstrate that AsV exposure of plants alters the metabolisms of carbohydrate and protein, and nodule formation (Mishra and Dubey, 2006, Lafuente et al., 2010). Long-term exposure of humans to As results in skin disorders and cancer (Duker et al., 2005). Furthermore, there is much agricultural land that is contaminated with As. For India, As concentrations from 3.34 to 105 mg kg−1 soil have been reported (Patel et al., 2005). Considering the damage to plants and human health, there is an urgent need for reliable and cost-effective methods that can reduce As toxicity to plants and also curtail As levels in food products.

Although AsV is a metalloid without redox activity it may induce the generation of reactive oxygen species (ROS) (Singh et al., 2013, Srivastava et al., 2013) through its inter-conversion from one ionic form to another (Mylona et al., 1998). In the absence of protective mechanisms, ROS can damage the cell's structure and function by oxidizing lipids, proteins and nucleic acids. Thus, the over production of cellular ROS generally causes loss of functions of macromolecules (Singh et al., 2013). However, ROS-mediated oxidative damage to the cell can be minimized by the various pathways that operate to keep the cellular concentration of free metalloid to a minimum (primary detoxification, e.g., thiol-mediated metalloid complexation; Requejo and Tena, 2012, Leão et al., 2014) and also prevent ROS-mediated damage to macromolecules (secondary detoxification, e.g. quenching of ROS by antioxidants; Namdjoyan and Kermanian, 2013, Singh et al., 2013, Gomes et al., 2014). Among various pathways of ROS detoxification, the ascorbate–glutathione cycle (AsA–GSH) is of prime importance. The AsA–GSH cycle consists of various enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reducatase (MDHAR), dehydrateascorbate reductase (DHAR) and glutathione reductase (GR) as well as non-enzymatic antioxidants such as glutathione and ascorbate (Foyer and Noctor, 2011). Various components of the AsA–GSH cycle act in a coordinated manner and protect the cell from oxidative damage.

Hydrogen sulfide (H2S), a colorless, soluble and flammable gas, is known for its toxic effects to different types of organisms. Interestingly, H2S has also been identified as an important metabolic regulator in plants within the last decade (Filippou et al., 2012, Christou et al., 2013, Li, 2013, Hancock and Whiteman, 2014). In mammals, it is considered third major endogenous gasotransmitter, besides nitric oxide (NO) and carbon monoxide (CO) (Wang, 2002, Olson, 2009, Li, 2013, Hancock and Whiteman, 2014) and also plays a central role as a stimulatory or inhibitory compound in gastrointestinal, inflammatory, cardiovascular, nervous, and endocrine systems by activating K+-ATP channels and modulating endothelial Ca2+ concentration (Bauer et al., 2010). In plants, H2S has also been reported to regulate growth and development (García-Mata and Lamattina, 2010, Hancock et al., 2011, Li, 2013, Li et al., 2013, Hancock and Whiteman, 2014). In plants, apart from sulfite reductase, there are at least four other enzymes capable of producing H2S (Calderwood and Kopriva, 2014). Among these enzymes, cysteine desulfhydrase appears to play a central role in H2S production and homeostasis (Riemenschneider et al., 2005). Besides developmental and regulatory roles, studies demonstrated that H2S can alleviate toxicities of abiotic stresses such as heavy metal, salinity, osmotic stress, heat, hypoxia, drought, etc. by affecting levels of antioxidants (Jin et al., 2011, Dawood et al., 2012, Cheng et al., 2013, Li, 2013, Li et al., 2013, Duan et al., 2015, Hancock and Whiteman, 2014). Furthermore, Xie et al. (2014) demonstrated that H2S can delay GA-mediated programmed cell death (PCD) in wheat aleurone layers by modulating glutathione homeostasis and expression of heme oxygenase-1. Although there are an increasing number of reports regarding the role of H2S in alleviating abiotic stresses in plants, however, the exact mechanisms by which H2S works remain unclear. Further, to our knowledge, there is no report on implications of H2S in the management of AsV toxicity in plants.

In India, As contamination in soil and water is a relevant problem, which has been worsened recently due to use of As-containing pesticides and contaminated irrigation water. Pea, an important legume crop, is widely cultivated for its protein content. As-toxicity results in reduced yield and contaminated seeds (Päivöke, 2003). The present study investigates whether NaHS is involved in the regulation of AsV toxicity in pea seedlings. We have measured various physiological and biochemical parameters such as growth, chlorophyll fluorescence, activities of L-cysteine desulfhydrase and nitrate reductase, contents of H2S, nitric oxide (NO) and cysteine, oxidative stress and damage, and components of the ascorbate–glutathione cycle (AsA–GSH cycle).

Section snippets

Plant material and growth conditions

Pea (Pisum sativum L. cv. Azad P-1) seeds were purchased from National Seed Corporation, New Delhi. Uniformly sized seeds were surface sterilized with 10% (v/v) sodium hypochlorite solution for 10 min, washed and soaked in distilled water for 4 h. After sterilization and soaking, healthy looking seeds were sown in plastic trays containing acid washed sterilized sand. Plastic trays were kept in the dark for seed germination at 26 ± 1 °C. After germination, seedlings were grown in a growth chamber

Effect of H2S on growth, total nitrogen and As accumulation

Growth was measured in terms of fresh and dry weights, and data are shown in Table 1. Treatment of AsV decreased (p < 0.05) fresh and dry weights by 29 and 27%, respectively. The NaHS alone had a stimulatory effect on growth as fresh and dry weights increased by 7 and 4%. Furthermore, application of NaHS together with AsV significantly (p < 0.05) ameliorated AsV-induced reduction of growth. For instance, under NaHS + AsV combination, declines in fresh and dry weights were only 12 and 10%,

Discussion

Arsenic toxicity constitutes one of major abiotic constraints that is affecting plant productivity and also threatening human health. Studies have demonstrated that exogenous application of certain chemicals may result in the mitigation of various abiotic stresses that could be important from both the theoretical and applied point of views (Christou et al., 2013, Singh et al., 2013, Sun et al., 2013, Lai et al., 2014). Among these chemicals, H2S is recently receiving increasing attention

Acknowledgments

Authors are grateful to the University Grants Commission, New Delhi, India for providing financial assistance to carry out this work. One of the authors, JK, is thankful to UGC for providing financial assistance as SRF under the scheme of RGNF-2012-13-SC-UTT-33185. Authors are thankful to the anonymous reviewers for their critical evaluation and improvement of the manuscript.

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