Research Article Influence of sulfur and cadmium on antioxidants, phytochelatins and growth in Indian mustard Humayra Bashir1, Mohamed M. Ibrahim2,3, Rita Bagheri1, Javed Ahmad1, Ibrahim A. Arif2,3, M. Affan Baig1 and M. Irfan Qureshi1* 1 Proteomics and Bioinformatics Lab, Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India 2 Department of Botany and Microbiology, Science College, King Saud University, PO Box 2455, Riyadh, Saudi Arabia 3 Department of Botany and Microbiology, Faculty of Science, Alexandria University, PO Box 21511, Alexandria, Egypt Received: 13 August 2014; Accepted: 16 December 2014; Published: 12 January 2015 Associate Editor: Wen-Hao Zhang Citation: Bashir H, Ibrahim MM, Bagheri R, Ahmad J, Arif IA, Baig MA, Qureshi MI. 2015. Influence of sulfur and cadmium on antioxidants, phytochelatins and growth in Indian mustard. AoB PLANTS 7: plv001; doi:10.1093/aobpla/plv001 Abstract. Soils in many parts of the world are contaminated with heavy metals, leading to multiple, deleterious effects on plants and threats to world food production efficiency. Cadmium (Cd) is one such metal, being toxic at rela- tively low concentrations as it is readily absorbed and translocated in plants. Sulfur-rich compounds are critical to the impact of Cd toxicity, enabling plants to increase their cellular defence and/or sequester Cd into vacuoles mediated by phytochelatins (PCs). The influence of sulfur on Cd-induced stress was studied in the hyperaccumulator plant Indian 2− 2− mustard (Brassica juncea) using two sulfur concentrations (+S, 300 mMSO4 and S-deficient 2S, 30 mMSO4 ) with and without the addition of Cd (100 mM CdCl2) at two different time intervals (7 and 14 days after treatment). Com- pared with control plants (+S/2Cd), levels of oxidative stress were higher in S-deficient (2S/2Cd) plants, and greatest in S-deficient Cd-treated (2S/+Cd) plants. However, additional S (+S/+Cd) helped plants cope with oxidative stress. Superoxide dismutase emerged as a key player against Cd stress under both 2S and +S conditions. The activity of ascorbate peroxidase, glutathione reductase and catalase declined in Cd-treated and S-deficient plants, but was up-regulated in the presence of sulfur. Sulfur deficiency mediated a decrease in ascorbate and glutathione (GSH) con- tent but changes in ascorbate (reduced : oxidized) and GSH (reduced : oxidized) ratios were alleviated by sulfur. Our data clearly indicate that a sulfur pool is needed for synthesis of GSH, non-protein thiols and PCs and is also important for growth. Sulfur-based defence mechanisms and the cellular antioxidant pathway, which are critical for tolerance and growth, collapsed as a result of a decline in the sulfur pool. Keywords: Antioxidants; cadmium; growth; oxidative stress; phytochelatins; sulfur. sulfur on agricultural land. Based on crop demand, Introduction fertilizer-use efficiency and current inputs, the worldwide In recent years, sulfur deficiency has become a major sulfur-deficit has been estimated to be 10.4 million problem for agricultural productivity, reducing both crop tonnes annually (Haneklaus et al. 2007). Increased food quality and yields. Legitimate approaches to reducing production will further raise sulfur requirements, elevat- emissions of sulfur into the atmosphere have resulted ing this sulfur-deficit to 12.5 million tonnes by 2015 in a concomitant decrease in atmospheric deposition of (Randazzo 2009). * Corresponding author’s e-mail address: [email protected]; [email protected] Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 1 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard As an essential macronutrient required for the proper reported that Cd induces the production of thiol com- growth and development of plants, sulfur plays a critical pounds and transcripts of the S-assimilation pathway. It role in a number of cellular processes, such as in protein is noteworthy that a PC-deficient Arabidopsis thaliana disulfide bridges (Saito 2000), mediating electron trans- mutant, cad1, exhibits Cd, As and Zn hypersensitivity port in iron–sulfur (Fe–S) clusters, the redox cycle, detoxi- (Cobbett and Goldsbrough 2002; Tennstedt et al. 2009). fication of heavy metals and xenobiotics, vitamin Cadmium activates the S-assimilation pathway respon- co-factors (Hell and Hillebrand 2001) and the metabolism sible for the synthesis of cysteine (Cys), a precursor of of secondary products (Hell 1997; Saito 2000). Sulfur is thus GSH biosynthesis. Glutathione (an NPT) acts as an import- a major plant nutrient (Takahashi et al. 1997; Leustek et al. ant antioxidant in mitigating Cd-induced stress (Meuwly 2000; Saito 2000; Lee and Korban 2002) that contributes to and Rauser 1992). It also plays an important role in PC an increase in crop yield, directly adding nutritional value synthesis, which has a proven role in Cd detoxification and improving the efficiency of use of other essential plant (Park et al. 2012). nutrients (Salvagiotti et al.2009), particularly nitrogen and Plants are also provided with an efficient mechanism phosphorus. Research on plant adaptations to S-related for protection against ROS and peroxidation reactions. stresses has shifted from an emphasis on excessive inputs The majority of ROS-scavenging pathways in plants and acidification to how deficiencies impact crop produc- involve superoxide dismutase (SOD), which is found in tion. Sulfur deficiency not only impacts crop quality and almost all cellular compartments, the water–water cycle yield, but it also raises the demand for adequate fertiliza- in chloroplasts and the ascorbic acid (AsA)–GSH cycle in tion to resist biotic and abiotic stresses. Exposure of plants chloroplasts, cytosol, mitochondria, apoplast and peroxi- to excessive toxic metals like cadmium (Cd) may affect the somes, in which ascorbate peroxidase (APX) and GR play uptake and metabolism of S and negatively impact the crucial roles. Catalase (CAT) removes H2O2 in peroxisomes yield and plant resistance to abiotic stresses (Gill and (Mittler 2002). Tuteja 2011). A varying degree of antioxidative response and toler- Cadmium is a non-essential heavy metal that is readily ance is exhibited by different plant species. Several studies absorbed and rapidly translocated in plants, which makes have demonstrated that most of the main S-responsive it highly bio-available and thus toxic, even at relatively low genes involved in sulfate assimilation are induced by Cd, concentrations. Cadmium exerts its phytotoxicity by inter- suggesting the existence of a general adaptive response fering with several basic events of plant growth, develop- to an increase in cellular demand for reduced S (Nocito ment and physiology (Qadir et al.2004). Cadmium induces et al.2006). Hence the availability of S to plants is crucial. oxidative stress leading to the overproduction of harmful Brassica juncea (L.) Czern. (Indian Mustard) belongs to reactive oxygen species (ROS) (Zhang et al. 2010). These the Brassicaceae family and is an important oilseed crop ROS may cause damage to cell membranes, proteins, that is known to tolerate considerable amounts of Cd DNA replication and repair. A major effect of this metal, (Qadir et al. 2004). In this study, we investigated the observed in most plants studied to date, is the inhibition effects of Cd, S-deficiency and their combination on of photosynthesis by altering chlorophyll synthesis (Pietrini S-assisted defence against oxidative stress, and changes et al.2003; Maksymiec and Krupa 2006) and the light- in cellular antioxidant activity, the contents of NPTs, PCs harvesting Chl a/Chl b protein complex II (Qureshi et al. and photosynthetic pigments, and growth parameters 2010), and interfering with RuBisCO activation (Prasad in B. juncea. 1995). Therefore, Cd-mediated reductions in photosyn- thetic activity lead to declines in crop yield (Ouzounidou Methods et al. 1997). Cadmium also interferes with processes such as carbohydrate and nitrogen metabolism (Sanita` di Toppi Experimental design and Gabbrielli 1999), enzyme catalysis (Van Assche and Seeds of mustard (B. juncea cv. Pusa Jaikisan), procured Clijsters 1990) and water balance (Perfus-Barbeoch et al. from the Indian Agriculture Research Institute (IARI), 2002). were treated with 1 % (v/v) sodium hypochlorite solution Sulfur plays a critical role in allowing plants to protect for 10 min followed by thorough washing in deionized themselves against heavy metal toxicity, especially Cd water. The seeds were germinated on moist soil (Soilritew; toxicity (Gill and Tuteja 2011). Numerous studies have 1 kg per pot). For S-nutrition, conditions were as described shown that S is involved in the biosynthesis of heavy in Abdallah et al. (2010). One set of plant cultures, consid- metal detoxification agents (Anjum et al. 2012), such as ered as the control, received Hoagland nutrient solution 2− non-protein thiols (NPTs) including phytochelatins (PCs), (Hoagland and Arnon 1950) with 300 mMSO4 (herein glutathione (GSH) (Noctor et al. 2011) and Cd -sulfide referred to as ‘S-sufficient’) and was designated as (+S). crystallites (Robinson et al. 1993). Harada et al. (2002) A second set received the same