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Journal of Hazardous Materials 298 (2015) 241–251

Journal of Hazardous Materials 298 (2015) 241–251 Contents lists available at ScienceDirect Journal of Hazardous

Contents lists available at ScienceDirect

Journal of Hazardous Materials

j o u r nal h o m e p a ge: www.elsevier.com/locate/jhazmat

j o u r nal h o m e p a ge: www.elsevier.com/locate/jhazmat Sulfur mediated reduction

Sulfur mediated reduction of arsenic toxicity involves efficient thiol metabolism and the antioxidant defense system in rice

Garima Dixit a , Amit Pal Singh a , Amit Kumar a , Pradyumna Kumar Singh a , Smita Kumar a , Sanjay Dwivedi a , Prabodh Kumar Trivedi a , Vivek Pandey a , Gareth John Norton b , Om Parkash Dhankher c , Rudra Deo Tripathi a,

, Om Parkash Dhankher c , Rudra Deo Tripathi a , ∗ a CSIR–National Botanical Research

a CSIR–National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, Uttar Pradesh, India

b Institute of Biological and Environmental Sciences, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, UK

c Stockbridge School of Agriculture, Paige Laboratory Room 318 (Office) and Room 320 (Lab), 161Holdsworth Way, University of Massachusetts, Amherst, MA 01003, USA

h i g

h l i g

h t s

Arsenic detoxification was mediated by PCs, NPTs and enzymes of S assimilatory pathway.

Sulfur supply results in immobilization of As in rice roots and low translocation to shoot.

Sulfur nutrition regulates arsenite and sulfate transporters in rice root.

High S ameliorates As toxicity by enhancing antioxidant enzymes activity.

a r

t i c

l e

i n f o

Article history:

Received 20 February 2015 Received in revised form 13 May 2015 Accepted 2 June 2015 Available online 5 June 2015

Keywords:

Antioxidant enzymes Arsenic Rice Sulfate and arsenic transporters Sulfur Thiol metabolism

a b s t r a c t

Arsenic (As) contamination is a global issue, with South Asia and South East Asia being worst affected. Rice is major crop in these regions and can potentially pose serious health risks due to its known As accumula- tion potential. Sulfur (S) is an essential macronutrient and a vital element to combat As toxicity. The aim of this study was to investigate the role of S with regards to As toxicity in rice under different S regimes. To achieve this aim, plants were stressed with AsIII and AsV under three different S conditions (low sul- fur (0.5 mM), normal sulfur (3.5 mM) and high sulfur (5.0 mM)). High S treatment resulted in increased root As accumulation, likely due to As complexation through enhanced synthesis of thiolic ligands, such as non-protein thiols and phytochelatins, which restricted As translocation to the shoots. Enzymes of S assimilatory pathways and downstream thiolic metabolites were up-regulated with increased S supple- mentation; however, to maintain optimum concentrations of S, transcript levels of sulfate transporters were down-regulated at high S concentration. Oxidative stress generated due to As was counterbalanced in the high S treatment by reducing hydrogen peroxide concentration and enhancing antioxidant enzyme activities. The high S concentration resulted in reduced transcript levels of Lsi2 (a known transporter of As). This reduction in Lsi2 expression level is a probable reason for low shoot As accumulation, which has potential implications in reducing the risk of As in the food chain.

© 2015 Published by Elsevier B.V.

Correspondence to: Chief Scientist & Professor, Plant Ecology and Environmental Science Division, C.S.I.R.–National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India. Tel.: +91 522 2297825; fax : +91 522 2205836. E-mail addresses: tripathi rd@rediffmail.com , tripathird@gmail.com

(R.D. Tripathi).

http://dx.doi.org/10.1016/j.jhazmat.2015.06.008

0304-3894/© 2015 Published by Elsevier B.V.

1. Introduction

Arsenic (As) contamination of drinking water affects the lives of 150 million people across the world [1]; rice is also a known source of As entering the human diet. Arsenic contamination is a major problem in parts of South Asia and South East Asia, where rice is a staple food. Rice is highly efficient in assimilating arsenic compared to other cereal crops, due to its cultivation in water logged conditions where arsenite (AsIII) predominates over arse- nate (AsV), and AsIII is efficiently transported in rice through the

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silicon transport systems [2]. Arsenite uptake in rice is mediated by aquaporins (nodulin 26-like intrinsic proteins: NIPs) and enters rice plants through the silicon uptake pathway [2], while AsV enters rice roots through phosphate transporters [3]. The NIP gene family consists of 10–13 genes in rice [4] which can be subdivided into three groups, NIP I, II, and III on the basis of their selectivity. Trans- port of AsIII from the root cells into the xylem is regulated by Lsi2 (a silicon/arsenite efflux carrier protein) [5]. In the cytosol, AsIII reacts with sulfhydryl groups of enzymes and proteins, affecting many biochemical functions [3,6]. Plants survive in As contaminated environments through metabolic adaptations. It is well recognized that AsIII and AsV induce reactive oxygen species (ROS) (superoxides, O 2 ; hydroxyl radicals, OH and H 2 O 2 ) in plants [7]. Therefore, the production of ROS needs to be balanced for the survival and growth of plants under stress conditions. To minimize ROS, plants are equipped with various antioxidant enzymes and thiolic compounds. There are a number of antioxidant enzymes involved in reducing cellular con- centrations of H 2 O 2 , which include ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT) [7]. Glutathione (GSH) plays various important functions in plants via its thiolic residue of Cysteine (Cys), therefore, acting as a key regulator of redox homeostasis [8,9]. Sulfur (S) rich low molecular weight non-protein thiols (Cys, GSH, phytochelatin), synthesized during S metabolism, play an important role in the detoxification of As [10]. Under stress condi- tions GSH detoxifies H 2 O 2 through the ascorbate-glutathione cycle and has the ability to chelate As in roots, therefore reducing As in the foliar part of the plant [10]. Up-regulation of sulfate transporters leads to a continuous supply of S which can be utilized in chelation and vacuolar sequestration of metals or metalloids in plants grown in a metal or metalloid rich environment [11]. Dedicated sulfate transporters [12], viz. high-affinity sulfate transporters (HASulTs), low-affinity vascular transporters (LASulTs), and vacuolar efflux transporters, are responsible for sulfate uptake and are regulated by the plants’ nutritional status [13]. HASulTs and LASulTs are responsible for sulfate uptake from the soil and transported to the xylem respectively. Vacuolar sulfate transporters are localized in the tonoplast, and facilitate the efflux of sulfate from the vacuole [14]. Sulfur deficiency or accessibility influences sulfate uptake to sustain homeostasis in the plants, and follows the demand driven control of S metabolism [15]. Hence, the S assimilation pathway is important in As detoxification in plants [16–18]. However, the S mediated attenuation of As toxicity involving S assimilation, and subsequent metabolism, has not been investigated in detail. The current study addresses the role of different S regimes on As and sulfate transporters, arsenic detoxification, and the antioxidant system during As stress.

of 210 mol cm 2 s 1 (16-h light/8-h dark) with relative humid- ity of 70%. The S conditions are abbreviated as follows: LS for the low sulfur conditions (0.5 mM), NS for standard sulfur conditions (3.5 mM), and HS for the high sulfur concentration (5.0 mM). All the experiments were conducted with three replicates for each treat- ment combination. Plants were harvested, washed three times with Milli-Q water, and the plant material was divided into different aliquots for the various analyses. In all the analyses, only plant roots were used, except in the determination of total S and As in which roots and shoots were used.

2.2. Determination of arsenic, sulfur, and root biomass

Prior to elemental analysis the root length of treated and untreated plants was measured, then oven dried at constant tem- perature (70 C) for determination of biomass. For analysis of total As, the analytical procedure was performed according to Dwivedi et al. (roots were washes with dithionite cit- rate bicarbonate (DCB) solution to remove surface adsorb metals or plaque) [23] using inductively coupled plasma-mass spectrometry (ICP-MS) (7500 cx; Agilent, Tokyo, Japan). Total S concentration was estimated as described by Chesnin and Yien [24].

2.3. Determination of transcript levels of arsenic and sulfate

transporters

Approximately 5 g RNase free DNase-treated total RNA iso- lated from roots of rice plants exposed to the various treatments were reverse-transcribed using SuperScriptII (Fermentas, USA), following the manufacturer’s recommendation. The synthesized cDNA was diluted 1:5 in RNase-free water and subjected to quan- titative RT-PCR (qRT-PCR) analysis. The qRT-PCR was performed using an ABI 7500 instrument (ABI Biosystems, USA) using gene specific primers (Table S-1). Each qPCR reaction contained 5 l of SYBR Green Supermix (ABI Biosystems, USA), 1 l of the diluted cDNA reaction mixture (corresponding to a starting amount of 5 g of RNA), and 10 pM of each primer in a total reaction volume of 10 l. qPCR reactions were performed under the following condi- tions: 10 min at 95 C and 40 cycles of the one step thermal cycling of 3 s at 95 C, and 30 s at 60 C in a 96-well reaction plate. The rice actin gene was used as an internal control to estimate the relative transcript levels of the target gene. Specificity of amplicons gener- ated in qPCR reactions was verified by melting curve analysis. Each qPCR reaction was performed in triplicate (technical replicates) for each biological replicate (three for each treatment). Relative gene expression was calculated using CT method [25–28].

2.4. Estimation of total non-protein thiol compounds

2. Materials and methods

2.1. Experiment design, hydroponic culture, and arsenic exposure

Rice seeds (Oryza sativa L.), cultivar IR-36, were surface steril- ized using 10% H 2 O 2 for 30s, followed by thorough washing with de-ionized water, and then were soaked in distilled water for 24 h. Seeds were germinated in the dark for 4 days at 37 ± 1 C. Uniform germinated seedlings were selected and transplanted to trays containing fixed PVC cups (4 cm diameter and 5 cm high, 10 plants per cup) and grown in modified Hewitt media [19] sup- plemented with low sulfur (0.5 mM), normal sulfur (3.5 mM) as used in standard Hewitt media, or high sulfur (5.0 mM) [7] for 10 days. Then, either no As was added (control), or AsIII (NaAsO 2 ; 25 M), or AsV (Na 2 HAsO 4 ; 50 M) [20–22] were added, for 7 days in a controlled growth environment at 28/21 C at light intensity

Total non-protein thiols (NPTs) and Cys content were measured following the method of Ellman [29] and Gaitonde [30]. The levels of reduced (GSH) and oxidized (GSSG) glutathione content were determined as described by Hissin and Hilf [31] using a Hitachi F 7000 fluorescence spectrophotometer (Japan). The concentration of total phytochelatins (PCs) was calculated as PCs = NPTs total GSH [32]. As–PC molar ratio has been calculated by following Sneller et al. and Dave et al. [33,34].

2.5. Enzymes of sulfur assimilation pathway and glutathione

metabolism

Assays for Cys synthase (CS; EC 2.5.1.47) and -glutamylcysteine synthetase ( ECS; EC 6.3.2.2) activities, homogenization, and reac- tion were performed following Saito et al. [35] and Seelig and Meister [36], respectively, with slight modifications [37]. For the assay of 5 -adenylylsulfate (APS) reductase (APR; EC1.8.4.9) and

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/ Journal of Hazardous Materials 298 (2015) 241–251 243 Fig. 1. Morphological alterations in the roots

Fig. 1. Morphological alterations in the roots of hydroponically grown rice under various S regimes and As stress. (A) LS control, (B) NS control, (C) HS control, (D) LS + AsIII 25 M, (E) NS + AsIII 25 M, (F) HS + AsIII 25 M, (G) LS + AsV 50 M, (H) NS + AsV 50 M, and (I) HS + AsV 50 M.

50 M, (H) NS + AsV 50 M, and (I) HS + AsV 50 M. Fig.

Fig. 2. Root length and dry weight of hydroponically grown rice under various S regimes and As stress. All the values are means of triplicate ±S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

serine acetyltransferase (SAT, EC2.3.1.30) homogenization was per- formed following Hartmann et al. [38], and APR activity was assayed according to Peck et al. [39], while the activity assay for SAT was performed following Blaszczyk et al. [40]. For assays of -glutamyl transpeptidase ( -GT; EC 2.3.2.2), the method by Orlowski and Meister [41] was followed. The GR activity was assayed by following the method of Smith et al. [42]. Glu- tathione S -transferase (GST; EC 2.5.1.18) activity was assayed as described by Habig and Jacoby [43].

2.6. Estimation of oxidative stress and antioxidant enzymes

The activity of superoxide dismutase (SOD; EC 1.15.1.1) was assayed following the method of Beauchamp and Fridovich [44]. Concentration of H 2 O 2 was assayed according to Tripathi et al. [3]. Ascorbate oxidase was assayed according to Esaka et al. [45]. Assay

of arsenate reductase (AR; EC 1.20.4.1) was performed as described by Shi et al. [46]. The activity of APX (EC 1.11.1.11) was measured according to the method of Nakano and Asada [47] by estimating the rate of ascorbate (ASC) oxidation (C -- = 2.8 mM 1 cm 1 ). GPX (EC 1.11.1.7) activity was assayed according to the method of Hemeda and Klein [48]. CAT activity was measured following the method of Aebi [49].

2.7. Statistical analyses

Differences among treatments were analyzed by one-way ANOVA followed by Duncan’s multiple range tests. Significance was measured at P 0.05. The correlation analysis was per- formed, which has been given within the text at relevant places (*** = P 0.001, ** = P 0.01, * = P 0.1, ns = non-significant).

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et al. / Journal of Hazardous Materials 298 (2015) 241–251 Fig. 3. Effect of As and

Fig. 3. Effect of As and different S doses on the concentration of As (A) and S (B) accumulation, root to shoot translocation factor of As (C) and As–PCs molar ratio (D) in Oryza sativa L. roots. All the values are means of triplicate ± S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

3. Results

3.1. Root morphology and biomass

Arsenite and AsV both influenced the root growth by alter- ing root thickness, length, and biomass with different S doses (Figs. 1 and 2). In the LS treatment root length was longer (38%) than compared to the NS treated plants. The HS treatment reduced root length in all the treatments compared to NS treated plants. Arsenite and AsV both decreased root length (Fig. 2A). Reduced S addition reduced root biomass. Arsenite and AsV both reduced root biomass significantly (Fig. 2B).

3.2. Arsenic and sulfur accumulation

Arsenic accumulation was determined in roots and shoots of the rice plants. The LS treatment had no significant impact on As accu- mulation in the roots. High sulfur (HS) supplementation enhanced As accumulation in the roots compared to the normal sulfur (NS) in both the AsIII and AsV treatments (Fig. 3A). Total S concentration in the roots was enhanced with As treat- ments compared to the respective controls. The LS treatment led to the lowest concentrations of S in the roots while the HS concen- tration had the highest concentration of S in the roots (Fig. 3B). In the shoots the As concentration decreased in both the AsIII and AsV treated plants with increasing S treatment (Fig S-1C). It is evident from translocation factor that LS condition enhances As translocation from root to shoot, while HS condition reduces As translocation to shoot both with AsIII and AsV (Fig. 3C). HS condition

decreased molar ratio of As–PCs than NS condition both AsIII and AsV treatments (Fig. 3D).

3.3. Expression of arsenic and sulfate transporters

The HS treatment down-regulated expression of OsNIP1;1, while the LS treatment enhanced expression of OsNIP1;1 compared to the NS treatment. Treatment with AsV down-regulated expres- sion of OsNIP1;1 at all the three S treatments (Fig. 4A). A similar pattern was observed with OsNIP3;1, with AsIII and AsV both reduc- ing its expression level significantly (Fig. 4B). Lsi1 expression was enhanced during AsIII (63%) stress while it was reduced with AsV (28%) stress compared to the control at NS supplementation. High S reduced Lsi1 expression both in AsIII (49%) and AsV (48%) compared to the HS control. Low S treatment enhanced Lsi1 expression level, while HS treatment reduced its expression compared to the NS control (Fig. 4C). Lsi2 expression was down-regulated under both AsIII (71%) and AsV (78%) stress compared to the control treatment under normal S condition. Lsi2 expression was greatly reduced (82%) under the HS and AsIII compared to the NS and AsIII treat- ment, while in the LS and AsIII treatment the expression of Lsi2 was up-regulated (38%) compared to the NS and AsIII treated plants; a similar pattern was found in the AsV treatments and with the HS and LS treatments (Fig. 4D). The HS and AsV treatment increased Lsi6 expression by 79% compared to the NS and AsV treated plants. Lsi6 expression increased in the LS and AsIII (236%) and LS and AsV (895%) compared to the NS and AsIII, and NS and AsV treated plants, respectively. Arsenite and AsV both reduced the expression level of Lsi6 significantly compared to the no arsenic controls (Fig. 4E).

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/ Journal of Hazardous Materials 298 (2015) 241–251 245 Fig. 4. Relative expression of As transporters

Fig. 4. Relative expression of As transporters in Oryza sativa L. roots during As stress under various S regimes (A) OsNIP1;1, (B) OsNIP3;1, (C) Lsi1, (D) Lsi2, and (E) Lsi6. All the values are means of triplicate ± S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

The expression of sulfate transporters in response to different concentrations of S with AsIII and AsV was done by determin- ing the expression of HASulTs, LASulTs, and vacuolar transporters. High affinity sulfate transporters (OsSultr1;1, OsSultr1;2, and OsSultr1;3) were down-regulated in the HS treatment compared to the NS treatment, with the highest reduction in expression observed in OsSultr1;3. OsSultr1;1, OsSultr1;2, and OsSultr 1;3 were significantly up-regulated in the LS treatments compared to the NS treatment. Low affinity sulfate transport (OsSultr 2;2) was also down-regulated in the HS treatment, while the AsIII and AsV treatments both enhanced OsSultr2;2 expression level. Vacuolar sulfate transporter, OsSultr4;1, expression significantly increased with AsIII (61%) and AsV (47%) treatment compared to the no As control. The HS treatment reduced vacuolar transporter expression (Fig. 5A–E).

3.4. Thiolic compounds and enzymes of sulfur assimilation

pathway and glutathione metabolism

The components of thiol metabolism were determined in the hydroponically grown rice roots under the various treatments (Fig. 6A–D). It was observed that the concentration of NPTs was significantly enhanced by AsIII and AsV, and that the NPT concen- tration also increased with increasing S concentration (Fig. 6A).

A similar pattern of response was observed for Cys, GSH, and PC

concentrations. Plant roots exposed to the HS and AsV treatment had higher concentrations of Cys (93%) than the NS and AsV treat- ment (Fig. 6B). The GSH concentration was greater (56%) in the HS and AsIII treatment compared to the plants treated with NS and AsIII (Fig. 6C). For GSSG, generally the plants treated with LS had higher concentrations than the plants treated with NS, and the plants treated with NS had higher concentrations than those treated with HS. The As treatments increased the concentrations

of GSSG (Fig. 6D). The concentration of PCs was lowest in the LS

treated plants and highest in the HS treated plants, and both AsIII and AsV increased PC concentration (Fig. 6E). Thiol metabolites had

significant positive correlation with As and S accumulation sepa-

rately; NPTs ( R = 0.913 for As and R = 0.958 for S), Cys ( R = 0.753 for As and R = 0.799 for S), GSH ( R = 0.892 for As and R = 0.941 for S)

( P 0.05), and PCs ( R = 0.909 for As and R = 0.933 for S) were highly enhanced with AsIII exposure compared to AsV exposure. To get a deeper insight into thiol metabolic response under As stress, enzymes involved in synthesis and consumption of thiols were evaluated. In the sulfate assimilation pathway conversion of APS to sulfite is mediated by APR. The highest activity of APR was found in the HS and AsIII treatment; in general increased S concen- tration increased APR activity (Fig. 7A). Activity of CS was correlated with Cys content ( R = 0.95, P 0.05) and significantly increased in

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et al. / Journal of Hazardous Materials 298 (2015) 241–251 Fig. 5. Relative expression of sulfate

Fig. 5. Relative expression of sulfate transporters in Oryza sativa L. roots during As stress under various S regimes (A) OsSultr1;1, (B) OsSultr1;2, (C) OsSultr1;3, (D) OsSultr2;2, and (E) OsSultr4;1. All the values are means of triplicate ± S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

the HS treatments (Fig. 7B). Activity of SAT was highest in the HS treatments than S control (Fig. 7C). The activity of -ECS was lower in the LS treatment, indicating its key role in glutathione synthesis (Fig. 7D). -GT and GST activity both decreased with increasing S treatments (Fig. 7E and F). The activity of GR was increased with increasing S treatments (Fig. 7G).

3.5. Oxidative stress and antioxidant enzymes

The concentration of H 2 O 2 deceased with increasing S treat- ment, and increased under AsIII and AsV treatment (Fig. 8A). SOD activity was low in the LS treatments and increased in the NS and HS treatments. SOD activity was decreased by the As treatments (compared to the no As control) in all the treatments except the HS with AsV treatments, where the As treatment was no different from the HS control (Fig. 8B). APX, GPX and CAT are all enzymes for defence against oxidative stress and degrade H 2 O 2 to water and oxygen. All these enzymes showed similar trends under S treatment and As treatment. Under As stress in the LS and NS treatments their activity increased. In the HS treatment the activity of APX and CAT increased under As stress but not GPX. There was a significant positive correlation between

CAT and H 2 O 2 ( R = 0.88, p 0.05). Under control (no As) conditions the highest activity of AR was in the LS treatment. Addition of As (either AsV or AsIII) increased activity of AR, with the highest activ- ity observed in the LS with AsIII treatment. AAO activity increased with increasing S in both the no As control and the AsIII treatment, while in the AsV treatment the activity of AAO was very similar across all three S treatments (Fig. 8C–G).

4. Discussion

The aim of the present study was to gain an insight of S mod- ulated As stress in rice roots, as roots are the entry point of the various nutrients and first point of contact for toxic metal(oid)s. After uptake into the roots a proportion of toxic metal(oid)s are complexed and compartmentalized into the root vacuoles [50–52], with thiols ( SH) playing a vital role during the process. Observed increases in root length during S deprivation may occur to increase root surface area to fulfill the demand of S. The higher reduction of root biomass in the LS treatment compared to the HS treatment under As stress is attributable to thinner roots in the LS treated plants. Therefore, analyzing the processes occurring

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/ Journal of Hazardous Materials 298 (2015) 241–251 247 Fig. 6. Effect of As on different

Fig. 6. Effect of As on different S doses on the level of (A) non-protein thiols (NPTs), (B) cysteine, (C) reduced glutathione (GSH), (D) oxidized glutathione (GSSG), and (E) phytochelatins (PCs) in Oryza sativa L. roots during arsenic stress in Oryza sativa L. roots. All the values are means of triplicate ± S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

in roots can provide insights into plant responses to As stress and mitigation through S containing compounds. The present study supports the hypothesis that adequate S may allow for the chelation of more As in plant roots, subsequently lim- iting its translocation from root to shoot [52]. High S with As leads to As accumulation in roots and inhibits its translocation to shoot, while during LS + As conditions more As translocated to shoot. Thus, from this data it is evident that S plays a crucial role in As immo- bilization in roots, thus, restricts its entry to shoot; similar results have been observed in previous studies [53,54]. Decreasing the con- centration of As being translocated is a desirable characteristic for rice cultivation in As contaminated areas [55]. Molar ratio of PC and As is crucial for determination of As toxicity (Fig S1C). In the current

study, at low S concentration, level of As and PCs both are low thus maintain the low As–PCs molar ratio than NS treated plants. How- ever, with high sulfur supplementation, molar ratio of PC and As decreased significantly than NS treated plants, that effect possibly because after sequestration of As–PCs complex into vacuole, PCs were degraded into their precursors (e.g. -EC and GSH). The GSH could then be shuttled back into the cytoplasm this is also evident by the enhanced level of GSH at HS condition. Similar result was also reported by Li et al. [56] where enhanced level of -EC was observed due to PCs degradation in Arabidopsis under As stress. OsNIP1;1 (NIP I) and OsNIP3;1 (NIP II) do not play major roles in AsIII uptake [2]. In the current study it has been observed that LS + AsIII enhanced OsNIP1;1 expression levels, which was not

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et al. / Journal of Hazardous Materials 298 (2015) 241–251 Fig. 7. Effect of As on

Fig. 7. Effect of As on the activity of 5 -adenylylsulfate reductase (APR) (A), cysteine synthase (CS) (B), serine acetyl transferase (SAT) (C), -glutamylcysteine synthetase ( -ECS) (D), -glutathione transpeptidase ( -GT) (E), glutathione-S-transferase (GST) (F), and glutathione reductase (GR) (G) in Oryza sativa L. roots under various sulfur regimes. All the values are means of replicate ± S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

related to As uptake. The expression level of OsNIP3;1 was down- regulated with As treatment under various S conditions. Previously it has been shown that expression of OsNIP1;1 and OsNIP3;1 were not induced by As treatment [2], but both OsNIP1;1 and OsNIP3;1 were involved in As uptake. Lsi1 transports AsIII into rice root cells, which is localized at the distal site, while Lsi2 mediates AsIII efflux into the xylem and is localized at the proximal side of both endodermis and exodermis of rice roots [2]. Lsi1 serves both as efflux and influx transporter for

AsIII in rice [57]; even if the plant is supplied with AsV exogenously, Lsi1 effluxes AsIII into the nutrient medium [5]. During the current study low accumulation of As in LS + As treated plant’s roots occurs potentially because uncomplexed AsIII may more easily be released to the external medium than loaded into the xylem in non hyperac- cumulator plants such as rice [5]. In the LS with AsIII treatment PC levels were found to be very low, indicating possibly low amounts of AsIII-PC complex formation. Therefore in this case uncomplexed AsIII may be released into the external medium through Lsi1, which

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/ Journal of Hazardous Materials 298 (2015) 241–251 249 Fig. 8. Effect of As on the

Fig. 8. Effect of As on the level of hydrogen peroxide (H 2 O 2 ) (A), superoxide dismutase (SOD) (B), ascorbate peroxidase (APX) (C), guiacol peroxidase (GPX) (D), catalase (CAT) (E), arsenate reductase (AR) (F), and ascorbate oxidase (AAO) (G) in Oryza sativa L. roots under various sulfur regimes. All the values are means of replicate ± S.D. ANOVA significant at P 0.01. Different capital letters indicate significantly different values among S treatments and small letters indicate significantly different values among As treatments (DMRT, P 0.05).

would lower the As accumulation in the root; Lsi1 is known to per- form bidirectional transport of AsIII in rice root [5]. At the same time, remaining uncomplexed AsIII may be unloaded to the xylem via Lsi2 [2]. The current study indicates that As accumulation in the shoot is directly proportional to Lsi2 transcript level; this relation- ship may be due to Lsi2 being responsible for xylem unloading in rice plants [2].

SULTR1;1, a high-affinity sulfate transporter, is highly regu- lated by S deficiency in the epidermis and cortex of Arabidopsis roots [58]. It is suggested that to meet the optimum need of S, plants express more HASulTs to internalize S from outside media. The expression of HASulTs increased in the LS with AsIII treat- ment to meet high S demand inside the plant by sensing low S conditions. Similarly, S starvation induced high affinity group

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1 sulfate transporters in Arabidopsis [58]. OsSult2;2 is a LASulTs,

which plays a role in involved in long-distance transport of sulfate. OsSult2;2 was highly expressed in the LS treatment and decreased

in expression with increasing S, which indicates that this gene could

be important for S acquisition in low S environments. Data also suggest that to meet increased S demand OsSult2;2 is further up- regulated in plants under the LS with As treatment. OsSult4;1 is

a vacuolar efflux transporter (transporting into the cytosol). The HS condition reduced the expression of this transporter, which could be to increase storage of S in the vacuole, which is indica- tive of adequate cytosolic S maintained through vacuolar efflux

[59].

Thiol-based antioxidant systems provide the second line of cellular defense against free radical induced damage [60]. The reduction of S in plants relies on multi dimensional functions of thiol containing compounds (Cys, GSH, PCs) in cellular homeosta- sis [61]. The observed response of the thiol metabolites to S and As treatments and the correlations between them suggest that As accumulation leads to stimulation of thiol metabolites and even- tually alters the sulfur assimilatory pathway [17]. The enhanced level of Cys and CS activity following metal(oid) s exposure may be due to the sulfate reduction pathways that stimulate the activ- ity of APR and SAT [7,61]. Enhanced activity of enzymes of the Cys assimilation pathway during metal stress may occur to ful- fill the demand of downstream peptides (GSH and PCs) [62], as is also evident by increased GSH and PC levels in the current study. Exposure of plants to both AsIII and AsV enhanced the level of GSH and the ratio of GSH/GSSG; S supplementation further stimulated this effect during the current study. Similarly, Srivas- tava and D’souza [7] reported enhanced thiol metabolism with As exposure, which led to enhanced -ECS and GR activities in the submerged aquatic plant Hydrilla verticillata. Enhanced activity of GST during As stress may occur to detoxify ROS and lipid perox- ides to prevent oxidative stress and membrane damage [63]. Heavy metal stress is also known to enhance GST expression levels in Arabidopsis [64]. To cope with stress, plants stimulate both the thiol biosynthesis pathway as well as pathways leading to their consumption [7,35]. The higher toxic effect seen in As exposed LS plants may be due to insufficient As chelation to thiols as compared to higher S supplied plants showing sufficient As-thiol chelation. The current study aimed to analyze the effect of S status on As stress in terms of reduced oxidative stress and the antioxi- dant system. SOD converts ROS into H 2 O 2, which is less reactive than ROS, with H 2 O 2 acting as a stress indicator [65]. Under As stress SOD activity was increased, although higher activity of SOD under AsV stress may be attributed to higher ROS gen- eration due to conversion of AsV to AsIII [32]. ROS mediated oxidative damage is a common indicator of As induced stress [66], as exposure of plants to AsIII and AsV stimulates the pro-

duction of the ROS species [67,68]. Hydrogen peroxide plays a dual role during stress, firstly as a highly reactive molecule which causes oxidative destruction, as well as a signaling molecule involved in the cellular stress response. During the current study, enhancement of H 2 O 2 in the LS with AsIII or AsV treatment appears to be a signal of high stress that may cause cellular destruction (Fig. 8A). Higher activity of SOD may be the cause of this H 2 O 2 production during the LS treatment or As stress;

S

supplementation seems to ameliorate this affect by reducing

H

2 O 2 levels. Catalase converts H 2 O 2 to water and oxygen, and

the observation of positive significant correlation between CAT and H 2 O 2 suggests its role in peroxide detoxification. Similarly, higher activity of APX in plants grown under LS indicates the defense mechanism mediated by antioxidants, thereby protecting

the cell membrane from hydroxyl radical induced lipid peroxida- tion [20].

5. Conclusion

Rice mitigates As stress by immobilizing a major amount of As in roots, by utilizing metalloid detoxifying phytochelatins and non- protein thiols, subsequently allowing its low translocation to shoot during high S conditions. It appears that various sulfate transporters (HASulTs, LASulTs, and vacuolar sulfate transporters) are regulated by sulfate availability. Effective thiol metabolism and antioxidant systems manage As stress in rice, despite the fact that As trans- porters do not play a significant role in As accumulation during different S regimes in rice. Low expression of Lsi2 is a possible way to reduce As accumulation in rice shoots, having an implication in reduced risk of food chain contamination.

Acknowledgements

The authors are thankful to Director, CSIR-National Botanical Research Institute (CSIR-NBRI), Lucknow for the facilities and for the financial support from the network projects (CSIR-INDEPTH and NWP-0111), New Delhi, India. The authors are grateful to the Joint Director, Rice Research Station (RRS), Chinsurah to provide rice germplasm. GD is thankful to Council of Scientific and Industrial Research, New Delhi, India for the award of Junior/Senior Research Fellowship and Academy of Scientific and Innovative Research (AcSIR) for her Ph.D. registration.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.jhazmat.2015.06.

008

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