Journal of Hazardous Materials 298 (2015) 241251

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Sulfur mediated reduction of arsenic toxicity involves efcient 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,
a

CSIRNational Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, Uttar Pradesh, India
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 (Ofce) and Room 320 (Lab), 161Holdsworth Way, University of Massachusetts, Amherst,
MA 01003, USA
b

h i g h l i g h t s

Arsenic detoxication 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 accumulation 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 sulfur (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 supplementation; 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.

1. Introduction

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.

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 efcient in assimilating arsenic
compared to other cereal crops, due to its cultivation in water
logged conditions where arsenite (AsIII) predominates over arsenate (AsV), and AsIII is efciently 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 1013 genes in rice [4] which can be subdivided into
three groups, NIP I, II, and III on the basis of their selectivity. Transport of AsIII from the root cells into the xylem is regulated by Lsi2
(a silicon/arsenite efux 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, O2 ; hydroxyl

radicals, OH and H2 O2 ) 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 concentrations of H2 O2 , 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 detoxication of As [10]. Under stress conditions GSH detoxies H2 O2 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-afnity sulfate transporters (HASulTs),
low-afnity vascular transporters (LASulTs), and vacuolar efux
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 efux of sulfate from the vacuole
[14]. Sulfur deciency or accessibility inuences 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 detoxication in plants [1618]. 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 detoxication, and the antioxidant
system during As stress.

2. Materials and methods

2.1. Experiment design, hydroponic culture, and arsenic exposure
Rice seeds (Oryza sativa L.), cultivar IR-36, were surface sterilized using 10% H2 O2 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 xed PVC cups (4 cm diameter and 5 cm high, 10
plants per cup) and grown in modied Hewitt media [19] supplemented 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 (NaAsO2 ;
25 M), or AsV (Na2 HAsO4 ; 50 M) [2022] were added, for 7 days
in a controlled growth environment at 28/21 C at light intensity

of 210 mol cm2 s1 (16-h light/8-h dark) with relative humidity 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 treatment 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 temperature (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 citrate 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 isolated from roots of rice plants exposed to the various treatments
were reverse-transcribed using SuperScriptII (Fermentas, USA),
following the manufacturers recommendation. The synthesized
cDNA was diluted 1:5 in RNase-free water and subjected to quantitative RT-PCR (qRT-PCR) analysis. The qRT-PCR was performed
using an ABI 7500 instrument (ABI Biosystems, USA) using gene
specic 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 conditions: 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. Specicity of amplicons generated in qPCR reactions was veried 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 [2528].
2.4. Estimation of total non-protein thiol compounds
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 uorescence spectrophotometer (Japan). The concentration
of total phytochelatins (PCs) was calculated as PCs = NPTs total
GSH [32]. AsPC 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 reaction were performed following Saito et al. [35] and Seelig and
Meister [36], respectively, with slight modications [37]. For the
assay of 5 -adenylylsulfate (APS) reductase (APR; EC1.8.4.9) and

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243

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.

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 signicant at
P 0.01. Different capital letters indicate signicantly different values among S treatments and small letters indicate signicantly different values among As treatments
(DMRT, P 0.05).

serine acetyltransferase (SAT, EC2.3.1.30) homogenization was performed 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]. Glutathione S-transferase (GST; EC 2.5.1.18) activity was assayed as
described by Habig and Jacoby [43].

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
-- = 2.8 mM1 cm1 ). GPX (EC
the rate of ascorbate (ASC) oxidation (C
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

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 H2 O2 was assayed according to Tripathi et al. [3].
Ascorbate oxidase was assayed according to Esaka et al. [45]. Assay

Differences among treatments were analyzed by one-way

ANOVA followed by Duncans multiple range tests. Signicance
was measured at P 0.05. The correlation analysis was performed, which has been given within the text at relevant places
(*** = P 0.001, ** = P 0.01, * = P 0.1, ns = non-signicant).

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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 AsPCs molar ratio (D) in Oryza
sativa L. roots. All the values are means of triplicate S.D. ANOVA signicant at P 0.01. Different capital letters indicate signicantly different values among S treatments
and small letters indicate signicantly different values among As treatments (DMRT, P 0.05).

3. Results

decreased molar ratio of AsPCs than NS condition both AsIII and

AsV treatments (Fig. 3D).

3.1. Root morphology and biomass

3.3. Expression of arsenic and sulfate transporters
Arsenite and AsV both inuenced the root growth by altering 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 signicantly (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 signicant impact on As accumulation 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 treatments compared to the respective controls. The LS treatment led
to the lowest concentrations of S in the roots while the HS concentration 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

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 expression of OsNIP1;1 at all the three S treatments (Fig. 4A). A similar
pattern was observed with OsNIP3;1, with AsIII and AsV both reducing its expression level signicantly (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 treatment, 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 signicantly compared to the no arsenic controls (Fig. 4E).

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245

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 signicant at P 0.01. Different capital letters indicate signicantly different values among S treatments and small letters
indicate signicantly 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 determining the expression of HASulTs, LASulTs, and vacuolar transporters.
High afnity 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 signicantly up-regulated in the LS treatments compared to
the NS treatment. Low afnity 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 signicantly increased
with AsIII (61%) and AsV (47%) treatment compared to the no As
control. The HS treatment reduced vacuolar transporter expression
(Fig. 5AE).
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. 6AD). It was observed that the concentration of NPTs was
signicantly enhanced by AsIII and AsV, and that the NPT concentration 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 treatment (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
signicant positive correlation with As and S accumulation separately; 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 sulte is mediated by APR. The highest activity of APR was
found in the HS and AsIII treatment; in general increased S concentration increased APR activity (Fig. 7A). Activity of CS was correlated
with Cys content (R = 0.95, P 0.05) and signicantly increased in

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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 signicant at P 0.01. Different capital letters indicate signicantly different values among S treatments
and small letters indicate signicantly 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).

CAT and H2 O2 (R = 0.88, p0.05). 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 activity 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. 8CG).

3.5. Oxidative stress and antioxidant enzymes

The concentration of H2 O2 deceased with increasing S treatment, 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 H2 O2 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 signicant positive correlation between

4. Discussion
The aim of the present study was to gain an insight of S modulated As stress in rice roots, as roots are the entry point of the
various nutrients and rst 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 [5052],
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 fulll 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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247

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 signicant at P 0.01. Different
capital letters indicate signicantly different values among S treatments and small letters indicate signicantly 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 limiting 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 immobilization in roots, thus, restricts its entry to shoot; similar results
have been observed in previous studies [53,54]. Decreasing the concentration 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 AsPCs molar ratio than NS treated plants. However, with high sulfur supplementation, molar ratio of PC and As
decreased signicantly than NS treated plants, that effect possibly
because after sequestration of AsPCs 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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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 signicant at P 0.01. Different capital letters indicate signicantly different values among S treatments and
small letters indicate signicantly different values among As treatments (DMRT, P 0.05).

related to As uptake. The expression level of OsNIP3;1 was downregulated 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 efux into the xylem and is
localized at the proximal side of both endodermis and exodermis
of rice roots [2]. Lsi1 serves both as efux and inux transporter for

AsIII in rice [57]; even if the plant is supplied with AsV exogenously,
Lsi1 efuxes AsIII into the nutrient medium [5]. During the current
study low accumulation of As in LS + As treated plants roots occurs
potentially because uncomplexed AsIII may more easily be released
to the external medium than loaded into the xylem in non hyperaccumulator 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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249

Fig. 8. Effect of As on the level of hydrogen peroxide (H2 O2 ) (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
signicant at P 0.01. Different capital letters indicate signicantly different values among S treatments and small letters indicate signicantly different values among As
treatments (DMRT, P 0.05).

would lower the As accumulation in the root; Lsi1 is known to perform 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 relationship may be due to Lsi2 being responsible for xylem unloading in
rice plants [2].

SULTR1;1, a high-afnity sulfate transporter, is highly regulated by S deciency 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 treatment to meet high S demand inside the plant by sensing low
S conditions. Similarly, S starvation induced high afnity 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 upregulated in plants under the LS with As treatment. OsSult4;1 is
a vacuolar efux 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 indicative of adequate cytosolic S maintained through vacuolar efux
[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 homeostasis [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 eventually 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 activity of APR and SAT [7,61]. Enhanced activity of enzymes of the
Cys assimilation pathway during metal stress may occur to fulll 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, Srivastava and Dsouza [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 peroxides 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 insufcient As chelation to thiols as
compared to higher S supplied plants showing sufcient 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 antioxidant system. SOD converts ROS into H2 O2, which is less reactive
than ROS, with H2 O2 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 generation 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 production of the ROS species [67,68]. Hydrogen peroxide plays a
dual role during stress, rstly 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 H2 O2 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 H2 O2 production during the LS treatment or As stress;
S supplementation seems to ameliorate this affect by reducing
H2 O2 levels. Catalase converts H2 O2 to water and oxygen, and
the observation of positive signicant correlation between CAT
and H2 O2 suggests its role in peroxide detoxication. 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 peroxidation [20].

5. Conclusion
Rice mitigates As stress by immobilizing a major amount of As
in roots, by utilizing metalloid detoxifying phytochelatins and nonprotein 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 transporters do not play a signicant 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 nancial 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 Scientic and Industrial
Research, New Delhi, India for the award of Junior/Senior Research
Fellowship and Academy of Scientic 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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