Ecotoxicology and Environmental Safety 124 (2016) 393–405

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Ecotoxicology and Environmental Safety

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

Response of two rice cultivars differing in their sensitivity towards

arsenic, differs in their expression of glutaredoxin and glutathione
S transferase genes and antioxidant usage
Arvind Kumar Dubey a, Navin Kumar a, Nayan Sahu a, Pankaj Kumar Verma b,
Debasis Chakrabarty b, Soumit K. Behera a, Shekhar Mallick a,n
a
Plant Ecology and Environmental Science Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India
b
Genetics and Molecular Biology Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India

art ic l e i nf o a b s t r a c t

Article history: Embodied study investigates the role of GRX and associated antioxidant enzymes in the detoxification
Received 3 June 2015 mechanism between arsenic (As) sensitive (Usar-3) and tolerant cultivar (Pant Dhan 11) of Oryza sativa
Received in revised form against As(III) and As(V), under GSH enriched, and GSH deprived conditions. The overall growth and
14 October 2015
physiological parameters in sensitive cultivar were lower than the tolerant cultivar, against various
Accepted 15 October 2015
treatments of As(III) and As(V). The As accumulation in sensitive cv. against both As(III) and As(V) was
lower than the corresponding treatments in tolerant cv. However, the As translocation against As(V) was
Keywords: lower (35% and 64%, resp.) than that of As(III), in both the cultivars. In sensitive cv. translocation of Zn
Arsenic and Cu was influenced by both As(V) and As(III) whereas, in tolerant cv. the translocation of Cu, Mn and
Oryza sativa
Zn was influenced only by As(III). Translocation of Fe was negatively influenced by translocation of As in
Glutaredoxin
sensitive cv. and positively in tolerant cv. Strong correlation between H2O2, SOD, GRX, GR, GST and GSH/
Tolerance
Sensitivity GSSG in sensitive cv. and between DHAR, APX, MDHAR and AsA in tolerant cv. demonstrates the un-
Glutathione-S-transferase derlying preference of GSH as electron donor for detoxification of H2O2 in sensitive cv. and AsA in tol-
erant cv. Higher expression of the four GRX and two GST genes in the sensitive cv. than tolerant cv,
suggests that under As stress, GRX are synthesized more in the sensitive cv. than tolerant cv. Also, the
expression of four GRX genes were higher against As(V) than As(III). The higher As accumulation in the
tolerant cv. is due to lower GST expression, is attributed to the absence of thiolation and sequestration of
As in roots, the translocation of As to shoots is higher.
& 2015 Elsevier Inc. All rights reserved.

1. Introduction and eukaryotic systems (Arabidopsis thaliana) arsenate reductase

Arsenic (As) is a non threshold class I carcinogen (Zhao et al.,
2010); which poses a great threat to the rice production and health
hazard to rice consumers in the As contaminated region of the
Indian subcontinent (Williams et al., 2009). Arsenic mostly exists
as inorganic arsenite [As(III)] and arsenate [As(V)], where As(III) is
predominant under anoxic paddy fields; that is readily taken up by
plants and its toxicity is several order higher than As(V). In almost
all the As resistant organisms, As(V) is either reduced to As(III) and
eventually exported outside the cell or sequestered into the va-
cuoles as thiolated As(III). GRX acts as transhydrogenase or thiol-
transferase, which are ubiquitous small heat-stable disulfide oxi-
do-reductases with a molecular mass between 10–15 kDa (Fo-
menko and Gladyshev, 2002). In both prokaryotic (Escherichia coli)
n
Corresponding author.
E-mail address: shekharm@nbri.res.in (S. Mallick).
(AR) uses the glutathione (GSH)/GRX system as a reductant for
reducing As(V) to As(III) (López-Maury et al., 2009), however, in
conversion of As(III) to As(V), apart from generation of reactive
oxygen species (ROS) two electrons are transferred, one from AR
and other from GSH (Fig. S1A in Supplementary material). Glu-
taredoxin/glutathione/glutathione reductase (GRX/GSH/GR) is one
of the major redox systems involved in maintaining the cellular
redox state. GRX can regulate a protein or enzyme activity by re-
versibly glutathionylating (S-glutathionylation) or reducing the
disulfide bonds in order to prevent their critical cysteine residues
from irreversible oxidation under oxidative stress (Meyer et al.,
2009; Rouhier et al., 2008). GRX uses the reducing power of GSH;
which is constantly regenerated by an NADPH-dependent GSH
reductase (GR) system (Rouhier et al., 2008; Zaffagnini et al., 2010,
2012), to glutathionylate the SH groups of redox-sensitive proteins
in order to prevent it from forming irreversible sulfinic (–SO2H) or
sulfonic acid (–SO3H) under oxidative stress. On the other hand,
GSTs are a ubiquitous family of multifunctional enzymes which are

http://dx.doi.org/10.1016/j.ecoenv.2015.10.017
0147-6513/& 2015 Elsevier Inc. All rights reserved.
394 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
implicated in combating different biotic and abiotic stresses in-
cluding heavy metal stress (Bartling et al., 1993). GST catalyzes-
GSH conjugation with xenobiotics or heavy metals, facilitating
transport of the conjugate into vacuoles through GSH pump
(Marrs, 1996). Hence, the overall tolerance of a plant towards As,
would be determined by the efficiency of the reduction of As(V) to
As(III), scavenging the ROS generated followed by glutathionyla-
tion of As(III) and eventually sequestering the glutathionylated As
(III) into the vacuole. This would be greatly dependent upon the
availability of the two major electron donor pool i.e. ascorbic acid
and GSH in the plant. Glutathione being the most abundant low
molecular-weight thiol is present in the range of 1–10 mM in most
living organisms (Alkhuder et al., 2009), whereas ascorbic acid is
reported to range widely between 3.4 and 20 mM g
1 fw in rice
(Chao et al., 2010; Guo et al., 2006) to 10–50 mM in Arabidopsis
leaves (Zechmann, 2011). Although, GRX does not directly depend
upon ascorbic acid for regeneration but dehydroascorbate re-
ductase (DHAR) uses GSH for reducing dehydro-ascorbate to As-
corbate (AsA).
Studies have revealed the role of GRX in imparting tolerance to
plants against oxidative stress. Recombinant E. coli exhibited tol-
erance towards 10 mM As(V) incorporating GRX gene (PvGRX5)
from As hyper-tolerant fern Pteris vitatta (Sundaram and Rathi-
nasabapathi, 2010; Sundaram et al., 2008, 2009). Silencing of
SlGRX1 gene in tomato plants led to susceptibility towards oxida-
tive, drought and salt stresses, whereas, over-expression of the
gene in Arabidopsis significantly increased the plant's resistance to
these stresses (Guo et al., 2010). Rice GRX (OsGrx) protects gluta-
mine synthetase from oxidative damage (Lee et al., 2002).
Plant GRX family is more extensive than other organisms i.e. 31
members in Arabidopsis (Laporte et al., 2012), 48 members in Or-
yza sativa (Garg et al., 2010) compared to 4 in E. coli and 6–7 in
Saccharomyces cerevisiae. Latest classification of GRX based upon
phylogenic and protein domain on 58 photosynthetic organisms,
identifies six classes of GRX (Rouhier, 2010), in which GRX be-
longing to Class III are of CC-type (containing, CxxC or CxxS in
their active site) present exclusively to land plants. Although, the
role of GRX in As reduction is well established in prokaryotic and
mammalian system (Meyer et al., 1999), however, reports about
expression of GRX genes in rice plants and more particularly
against As stress, are very few. The most documented functions of
GRXs in plants against metal related oxidative stress, are against
Cd (Aina et al., 2007; Schwarzländer et al., 2009). Rai et al. (2011),
studied the expression of genes associated in sulfur assimilation
pathway in four different cultivars of rice against 5 and 25 mM As
(III). Among the up-regulation of many sulfur assimilation genes in
these cultivars, two GRX genes i.e. Os01g27140, Os02g40500 were
found to be up-regulated against As(III) stress, whereas only one
(Os02g40500) against As(V) stress. Similar observation was also
made from another study (Chakrabarty et al., 2009). Although, the
upregulation of GRX and enzymes involved in detoxification
pathway (GR, APX, GST) in As treated rice plants have been re-
ported (Rai et al., 2011), however, the expression of GRX genes
against As(III) and As(V) stress in rice cultivars differing in sensi-
tivity towards As, and the underlying variation in detoxification
mechanism involving the two major antioxidant pool, has not
been investigated in greater detail. The present study attempts to
investigate the expression of four GRX genes against both As(III)
and As(V) stress under GSH deprived and GSH enriched condi-
tions, in two rice cultivar which differs in sensitivity towards As.
The study also attempts to highlight the fundamental difference in
the uptake of As and antioxidative defense mechanism, between
the two cultivars against As(III) and As(V).
2. Materials and methods
2.1. Growth conditions and experimental design
The experimental design consists of treatments with various
regimes of GSH treatment with As(III) and As(V), As(III) and As
(V) along with GSH synthesis inhibitor (buthionine sulfoximine,
BSO), in a pair of As tolerant and sensitive rice cultivars towards As
(III) along with their treatment controls. Two rice cultivars, namely
Usar-3 (sensitive cv. U) and Pant Dhan 11 (tolerant cv. PD) were
obtained from Narendra Dev Agricultural University, Faizabad,
Uttar Pradesh, and from Govind Ballav Pant Agricultural Uni-
versity, Pantnagar, Uttarakhand, respectively. The selection of the
tolerant and sensitive cultivar of rice was based upon the growth
performance of 100 cultivars of rice against 3 mg ml
1 of As(III)
(Supplementary Fig. S2). The seeds were germinated and cultured
as explained in (Kumar et al., 2013). Briefly, 25 uniform [10 cm
(cm)] seedlings were placed in a 150 ml beaker, containing 100 ml
of 100% Hewitt nutrient medium (HNM), prepared with Milli-Q
water (pH 6.8–7.0) (Hewitt, 1966). The composition (mg ml
1) of
Hewitt nutrient solution included N (168), P (41), K (156), Mg (36),
Ca (160), S (48), Fe (2.8), Mn (0.55), B (0.54), Cu (0.064), Zn (0.065),
Mo (0.048). There were 11 treatments in total which were, [“As
(III)”] abbreviated for 4 mg ml
1 of arsenite (  53.3 mM), “As(III) þ
GSH” for As(III) and 2000 mM GSH, “As(III) þBSO” for As(III) (4 m
g ml
1) and 200 mM BSO, “As(III) þ BSOþGSH” for As(III) (4 m
g ml
1) þ2000 mM GSH þ200 mM BSO. Similarly, “As(V)” for ar-
senate 4 mg ml
1 ( 53.3 mM), “As(V)þ GSH” for As(V) and
2000 mM GSH, “As(V) þBSO” for As(V)(4 mg ml
1) and 200 mM
BSO, “As(V)þBSO þGSH” for As(V)(4 mg ml
1) þ2000 mM
GSHþ200 mM BSO] including one experimental control (“C”) and
two treatment controls [“C þGSH” control with GSH (2000 mM)
and “C þ BSO”; control with (200 mM BSO)]. After 7 days (7 d) of
growth in the nutrient solution, the different treatments were
provided using salts of NaAsO2, NaHAsO4  7H2O (Sigma-Aldrich),
L-buthionine sulfoximine (Sigma-Aldrich), glutathione (Sigma-Al-
drich) and harvested after 7 d after treatment. All the treatments
consisted of four biological replicates.
2.2. Plant physiological and growth parameters
The effect of different treatments on the physiological perfor-
mance of the cv. Usar-3 and Pant dhan-11 plants were evaluated by
open flow gas exchange system (Li-Cor 6400XT; Li-Cor, Inc., Lin-
coln, NE, USA). All the physiological measurements were per-
formed on 7 d of treatment with constant air flow rate. The re-
lative humidity, Tleaf, vapor pressure deficit (VPD), and CO2 conc.
were kept constant for both the cultivars at 55%, 28 °C, 1.5 KPa and
400 mmol m
2 s
1 respectively. The plantlets of both the cultivars
were subjected to saturating photosynthetic photon flux density
(PPFD) of 1200 mmol m
2 s
1 (provided by artificial LED light
source Li-Cor 6400-02B). Prior to recording of each measurement,
the leaves were dark adapted by using dark adapting clips (Li-Cor
9964-091) for 20 min.
All the growth parameters and biochemical parameters were
measured after 7 d of treatments. The fresh-weight (fw) of the
plants were measured after 7 d of treatment by blot-drying the
roots, on an electrical balance (Mettler-Toledo) in milligrams (mg),
shoot length and roots in cm.
2.3. Biochemical analysis
Chlorophyll content was estimated by Arnon (1949) and total
soluble protein was estimated using Bradford reagent (Sigma-Al-
drich, USA) as per manufacturer defined method. TBARS content
(mmol g
1 fw) was estimated following (Heath and Packer, 1968)
 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
using ε ¼ 0.155 M g
1 fw for MDA-TBA adduct. H2O2 content
(nmol g
1 fw) by Velikova et al. (2000) and ascorbic acid (mM g
1
fw) by Shukla et al. (1979). Reduced and total glutathione (GSH
and total GSH) were estimated following Anderson (1985) by es-
timating the stoichiometric formation of 5-thio-2-nitrobenzioc
acid (TNB) at λ ¼412 nm and comparing with a standard curve
prepared with GSH (Sigma).
For estimation of antioxidant enzyme activities, fresh samples
of leaves ( 300 mg each) were used following Kumar et al. (2013).
Superoxide dismutase (SOD) (EC 1.15.1.1) activity was measured
spectrophotometrically at 560 nm following Beauchamp and Fri-
dovich (1971) and presented as U mg
1 protein, where 1 U of SOD
activity is the amount of protein required to inhibit 50% of the
initial reduction of nitro-blue tetrazolium (NBT) activity under
light. Ascorbate peroxidase (APX) (EC 1.11.1.11) activity was mea-
sured following Nakano (1981) using ε ¼ 2.8 mM
1 cm
1 and the
enzyme activity was expressed as μ moles of ascorbate oxidized
min
1 mg
1 protein. Catalase (CAT) (EC 1.11.1.6) activity was
measured following Scandalios et al. (1983) and was expressed as
mM min
1 mg
1 protein. Guaiacol peroxidase (POD) (EC 1.11.1.7)
activity was assayed at 470 nm following Kato and Shimizu (1987)
using ε ¼26.6 mM
1 cm
1 and expressed as millimoles of guaia-
col oxidized min
1 mg
1 protein. Glutathione reductase (GR) (EC
1.6.4.2) activity was assayed following Smith et al. (1988) and re-
presented as U mg
1 protein, where 1 U is the conversion of 1 mM
of oxidized glutathione (GSSG) min
1 to reduced glutathione
(GSH). Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) ac-
tivity was assayed following Vanacker et al. (1998) by monitoring
the rate of formation of monodehydroascorbate at λ ¼ 340 nm, due
to the action of ascorbate oxidase (0.4 unit; 1 unit; 1 mmol of
ascorbate oxidized per min) and represented as mM mg
1 protein.
Dehydroascorbate reductase (DHAR; EC 1.8.5.1) activity by Doulis
et al. (1997) at λ ¼265 nm using ε ¼ 7.0 mM
1 cm
1 and re-
presented as mM mg
1 protein. Glutathione S-transferase (GST; EC
2.5.1.13) activity by Habig et al. (1974) using the ε
¼9.6 mM
1 cm
1 of the product formed and is expressed as mM
of CDNB conjugated mg
1 protein.
2.4. Elemental analysis
The metal(loid) contents (As) were determined following Ku-
mar et al. (2013). Plant tissues (leaves  200 mg and roots  80 mg)
were digested on a hot plate using HNO3:HClO4 (3:1). Cu, Fe, Zn
and Mn (mg ml
1) were analyzed using AAS (GBC Avanta ∑),
whereas, for As (mg l
1) estimation, AAS was fitted with a hydrate
generator (MDS 2000) using NaH2BO4 þNaOH (3 M) and HCl
(3 M). The values are presented as mg g
1 dw (dry weight) and the
translocation factor (TF) is the ratio of the level of elements in
shoots and roots.
2.5. Expression analysis using RT-PCR
After 7 d of treatment, total RNA from the shoots was extracted
using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, USA), fol-
lowed by treatment with RNAase-free DNase (Sigma-Aldrich, USA).
RT-PCR was carried out using 2 mL of the cDNA corresponding to the
set of selected genes in a reaction containing 2  PCR Master mix
(Thermo Scientific, USA). Two CC type GRX (Os01g27140,
Os01g13950), one CPYC Type (Os02g40500) and one GRL type (LO-
C_Os01g61350) GRX were taken for the study. The expression of
two glutathione-S-transferase (GST) genes, namely (Os09g20220,
Os02g38160) were also studied using specific primers. Three tech-
nical replicates of each treatment were analyzed for real-time PCR
analysis. The primers for rice ubiquitin gene were used as an in-
ternal control to ensure that equal amounts of cDNA were used in
all the reactions. The PCR reaction was carried out using the
395
following cycle conditions: an initial denaturation at 94 °C for
2 min, number of PCR cycles provided in at 94 °C for 30 s, 55 °C for
30 s, and 72 °C for 30 s, followed by a final 5-min extension at 72 °C.
After obtaining the Ct value for each reaction, the relative expres-
sion was calculated by ΔΔCt method. The list of selected genes and
oligonucleotide primers (Eurofins, India) used for each gene are
listed in the additional file (Supplementary Table S1).
2.6. Statistical analysis and analytical quality control
The whole experiment was set in completely randomized de-
sign. The data were subjected to one way ANOVA through Dun-
can's Multiple Range Test (DMRT) for the analysis of significant
difference between the treatments. Principal component analysis
(PCA) attempts to explain the variance of a large set of inter-cor-
related variables and transforming into a smaller set of in-
dependent (uncorrelated) variables (principal components). PCA
was applied to experimental data standardized through z-scale
transformation in order to avoid mis-classification due to wide
differences in data dimensionality using Statistica 7.0. Data sets
analyzed for PCA comprised of; (i) 5 variables (Translocation fac-
tors of Cu, As, Zn, Mn and Fe); (ii) 10 variables (antioxidant en-
zymes activities i.e. SOD, MDHAR, APX, DHAR, GPX, GST, GR, GRX
and stress markers (TBARS, H2O2, GSH/GSSG), (iii) 7 variables of
plant physiological parameters [non-photochemical quenching
(NPQ), water use efficiency (WUE), photochemical quenching (qP),
photosynthesis rate (A), stomatal conductance (G), electron
transport rate (ETR) and transpiration (E)]. For each set of PCA, the
data analysis were made separately for each cultivar.
The analytical data quality of the element analysis was ensured
through repeated analysis (n ¼6) of Standard Reference Material
(CRM 028–050) Resource Technology Corporation, USA (Lot no. IH
028). The values obtained (10 times) varied between
3.97% and
22.86% error. The blanks were used to eliminate background noise.
3. Results
3.1. Growth and physiological response
The As(V) treatments were relatively less toxic than the As(III)
treatments towards either of the cultivars, as evident from the
greater reduction of growth parameters against As(III) (Fig. S3 in
Supplementary material). The fresh-weights and shoot-lengths
were significantly higher in the tolerant cv. than the sensitive cv.,
both in controls and against different treatments, except in treat-
ments where BSO were added [As(V) þ BSO and As(V) þBSO þGSH]
(Fig. S3A and C in Supplementary material). The root lengths of
both the cultivars were higher (29.6% and 28.9%, respectively)
against As(III) compared to C þBSO, whereas, there was no sig-
nificant difference between the root lengths of C and C þBSO or C
and As(III) (Fig. S3B in Supplementary material). In the tolerant cv.,
the root lengths of As(III) þGSH was significantly higher than its
control [C(PD)], C þBSO and As(III), whereas, it was significantly
lower in As(III) þBSO and As(III) þBSO þGSH. When compared
between the As(V) treatments, root lengths were significantly
higher in As(V)þ GSH than As(V) and also in As(V) þBSO than As
(V)þ BSOþGSH, in both the cultivars. In tolerant cv., the shoot
lengths of all the treated plants receiving As(III) and As(V) were
significantly lower than its control [C(PD)], whereas, in the sen-
sitive cv., it was significantly lower in As(III) þ GSH, As(III) þ BSO
and As(III) þBSO þGSH than its control [C(U)] (Fig. S3A in Sup-
plementary material). The fresh weights of tolerant cv., decreased
significantly with As(III) and As(V) treatments (17.6% and 14.7%,
respectively), as compared to C(PD), however, it significantly in-
creased when As(III) and As(V) were co-applied with 2000 mM of
396 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
GSH (14.2% and 13.8%, respectively) (Fig. S3C in Supplementary
material).
Photosynthetic assimilation rate (A) per unit leaf area (m
mol m
2 s
1) in the tolerant cv. varied from a maximum of
15.1 71.5 in C(PD) to a minimum of 0.37 70.12 in As(III) þBSO (Fig.
S4C in Supplementary material). While in sensitive cv., the max-
imum assimilation rate (A) varied from 10.6 72.4 mmol m
2 s
1 in
C(U) to a minimum of 0.04 70.001 mmol m
2 s
1 in As(III) þ BSO
(Fig. S4C in Supplementary material). Seedlings of sensitive cv.,
receiving As(III) exhibited negative assimilation rate
(
0.70 70.01 mmol m
2 s
1). Transpiration rate (E) was sig-
nificantly decreased in both the cultivars exposed to As(III), As
(III) þGSH and As(III) þGSH þBSO, whereas, against As(V) the rate
of decrease was lower (Fig. S4D in Supplementary material). The
pattern of stomatal conductance (Gs; molH2O m
2 s
1) was also
similar to that of assimilation rate where the maximum rates i.e.
0.34 70.02 and 0.21 70.01 were observed in the controls of both
tolerant and sensitive cv., respectively and the values in treated
plants were lower (Fig. S4B in Supplementary material). In both
the cultivars, the rate of Gs significantly declined in plants treated
with As(III) as compared to their respective controls. Photo-
chemical quenching qP was maximum in control plants both the
cultivars (Fig. S4F in Supplementary material), whereas, non-
photochemical quenching (NPQ) was minimum in As(III) þBSO of
both the cultivars, respectively, (Fig. S4E in Supplementary mate-
rial). Electron transport rate (ETR; mmol e
m
2 s
1) was max-
imum in the control plants of either of the cultivars, which was
higher in the tolerant cv. (119.8 76.9), than in sensitive cv.
(89.4 71.9 mmol e
m
2 s
1) (Fig. S4G in Supplementary materi-
al). Leaf transpiration rate (mmolH2O m
2 s
1) was minimum
(0.57 0.02 and 0.637 0.01) in As(III) þBSO treated plants of both
the tolerant and sensitive cultivars, respectively. Instantaneous
water use efficiency (WUE), ranged between a minimum of
0.07 70.02 in As(III) þBSO and maximum of 0.34 70.06 in As
(III) þGSH in tolerant cv. While in sensitive cv., it varied between a
minimum of
0.18 70.001 in As(III) and a maximum of
0.33 70.06 in its control [C(U)]. Overall, the tolerant cv. exhibited
more resistance towards As(III) toxicity (both stand alone and in
combination) as compared to the sensitive cv.
3.2. Accumulation and translocation of elements
Accumulation of both the species of As i.e. As(III) and As(V),
was higher in the tolerant than in the sensitive cv. (Table 1). Also,
the accumulation against As(V) was higher in both the cultivars
(16.44 72.05 and 24.04 71.08) than As(III), however, the translo-
cation of As were higher against As(III), which was 0.14 and 0.17 in
tolerant and sensitive cultivars, respectively. Application of GSH
lowered the translocation of As(III) to shoots (0.06) in tolerant cv.,
on the contrary, in sensitive cv. it increased (0.13), in comparison
to its As(V) translocation. On the other hand, application of BSO
[As(III) þBSO] increased the translocation of As (0.34) by 100% in
tolerant cv. and by 114.2% in sensitive cv., it (0.30), in comparison
to their respective As(III) treatments (Supplementary Table S2). In
support of this, the level of As (20.4 72.2 and 55.7 77.4) in the
roots of the As(III) þ BSO treated plants, in both the cultivars was
lower than their respective As(III) treatments. Similarly, the ac-
cumulations of As (82.5 74.8 and 138.97 19.4) in the roots of As
(V) þBSO, was also lower compared to As(V) values of sensitive
and tolerant cultivars, respectively. Despite decline in uptake of As
in roots of As(V)þGSH in both the cultivars against their re-
spective As(V) treatments, the translocation of As in As(V)þ BSO
was higher (200%) in sensitive cv. (0.27) than in the tolerant cv.,
(0.11), compared to their respective As(V) treatments. Further-
more, comparison of the translocation of As against As(V) between
the two cultivars, show that in As(V)þ GSH of sensitive cv., it was
44.4% higher than the tolerant cv. (33.3%) (Table S1).
PCA performed on the data pertaining to the elemental trans-
location of the sensitive and tolerant cv. against the various
treatments, yielded two significant PCs capturing 76.7% and 75.2%
variance, respectively (Table S2). In case of sensitive cv., PC1
(47.7%) showed strong positive (4 0.7) loadings on AsTF (0.87) and
strong negative loadings on Fe (
0.94), whereas, PC2 (29.3%) ex-
hibited strong positive loadings on Mn (0.71) and Zn (0.85). In case
of the tolerant cv., the MnTF (0.88) and FeTF (0.93) on PC1 (41.8%),
AsTF (0.87) and ZnTF (0.83) on PC2 (24.3%) exhibited strong and
positive loadings (Table S2). A simultaneous analysis of the re-
spective loadings and score plots (Fig. 1) of both the sensitive and
tolerant cultivars revealed, that AsTF were significant in the As
(III) þBSO þGSH, As(V)þ BSOþGSH and As(III) þBSO; TF of Zn and
Cu were significant in As(III) þ GSH, As(III) þ BSO and As(III),
whereas, TF of Fe was significant in C þBSO. Translocation of none
of the elements was influenced in the controls of either of the
cultivars, whereas, in the tolerant cv., As(V) exhibited no influence
over the TF of As, Cu, Fe, Mn and Zn (Fig. 1). Similarly, the plot
between PC1 vs. PC2 (Fig. 1A) exhibits a positive correlation be-
tween TF of As, Cu, Mn, and Zn but negative with Fe.
3.3. Toxicity and antioxidant response
Although there was no significant difference between the levels
of H2O2 between the two cultivars, as evident by its level in control
plants, however, the level of TBARS in the tolerant cv. was higher
than that of the sensitive cv. (Supplementary Fig. S3H). Conse-
quently, the overall levels of TBARS in all the treatments of tolerant
cv. were higher than in sensitive cv. When compared with the
TBARS levels of the respective control, the level was 76% higher in
As(III) treated plants in sensitive cv., whereas, it was 91.3% higher
in tolerant cv. On the contrary, there was no significant increase in
TBARS levels against As(V), in either of the cultivars with respect
to control. In the sensitive cv., the plants treated with As(III) þ
BSOþGSH were showing lower TBARS levels than in As(III) þBSO.
Similarly, the higher level of ascorbate (AsA) was observed in As
(III) and As(V) in sensitive cv. against both the species of As, than
that of the tolerant cv. (Fig. S3F in Supplementary material).
The GSH/GSSG ratio in the tolerant cv. was higher than in the
corresponding treatments in sensitive cv. (Fig. S3I in Supplemen-
tary material). In the tolerant cv., the GSH/GSSG in As(V) þ
BSOþGSH and in As(III) þBSO þGSH was higher than As(V)þBSO
and As(III) þBSO, respectively.
3.4. Effect on glutathione metabolism pathway
The activity of GST and GR was higher in tolerant cv. than that
in the sensitive cv. however, the activity reduced in As(III) and As
(III) þBSO treated plants in both the cultivars (Fig. S5B and C of
Supplementary material). On the other hand, the activity of GR in
Cþ BSO was significantly higher than their respective control, in
both the cultivars.
PCA was performed on the various antioxidant enzymes ac-
tivities and level of antioxidant, in order to identify the prime
component influencing the defence mechanism between the
contrasting cultivars. The PCA captured 87.0% and 90.3% of data
variance in the sensitive and tolerant cv., respectively, with 5 sig-
nificant PCs in each cultivars (Table 2). In case of sensitive cv., GSH/
GSSG (0.78) and CAT (0.86) exhibited strong positive loadings and
AsA (
0.77) negative loadings on PC1 (21.9%). Similarly, strong
loadings (eigenvalues 40.7) were exhibited on TBARS (0.82) and
POD (0.79) on PC2 (17.9%); H2O2 (0.87) and GRX (0.86) on PC3
(18.3%); GR (0.93) and GST (0.83) on PC4 (14.2%) and SOD (0.88) on
PC5 (14.9%). In case of tolerant cv., AsA (0.93) and GRX (0.88) ex-
hibited strong positive loadings (40.7) on PC1 (21.4) whereas,
Table 1
Level of As, Cu, Mn, Zn, and Fe in roots and shoots of two cv. of Oryza sativa, Usar-3 and PD-11 against As(III) and As(V) treatments under GSH enriched and deprived regimes. All values are mean of four replicates 7SD. Significance
of the mean values have been compared within the column where values marked with same alphabets are not significantly different (Duncan's test, p o 0.05).

A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
Accumulation (lg g
1 dw)

Treatments As root As shoot Cu root Cu shoot Mn root Mn shoot Zn root Zn shoot Fe root Fe shoot

Usar-3 C – – 204.317 27.96c 19.72 71.61abc 32.54 7 0.82ab 85.03 7 4.85bcd 121.49 7 9.58hi 53.32 7 3.56bcdef 353.767 40.83abc 219.92 7 7.25e
C þ GSH – – 128.63 7 15.10d 17.01 72.28a 32.62 7 5.07ab 54.69 7 4.40a 116.50 7 6.76ghi 42.277 4.56a 297.92 7 42.80ab 170.56 7 22.3abc
C þ BSO – – 89.89 7 12.27bc 19.75 71.81abc 28.59 7 4.33a 66.167 8.82ab 81.66 7 11.61abc 45.077 3.98ab 262.65 7 9.41a 154.337 22.9a
As(III) 58.60 7 8.83b 8.15 71.05a 61.87 7 2.41ab 19.41 72.84abc 34.30 7 4.48abc 87.767 11.84bcd 102.727 2.99defgh 46.137 4.27abc 460.58 7 11.84def 202.80 7 9.32cde
As(III) þGSH 80.23 7 12.60bc 13.20 7 2.18abc 90.04 7 8.40bc 24.81 72.36fg 45.917 3.75efg 103.80 7 9.76defg 112.58 7 9.46fghi 60.147 3.49efg 568.65 7 88.78fghij 222.45 7 20.5e
As(V) 188.63 7 23.02e 16.44 72.05abcd 72.007 9.49abc 23.59 70.90ef 44.23 7 3.59defg 93.82 7 11.25cdef 102.007 14.02defg 69.99 7 8.56h 736.377 10.51l 181.107 16.8abcd
As(V) þ GSH 178.45 7 20.95e 22.36 7 1.92bcd 94.677 12.65bc 19.78 72.63abc 42.717 7.04cdefg 158.077 13.07jk 107.357 7.15efgh 52.69 7 4.18bcdef 609.447 84.23ijk 189.88 7 18.0abcde
As(III) þBSO 20.35 72.22a 6.077 0.59ab 74.46 7 3.92a 17.58 71.17ab 57.84 7 6.12hi 76.117 11.51abc 117.32 7 17.98gh 50.58 7 2.69abcd 1113.48 7 81.07m 176.93 7 20.1abcd
As(III) þBSO þGSH 65.117 9.10bc 20.90 7 2.22bcd 82.357 5.18bc 22.15 71.91cdef 44.49 7 3.65defg 71.79 7 8.09abc 112.517 9.85fghi 51.39 7 2.60abcd 674.28 7 64.55jkl 158.63 7 4.65ab
As(V) þ BSO 82.45 7 4.77bc 21.90 7 2.92bcd 81.727 6.69bc 21.39 70.82cdef 43.40 7 1.26cdefg 169.917 15.63k 110.447 17.17efgh 62.167 8.10fgh 520.177 55.91efghi 198.85 7 20.0cde
As(V) þ BSO þGSH 64.22 7 4.02bc 12.107 0.57ab 84.75 7 4.02bc 20.79 71.76bcde 58.377 6.87hi 124.337 20.96gh 96.117 14.18bcde 51.02 7 2.22abcde 573.147 68.15ghij 180.08 7 22.1abcd

PD-11 C – – 99.137 9.89bc 23.60 70.91ef 52.02 7 1.14gh 107.60 7 2.04defgh 100.447 12.05cdefg 53.95 7 2.09bcdefg 440.82 7 32.67cde 182.167 25.0abcd
C þ GSH – – 57.52 7 5.82ab 23.24 72.35def 47.29 7 0.72fg 151.48 7 21.53jk 106.727 6.12efgh 56.38 7 3.32defg 471.377 25.41efgh 212.03 7 21.8de
C þ BSO – – 77.357 8.67bc 21.46 71.38cdef 36.89 7 5.78abcde 110.38 7 14.68efgh 91.65 7 4.24abcde 53.64 7 7.89bcdefg 389.337 36.13bcd 191.23 7 3.28abcd
As(III) 93.617 13.30c 15.93 7 2.05abcd 81.03 7 11.13abc 24.80 71.80fg 30.977 2.25ab 129.04 7 9.67hi 79.357 8.39ab 55.377 7.72cdefg 454.96 7 77.03cde 222.40 7 29.7e
As(III) þGSH 243.47 712.50f 15.80 7 1.39abcd 72.95 7 10.88bc 22.23 72.05cdef 35.107 4.73abcd 107.197 12.63defgh 76.54 7 9.26a 52.80 7 3.91bcdef 487.577 40.67defgh 194.677 15.6bcde
As(V) 373.52 730.35h 24.04 7 1.08d 87.53 7 9.04abc 19.66 71.51abc 38.94 7 2.35bcdef 113.39 7 14.34fgh 95.677 14.85bcdef 55.75 7 5.27defg 605.46 7 59.41ijk 192.157 14.8abcde
As(V) þ GSH 273.43 7 15.68j 23.03 7 1.93cd 72.80 7 6.75abc 19.97 72.50abcd 29.52 7 2.51ab 74.48 7 7.51abc 124.32 7 15.14i 54.25 7 3.48bcdefg 594.08 7 83.49hij 166.58 7 16.6abc
As(III) þBSO 55.67 77.36b 19.167 2.20bcd 82.69 7 9.91bc 27.13 71.80g 63.94 7 9.98i 146.83 7 15.43ij 86.63 7 9.09abcd 62.81 7 8.11gh 727.717 68.91l 212.447 26.3de
As(III) þBSO þGSH 85.017 11.19abc 15.317 1.30abcd 100.02 7 13.05c 22.79 72.69cdefg 44.69 7 6.69defg 92.077 9.32cdef 93.017 9.85abcde 52.92 7 4.85bcdef 703.36 7 105.26kl 173.777 22.1abc
As(V) þ BSO 138.85 7 19.39d 15.52 7 0.70abcd 59.197 6.62abc 21.13 72.37cde 37.177 4.49abcde 109.32 7 16.74efgh 91.447 3.20abcde 57.29 7 2.33defg 502.577 1.74efghi 188.197 16.0abcde
As(V) þ BSO þGSH 88.94 7 11.14c 15.52 7 1.31abcd 65.02 7 5.91ab 23.37 71.66def 47.677 7.85fg 113.48 7 4.81fgh 98.747 5.23cdefg 56.417 5.62defg 667.28 7 18.15jkl 184.83 7 25.1abcde

397
398 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405

Fig. 1. Plot between the loadings of (A) PC1 vs. PC2 and (B) score plot of the translocation factors of Cu, Zn, Fe, Mn, and As in rice cv. PD11 (tolerant) against As(III) and As
(V) under GSH enriched and deprived conditions, (C) PC1 vs. PC2 and (D) Score plot of the rice cv. Usar (sensitive) against As(III) and As(V) under GSH enriched and deprived
conditions.

Table 2
Factor loadings obtained from the principal component analysis performed with the different metabolites and activities of antioxidant enzymes in two cv. Usar-3 (sensitive)
and PD-11 (tolerant) of Oryza sativa against As(III) and As(V) under GSH deprived and enriched conditions.

Factor loadings of Usar-3 (Varimax normalized) Factor loadings of PD-11 (Varimax normalized)

Extraction: principal components (marked loadings are 40.700000) Extraction: principal components (marked loadings are 40.700000)

Parameters Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 1 Factor 2 Factor 3 Factor 4 Factor 5

TBARS 0.252642 0.820218
0.318294 0.150505
0.218131 0.363220 0.318952 0.434328
0.547772 0.072461
GSH/GSSG 0.784369
0.503739
0.093884 0.087582
0.266782 0.470748 0.349221
0.747817 0.022773
0.244925
AsA
0.775154 0.124434
0.363671 0.075124
0.158528 0.933997 0.086984 0.128859
0.083231 0.058582
H2O2
0.138914
0.026203 0.876388 0.270011 0.201912
0.389265 0.370999
0.100835
0.686149
0.258557
CAT 0.861184 0.248800 0.057883
0.003830 0.057720 0.315382 0.486626 0.745236 0.234163
0.136712
POD
0.209351 0.796628 0.154750
0.056411 0.363014 0.277574 0.251658 0.883075 0.215570 0.087913
SOD
0.030494 0.078333 0.224237
0.221795 0.885545 0.666084 0.646693 0.108129 0.174705
0.223975
GRX 0.353774 0.142028 0.869495
0.126399 0.060639 0.879208
0.130767 0.306988 0.047236
0.117195
GPX 0.534966 0.068355
0.177471 0.059118 0.691511 0.179221 0.616392 0.173539
0.117661 0.653722
GR
0.035783
0.129622 0.110224 0.936216 0.046339
0.088601 0.898981
0.109304
0.070614 0.099370
GST 0.003180 0.141158
0.114591 0.831429
0.329131
0.238574 0.293504
0.754779 0.373279 0.148299
APX 0.225190
0.509278 0.623136
0.301228
0.327104
0.042093 0.170185 0.130617 0.959642
0.127782
MDHAR 0.668692 0.336556 0.172721
0.234461 0.535981
0.110694
0.039539
0.116828 0.028214 0.942033
DHAR
0.285937
0.662846
0.505050 0.307970
0.068068 0.346159
0.169808 0.739149 0.085406
0.482649
Expl.Var 3.077875 2.518904 2.574022 1.989063 2.095200 3.001337 2.437952 3.404580 1.999526 1.821764
Prp.Totl 0.219848 0.179922 0.183859 0.142076 0.149657 0.214381 0.174139 0.243184 0.142823 0.130126
 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405 399

Fig. 2. Factor loadings of the principal components obtained for the various metabolites and antioxidant enzyme activities in rice cv. Usar (sensitive) against As(III) and As(V),
(A) PC2 vs. PC3; (B) PC4 vs. PC3; (C) PC3 vs. PC5 along their respective score plots (D) PC2 vs. PC3; (E) PC4 vs. PC3; (F) PC3 vs. PC5.
400 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405

Fig. 3. Factor loadings of the principal components obtained for the various metabolites and antioxidant enzyme activities in rice cv. PD11 (tolerant) against As(III) and As(V),
(A) PC1 vs. PC3; (B) PC1 vs. PC4; (C) PC1 vs. PC5; (D) PC3 vs. PC4 along their respective score plots (E) PC1 vs. PC3; (F) PC1 vs. PC4; (G) PC1 vs. PC5; (H) PC3 vs. PC4.
 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405 401

SOD (0.66) exhibited moderate loadings. GR (0.89) exhibited
strong positive loadings on PC2 (17.4%) whereas, moderate positive
loadings exhibited by GPX (0.61). CAT (0.74), POD (0.88) and DHAR
(0.73) exhibited strong positive loadings on PC3 (24.3%), whereas,
GSH/GSSG (
0.74) and GST (
0.75) exhibited strong negative
loadings. On PC4 (14.2%). APX (0.95) and MDHAR (0.94) on PC5
(13.0%), exhibited strong positive loadings, whereas, H2O2 (
0.68)
and GPX (0.65) exhibited moderate negative loading in PC4 and
moderate positive loading on PC5, respectively.
The PCA shows a marked difference between the sensitive and
tolerant cv. in their biochemical response against different As ex-
posure. In tolerant cv., H2O2 and TBARS did not exhibit strong
loadings in any of the PCs, whereas, in sensitive cv., both of them
exhibited strong loadings on PC2 and PC3, respectively, signifying
that sensitive cv. is more prone to As induced lipid peroxidation
than tolerant cv. at equimolar concentration of As (Table 2). A si-
multaneous analysis of the loadings and respective score plots of
the sensitive cv. reveals that, AsA has a negative relationship with
CAT and GRX, while GRX was significantly influenced by As(III) þ
GSH, As(III) þBSO and As(V)þ GSH; (Fig. 2A and E). Factor loadings
between PC2 vs. PC3 shows, GRX was significantly influenced by
As(III) þBSO þGSH and As(V)þ BSOþ GSH, whereas, the GSH/GSSG
is significant in As(III), As(V) and As(III) þGSH (Fig. 2B and F). Score
plot between PC3 vs. PC4, reveals that GST is significantly influ-
enced in As(V), As(III) þGSH and As(V)þGSH, whereas, the GR and
H2O2 is influenced in As(V) þBSO þGSH and As(III) þBSOþ GSH
(Fig. 2C and G). A strong relationship was also exhibited between
H2O2 and SOD (Fig. 2D). Both the APX and GPX activities have also
exhibited moderate positive loadings on PC3 and PC5, respectively
(Fig. 2D and H). CAT and H2O2 have exhibited strong positive
loadings on PC1 and PC3, respectively in (Fig. 2A). Moderate po-
sitive loadings exhibited by APX, DHAR and MDHAR on PC1, PC2
and PC5, respectively, shows preference of AsA over GSH (Table 2).
Coincidently, the activities of major enzymes involved in AsA

Fig. 4. Expression of GRX and GST genes in two cultivar of rice viz. Usar (sensitive) and PD11 (tolerant) through qRT-PCR against As(III) and As(V) under GSH enriched and
GSH deprived conditions; (A) GRX 40500; (B) GRX 61350; (C) GRX 13950; (D) GRX 27140; (E) GRX 27140; (F) GST 38160.
402 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
pathway were also less (Fig. S5E, I and J of Supplementary mate-
rial). Alternately, the enzymes and metabolites of GSH pathway,
i.e. GRX, GPX, GR, GST, GSH/GSSG have shown strong and positive
loadings, suggesting that GSH mediated detoxification was domi-
nant over AsA. Plot between PC1 vs. PC3 (Fig. 3A) and between PC3
vs. PC4 (Fig. 2C), exhibits a strong and positive correlation between
GSH/GSSG and GPX, and between H2O2 and GRX in plot between
PC3 vs. PC5 (Fig. 2D),
Contrarily, in tolerant cv., strong positive loadings of AsA (0.93),
DHAR (0.74), APX (0.95), MDHAR (0.94), were exhibited on PC1,
PC3, PC4, and PC5, respectively whereas, there was strong negative
loadings of GSH/GSSG (
0.74), GST (
0.75) on PC3, (Table 2).
Incidentally, DHAR uses GSH as reductant for converting dehydro-
ascorbate to ascorbate. Negative moderate loadings for H2O2
(
0.68) exhibited on PC4 along with strong and positive loadings
of CAT (0.74) and POD (0.88) on PC3, can be attributed towards
action of these enzymes in detoxification of H2O2. This is also
evident from the factor loading between PC3 and PC4, where the
activity of POD and CAT has exhibited a close association (Fig. 3D).
The strong negative loading on GST (
0.75), can be explained in
light of the higher translocation of As in tolerant cv., where the
lower expression of GST resulted into less As sequestration in roots
(Fig. 4J and L). The strong positive loading of GRX (0.87) on PC1, GR
(0.89) on PC2, resulting into exhausting GSH pool, coincides with
the negative loading on GSH/GSSG.
3.5. Expression of glutaredoxin genes against As(III) and As(V)
Among the 48 members of the GRX gene family in rice (Garg
et al., 2010), expression of four genes. i.e. two CC type GRX i.e.
Os01g27140, Os01g13950, one CPYC type i.e. Os02g40500 (Fig. 4A
and B) and one GRL type i.e. Os01g61350 (Fig. 4B and C) were
studied against As(III) and As(V). Overall, the expression of all the
GRX genes were higher in sensitive cv. than in the tolerant cv.,
moreover, they were higher in both cultivars against As(V) than As
(III) treatments. The expression of CC type GRX gene Os01g 27140,
was up-regulated by 7 folds against As(V) in the sensitive cv.,
while, there was no significant expression in the tolerant cv.
against same treatment. On the contrary, the same gene was
highly expressed with exogenous GSH application in the tolerant
cv. by 12 fold, while there is no expression in sensitive cv. against
the same treatment. With BSO treatment (C þBSO), and also in As
(III) treated plants, the expression of the gene Os01g27140 in the
sensitive cv. was higher than in its control [C(U)]. Whereas, in the
tolerant cv. higher level of expression of the gene was only ob-
served against As(III) treatment, as compared to its control [C(PD)].
The expression of the second gene, viz. CC type GRX i.e.
Os01g13950 was upregulated against As(V) in the sensitive cv.,
while, it had no significant expression in tolerant cv. (Fig. 4F).
Among the two GST genes studied the expression of both the
genes in sensitive cultivar was tens of fold higher than tolerant cv.
In the sensitive cv. the expression of Os02g38160 was high against
both As(III) and As(V) treatments, whereas, the expression of
Os02g20220 was twice against As(III) than that against As(V).
4. Discussion
The presented study highlights differential response between
two rice cultivars differing in their sensitivity towards As, in terms
of differential expression of GRX genes and preference of AsA or
GSH for detoxification. It also showed that absence of free thiol
leads to increase in translocation of both As(III) and As(V) to
shoots irrespective of plants being tolerant towards As. The bio-
chemical response is also consistent with the response of the
morphological parameters, where the root lengths of plants
receiving As(III) and devoid of free thiols (GSH), were significantly
reduced with respect to the plants receiving only As(III). Hence,
the toxicity towards the root due to As(III), were aggravated in
absence of thiols. On the contrary, in the tolerant cv., amelioration
of As toxicity by GSH is evident as the root length in the plants was
less affected when As(III) was applied along with GSH, than in the
control plants [C(PD)], BSO treated plants (C þ BSO) or As(III)
treated plants. Also, the root lengths in plants receiving As(III) þ
BSO or As(III) þBSO þGSH were significantly reduced, showing the
aggravation of As(III) toxicity in absence of GSH. Additionally,
significant decline in the shoot length in sensitive cv., against As
(III) þGSH, As(III) þBSO and As(III) þBSO þGSH than in As(III),
signifies that cv. U being sensitive was unable to overcome the As
(III) toxicity in absence of GSH and the exogenous GSH application
could not replenish the cellular GSH.
Relatively higher physiological parameters such as photo-
synthetic assimilation rate (A), stomatal conductance (Gs) photo-
chemical quenching (qP), leaf transpiration rate, electron transport
rate (ETR), photochemical quenching (qP), transpiration rate (E),
photosynthetic rate (A) and stomatal conductance (Gs) in cv. PD11,
also supports it being tolerant. Whereas, the non photochemical
quenching (NPQ) representing the loss of energy towards heating,
was higher in sensitive cv. than in the tolerant cv., which supports
that cv. U being sensitive was wasting more energy towards
heating. The stomatal conductance (Gs) in both the cultivars
treated with As(III) and As(V) decreased significantly, as compared
to their respective controls. The lower rate of A and NPQ in As
(III) þBSO in both the cultivars, indicates minimum heat dissipa-
tion as a result of toxicity manifestation due to absence of GSH,
which being higher in As(III) than As(V). Also, the rate of Gs in As
(III) þBSO, was lower than As(III), whereas, it was higher in As
(V)þBSO, demonstrates the aggravation of toxicity against As(III)
in absence of GSH. This is also congruent with lower rate of
transpiration (E) in As(III) than As(V) treated plants. On the con-
trary, addition of GSH with As(III) and As(V), increased the rates of
Gs and E marginally, which shows exogenous GSH supplementa-
tion had little recovery. Photosynthetic rate (A) in As(III) and As
(V) treated plants was significantly lower in both the cultivars, and
the rate against As(III) was lower than As(V), thus proving that
photosynthesis is affected more with As(III). In the sensitive cv.,
the photosynthetic rate stopped when As(III) was supplemented
with BSO; however, marginal recovery was observed with the
exogenous addition of GSH to As(III) þBSO i.e. As(III) þBSO þGSH.
The photochemical quenching, which was higher in the tolerant
cv. than the sensitive cv., shows cv., PD 11 being tolerant, it was
efficient in harvesting light energy under As stress. The addition of
BSOþGSH did not show any significant recovery in photosynthetic
rate either over As(III) or As(V) treatments. Therefore, it can be
argued that exogenous GSH does not significantly ameliorate the
As(V) toxicity, whereas, the As(III) induced toxicity could be mar-
ginally ameliorated by exogenous GSH, as the rates of ETR, E, A, Gs
and WUE were higher in As(III) þBSO þGSH than As(III) þBSO.
GSH thiolates As(III) and not As(V), hence the observed ameli-
oration in As(III) toxicity due to exogenous GSH supplementation,
could be attributed to sequestration of thiolated As(III) in roots,
thereby restricting its translocation to the shoots.
4.1. Accumulation and translocation of elements
Genetic variation in As accumulation exists in different geno-
types (Norton et al., 2009), it was evident from the higher accu-
mulation of As in tolerant cultivar than the sensitive cv., both
against As(III) and As(V). With blockage of GSH synthesis, required
for thiolation of As(III); larger amount of As(III) was translocated to
the shoots, which was more in the sensitive cv. than in the tolerant
cv. This also explains the inherent tolerance mechanism of cv.
 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
PD11, as it was able to tolerate higher amount of As than the
sensitive cv. Similarly, with As(V) treatments, higher translocation
of As in As(V) þBSO treated plants of sensitive cv., compared to the
same treatment in tolerant cv., demonstrates the ability of sensi-
tive cultivar to restrict translocation of As(V), is relatively less in
absence of GSH. Several studies have reported conflicting results
with respect to the translocation of As in plants with application of
BSO. Recently, Lei et al. (2013) and Watanabe et al. (2014) reported
a decrease in As accumulation in the shoots of As hyper-accumu-
lating plant P. vittata with BSO treatment. However, in the present
study exogenous application of GSH could not decrease the As
(V) translocation significantly, as evident from the higher level of
As in the shoots of As(V)þ BSOþ GSH treatments in both the cul-
tivars. Thus, it can be argued that exogenous GSH application, fa-
cilitates higher As(III)-GSH binding, preventing the translocation
of As(III) from the root to shoot.
The translocation of As in plants is reported to influence the
translocation of other essential elements (Dwivedi et al., 2010a).
This has been captured by the PCA performed on the TFs of various
elements. It shows that in sensitive cv., the TF of Zn and Cu was
highly influenced by both As(V) and As(III) whereas, in tolerant cv.
only As(III) exerted influence over the TF of Cu, Mn and Zn. The TF
of Fe was negatively influenced by AsTF in sensitive cv., whereas,
in tolerant cv., TF of As was positively influenced (Check). Dwivedi
et al. (2010b) reported that the lower levels Fe in the shoots of two
rice cultivars viz. Triguna and IET-4786 which accumulated higher
As [As(V); 12 mg l
1], in contrast to the higher Fe level in the other
two cultivars viz. IR-36 and PNR519, where As accumulations were
low. The present study shows inverse relationship between As and
Fe accumulation in rice plants. The tolerant cv. exhibited strong
and positive loadings on MnTF and FeTF in PC1, AsTF and ZnTF on
PC2, which demonstrates that As positively influences the trans-
location of Fe and Mn in tolerant cv. Hence, it can be said that the
translocation of Cu was positively and Fe was negatively influ-
enced by As in the sensitive cv., whereas, in the tolerant cv., As
positively influenced the translocation of Fe while, TF of Mn and
Zn were positively influenced by As in either of the cultivars.
4.2. Toxicity and antioxidant response
Inspite of higher lipid peroxidation in sensitive cultivars against
As stress, as reported in other studies (Kumar et al., 2013; Tripathi
et al., 2012), however, higher level of TBARS exhibited in the tol-
erant cv. in the present study, is attributed to inherent character-
istic of the cultivar, as the level was also higher in the control
plants than that in sensitive cv. As(III) is more toxic than As(V) by
several orders (Mascher et al., 2002), consequently the level of
lipid peroxidation was higher in plants receiving As(III) than As(V),
which was higher in the tolerant cv. than sensitive cv. In tolerant
cv., the higher levels of TBARS in As(III) þBSOþ GSH and As(V)þ
BSO þGSH, in comparison to As(III) þBSO and As(V) þBSO, re-
spectively, establishes that BSO and GSH together causes toxicity
both with As(III) or As(V), than when the plants are treated either
with GSH or BSO alone. Thus, exogenous application of GSH has
little effect to ameliorate the toxicity due to absence of internal
GSH synthesis mechanism. There are studies demonstrating the
beneficial effect of exogenous GSH application ranging from
300 mM to 10 mM (Teh et al., 2015) in rice plants without any toxic
effect. Also, in tolerant cv. the levels of TBARS in Cþ BSO, As(III) þ
BSO and As(V) þBSO were lower than its control, whereas, in
sensitive cv. the levels in the corresponding treatments were
higher than its control as well as its C þBSO. Hence, the lipid
peroxidation inflicted in tolerant cv. due to BSO, As(III) þ BSO, and
As(V) þBSO were lower by virtue of its tolerance towards As. The
ascorbate level in tolerant cultivar was relatively higher in tolerant
cv. than the sensitive cv. This is also supported by the increase in
403
the activity of AsA mediated enzymes viz. MDHAR, DHAR, and
APX. Interestingly, the application of the GSH decreased the AsA
level in the C þ GSH, As(III) þ GSH, As(V) þGSH as compared to C,
As(III) and As(V), respectively. This also suggests that in the ab-
sence of GSH, the antioxidant defense mechanism in tolerant cv. is
largely dependent on AsA, as a result the level of AsA increases to
overcome the toxicity. Whereas, in case of BSO treated plants,
where the synthesis of GSH was stopped or diminished, the AsA
levels were lower than that of the GSH treated plants. This can be
attributed to the fact that DHAR uses GSH for reduction of dehy-
droascorbate to ascorbate, and in absence of GSH, the AsA pool
was less replenished. The GSH/GSSG ratio is a measure of the
overall reduced GSH level against toxic response, higher the oxi-
dative stress the lesser will be GSH level. In tolerant cv., the higher
GSH/GSSG in As(V)þ BSOþ GSH and in As(III) þBSO þGSH than As
(V)þ BSO and As(III) þBSO, respectively. It shows that, external
GSH contributed towards the overall GSH built up in the tolerant
cv., whereas, in absence of such trend in sensitive cv. supports that
detoxification is less dependent upon GSH in sensitive cv.
4.3. Dominance of ascorbate or glutathione pathway between
cultivars
The antioxidant enzymes involved in the GSH metabolism
pathway are GR, GST, GPX and GSH dependent small redox-pep-
tides like GRX. The higher activity of GST in tolerant cv. against As
(V) than As(III), is congruent with the reports of Tripathi et al.
(2012), where the activity of GST in a tolerant cv., (Triguna) was
higher than sensitive cv. (IET-4786) against 20 mM of As(V). The
PCA shows a marked difference between the sensitive and tolerant
cv. in their biochemical response against As exposure. In light of
the strong loading on SOD and H2O2 in sensitive cv., it can be said
that the prevailing detoxification of As induced ROS (Fig. S1C in
Supplementary material), is via dismutation of O−2 · to H2O2 by SOD,
which further by the action of either POD or APX, H2O2 is con-
verted to H2O. Moderate positive loadings both on APX and GPX
compared to strong and positive loadings on CAT and H2O2 ac-
tivities, signifies that CAT played a prime role in detoxifying H2O2.
In support of this, the moderate positive loadings on DHAR, APX
and negative loadings on MDHAR, suggests that the detoxification
of H2O2 was lesser using AsA as electron donor in sensitive cv.
Alternately, the strong and positive loadings on enzymes and
metabolites of GSH pathway, i.e. GRX, GPX, GR, GST, GSH/GSSG
suggests that GSH mediated detoxification was dominant. Also, the
strong and positive correlation between GSH/GSSG and GPX,
whereas, close association between H2O2 and GRX suggests, ex-
pression of GPX could be triggered by H2O2.
On the contrary, in the tolerant cv., the strong and positive
loadings exhibited on DHAR (0.74), APX, MDHAR, and AsA, and
negative loadings on GSH/GSSG, GST, indicate the dominance of
AsA based detoxification. The strong and negative loadings on
GSH, demonstrate that GSH pool was less utilized for H2O2 de-
toxification. Similarly, the strong and negative loading on GST, can
be explained in light of the higher translocation of As in tolerant
cv., where the lower expression of GST resulted into less As se-
questration in roots. In roots, the As(III) are thiolated by conjuga-
tion with GSH and further GSH–As(III) conjugate is sequestered
into root vacuoles by the activity of ABC transporters located on
tonoplast, thereby restricting the translocation of As(III) to shoots.
Thus, in absence of the efficient GST activity, most of the As(III) got
translocated to the shoots. Glutaredoxins are efficient disulfide
reducers, which uses GSH as electron donor, in the process, GSH is
converted to GSSG, subsequently the GSSG gets reduced by GR to
replenish the GSH pool (Laporte et al., 2012; Rouhier et al., 2002).
The strong and positive loading of GRX (0.87) on PC1, GR (0.89) on
PC2, and negative loading on GSH/GSSG, suggests that due to the
404 A.K. Dubey et al. / Ecotoxicology and Environmental Safety 124 (2016) 393–405
action of GRX, the GSH pool got exhausted. Contrarily, the positive
loading on GR shows its enhanced activity for conversion of GSSG
to GSH. These two reactions are coupled hence, there is a positive
correlation between them (Fig. S1B in Supplementary material). A
recombinant E. coli using GRX gene (PvGRX5) extracted from the
fronds of As hyper-tolerant fern P. vitatta, was reported to exhibit
tolerance towards 10 mM As(V), high temperature, and oxidative
stresses (Sundaram and Rathinasabapathi, 2010; Sundaram et al.,
2008, 2009). Guo et al. (2010) found that silencing of SlGRX1 gene
in tomato plants led to the plant being susceptible towards oxi-
dative, drought and salt stresses, whereas over-expression of
SlGRX1 in Arabidopsis significantly increased the plant's resistance
to these stresses. In addition to oxido-reductase activity of GRX,
Lee et al. (2002) have reported that a rice GRX (OsGrx) can also
function as a GSH-dependent peroxidation thereby protecting
glutamine synthetase from oxidative damage.
4.4. Differential expression of glutaredoxin genes against As(III) and
As(V)
GRXs are important peptides in plants for regeneration of an-
tioxidant enzymes and their deglutathionylation activity during
oxidative stress. The expression of the four GRX genes presented
in the present study were also reported by Garg et al. (2010) and
represented as OsGRX4, OsGRX3, OsGRX9 and OsGRL2, in which
OsGRX3 and OsGRX4 were found to be higher at all the seed de-
velopment stages, whereas, OsGRX4, OsGRL2 and OsGRX9 in
young leaves. The CC type GRXs have been reported to be ex-
clusively found in the land plants (Rouhier, 2010), and expression
of the gene in the sensitive cv. against As(III) demonstrates its
specificity against As(III). In agreement to these observation, dif-
ferential expression of the GRX gene Os01g13950 as represented by
OsGRX3, was found to be higher in a As(V) sensitive rice cultivar
(Azucena) than in As(V) tolerant (Bala), whereas, expression of
Os01g40500 (OsGRX9) was higher in both the cultivars (Garg et al.,
2010). The CPYC type GRX i.e. Os01g40500 was highly expressed
against As(V) treatment in both the cultivars, which shows the
specificity of the gene towards As(V). This is congruent with the
findings made by Chakrabarty et al. (2009), where they found
higher expression of Os01g40500 against both As(III) and As(V).
The expression of the GRL type GRX gene, Os01g61350 exhibited
significant upregulation (30 fold) against As(V) in the sensitive cv.,
whereas, in the tolerant cv. it exhibited no expression against any
treatments. The higher expression of GRX CC type gene
Os01g27140 with exogenous GSH treatment shows its dependency
on GSH but with BSO treatment (C þBSO), upregulation of both the
CC type GRX i.e. Os01g27140 and Os01g13950, indicate that an al-
ternate electron donor other than GSH could be involved. Hence,
in the light of the relatively higher expression of all the four GRX
genes in sensitive cultivars against As(V), it can be argued that in
sensitive cv., GRX plays a role towards As(V) detoxification.
GST is a cytoplasmic enzyme which thiolates As(III) and targets
them for ATP-dependent transport into the vacuole (Tripathi et al.,
2012). Also, in plants, GSTs are known to be induced by heavy metal
exposure (Chakrabarty et al., 2009; Moons, 2003). Plant GSTs can also
act as glutathione peroxidases (Bartling et al., 1993), protect cells
from oxygen toxicity (Hayes and McLellan, 1999). Out of the 79 GST
genes reported (Jain et al., 2010), expression of two GST genes i.e.
Os09g20220 and Os02g38160 (TAU class) were studied to find out the
role of GST in absence of GSH against As(III) and As(V). The higher
expression of GST against As(III)þGSH, and no expression against
BSO treatments (CþBSO, As(III)þBSO), supports that absence of GSH
represses the expression of this GST gene. Contrary to the report of
up-regulation of 10 GSTs upon As(V) exposure (Chakrabarty et al.,
2009), the expression of Os09g20220 in the tolerant cv. was higher
against As(III) than As(V), whereas, no change in the expression of
Os02g38160 in sensitive cv. suggests active role of GST in the tolerant
cv. towards countering As toxicity. In agreement with the present
study, where higher expression of Os09g20220 was observed in the
sensitive cv., (Jain et al., 2010) had also found 222.36 fold change in
the same gene represented as OsGSTU5, in another sensitive cv. of
rice (Azucena), than 94.03 fold in the tolerant cv. (Bala) against As
(V) treatment. Hence, the obtained results of the GST expression
shows that the expression was higher in sensitive cv. than the tol-
erant cv. against As(III) than As(V). This also supports the lower
translocation of As in sensitive cv. than in tolerant cv., where the
lower translocation could be due to higher expression of GST genes.
5. Conclusion
Analysis of the obtained results involving a pair of contrasting
rice cultivars i.e. Usar-3 and Pant Dhan 11, against As(III) and As(V),
under GSH enriched and GSH deprived conditions, show that the
morphological and physiological parameters in the tolerant cv.
were less affected than the sensitive cv. Toxicity due to As(III) was
higher than that of As(V) in both the cultivars, and blocking of GSH
synthesis aggravated the toxicity, whereas, exogenous GSH treat-
ment does not overcome the toxicity. The As accumulation against
both As(III) and As(V) in the sensitive cv. was lower than that in
the tolerant cv., moreover, the As translocation in As(V) was higher
than that of As(III), in both the cultivars. In the sensitive cv., the
translocation of As and Fe was positively influenced by As
(V) whereas, the translocation of Zn and Mn was influenced by As
(III). In the tolerant cv., As positively influenced the translocation
of Fe and Mn. Based upon the strong and positive correlation be-
tween H2O2, SOD, GRX, GR, GST and GSH/GSSG in the sensitive cv.
and between DHAR, APX, MDHAR and AsA in the tolerant cv.; it
can be said GSH is primarily used as electron donor for H2O2 de-
toxification in sensitive cv., whereas, AsA is preferred in tolerant
cv. The higher activity of GRX, GR, GST in the sensitive cv. than in
the tolerant cv., in conjecture with the higher expression of the
four GRX genes, demonstrates that GRX plays a significant role in
As sensitive rice cultivars. The higher As accumulation in the tol-
erant cultivar is due to lower GST expression, leading to higher
translocation to shoot in absence of As thiolation mechanism.
Acknowledgment
The authors are thankful to Director, CSIR-NBRI, Lucknow, for
providing the infrastructural support to carry out the research. The
work is supported by research grant provided by SERC-DST, GOI,
New Delhi (SR/SO/PS/81/2010). AKD acknowledges SERC-DST, GOI
for junior research fellowship. Special thanks are due to Dr. K.
P. Singh, IITR, Lucknow for his valuable inputs while performing
PCA on the data sets.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.10.017.
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