lable at ScienceDirect

Plant Physiology and Biochemistry 99 (2016) 86e96

Contents lists avai

Plant Physiology and Biochemistry

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

Research article

Reduced arsenic accumulation in rice (Oryza sativa L.) shoot involvessulfur mediated improved thiol metabolism, antioxidant system andaltered arsenic transporters

Garima Dixit a, Amit Pal Singh a, Amit Kumar b, Seema Mishra a, Sanjay Dwivedi a,Smita Kumar a, Prabodh Kumar Trivedi a, Vivek Pandey a, Rudra Deo Tripathi a, *

a CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, Uttar Pradesh, Indiab Department of Botany, University of Lucknow, Lucknow, India

a r t i c l e i n f o

Article history:Received 31 August 2015Received in revised form6 November 2015Accepted 6 November 2015Available online 11 November 2015

Keywords:ArsenicRiceSulfate and arsenic transportersSulfurThiol metabolism

* Corresponding author.E-mail addresses: tripathird@gmail.com,

(R.D. Tripathi).

http://dx.doi.org/10.1016/j.plaphy.2015.11.0050981-9428/© 2015 Published by Elsevier Masson SAS

a b s t r a c t

Arsenic (As) contamination in rice is at alarming level as majority of rice growing regions are Ascontaminated such as South East Asia. Restricting the As in aerial parts of rice plant may be an effectivestrategy to reduce As contamination in food chain. Sulfur (S), an essential element for plant growth anddevelopment, plays a crucial role in diminishing heavy metal toxicity. Current study is designed toinvestigate the role of S to mitigate As toxicity in rice under different S regimes. High S (5 mM) treatmentresulted in enhanced root As accumulation as well as prevented its entry in to shoot. Results of thiolmetabolism indicate that As was complexed in plant roots through enhanced synthesis of phytochelatins.High S treatment also reduced the expression of OsLsi1 and OsLsi2, the potent transporters of As in rice.High S treatment enhanced the activities of antioxidant enzymes and mitigated the As induced oxidativestress. Thus from present study it is evident that proper supply of S nutrition may be helpful in pre-vention of As accumulation in aerial parts of plant as well as As induced toxicity.

© 2015 Published by Elsevier Masson SAS.

1. Introduction

Arsenic (As) is ubiquitous toxic element and present in all typesof soil. Arsenic is posing serious health concerns in South East Asiawhere elevated concentration of As, up to 3200 mg l�1 in drinkingwater has been reported (McCarty et al., 2011) against the safe limitof 10 mg l�1 recommended by World Health Organization (WHO).Arsenic contaminated water serves as principal source of Asexposure followed by food. Arsenic exposure from food becomemore important for the people based on rice as subsistence diets,where dietary As exposure from rice alone can be considerable(Williams et al., 2007). South East Asia especially the Bangladeshand West Bengal in India are worst affected areas by As contami-nation. Rice, the staple food for more than 50% of the world'spopulation (IRRI, 1993), is the main crop of As contaminated areaand posing problem because of its high As uptake property.

Arsenate (AsV) and arsenite (AsIII) are two inorganic chemical

tripathi_rd@rediffmail.com

.

forms of As occurring in soil. Arsenate is chemically analogous tophosphate and thus competes with it for the uptake by the plantsthrough phosphate transporters (Zhao et al., 2010b). Aquaporins(nodulin 26-like intrinsic proteins: NIPs), the silicon uptakepathway, are responsible for AsIII uptake in rice (Ma et al., 2008).Transport of As from the cells into the xylem is regulated by OsLsi2(a silicon/arsenite efflux protein) (Zhao et al., 2010a). Arsenic is nonessential element for plant growth and development and hampersthe plant growth in various ways (Singh et al., 2015). Severalphysiological functions of plant are fugitive for As induced toxicity.

One of common mode of toxicity of As is the induction ofoxidative stress and disturbance of redox state leading to damage tomembranes, proteins, lipids and ultimately cell death (Srivastavaet al., 2007, 2011). Several antioxidant enzymes and metabolitesare involved in the defense pathways of plants against As-inducedoxidative stress. Glutathione is a redox buffer that protects cellagainst reactive oxygen species (ROS), which accumulate inresponse to heavy metal stress (Mullineaux, 2005). It functionsthrough the ascorbateeGSH-cycle (AGC) and glutathione S-trans-ferase (GST) based detoxification mechanisms (Labrou et al., 2015).Moreover, GSH also serves as the precursor of phytochelatins (PCs),

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e96 87

the cysteine-rich peptides synthesized via phytochelatin synthase(PCS) under heavy-metal exposure (Scheller et al., 1987).

Sulfur has various vital functions in plant system. After uptakesulfur is converted to S-containing compounds such as cysteine(Cys) and methionine (Capaldi et al., 2015), this cys is important inthe formation of sulfhydryl (SeH) and disulfide bonds (SeS) inproteins and many enzymes possess thiol groups at their activecenters (Saito, 2000). Further, sulfur-containing compounds play animportant role in plant stress defense; however, only a little isknown about the molecular mechanisms of regulation of sulfateassimilation during As stress. Sulfate assimilation is highly regu-lated in a demand-driven manner (Davidian and Kopriva, 2010). Atlow sulfate availability, plants increase sulfate transport and therate of reduction. Dedicated sulfate transporters viz., high-affinitysulfate transporters (HASulTs), low-affinity vascular transporters(LASulTs), and vacuolar efflux transporters (OsSultr4;1, OsSultr4;2),are responsible for sulfate uptake, translocation and cellularcompartmentalization (Hawkesford, 2003), and are regulated bythe plants' nutritional status (Davidian and Kopriva, 2010). Sulfurdeficiency or accessibility influences sulfate uptake to sustain ho-meostasis in the plants and follows the demand driven control of Sdistribution and metabolism (Chan et al., 2013).

The current study investigates the role of different S regimes onAs and S uptake related to physiological and molecular changesduring As and S interaction in rice plants. To get insight of thesechanges we have examined As and sulfate transporters, sulfurassimilation, As detoxification and the antioxidant system duringAsV stress.

2. Materials and methods

2.1. Experiment design, hydroponic culture and arsenic exposure

Rice seeds (Oryza sativa L.), cultivar BRG-20, were surface ster-ilized using 10% H2O2 for 30 s, and washed with double distilledwater. Seeds were germinated in moist pre-sterilized blottingsheets on a tray in seed germinator for 4 d at 37 �C and 65% relativehumidity. Uniform germinated seedlings were selected and trans-planted to trays containing fixed PVC cups (4 cm diameter and 5 cmhigh, 10 plants per cup) and grown in modified Hewitt media (Liuet al., 2004) supplemented with low sulfur (LS; 0.5 mM), normalsulfur (NS; 3.5 mM) used as standard Hewitt media, or high sulfur(HS; 5.0 mM) (Srivastava and D'souza, 2009) for 10 d prior to AsVexposure. Arsenic is provided as (Na2HAsO4; 25 mMand 50 mMAsV)while the plants not exposed to AsV served as control and grownfor 7 d in a controlled growth environment at 28/21 �C at lightintensity of 210 m mol cm�2 s�1 (16-h light/8-h dark) with relativehumidity of 70%. All the experiments were conducted with threereplicates for each treatment. After 7 d of treatments plants wereharvested for analysis. Morphological parameters (root length,shoot length, weight and chlorophyll content) were measured.

2.2. Determination of arsenic and sulfur content

Arsenic content was determined by Dwivedi et al., 2010 usinginductively coupled plasma-mass spectrometry (ICP-MS) (7500 cx;Agilent, Tokyo, Japan) For the estimation of As, 0.5 g oven driedtissue was taken and digested in 3 ml of HNO3 at 80 �C for 2 h and100 �C for 4 h. Then filtered in 10 ml of Milli Q water and stored at4 �C till the estimation. These metalloids (As and Se) were quan-tified with the help of Inductively Coupled Plasma Mass Spec-trometer (ICP-MS, Agilent 7500 cx). The standard solution materialof As and Se (Agilent, Part # 8500e6940) was used for the cali-bration and quality assurance for each analytical batch. Recovery ofAs rice flour NIST 1568a was used as a reference material with

known spiked samples and recovery of total As were 95.3% (±2.8;n¼ 5) and 92.5% (±3.1; n¼ 5) respectively. The detection limit of Aswas 1 mg L�1. Total S concentration was estimated as described byChesmin and Yien (1957). A suitable aliquot was taken in 50 ml testtube and volumewas made 2.5 ml with glass distilled water. To thisaliquot, 2.5 ml sodium acetate buffer pH 4.8, 2.0 ml of 50% glyceroland 5.0 ml of 20% barium chloride were added one after theaddition of barium chloride. A reagent blank was also runwith eachset of estimation. The turbidity of the solutionwasmeasuredwithinhalf an hour of reaction using violet filter on spectrophotometer.Concentration of sulfur was estimated by referring the reading to astandard calibration curve prepared from AR grade sodium sulfatewithin the range of 50e250 mg sulfur.

2.3. Determination of transcript levels of arsenic and sulfatetransporters

Approximately, 5 mg RNase free DNase-treated total RNA (5 mg)isolated from roots of rice plants exposed to the various treatmentswas reverse-transcribed using SuperScriptII (Fermentas, USA),following the manufacturers recommendation. The synthesizedcDNA was diluted 1:5 in RNase-free water and subjected to quan-titative RT-PCR (qRT-PCR) analysis. The qRT-PCR was performedusing an ABI 7500 instrument (ABI Biosystems, USA) using genespecific primers (Tables Se1). Each qPCR reaction contained 5 ml ofSYBR Green Supermix (ABI Biosystems, USA), 1 ml of the dilutedcDNA reactionmixture (corresponding to at starting amount of 5 ngof RNA) and 10 pM of each primer in a total reaction volume of 10 mlqPCR reactions were performed under the following conditions:10 min at 95 �C and 40 cycles of the one step thermal cycling of3 s at 95 �C and 30 s at 60 �C in a 96-well reaction plate. The riceactin gene was used as an internal control to estimate the relativetranscript levels of the target gene. Specificity of ampliconsgenerated in qPCR reactions was verified by melting curve analysis.Each qPCR reaction was performed in triplicate (technical repli-cates) for each biological replicate (three for each treatment).Relative gene expression was calculated using DCT method (Livakand Schmittgen, 2001).

2.4. Estimation of total non protein thiol compounds

Total non protein thiols (NPTs) and Cys content were measuredfollowing the method of Ellman's (1959) and Gaitonde (1967). Thelevel of reduced (GSH) and oxidized (GSSG) glutathione contentwere determined as described by Hissin and Hilf (1976) using aHitachi F 7000 fluorescence spectrophotometer (Japan). The con-centration of total PCs was calculated as PCs¼NPTs � total GSH(Duan et al., 2011).

2.5. Activity of enzymes of sulfur assimilation pathway andglutathione metabolism

Assay of cysteine synthase (CS; EC 2.5.1.47) and g-gluta-mylcysteine synthetase (gECS; EC 6.3.2.2) activities performedfollowing Saito et al. (1994) and Seelig and Meister (1984),respectively, with slight modifications (Kumar et al., 2014b). For theassay of 50-adenylylsulfate (APS) reductase (APR; EC1.8.4.9) andserine acetyl transferase (SAT, EC 2.3.1.30) activities, plant materialwas homogenized as described in Hartmann et al. (2000). Thehomogenate after centrifugation was used for APR activityfollowing the method of Peck et al. (1965), while SAT activity wasperformed following the method of Błaszczyk et al. (2002).

For assays of g-glutamyl transpeptidase (g-GT; EC 2.3.2.2), themethod by Orlowski and Meister (1973) was followed. The gluta-thione reductase (GR) activity was assayed by following the

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e9688

method of Smith et al. (1988). Glutathione S-transferase (GST; EC2.5.1.18) activity was assayed as described by Habig and Jakoby(1981).

2.6. Estimation of oxidative stress and activity of antioxidantenzymes and arsenate reductase

The activity of superoxide dismutase (SOD; EC 1.15.1.1) wasassayed following the method of Beauchamp and Fridovich (1971).Concentration of H2O2 was assayed according to Kumar et al.(2014a). Ascorbate oxidase was assayed according to Esaka et al.(1988). Assay of arsenate reductase (AR; EC 1.20.4.1) was per-formed as described by Shi et al. (1999). The activity of ascorbateperoxidase (APX,EC 1.11.1.11) was measured according to themethod of Nakano and Asada (1981) by estimating the rate ofascorbate (ASC) oxidation (є¼ 2.8 mM�1 cm�1). Guaicol peroxidase(GPX, EC 1.11.1.7) activity was assayed according to the method ofHemeda and Klein (1990). Catalase (CAT) activity was measuredfollowing the method of Aebi (1974).

2.7. Statistical analyses

Analysis of variance (ANOVA), Duncan's multiple range test(DMRT) analysis was performed to determine the significant dif-ference between treatments at 95% confidence level by using SPSS17.0 software. The correlation analysis was performed, which hasbeen given within the text at relevant places (*** ¼ P� 0.001,** ¼ P� 0.01, * ¼ P� 0.1, ns ¼ non-significant).

3. Results

3.1. Rice plant morphology and biomass

Arsenate hindered the plant growth by hampering plant lengthand weight. Application of different sulfur treatments had variedimpact on plant growth against AsV toxicity. Root length enhancedwith LS treatment while reduced with HS treatment than control inabsence of As toxicity. Root length was decreased in HS treatedplants while shoot length was decreased in LS than respective NStreatment with or without AsV exposed plants. AsV stress showedthe dose dependent decrease in root and shoot length. Contradic-tory results were observed in roots where root weight wasdecreased despite of increase in root length in LS treated plantsirrespective of AsV presence (Fig. S1A, B).

3.2. Arsenic and sulfur accumulation

Arsenic accumulation was enhanced in root and shoot in dosedependent manner. HS has enhanced the As accumulation in rootwhile decreased in shoot than NS at both the As exposures(Fig. 1AeD). In terms of As distribution in root and shoot, during NStreatment ca. 80% As was confined to root and ca. 20% translocatedto shoot while in HS, ca. 90% As was confined to root and 10% wastranslocated to shoot. Thus sulfur supplementation has changedthe spatial distribution of As. A parallel changewas also observed inTF of As. During NS treatment, TF was ca. 0.2 while in LS treatment,TF was enhanced to ca. 0.5 and in HS, TF was decreased to ca. 0.1.Thus Translocation factor (root/shoot) provided clearer picture ofAs accumulation. HS has reduced the As translocation factor approxhalf than NS, that indicates that in HS treated plants most of the Asremained confined to the root and lesser is translocated to theshoot than NS, while in LS plants translocation factor was enhancedthan NS that indicates more As is translocated to the shoot than NS.

Arsenic exposure enhanced the S accumulation in both root andshoot in dose dependent manner than the plants not exposed to As.

Sulfur accumulation in root and shoot was allied to S treatment inthe nutrient solution. Sulfur accumulation in root was positivelycorrelated (R ¼ 0.999 at 25 mM AsV; R ¼ 0.857 at 50 mM AsV) withAs accumulation while in shoot there was negative correlation(R¼�0.971 at 25 mMAsV; R¼�0.933 at 50 mMAsV). In presence ofAs, there was negative correlation (R ¼ �0.993 at 25 mM AsV;R ¼ �0.988 at 50 mM AsV) between As and S translocation factors.

3.3. Thiolic compounds and enzymes of sulfur assimilation pathwayand glutathione metabolism

The components of thiol metabolism were determined in thehydroponically grown rice exposed to As under the various S con-ditions (Fig. 2AeE). It was observed that concentration of NPTs wassignificantly enhanced by both the As concentrations and HS sup-plementation in both root and shoot (Fig. 2A). Cys and PCs also havesimilar pattern in both root and shoot. Plant root exposed to theHS þ As treatment has higher concentrations of Cys (40% at 25 and63% at 50 mM As respectively) than the NS þ As treatment (Fig. 2B).As treatment highly increased the PC concentration than As un-treated plants (Fig. 2E). The concentration of PCs was lowest in theLS treated plants and highest in the HS treated plants in all treat-ments. The level of GSH was lower at LS treatment both in root(36%) and shoot (37%) than NS and without As treatment. Theconcentration of GSH was greater (109% and 61% in root and shootrespectively) in the HS þ As treatment at higher dose of Ascompared to NSþAs treated plants (Fig. 2C). In contrast, the level ofGSSG was in the order LS > NS > HS both in As treated and un-treated plants, while in terms of As stress, level of GSSG enhancedthan As untreated plants (Fig. 2D). Ratio of GSH to GSSG is reducedwith As concentration in dose dependent manner. HS has restoredthis ratio both in root and shoot (Fig. 2F).

To get a deeper insight in to thiol metabolic response under Asstress, enzymes involved in synthesis and consumption of thiolswere also evaluated. In the sulfate assimilation pathway conversionof APS to sulfite is mediated by APR. APR activity maximumenhanced in HS treated (290% and 102% in root and shoot respec-tively) plants as compared to NS without As treatments (Fig. 3A).Activity of CS was significantly increased in the HS treatments andshowed strong positive correlation with Cys content in both root(R ¼ 0.909) and shoot (R ¼ 0.977) (Fig. 3B). Activities of SAT and g-ECS were also increased with increased S supply with highest ac-tivities in the HS treatments and were also positively correlatedwith As treatments (Fig. 3C and D). g-GT and GST activities bothdecreased with increasing S treatments (Fig. 3E and F). The activityof GR was increased with increasing S treatments (Fig. 3G).

3.4. Oxidative stress and antioxidant enzymes

SOD activity enhanced in root with S and As concentration indose dependent manner. However, in shoot, though the activity ofSODwas enhanced upon As exposure in dose dependent manner, itdecreased with increasing S treatments at As treatments (Fig. 4A).The H2O2 accumulationwas decreased with increasing S treatment,and increased under As treatments both in root and shoot (Fig. 4B).APX, GPX and CAT are all enzymes for defence against oxidativestress and degrade H2O2 to water and oxygen. All these enzymesshowed similar trend with both S and As treatments. APX showedlower activity in root than shoot in all the treatments. LS treatmentenhanced APX activity than NS both in root and shoot irrespectiveof As stress. GPX activity enhanced with increasing S treatments inroot, while in shoot opposite trendwas observed in non As exposedplants. CAT activity was enhanced with As concentration in LStreated plants, NS and HS the activity was decreased than in theroot of respective controls. CAT activity was higher in shoot than

Fig. 1. Effect of As and different S doses on the treatment of As (A) and S (B) accumulation, and root to shoot translocation factor of As (C) and S (D) in Oryza sativa. All the values aremeans of triplicate± S.D. ANOVA significant at p� 0.01. Different letters indicate significantly different values at a particular treatment (DMRT, p� 0.05).

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e96 89

root. The activity of AR decreased upon increased S supply in all thetreatments while, increased by As exposure in both root and inshoot. Ascorbate oxidase (AAO) activity increased with increasing Streatment and As treatment in root (Fig. 4CeG).

3.5. Expression of sulfate and arsenic transporters in rice roots

The effect of different treatments of S and As on sulfate trans-porters was determined by the expression analysis of high affinitysulfate transporters (HASulTs: OsSultr1;1, OsSultr1;2 and OsSultr1;3),low affinity sulfate transporters (LASulTs; OsSultr2;2) and vacuolartransporters (OsSultr4;1). In general all the transporters were upregulated upon exposure to As in comparison to their respectivecontrols except for OsSultr1;3which did not significantly alter at NSand HS. With respect to S treatments, the sulfate transporters wereupregulated at LS and were down regulated at HS in comparison toNS (Fig. 5AeE).

The expression of OsNIP1;1 was down regulated by As treat-ment. The HS treatment down regulated the expression ofOsNIP1;1, while the LS treatment enhanced expression of OsNIP1;1compared to the NS treatment (Fig. 6A). A similar pattern wasobserved with OsNIP3;1 (Fig. 6B). The expression of Lsi1 wasincreased upon As exposure while the expression of OsLsi2 wasdecreased. Expression of both OsLsi1 and OsLsi2was enhanced in LStreatment, but were reduced by HS treatment than NS irrespectiveof As (Fig. 6C and D). Arsenic exposure significantly reduced theexpression level of OsLsi6 than respective controls. LS treatment hasenhanced while HS has reduced the expression of OsLsi6 than NS

irrespective of As treatment (Fig. 6E).

4. Discussion

Sulfur is known to interact with As in various environmentalconditions, from biogeochemical level to inside biological entities(Dixit et al., 2015). Arsenic stress hampered rice plant growth indose dependent manner and sulfur restored the growth. Enhancedsupply of sulfur (HS) in nutrient solution enhanced growth ofplants exposed to AsV. Low sulfur supply has reduced, while highsulfur enhanced the plant biomass in comparison to NS, irre-spective of AsV exposure. Similar trendwas observed in chlorophyllcontent (Fig. S1C). High sulfur treated plants also experienced lessAsV toxicity. Thus frommorphological studies it is evident that highsulfur supply is helpful in mitigation of AsV induced toxicity in rice.Low sulfur supply reduced the plant biomass, probably due to thereduced level of photosynthetic pigments and low availability of S.The enhanced plant growth under HS could directly be related tolow As accumulation and eventually enhanced photosyntheticpigments in comparison to NS and LS (Fig. S1C). Sulfur nutrition hasbeen shown to directly affect the photosynthetic pigments. Muneeret al. (2013) showed reduction in photosynthetic pigment at low Scondition in Brassica napus. Thus, improving S nutrition mightimprove plant's tolerance for As either by lowering free As or byimproving the level of photosynthetic pigments. Sulfur deprivationenhances plant root length probably to enhance root surface areafor nutrient availability. Zhao et al. (2014) reported that low sulfuravailability promotes the primary root elongation in Arabidopsis.

Fig. 2. 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), (E) GSH/GSSG ratioand (F) phytochelatins (PCs) in Oryza sativa. All the values are means of triplicate ± S.D. ANOVA significant at p� 0.01. Different letters indicate significantly different values at aparticular treatment (DMRT, p� 0.05).

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e9690

Kutz et al., 2002 reported that in the absence of an external sulfatesupply to Arabidopsis, plants developed longer roots with a highernumber of lateral roots. They explained that the increased growthof the root system occurred at the expense of shoot growth whichwas retarded under conditions of sulfur starvation. It is suggestedthat a regulatory loop appears to exist by which sulfate deficiency,through an increase in glucobrassicin turnover and nitrilase 3accumulation, initiates the production of extra auxin leading toncreased root growth and branching, thus allowing the root systemto penetrate new areas of soil effectively to gain access to freshsupplies of sulfur.

Total As accumulation (root þ shoot) was enhanced with the HStreatment, while contrasting behavior was observed with the Asdistribution. Most As was found to restricted in root and lesser

amount of As was translocated in to shoot and probably in to ricegrains. This may occur due to As compartmentalization. In thepresent study it has been observed that LS enhanced expressionlevel of arsenic transporters while AsV treatment has reduced theexpression levels of As transportes. Previously it has been shown byMa et al., 2008 that expression of OsNIP1;1 and OsNIP3;1 were notinduced by As treatment, but from oocyte study, it was proved thatOsNIP3;1 expression caused very less uptakes of As than OsLsi1 orOsNIP1;1 (Ma et al., 2008). Thus OsNIP3;1 does not play a signifi-cant role in As uptake. OsLsi1 serves both as efflux and influxtransporter for AsIII in rice (Ali et al., 2009); even if the plant issupplied with AsV exogenously, OsLsi1 effluxes AsIII into thenutrient medium (Zhao et al., 2010a). OsLsi1 is localized at thedistal site of rice root and transports AsIII into rice root cells, while

Fig. 3. Effect of As on the activity of 50-adenylylsulfate reductase (APR) (A), cysteine synthase (CS) (B), serine acetyl transferase (SAT) (C), g-glutamylcysteine synthetase (g-ECS) (D),g-glutathione transpeptidase (g-GT) (E), glutathione-S-transferase (GST) (F), and glutathione reductase (GR) (G) in Oryza sativa under various sulfur regimes. All the values aremeans of replicate± S.D. ANOVA significant at p� 0.01. Different letters indicate significantly different values at a particular treatment (DMRT, p� 0.05).

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e96 91

OsLsi2 is responsible for AsIII efflux into the xylem and is localizedat the proximal side of both endodermis and exodermis of rice roots(Ma et al., 2008). In present study low accumulation of As in LS andAs treated plant's roots occurs potentially because uncomplexedAsIII was released to the external medium. According to Zhao et al.(2010a), OsLsi1 is responsible for AsIII efflux in to external medium.Remaining uncomplexed AsIII may be unloaded to the xylem viaOsLsi2 (Ma et al., 2008). The present study indicates that As accu-mulation in the shoot is directly proportional to OsLsi2 transcript

level, that is close agreement with previous findings that OsLsi2was responsible for xylem unloading of As in rice plants (Ma et al.,2008).

Inside the plant exogenously supplied sulfur gets reduced to Cys(Anderson, 2014) that plays a crucial role in GSH and PCs synthesis.Inside the plant, AsV gets reduced to AsIII that forms PC-AsIIIcomplex. PC-AsIII complex is transported to vacuole that reducesthe free As concentration in cytoplasm (Meadows, 2014). SimilarlyGSH also binds with AsIII (Spuches et al., 2005). In present study,

Fig. 4. Effect of As on the level of superoxide dismutase (SOD) (A), hydrogen peroxide (H2O2) (B), ascorbate peroxidase (APX) (C), guiacol peroxidase (GPX) (D), catalase (CAT) (E),arsenate reductase (AR) (F), and ascorbate oxidase (AAO) (G) in Oryza sativa under various sulfur regimes. All the values are means of replicate± S.D. ANOVA significant at p� 0.01.Different letters indicate significantly different values at a particular treatment (DMRT, p� 0.05).

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e9692

Cys and PCs level was enhancedwith the enhanced supply of sulfur.Therefore, proper S fertilization can promote the compartmentali-zation of As in roots, resultant in lesser load to the above groundpart of the plant (Duan et al., 2013). During the current study ac-tivity of AR was also enhanced with HS and AsV treatments, thismay reduce AsV to AsIII, facilitating more AsIII complexation. Sulfuris incorporated into organic molecules in plants and is located inthiol (eSH) groups in proteins (Cys-residues) or NPTs. Enhance-ment of NPTs under HS condition and As stress showing its role inAs detoxification. Non protein thiols mainly constitute glutathioneand PCs. Sulfur assimilation regulates biosynthesis of the NPTs. Cys

is the final product of assimilatory sulfate reduction, enhancementof Cys during HS condition and As stress stimulate may synthesis ofGSH through the increase in activities of key enzymes responsiblefor S-assimilation (Dhankher et al., 2002). Enhanced level of g-ECSplays a central role in As detoxification in Arabidopsis thaliana(Dhankher et al., 2002). Arsenic exposure induced the activity of g-ECS in dose dependent manner as previously reported in A. thaliana(Sarry et al., 2006).

High S treatment enhanced the activity of enzymes involved in Sassimilatory pathway and thiol metabolism, thus contributing inenhanced GSH production. Glutathione is well known for the

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

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e96 93

removal of excess ROS (Rausch et al., 2007), and protects plantsfrom oxidative damage. The homeostasis of GSH and GSSG main-tains signaling of stress responsive proteins and regulates oxidativestress. Since GSH enhanced under HS and As stress leading toprotection against oxidative stress in rice plants, thus HS plays animportant role in As detoxification in rice. GSH may be involved inAs binding, so that its degrading enzymes (gamma-GT and GST) donot sense GSH as its enhanced substrate. Thus despite the enhancedGSH activities of gamma-GT and GST decreased.

Present study showed that As treatment resulted in oxidativestress in terms of enhanced H2O2. Antioxidant enzymes such asSOD, CAT, APX and GPX were also enhanced in both root and shoot,to counteract the oxidative stress caused by As. Sulfur supple-mentation with As caused a substantive reduction in the level ofH2O2, which indicates the ameliorating effect of S on As inducedoxidative stress. SOD, APX and GPX are the major antioxidant en-zymes associated with scavenging ROS and SOD is likely to becentral in the defense (Singh et al., 2015; Dave et al., 2013; Kumaret al., 2014a). Enhancement of H2O2 in the LS with As treatmentappears to be a signal of high stress (Fig. 4B). Higher activity of SODmay be the cause of this H2O2 production during the LS treatment

or As stress; S supplementation seems to ameliorate this affect byreducing H2O2 levels. Further, HS supplementation to As, reducedthe activity of antioxidant enzymes than in LS or S treated plants. Itagain confirms the protective role of S against As induced oxidativestress. Increased AAO activity with S supplementation and Astreatment also indicates its stress protective role by synthesizingascorbate.

The role of sulfate transporters has been implicated in As stressresponse in rice (Norton et al., 2008). Sulfur availability also regu-lates sulfate transporters (Takahashi et al., 2011). SULTR1;1, a high-affinity sulfate transporter, is highly regulated by S deficiency in theepidermis and cortex of Arabidopsis roots (Yoshimoto et al., 2002). Itis suggested that to meet the optimum need of S, plants expressmore HASulTs to internalize S from outside media. Sulfate trans-porters were up regulated by AsV treatment. Arsenic mediated upregulation of sulfur transporters was previously reported by Duanet al. (Duan et al., 2013). Arsenate exposure enhanced the HASulTsunder normal and high sulfur condition. This indicates enhancedrequirement of S in AsV exposed plants to synthesize more Cys,GSH and PCs for chelation of As after reduction to AsIII (Mishraet al., 2013). The expression of HASulTs increased in the LS with

Fig. 6. Relative expression of As transporters in Oryza sativa during As stress under various S regimes (A) OsNIP1;1, (B) OsNIP3;1, (C) OsLsi1, (D) OsLsi2, and (E) OsLsi6. All the valuesare means of triplicate± S.D. ANOVA significant at p� 0.01. Different letters indicate significantly different values at a particular treatment (DMRT, p� 0.05).

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e9694

As treatment to meet high S demand inside the plant by sensinglow S conditions. Similarly, S starvation induced high affinity group1 sulfate transporters in Arabidopsis (Yoshimoto et al., 2002).OsSult2;2 is a LASulTs, which plays a role in the transport of sulfatevia root phloem (Davidian and Kopriva, 2010). OsSult2;2 expressionwas up regulated under low sulfur conditions while down regu-lated under high sulfur. It indicates that group 2 sulfate transportersplay important role in S acquisition during S deficient conditions(Maruyama-Nakashita et al., 2015). OsSult4;1 is a vacuolar effluxtransporter (transporting S into the cytosol) of sulfur. High sulfurcondition reduced the expression of OsSult4;1, which indicatespresence of sufficient sulfur in cytosol to support increased sulfurrequirement under As stress, thus, restoring vacuolar S (Kataokaet al., 2004).

5. Conclusion

Present study clearly showed that high S condition resulted inlow As accumulation in rice shoot, and improved plant growth

through antioxidant defense system and enhance thiol metabolism.The lower translocation of As to shoot is not only dependent onthiol based complexation in root but also to alter As transportthrough down regulation of transporters. Localization of As in rootsunder high S condition provides a tool to produce rice with lowgrain As, through improved S nutrition either through genetic en-gineering methods or agronomic practices.

Contribution

RDT, PKT, VP and SM designed experiments and reviewedmanuscript. APS, GD performed experimental work and preparedfigures. AK and SD helped in elemental analysis, and SK performedqRT-PCR analysis. All authors have read and approved themanuscript.

Acknowledgment

The authors are thankful to Director, CSIR-National Botanical

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e96 95

Research Institute (CSIR-NBRI), Lucknow for the facilities and forthe financial support from the network projects (CSIR-INDEPTH,NWP-0111), New Delhi, India. The authors are grateful to the JointDirector, Rice Research Station (RRS), Chinsurah to provide ricegermplasm. GD is thankful to Council of Scientific and IndustrialResearch, New Delhi, India for the award of Junior/Senior ResearchFellowship and Academy of Scientific and Innovative Research(AcSIR) for her Ph.D. registration. Amit Kumar is thankful toDSKPDF Cell, Pune, India, and University Grant Commission, NewDelhi, India, for the award of the D.S. Kothari PostdoctoralFellowship.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2015.11.005.

References

Aebi, H., 1974. Catalases. Methods Enzym. Anal. 2, 673e684.Ali, W., Isayenkov, S.V., Zhao, F.J., Maathuis, F.J., 2009. Arsenite transport in plants.

Cell Mol. Life Sci. 66, 2329e2339.Anderson, J.W., 2014. Assimilation of inorganic sulfate into cysteine. Biochem.

Plants 5, 203e223.Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an

assay applicable to acrylamide gels. Anal. Biochem. 44, 276e287.Błaszczyk, A., Sirko, L., Hawkesford, M.J., Sirko, A., 2002. Biochemical analysis of

transgenic tobacco lines producing bacterial serine acetyltransferase. Plant Sci.162 (4), 589e597.

Capaldi, F.R., Grat~ao, P.L., Reis, A.R., Lima, L.W., Azevedo, R.A., 2015. Sulfur meta-bolism and stress defense responses in plants. Trop. Plant Biol. 1e14.

Chan, K.X., Wirtz, M., Phua, S.Y., Estavillo, G.M., Pogson, B.J., 2013. Balancing me-tabolites in drought: the sulfur assimilation conundrum. Trends Plant Sci. 18 (1),18e29.

Chesmin, L., Yien, C.H., 1957. Turbidimetric determination of available sulphate. In:Soil Sci. Soc. Am. Proc. 15, vol. 14.

Dave, R., Tripathi, R.D., Dwivedi, S., Tripathi, P., Dixit, G., Sharma, Y.K.,Chakrabarty, D., 2013. Arsenate and arsenite exposure modulate antioxidantsand amino acids in contrasting arsenic accumulating rice (Oryza sativa L.) ge-notypes. J. Hazard Mater. 262, 1123e1131.

Davidian, J.C., Kopriva, S., 2010. Regulation of sulfate uptake and assimilationdthesame or not the same? Mol. Plant ssq001.

Dhankher, O.P., Li, Y., Rosen, B.P., Shi, J., Salt, D., Senecoff, J.F., Meagher, R.B., 2002.Engineering tolerance and hyperaccumulation of arsenic in plants by combiningarsenate reductase and g-glutamylcysteine synthetase expression. Nat. Bio-technol. 20 (11), 1140e1145.

Dixit, G., Singh, A.P., Kumar, A., Singh, P.K., Kumar, S., Dwivedi, S., Tripathi, R.D.,2015. Sulfur mediated reduction of arsenic toxicity involves efficient thiolmetabolism and the antioxidant defense system in rice. J. Hazard Mater. 298,241e251.

Duan, G., Liu, W., Chen, X., Hu, Y., Zhu, Y., 2013. Association of arsenic with nutrientelements in rice plants. Metallomics 5, 784e792.

Duan, G.L., Hu, Y., Liu, W.J., Kneer, R., Zhao, F.J., Zhu, Y.G., 2011. Evidence for a role ofphytochelatins in regulating arsenic accumulation in rice grain. Environ. Exp.Bot. 71, 416e421.

Dwivedi, S., Tripathi, R.D., Srivastava, S., Singh, R., Kumar, A., Tripathi, P., Bag, M.K.,2010. Arsenic affects mineral nutrients in grains of various Indian rice (Oryzasativa L.) genotypes grown on arsenic-contaminated soils of West Bengal.Protoplasma 245, 113e124.

Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70e77.Esaka, M., Imagi, J., Suzuki, K., Kubota, K., 1988. Formation of ascorbate oxidase in

cultured pumpkin cells. Plant Cell Physiol. 29, 231e235.Gaitonde, M.K., 1967. A spectrophotometric method for the direct determination of

cysteine in the presence of other naturally occurring amino acids. Biochem. J.104, 627e633.

Habig, W.H., Jakoby, W.B., 1981. Assays for differentiation of glutathione S-Trans-ferases. Methods Enzymol. 77, 398e405.

Hartmann, T., Mult, S., Suter, M., Rennenberg, H., Herschbach, C., 2000. Leaf age-dependent differences in sulphur assimilation and allocation in poplar (Pop-ulus tremula� P. alba) leaves. J. Exp. Bot. 51, 1077e1088.

Hawkesford, M.J., 2003. Transporter gene families in plants: the sulphate trans-porter gene familydredundancy or specialization? Physiol. Planta 117, 155e163.

Hemeda, H.M., Klein, B.P., 1990. Effects of naturally occurring antioxidants onperoxidase activity of vegetable extracts. J. Food Sci. 55, 184e185.

Hissin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidized andreduced glutathione in tissues. Anal. Biochem. 74, 214e226.

IRRI, 1993. IRRI Rice Almanac 1993e95. International Rice Research Institute, LosBanos, The Philippines, 142pp.

Kataoka, T., Watanabe-Takahashi, A., Hayashi, N., Ohnishi, M., Mimura, T.,

Buchner, P., Takahashi, H., 2004. Vacuolar sulfate transporters are essentialdeterminants controlling internal distribution of sulfate in Arabidopsis. PlantCell 16, 2693e2704.

Kumar, A., Singh, R.P., Singh, P.K., Awasthi, S., Chakrabarty, D., Trivedi, P.K.,Tripathi, R.D., 2014a. Selenium ameliorates arsenic induced oxidative stressthrough modulation of antioxidant enzymes and thiols in rice (Oryza sativa L.).Ecotoxicol 23, 1153e1163.

Kumar, A., Dwivedi, S., Singh, R.P., Chakrabarty, D., Mallick, S., Trivedi, P.K.,Tripathi, R.D., 2014b. Evaluation of amino acid profile in contrasting arsenicaccumulating rice genotypes under arsenic stress. Biol. Plant. 58, 733e742.

Kutz, A., Müller, A., Hennig, P., Kaiser, W.M., Piotrowski, M., Weiler, E.W., 2002.A role for nitrilase 3 in the regulation of root morphology in sulphur-starvingArabidopsis thaliana. Plant J. 30, 95e106.

Labrou, N.E., Papageorgiou, A.C., Pavli, O., Flemetakis, E., 2015. Plant GSTome:structure and functional role in xenome network and plant stress response.Curr. Opin. Biotech. 32, 186e194.

Liu, W.J., Zhu, Y.G., Smith, F.A., Smith, S.E., 2004. Do phosphorus nutrition and ironplaque alter arsenate (As) uptake by rice seedlings in hydroponic culture? NewPhytol. 162, 481e488.

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2�DDCT method. Methods 25, 402e408.

Ma, J.F., Yamaji, N., Mitani, N., Xu, X.Y., Su, Y.H., McGrath, S.P., Zhao, F.J., 2008.Transporters of arsenite in rice and their role in arsenic accumulation in ricegrain. Proc. Natl. Acad. Sci. U. S. A. 105, 9931e9935.

Maruyama-Nakashita, A., Watanabe-Takahashi, A., Inoue, E., Yamaya, T., Saito, K.,Takahashi, H., 2015. Sulfur-responsive elements in the 30-nontranscribedintergenic region are essential for the induction of sulfate transporter 2; 1 geneexpression in arabidopsis roots under sulfur deficiency. Plant Cell 27,1279e1296.

McCarty, K.M., Hanh, H.T., Kim, K.W., 2011. Arsenic geochemistry and human healthin South East Asia. Rev. Environ. Health 26, 71e78.

Meadows, R., 2014. How plants control arsenic accumulation. PLoS Biol. 12,e1002008.

Mishra, S., Wellenreuther, G., Mattusch, J., St€ark, H.J., Küpper, H., 2013. Speciationand distribution of arsenic in the nonhyperaccumulator macrophyte Cerato-phyllum demersum. Plant Physiol. 163, 1396e1408.

Muneer, S., Lee, B.R., Bae, D.W., Kim, T.H., 2013. Changes in expression of proteinsinvolved in alleviation of Fe-deficiency by sulfur nutrition in Brassica napus L.Acta Physiol. Plant. 35, 3037e3045.

Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specificperoxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867e880.

Norton, G.J., Lou-Hing, D.E., Meharg, A.A., Price, A.H., 2008. Riceearsenate in-teractions in hydroponics: whole genome transcriptional analysis. J. Exp. Bot.59, 2267e2276.

Orlowski, M., Meister, A., 1973. g-Glutamyl cyclotransferase distribution, isozymicforms, and specificity. J. Biol. Chem. 248, 2836e2844.

Peck, H.D., Deacon, T.E., Davidson, J.T., 1965. Studies on adenosine 50-phosphosulfatereductase from desulfovibrio desulfuricans and thiobacillus thioparus I. Theassay and purification. Biochim. Biophys. Acta Nucleic Acids Protein Synthesis96, 429e446.

Rausch, T., Gromes, R., Liedschulte, V., Müller, I., Bogs, J., Galovic, V., Wachter, A.,2007. Novel insight into the regulation of GSH biosynthesis in higher plants.Plant Biol. 9, 565e572.

Saito, K., Kurosawa, M., Tatsuguchi, K., Takagi, Y., Murakoshi, I., 1994. Modulation ofcysteine biosynthesis in chloroplasts of transgenic tobacco overexpressingcysteine synthase (O-acetylserine (thiol)-Iyase). Plant Physiol. 106, 887e895.

Saito, K., 2000. Regulation of sulfate transport and synthesis of sulfur-containingamino acids. Curr. Opin. Plant Biol. 3, 188e195.

Sarry, J.E., Kuhn, L., Ducruix, C., Lafaye, A., Junot, C., Hugouvieux, V., Bourguignon, J.,2006. The early responses of Arabidopsis thaliana cells to cadmium exposureexplored by protein and metabolite profiling analyses. Proteomics 6,2180e2198.

Seelig, G.F., Meister, A., 1984. Gamma-glutamylcysteine synthetase. Interactions ofan essential sulfhydryl group. J. Biol. Chem. 259, 3534e3538.

Shi, J., Vlamis-Gardikas, A., Åslund, F., Holmgren, A., Rosen, B.P., 1999. Reactivity ofglutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is theprimary hydrogen donor to ArsC-catalyzed arsenate reduction. J. Biol. Chem.274, 36039e36042.

Singh, A.P., Dixit, G., Mishra, S., Dwivedi, S., Tiwari, M., Mallick, S., Tripathi, R.D.,2015. Salicylic acid modulates arsenic toxicity by reducing its root to shoottranslocation in rice (Oryza sativa L.). Front. Plant Sci. 6, 340.

Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase incrude tissue homogenates using 5, 50-dithiobis (2-nitrobenzoic acid). Anal.Bioch. 175, 408e413.

Scheller, H.V., Huang, B., Hatch, E., Goldsbrough, P.B., 1987. Phytochelatin synthesisand glutathione levels in response to heavy metals in tomato cells. PlantPhysiol. 85, 1031e1035.

Srivastava, S., D'souza, S.F., 2009. Increasing sulfur supply enhances tolerance toarsenic and its accumulation in Hydrilla verticillata (Lf) Royle. Environ. Sci.Technol. 43, 6308e6313.

Srivastava, S., Mishra, S., Tripathi, R.D., Dwivedi, S., Trivedi, P.K., Tandon, P.K., 2007.Phytochelatins and antioxidant systems respond differentially during arseniteand arsenate stress in Hydrilla verticillata (Lf) Royle. Environ. Sci. Technol. 41,2930e2936.

Srivastava, S., Suprasanna, P., D'Souza, S.F., 2011. Redox state and energetic

G. Dixit et al. / Plant Physiology and Biochemistry 99 (2016) 86e9696

equilibrium determine the magnitude of stress in Hydrilla verticillata uponexposure to arsenate. Protoplasma 248, 805e815.

Spuches, A.M., Kruszyna, H.G., Rich, A.M., Wilcox, D.E., 2005. Thermodynamics ofthe As (III)-thiol interaction: arsenite and monomethylarsenite complexes withglutathione, dihydrolipoic acid, and other thiol ligands. Inorg. Chem. 44 (8),2964e2972.

Takahashi, H., Kopriva, S., Giordano, M., Saito, K., Hell, R., 2011. Sulfur assimilation inphotosynthetic organisms: molecular functions and regulations of transportersand assimilatory enzymes. Annu. Rev. Plant Biol. 62, 157e184.

Williams, P.N., Villada, A., Deacon, C., Raab, A., Figuerola, J., Green, A.J., Meharg, A.A.,2007. Greatly enhanced arsenic shoot assimilation in rice leads to elevated grainlevels compared to wheat and barley. Environ. Sci. Technol. 41, 6854e6859.

Yoshimoto, N., Takahashi, H., Smith, F.W., Yamaya, T., Saito, K., 2002. Two distincthigh-affinity sulfate transporters with different inducibilities mediate uptake ofsulfate in Arabidopsis roots. Plant J. 29, 465e473.

Zhao, F.J., Ago, Y., Mitani, N., Li, R.Y., Su, Y.H., Yamaji, N., Ma, J.F., 2010a. The role ofthe rice aquaporin Lsi1 in arsenite efflux from roots. New Phytol. 186, 392e399.

Zhao, F.J., McGrath, S.P., Meharg, A.A., 2010b. Arsenic as a food chain contaminant:mechanisms of plant uptake and metabolism and mitigation strategies. Annu.Rev. Plant Biol. 61, 535e559.

Zhao, Q., Wu, Y., Gao, L., Ma, J., Li, C.Y., Xiang, C.B., 2014. Sulfur nutrient availabilityregulates root elongation by affecting root indole-3-acetic acid levels and thestem cell niche. J. Integr. Plant Biol. 56, 1151e1163.