Arsenic tolerances in rice ( Oryza sativa) have a predominant role in transcriptional regulation of...

Chemosphere 82 (2011) 986–995

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Chemosphere

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Arsenic tolerances in rice (Oryza sativa) have a predominant role intranscriptional regulation of a set of genes including sulphur assimilationpathway and antioxidant system

Arti Rai a,1, Preeti Tripathi a,1, Sanjay Dwivedi a, Sonali Dubey a, Manju Shri a, Smita Kumar a,Pankaj Kumar Tripathi a, Richa Dave a, Amit Kumar a, Ragini Singh a, Bijan Adhikari b, Manas Bag b,Rudra Deo Tripathi a,⇑, Prabodh K. Trivedi a,⇑, Debasis Chakrabarty a,⇑,2, Rakesh Tuli a,c

a National Botanical Research Institute (CSIR), Lucknow 226 001, UP, Indiab Rice Research Station, Chinsurah, West Bengal, Indiac National Agri-Food Biotechnology Institute, C-127, Industrial Area, SAS Nagar, Phase 8, Mohali 160 071, Punjab, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 July 2010Received in revised form 8 October 2010Accepted 21 October 2010Available online 13 November 2010

Keywords:ArsenicAntioxidant enzymesHeat shock proteinRiceSulphate transporterMetallothionine

0045-6535/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2010.10.070

⇑ Corresponding authors.E-mail addresses: [email protected]

hotmail.com (P.K. Trivedi), [email protected] Contributed equally.2 Tel.:+91 9451245572.

World wide arsenic (As) contamination of rice has raised much concern as it is the staple crop for mil-lions. Four most commonly cultivated rice cultivars, Triguna, IR-36, PNR-519 and IET-4786, of the WestBengal region were taken for a hydroponic study to examine the effect of arsenate (AsV) and arsenite(AsIII) on growth response, expression of genes and antioxidants vis-à-vis As accumulation. The rice geno-types responded differentially under AsV and AsIII stress in terms of gene expression and antioxidantdefences. Some of the transporters were up-regulated in all rice cultivars at lower doses of As species,except IET-4786. Phytochelatin synthase, GST and c-ECS showed considerable variation in their expres-sion pattern in all genotypes, however in IET-4786 they were generally down-regulated in higher AsIII

stress. Similarly, most of antioxidants such as superoxide dismutase (SOD), ascorbate peroxidase(APX), guaiacol peroxidase (GPX), catalase (CAT), monodehydroascorbate reductase (MDHAR) and dehy-droascorbate reductase (DHAR) increased significantly in Triguna, IR-36 and PNR-519 and decreased inIET-4786. Our study suggests that Triguna, IR-36 and PNR-519 are tolerant rice cultivars accumulatinghigher arsenic; however IET-4786 is susceptible to As-stress and accumulates less arsenic than othercultivars.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Arsenic (As) contamination of groundwater is a problem in manycountries and has received increased attention because of itschronic and epidemic effects on human health (Abernathy et al.,1999). In coastal belts of Bangladesh, India and China (Rosen andLiu, 2009) arsenic in drinking water exceeds the safe limit of10 lg L�1 described by WHO (WHO, 2001) and even 50 lg L�1 spec-ified in India and Bangladesh (Tuli et al., 2010). Widespread use ofarsenic contaminated groundwater for irrigation in rice field ele-vates its concentration in surface soil and eventually into rice plantsand grains which is one of the major staple food around the world.Field trials conducted for screening of rice germplasm with respectto grain arsenic accumulation showed major differences among

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(R.D. Tripathi), prabodht@in (D. Chakrabarty).

accessions which suggest that arsenic accumulation in rice grainmay also be dependent on genetic differences of different cultivars(Norton et al., 2009; Tuli et al., 2010). Four rice cultivars that arecommonly cultivated in West Bengal in India were selected for thisstudy which showed Specific Arsenic Uptake (SAU) in the order Tri-guna (134) > IR-36 (71.5) > PNR-519 (53) > IET-4786 (29), however,the grain As order was IR-36 (1.5) > Triguna (1) > PNR-519(0.5) > IET-4786 (0.3) in a simulated pot experiment (Dwivediet al., in press).

Although there have been several studies related to arsenic inrice, mechanisms involved in arsenic uptake, efflux from roots,loading into xylem, transport, partitioning, arsenate reduction, ar-senic sequestration in vacuoles, volatilization from leaves, accu-mulation in grains etc. are still poorly understood (Tuli et al.,2010). Our previous studies on the protective effect of reduced glu-tathione (GSH) and cysteine (Cys) to As(III)/As(V) stressed seed-lings showed that both GSH and Cys imparted enhancedtolerance to seedlings against arsenic stress. Germination percent-age and seedling growth improved while the level of malondialde-hyde (MDA) declined significantly when GSH and Cys were

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A. Rai et al. / Chemosphere 82 (2011) 986–995 987

supplemented to As(III)/As(V) treatments suggesting GSH and Cys-mediated protection against oxidative stress (Shri et al., 2009). Theeffect of arsenic (As) exposure on genome-wide expression exam-ined in rice (Chakrabarty et al., 2009) suggested that a group of de-fence and stress-responsive transporters, heat-shock proteins,metallothioneins, sulfate-metabolizing proteins, and regulatorygenes are differentially expressed in rice seedlings challenged witharsenate (AsV) and arsenite (AsIII). Interestingly, one of the mem-bers of the sulfate transporter gene family (Os03g09970) was ob-served to be up-regulated more than 10-fold in both As(V) andAs(III) stresses suggesting its role in arsenic accumulation in rice.The members of this gene family are known to differ in their intra-cellular locations, expression patterns, and kinetic properties andhelp in transport of sulfate (Nocito et al., 2006) leading to en-hanced production of S-rich metal-binding peptides (such asGSH, Cys and phytochelatins), thus providing metal tolerance andresulting in metal accumulation (Mishra et al., 2009). Apart fromone Nramp1, two glutathione-related transporters, two MATE-ef-flux family proteins, and one zinc/iron-transport family proteinwere also shown to be up-regulated specifically in AsV stress in rice(Chakrabarty et al., 2009). The study also suggested that expressionof genes encoding metallothioneins (MTs) are differentially ex-pressed during AsV and AsIII stresses (Chakrabarty et al., 2009).

In order to elucidate mechanism of differential arsenic accumu-lation in different rice cultivars, we studied different antioxidantenzymes and expression of set of genes in different contrastinggermplasms. Here we present, our findings related to gene expres-sion and responses of antioxidants of four commonly cultivatedrice cultivars showing variability of different degree up to contrast-ing type for specific arsenic uptake.

2. Materials and methods

2.1. Experimental conditions

Rice seeds of four selected cultivars (Triguna, IR-36, PNR-519and IET-4786) obtained from Rice Research Station, Chinsurah,West Bengal were screened in hydroponics. Seeds were disinfectedin 0.1% HgCl2 solution for 30 s, followed by thorough washing withdeionized water and soaking in milli-Q for 24 h. These seeds werethen transferred to Petri-dishes (3–4 d) kept in culture room at26 �C in dark for proper germination. The plants were grown inhydroponic medium for 10 d before treatment and then exposedto AsV (Na2HAsO4; 0, 10, 20, 50 lM) and AsIII (NaAsO2; 0, 5, 10,25 lM) for 7 d. After that plants were harvested, washed withmilli-Q, blotted and used for the study of various parameters. Rootand shoot length were measured by metric scale.

2.2. Arsenic quantification and quality control

For estimation of total As, dried plant samples (1 g) were pow-dered and digested in 3 mL HNO3 at 120 �C for 6 h (Smith et al.,2008). The level of As was quantified by Inductively Coupled Plas-ma Mass Spectrometer (ICP-MS, Agilent 7500 ce). Specific ArsenicUptake (SAU) indicates the ability of As uptake by rice genotypesper unit root under AsV and AsIII exposure (Zhang and Duan, 2008).

SAU ¼ ðTroot-As þ Tshoot-AsÞ=root biomass

The standard reference material of As (E-Merck, Germany) wasused for the calibration and quality assurance for each analyticalbatch. Rice flour NIST 1568a was used as a reference material withknown spiked samples and recovery of total AS was 85.3% (±2.8;n = 5) and 89.5% (±3.1; n = 5) respectively. The detection limit ofAs was 1 lg L�1.

2.3. Growth and non-protein thiol measurement

Plant growth was measured with metric scale in terms of rootand shoot length. NP-SH content was measured by following themethod of Ellman (1959). For which, plant material (700 mg)was homogenized in 6.67% 50-sulfosalicylic acid. After centrifuga-tion at 10 000g for 10 min at 4 �C, NP-SH content was measuredin the supernatant by reaction with Ellman reagent.

2.4. Assay of antioxidant enzymes

Plant material (500 mg) was homogenized in 100 mM chilledpotassium phosphate buffer (pH 7), containing 0.1 mM EDTA and1% PVP (w/v) at 4 �C. Homogenate was squeezed through four lay-ers of cheese cloth and extract thus obtained was centrifuged at10 000g for 15 min at 4 �C. Supernatant thus obtained was usedto measure the activities of various enzymes which, were ex-pressed as units mg�1 protein.

APX (EC 1.11.1.11) activity was measured according to themethod of Nakano and Asada (1981) by estimating the rate ofascorbate (ASC) oxidation (€ = 2.8 mM�1 cm�1). Enzyme activitywas expressed as lmoles ASC oxidised min�1 mg�1 protein.

GPX (EC 1.11.1.7) activity was assayed according to Hemedaand Klein (1990). A 100 mL of reaction mixture was prepared byadding 10 mL of 1% guaiacol (v/v), 10 mL of 0.3% H2O2 and 80 mLof 50 mM phosphate buffer (pH 6.6). Enzyme extract (75 lL) wasadded to reaction mixture in a final volume of 3 mL. The increasein absorbance due to oxidation of guaiacol (€ = 26.6 mM�1 cm�1)was monitored at 470 nm. Enzyme activity was expressed aslmoles guaiacol oxidised min�1 mg�1 protein.

Activity of GR (EC 1.6.4.2) was assayed by following the methodof Smith et al. (1988). The reaction mixture contained 1.0 mL of0.2 M potassium phosphate buffer (pH 7.5) containing 1 mM EDTA,0.5 mL 3 mM 5,50-dithiobis (2-nitrobenzoic acid) in 0.01 M phos-phate buffer (pH 7.5), 0.25 mL H2O, 0.1 mL 2 mM NADPH,0.05 mL enzyme extract and 0.1 mL 20 mM GSSG. The componentswere added in the order as above directly to a cuvette and the reac-tion was started by the addition of GSSG. The increase in absor-bance was monitored for 5 min at 412 nm. The rate of enzymeactivity was calculated using standard curve prepared by knownamounts of GR (Sigma, USA). The rate of enzyme activity was ex-pressed as lmoles GSSG reduced min�1 mg�1 protein.

DHAR (EC 1.8.5.1) activity was assayed by formation of ASC at265 nm (€ = 14 mM�1cm�1) in a reaction mixture containing0.1 M Na-phosphate buffer (pH 6.2), 2 mM GSH, 1 mM dehydroa-scorbic acid (DHA) and 100 lg protein with enzyme extract (DeTullio et al., 1998). The rate of non-enzymatic DHA reductionwas corrected by subtracting the values obtained in the absenceof enzyme extract. The rate of enzyme activity was expressed aslmoles DHA reduced min�1 mg�1 protein.

MDHAR activity was assayed by monitoring NADPH oxidationat 340 nm (€ = 6.2 mM�1 cm�1) in a 3 mL of reaction mixture con-taining 0.1 mM NADPH, 2.5 mM ASC, 50 mM Na-phosphate buffer(pH 7.6) and 100 lg protein. The reaction was started by the addi-tion of four units of ascorbate oxidase (Drazkiewicz et al., 2003).Enzyme activity was expressed as lmoles MDHA reduced min-1 mg�1 protein.

For the measurement of CAT (EC 1.11.1.6) activity, extractionwas done in buffer containing Tris HCl (pH 7), 1 mM EDTA, 1 mMPMSF and 0.3 g g�1 fw PVP. Activity was measured by method ofAebi (1974). The assay system comprised of 50 mM Na2PO4 buffer(pH 7), 20 mM H2O2 (€ = 0.04 cm2 lmole�1) and suitable aliquot ofenzyme in final volume of 3 mL. Decrease in the absorbance wasrecorded at 240 nm. Enzyme activity was expressed as lmolesH2O2 degradation min�1 mg�1 protein.

988 A. Rai et al. / Chemosphere 82 (2011) 986–995

The activity of SOD (EC 1.15.1.1) was assayed following themethod of Beauchamp and Fridovich (1971) by measuring its abil-ity to inhibit the photochemical reduction of nitrobluetetrazolium(NBT). One unit was the amount of protein required to inhibit 50%initial reduction of NBT under light.

2.5. Statistical analysis

Two-way analysis of variance (ANOVA) and Duncan’s multiplerange test (DMRT) were performed to determine the significantdifference between treatments and genotypes. Correlation analysiswas performed for all the data at each duration with respect tochange in metal content or between parameters, which has beengiven within text at relevant places (p < 0.001��; p < 0.01��;p < 0.1�; NS non significant) (Gomez and Gomez, 1984).

2.6. Expression analysis using semiquantitative RT-PCR

Total RNA from the root and the shoot of 17-d-old seedlings wasextracted using the QIAGEN RNeasy Plant Maxi Kit (QIAGEN, MD),followed by treatment with RNAase-free DNase (Fermantas, LifeSciences, Ontario, Canada). RTPCR was carried out using 20 lL ofthe cDNA corresponding to the set of selected genes in a reactioncontaining 2X PCR Master mix (Fermantas, Life Sciences, Ontario,Canada). The list of selected genes and oligonucleotide primers(MWG, India) used in the study is provided as SupplementaryTable S1. The primers for rice ubiquitin gene were used as loadingcontrol to ensure that equal amounts of cDNA were used in all thereactions. The PCR reaction was carried out using the following cy-cle conditions: an initial denaturation at 94 �C for 2 min, number ofPCR cycles provided in Table S1 at 94 �C for 30 s, 55 �C for 30 s, and72 �C for 30 s, followed by a final 5-min extension at 72 �C.

3. Results

3.1. Effect of arsenite and arsenate on growth in four rice lines withvarying degree of grain As accumulation

In AsV exposed plants, root length (Fig. S1A) was significantlyenhanced in Triguna (30% at 10 lM, R = 0.998��). IET-4786 showedsignificant inhibition in root elongation (27% at 50 lM,R = �0.719�). No significant change was observed in root lengthof other two cultivars, IR-36 and PNR-519, at different concentra-tions of AsV. However, under AsIII exposed plants, all the cultivarsshowed a sharp decline in root length which was maximum inIET-4786 (32% at 25 lM, R = �0.949��; Fig. S1C). In general, lowerdose (10 lM) of AsV enhanced the shoot length (Fig. S1B) of allthe rice genotypes except IR-36. However, higher doses of AsV sig-nificantly reduced the shoot growth of the three cultivars withmaximum reduction in IR-36 (26%, at 50 lM, R = �0.769�). Inter-estingly, no decease in shoot length was observed in PNR-519 evenat higher AsV concentration. In AsIII exposed plants, increase inshoot length was observed in all the cultivars with maximum in-crease in Triguna (22% at 10 lM, R = 0.998��) at lower concentra-tion. At higher concentration decrease in shoot length wasobserved in all the cultivars with maximum decrease in IET-4786(31% at 25 lM, R = �0.608NS).

3.2. Arsenic accumulation in four contrasting lines

A significant genotypic variation was observed in total As accu-mulation which varied significantly depending on the As species.In roots, total As accumulation (Fig. 1A and D) was two fold higherin AsV exposed rice genotypes. In shoot (Fig. 1B and E), a concentra-tion dependent increase was observed in all the cultivars in case of

AsV exposure. In AsIII treatment, increase in As accumulation wasobserved till 10 lM concentration followed by decrease at 25 lMexcept in IET-4786. Total As (mg kg�1 dw) accumulation was foundto be maximum in Triguna (956 at 50 lM AsV, 412 at 10 lM AsIII)and minimum in PNR-519 (501 at 50 lM AsV) and IET-4786 (108 at10 lM AsIII) for AsV and AsIII exposed plants, respectively. Similarly,a significant genotypic variation in SAU (Fig. 1C and F) was ob-served in both the species of As.

3.3. Expression analysis using semiquantitative RT-PCR

Expression analyses of four different cultivars showed that onlower concentration of AsIII majority of transporters were up-regu-lated which included sulphate transporter and MATE. On higherconcentration (25 lM) of AsIII treatment, all transporters weredown-regulated except oligopeptide transporter (OPT) and multi-drug efflux transporter (MATE) (Os03g08900) in Triguna andPNR-519. MATE (Os03g08900) was also up-regulated in IR-36 athigher AsIII concentration. No detectable expression of sulphatetransporter, Nramp and OPT was found in IET-4786 (Fig. 2) at25 lM AsIII. However, differential expression profile was observedin AsV stress in comparison to AsIII. It was found that there is up-regulation of sulphate transporter at higher concentration of AsV

(50 lM) in all the cultivars. However, all other transportersshowed differential expression pattern in different cultivars. MATE(Os10g20350) showed up-regulation in three cultivars (Triguna,IR-36 and IET-4786) (Fig. 3). Similarly, significant decrease inMATE (Os03g08900) transcript level was observed in Triguna,PNR-519 and IET-4786.

Similarly, expression of glutaredoxin and cytochrome p-450showed differential expression pattern in different cultivars. Dur-ing AsIII and AsV stress these genes were down-regulated in IET-4786 and PNR-519. However, in Triguna and IR-36 these genesare up-regulated at lower concentration in comparison to control(Figs. 2 and 3).

All the heat-shock proteins (HSPs) were up-regulated at lowerconcentrations of arsenic in Triguna but showed differentialexpression pattern in all other cultivars. For example,Os03g16020 was up-regulated in IET-4786 but all others weredown-regulated in AsIII, where as in AsV treatment all the HSPswere down-regulated in the same cultivar (Figs. 2 and 3).

Decrease in expression of APX and MDHAR was observed in IET-4786 at higher AsIII and AsV treatment (Figs. 2 and 3), However, nosignificant changes were observed in these genes in other threecultivars (Figs. 2 and 3). GST and c-ECS were up-regulated duringAsIII stress in Triguna, PNR-519 and IR-36 but in case of IET-4786they were all down-regulated (Figs. 2 and 3). Similarly, phytochel-atin synthase was up-regulated in Triguna and IR-36 during AsIII

stress. A decrease in expression of phytochelatin synthase was ob-served in other two cultivars. In case of AsV stress, GST was up-reg-ulated in all the cultivars. However, phytochelatin synthase down-regulated in all the cultivars during AsV stress. No significantchange in the transcript level of c-ECS was observed in all the cul-tivars in all the treatments.

3.4. Antioxidant response in four rice cultivars with varying degree ofgrain As accumulation

The selected rice cultivars exhibited variable responses for anti-oxidant enzymes when exposed to As. Increase in the activity ofvarious enzymes was more pronounced in plants exposed to AsIII

then AsV. In all the rice genotypes, the significant stimulationswas observed in the activities of root-APX (PNR-519, 413%) andGPX (PNR-519, 133%) under AsIII stress except IET-4786 whichshowed concentration dependent decline in these enzymes.

Cb Cb Cb

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Fig. 1. (A–F): Total arsenic accumulation in rice cultivars exposed to different concentrations of arsenate [root (A), shoot (B), SAU (C)] and arsenite [root (D), shoot (E), SAU(F)]. All the values are mean of triplicates ±S.D. ANOVA significant at p 6 0.01. Different capital letters indicate significantly different values among As treatments in aparticular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p 6 0.05).


However, GR activity of IET-4786 increased significantly when ex-posed to different concentrations of AsIII. A concentration depen-dent increase in root-APX was observed in AsV (Fig. 4C) exposedplants, except IET-4786 (78%, R = �0.981��) which showed contin-uous inhibition upon As exposure however, higher dose (25 lM) ofAsIII (Fig. 4G) inhibited the activity of APX. Under AsV stress, shootAPX activity (Fig. 4D) showed a different trend instead of concen-tration dependent increase, inhibition was noticed in shoot APXactivity of Triguna (44%) and IR-36 (23%) while continuousdecrease was observed in IET-4786 (77%, R = �0.971��). In contrast,PNR-519 (52%, R = 0.792�) showed concentration dependent induc-tion. A concentration dependent increase in shoot APX activity wasobserved in AsIII (Fig. 4H) exposed plants which was maximum inIR-36 (466%, R = 0.873�) while, IET-4786 (25%, R = �0.918��)showed inhibitory response upon As treatment.

Although the lower dose of AsV enhanced the activity of rootGPX in all the cultivars (Fig. 5A), the lower dose of AsIII (Fig. 5E) en-hanced the activity of the enzyme in all the cultivars except IET-4786 (34%, R = �0.952��) which showed concentration dependentdecrease under AsIII stress. The shoot-GPX activity increased signif-icantly under AsIII (Fig. 5F) exposure while, under AsV (Fig. 5B)stress concentration dependent inhibition was observed in IR-36(40%, R = �0.953��).

AsIII stress significantly enhances the root-GR activity (Fig. 5G)in comparison to AsV (Fig. 5C), which was more in PNR-519(294%, R = 0.804�) and Triguna (183%, R = 0.62�) minimum in IET-4786 (63%, R = 0.956��), however, at higher doses shoot-GR(Fig. 5H) activity was decreased in PNR-519 (27%) and IR-36(54%). A concentration dependent decrease was observed in AsV

exposed shoot-GR activity of IET-4786 (18%, R = �0.990��,

Os01g43740 (CYTOCHROME P450)


TRIGUNA AsIII

0µm 5µm 25µm 0µm 5µm 25µm 0µm 5µm 25µm 0µm 5µm 25µm

IR-36 AsIII PNR-519 AsIII IET-4786 AsIII

Os03g09970 (ST)

Os03g54000 (OPT)

Os07G15460(Nramp)

Os12g38300 (MT)

Os12g38290 (MT)

Os12g38064 (MT)

Os01g26912 (GLUTAREDOXIN)


Os03g16030 (HSP)

Os03g16920 (HSP)

DQ323738 (GST)

Os07g27790(γγ ECS)

Os05g02530 (DHAR)

AF439787.2 (PCS)

Os03g16020 (HSP)

Os01g08860 (HSP)

Os03g08900 (MATE)

Os10g20350 (MATE)

Ubiquitin

Os03g17690 (APX)

Os09g39380 (MDHR)

Fig. 2. Expression analysis of selected genes in rice cultivars exposed to different concentrations of arsenite.


Fig. 5D) while maximum induction was observed in Triguna(169%).

The MDHAR (Fig. 6A, B, E, and F) and DHAR (Fig. 6C, D, G, and H)activity in root and shoot showed a similar trend for AsV and AsIII

treatment. It increased up to 20 lM in all the cultivars followedby a decline at 50 lM except IET-4786 which showed a concentra-tion dependent decline upon exposure to both AsV and AsIII.

The significant induction in CAT was observed in roots of Trigu-na, under AsV (165%, R = 0.976��) and AsIII (318%) stress, while,PNR-519 (155%, R = 0.883�) showed maximum induction inshoot-CAT activity under AsV stress. However, a concentrationdependent decrease was observed in root and shoot-CAT activityof IET-4786 upon AsV (Fig. S2A and B) and AsIII (Fig. S2E and F)exposure. The maximum increase in root-SOD activity was ob-served in PNR-519 (651%, R = 0.960��) under AsV and AsIII (66%,R = 0.905��) stress, however, in shoot, it was maximum in Triguna(729%, R = 0.805�) under AsIII stress.

3.5. Non-protein thiols response

A significant genotypic variation was observed in the NP-SH re-sponse of selected cultivars, under both As species stress. No changein NP-SH content was observed in AsV treated Triguna and IR-36 cul-tivars. Significant induction in level of NP-SH content was observedin AsV exposed roots (Fig. 4A) of PNR-519 (93% at 10 lM,R = 0.997��) which subsequently decreased at higher concentra-tion. IET-4786 showed a concentration dependent decline (63% at50 lM, R = �0.903��). However, in AsIII (Fig. 4E) exposed roots, morethan 3-fold increase was observed in Triguna, while IET-4786showed a decline of 86% at highest concentration (25 lM AsIII) whencompare to control.

NP-SH level in shoot of AsV exposed plants showed a concentra-tion dependent increase in Triguna and IR-36. However, a slight in-

crease in NP-SH level at lower concentration followed by decreaseat higher concentration was observed in PNR-519 and IET-4786(Fig. 4B). In contrast, a concentration dependent decrease in NP-SH level was observed in all the cultivars under AsIII exposed plants(Fig. 4F), which was more pronounced in IET-4786 (7% at 50 lMAsV; 49% at 25 lM AsIII, R = �0.993��).

4. Discussion

Arsenic is a carcinogenic metalloid, ubiquitous in the environ-ment with both natural and anthropogenic sources. Its level in ricegrain is dominated by inorganic As and dimethylarsinic acid (DMA)(Meharg et al., 2008). The toxicity of As is dependent on its speci-ation with inorganic AsIII and AsV thought to be more toxic thanmethylated species (Tuli et al., in press). Similarly, during the pres-ent study, AsIII caused more toxicity which was evident by thereduction in the root and shoot elongation. Reduced root and shootlength in response to As has been reported by a number of workers(Hartley-Whitaker et al., 2001; Sneller et al., 1999; Liu et al., 2005).Recently, Mokgalaka-Matlala et al. (2009) observed that root elon-gation decreased significantly with increasing AsV and AsIII concen-trations, who also concluded that reduction was more pronouncedin the roots of Mesquite (Prosopis juliflora) plant exposed to AsIII.Total As accumulation in roots and shoot of rice genotypes in-creased with increasing concentration of AsV and AsIII in the nutri-ent medium, except higher (25 lM) dose of AsIII, where the rootgrowth of all rice cultivars were significantly affected, thus reduc-ing the transport of As. Accumulation of total As was higher inroots in comparison to shoot upon AsV and AsIII treatments, whileTF was higher in shoots of AsIII exposed plants. Higher retention ofAsV and AsIII in the roots might be attributed to its compartmental-ization in root vacuoles. The significant genotypic difference in Asuptake and translocation was observed and it was found that

TRIGUNA AsV

0µm 10µm 50µm 0µm 10µm 50µm 0µm 10µm 50µm 0µm 10µm 50µm

IR-36 AsV PNR-519 AsV IET-4786 AsV

Os03g09970 (ST)

Os03g54000 (OPT)

Os07G15460(Nramp)

Os12g38300 (MT)

Os12g38290 (MT)

Os12g38064 (MT)





Os03g16030 (HSP)

Os03g16920 (HSP)

DQ323738 (GST)

Os07g27790(γ ECS)

Os05g02530 (DHAR)AF439787.2 (PCS)

Os03g16020 (HSP)

Os01g08860 (HSP)

Os03g08900 (MATE)

Os10g20350 (MATE)

Ubiquitin

Os03g17690 (APX)

Os09g39380 (MDHR)

Fig. 3. Expression analysis of selected genes in rice cultivars exposed to different concentrations of arsenate.


cultivars IET-4786 had the lowest SAU, in comparison to Triguna(Dwivedi et al., in press). Similarly, Zhang and Duan (2008)reported significant difference in As and Cd uptake and foundtwo fold lower SAU in JX17 rice cultivars, when compared toZYQ8. Furthermore, the genetic variability in rice has also beenreported during iron plaque formation (Liu et al., 2006), arsenicspeciation and even at molecular level (Norton et al., 2008).

Some studies have been conducted to analyze induction of theantioxidant system under As stress (Shri et al., 2009), however,only a few compared the responses of antioxidants to AsV and AsIII

(Mylona et al., 1998). Significant increase in APX, MDHAR andDHAR with GR would have maintained the function of the ascor-bate–glutathione cycle to combat enhanced generation of ROS(Singh et al., 2006). An increase in the level of antioxidants en-zymes may be attributed to the induced transcription of theirgenes, as evident during transcriptomic analysis. SOD plays animportant role in dismutation of free superoxide radicals (O��2 ). Sig-nificant increase in SOD activity was found in Triguna, IR-36 andPNR-519 under both As species while inhibition was observed inroots of IET-4786, which may be due to de novo reduced synthesisof enzyme protein, as previously demonstrated in Holcus (Hartley-Whitaker et al., 2001), Ceratophyllum (Mishra et al., 2008), and rice(Shri et al., 2009). Thus significant induction in SOD and GR activityin Triguna, IR-36 and PNR-519 suggested that these cultivars re-sponded positively to As stress providing sufficient GSH to supportantioxidant system viz., ascorbate–glutathione (ASC–GSH) cycle.APX activity also increased in Triguna, IR-36 and PNR-519 showingactive participation of ASC–GSH cycle in these cultivars. However,IET-4786 exhibited decreased APX activity in both protein level aswell as gene level. The other two peroxide degrading enzymes

(GPX and CAT) showed significant decrease in their activity. Thismight be due to less availability of H2O2 because of its efficientbreakdown in ASC–GSH cycle or due to inactivation of enzyme di-rectly by As or ROS.

Similarly, other genes related to arsenic stress (HSPs, glutare-doxin and CYT-P450) showed that Triguna, IR-36 and PNR-519have higher level of gene expression compared to IET-4786. Athigher concentration both AsIII and AsV reduce the expression pat-tern of several genes in IET-4786 that are directly related to arsenicstress demonstrated earlier (Chakrabarty et al., 2009). Interest-ingly, no significant change was observed in MTs expression in fourcultivars under both AsIII and AsV treatments. This pattern of geneexpression as well as defence related enzyme activities indicatedthat IET-4786 is a very sensitive cultivar to arsenic. It accumulateslow arsenic, but at higher concentration significantly reduced itsgrowth as a result of down-regulation of several defence relatedgenes reported earlier (Chakrabarty et al., 2009).

While, antioxidant enzymes are considered to be an importantdefence system against oxidative stress caused by metals (Weckand CliJsters, 1996; Shri et al., 2009), NPTs and glutathione (GSH)are important non-enzymatic antioxidants. Triguna and PNR-519showed a continuous increase in NPTs content in roots under boththe species of As, while it significantly enhanced in shoot under AsV

stress. This might be attributed to significant stimulation of sul-phate uptake and cysteine biosynthetic as well as consumptionpathways (Mishra et al., 2008). NPTs are considered as an indexof GSH and PCs in plants, therefore, an increase in NPTs contentof Triguna, IR-36 and PNR-519 upon AsV treatment may suggestan induction of GSH and PCs for protection of plant cells throughdirect chelation with As (Schmoger et al., 2000). The expression

Ac BcDb

Aa

Ac Ac

Ab

Ba

Ab Bb

Ba Ca

Ab BbCa Da

0

0.03

0.06

0.09

0.12

0.15N

PSH

(µ m

ol g

-1 fw

)N

PSH

(µ m

ol g

-1 fw

)

NPS

H (µ

mol

g-1

fw)

NPS

H (µ

mol

g-1

fw)

0 10 20 50 µM

Cb Ab Cb

Ba

CcBc

Bb

Aa

Bc

Bd

AaCb

Aa

Cc Db Db

0

0.03

0.06

0.09

0.12

0.15 0 5 10 25 µM

Cb DbCa BaBc

Cb AaAb

Bc

Ba BaBb

Ab

AaCb

Cb

0

0.03

0.06

0.09

0.12

0.15 0 10 20 50 µM

AbAb

Aa Aa

BcBd

BbBa

CbBc

Ca Ca

Db Cc

Db Da

0

0.03

0.06

0.09

0.12

0.15 0 5 10 25 µM

DbDc Cc

Aa

Ca CbCc

BaBa BbBc

BbAa Ab

Ab

Cc

0

0.1

0.2

0.3

0.4

APX

(uni

t mg

-1 p

rote

in)

APX

(uni

t mg

-1 p

rote

in)

APX

(uni

t mg

-1 p

rote

in)

APX

(uni

t mg

-1 p

rote

in)

0 10 20 50 µM

Aa

DcBcDb

AaBa

Bb

Ca Bb

Aa

Ac

Aa

Cc

Cb

Cd

Ba

0

0.1

0.2

0.3

0.4 0 5 10 25 µM

Aa

BbCb

BaBc

Bc

Ab

Aa

CcBb

BbCa

Dc

AaCcDb

0

0.04

0.08

0.12

0.16

Rice varieties

0 10 20 50 µM

Db

DdDc

AaCa CbCb

BbBb

Aa

BcBc

Aa

Bb Ab

Cc

0

0.04

0.08

0.12

0.16

Triguna IR-36 PNR-519 IET-4786 Triguna IR-36 PNR-519 IET-4786







0 5 10 25 µM

HD

C G

A E

FB

Fig. 4. (A–H): NP-SH content and APX activity in rice cultivars exposed to different concentrations of arsenate [root-NPSH (A), shoot-NPSH (B), root-APX (C), shoot-APX (D)]and arsenite [root-NPSH (E), shoot-NPSH (F), root-APX (G), shoot-APX (H)]. All the values are mean of triplicates ±S.D. ANOVA significant at p 6 0.01. Different capital lettersindicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at aparticular treatment (DMRT, p 6 0.05).


Cb Ba

Cc

Ba

Ca

Ab

Bc

AbAa

CcAb CbBa

Db Db

Da

0

1

2

3

4


GPX

(uni

t mg-1 p

rote

in)

0 10 20 50 µM

CbCa Ca

AaAc

Ab

Aa

BdAb Bb

Aa

CcBb Cb

Ba

Dc

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GPX

(uni

t mg-

1 pro

tein

)

0 5 10 25 µM

Bc

AaBc

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CbCa Ba Ca

0

0.3

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1.2


GPX

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t mg-1

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tein

)

0 10 20 50 µM

Cc

Da

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AbAc

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GPX

(uni

t mg-1

pro

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)

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Bb BbAc

AaBa

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AbBaBb

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GR

(uni

t mg

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rote

in)

0 10 20 50 µM

Db DbDc

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GR

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t mg-

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tein

)

0 5 10 25 µM

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CcBcBb BbBbAb

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BbAbAb

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Rice varieties

GR

(uni

t mg-1

pro

tein

)

0 10 20 50 µM

DbAc Bc

DaCb

AcAb

CaBa

AbBb

BaAa

Bc Cb

Aa

0

0.1

0.2

0.3


GR

(uni

t mg-1

pro

tein

)

0 5 10 25 µM

A E

D H

GC

B F

Fig. 5. (A–H): GPX and GR activity in rice cultivars exposed to different concentrations of arsenate [root-GPX (A), shoot-GPX (B), root-GR (C), shoot-GR (D)] and arsenite [root-GPX (E), shoot-GPX (F), root-GR (G), shoot-GR (H)]. All the values are mean of triplicates ±S.D. ANOVA significant at p 6 0.01. Different capital letters indicate significantlydifferent values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT,p 6 0.05).


CbCc Dc

Ba

Aa

Bc

Bb AbBaAb

AaCb

Ca Ba Ca Da

0

0.03

0.06

0.09


MD

HA

R (u

nit m

g-1 p

rote

in)

0 10 20 50 µM

Cb Cb

Bc

AaCbBa

Bc Bc

AbAa

BcCd

Ba

Db Ab

Dc

0

0.03

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MD

HA

R (u

nit m

g-1 p

rote

in)

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BbBc Dc

AaAa

BcCb

Ba

CbAa Ba Ba

Dd

AbAa

Cc

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MD

HA

R (u

nit m

g-1 pr

otei

n)

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DbDc

Dc

Aa

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nit m

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in)

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Db CbAc

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AR

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CbCb Ab

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Bd

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AR

(uni

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0 5 10 25 µM

BcCc

Ab

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AbBb

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AbAa

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CbCa

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0.02

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DH

AR

(uni

t mg-

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in)

0 10 20 50 µM

BcCc

Ab

Aa

AbBb

Bb

Aa

AbAa

Cc

Ba

Bb

Ba

CbCa

0

0.02

0.04

0.06

0.08


DH

AR

(uni

t mg-1

pro

tein

)

A E

D H

GC

B F

Fig. 6. (A–H): MDHAR and DHAR activity in rice cultivars exposed to different concentrations of arsenate [root-MDHAR (A), shoot-MDHAR (B), root-DHAR (C), shoot-DHAR(D)] and arsenite [root-MDHAR (E), shoot-MDHAR (F), root-DHAR (G), shoot-DHAR (H)]. All the values are mean of triplicates ±S.D. ANOVA significant at p 6 0.01. Differentcapital letters indicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among ricecultivars at a particular treatment (DMRT, p 6 0.05).



data of sulphate transporter also validate our results. However,IET-4786 is very sensitive cultivar as there was decrease in allthe antioxidant enzyme activities as well as expression of all thosegenes related to sulphate and glutathaione metabolism. IncreasingAs concentration significantly decreased the NPT level indicatingthat As caused the toxicity in plants cells and thus reduced thenon-protein thiols (Chakrabarty et al., 2009).

5. Conclusions

Considerable variations are found among different varieties ofrice cultivars with respect to tolerance of arsenic stress. Data re-vealed that the rice cultivar (IET-4786) is very sensitive to arsenicstress due to reduction of both sulphate assimilation pathway andantioxidant defence enzymes in As-detoxification. However, theother two varieties, Triguna and IR-36 showed considerable detox-ification mechanism due to up-regulation of several of these genesduring arsenic stress.

Acknowledgements

The authors are thankful to Director, National Botanical Re-search Institute, Lucknow for the facilities and for the financialsupport from the network projects (CSIR), New Delhi, India.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.chemosphere.2010.10.070.

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