arsenic stress in rice: redox consequences and regulation by iron

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Plant Physiology and Biochemistry

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Research article 656667686970717273
Arsenic stress in rice: Redox consequences and regulation by iron

Shwetosmita Nath a, Piyalee Panda a, Sagarika Mishra b, Mohitosh Dey b,Shuvasish Choudhury c,*, Lingaraj Sahoo b, Sanjib Kumar Panda a

a Plant Molecular Biotechnology Laboratory, Department of Life Science and Bioinformatics, Assam University, Silchar 788 011, IndiabDepartment of Biotechnology, Indian Institute of Technology, Guwahati, Assam, IndiacCentral Instrumentation Laboratory, Assam University, Silchar 788 011, India

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a r t i c l e i n f o

Article history:Received 30 October 2013Accepted 14 April 2014Available online xxx

Keywords:AntioxidantsArsenicGene expressionIronOxidative stressOryza sativa

* Corresponding author. Tel.: þ91 94351 75587.E-mail addresses: [email protected], sh

(S. Choudhury).

http://dx.doi.org/10.1016/j.plaphy.2014.04.0130981-9428/� 2014 Published by Elsevier Masson SAS

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Please cite this article in press as: Nath, S.Biochemistry (2014), http://dx.doi.org/10.10

a b s t r a c t

Arsenic (As) contamination is a serious hazard to human health and agriculture. It has emerged as animportant threat for rice cultivation mainly in South Asian countries. In this study, we investigated theeffect of iron (Fe) supplementation on arsenic (AsV) induced oxidative stress responses in rice (Oryzasativa L.). Rice seedlings treated with AsV for 24 and 48 h in presence or absence of 2.5 mM Fe after whichthe root and shoot tissues were harvested for analysis. The results indicate significant (p � 0.05)reduction in root and shoot length/dry biomass. Supplementation of Fe showed improved growth re-sponses under stress as compared to AsV alone. The scanning electron microscopy (SEM) analysis of rootsunder AsV treatment for 48 h showed major alterations in root structure and integrity, although nonoticeable changes were observed in Fe e supplemented seedlings. Significantly high (p � 0.05) accu-mulation of AsV was observed in root and shoot after 24 and 48 h of stress. However, under Fe e sup-plementation As accumulation in root and shoot were considerably low after 24 and 48 h of AsV

treatment. The hydrogen peroxide (H2O2) and malondialdehyde (MDA) content in both root and shootincreased significantly (p � 0.05) after 24 and 48 h of AsV treatment. In Fe e supplemented seedlings, thelevels of H2O2 and MDA were considerably low as compared to AsV alone. Ascorbate (AsA) and gluta-thione (GSH) levels also increased significantly (p � 0.05) under AsV stress as compared to control andFe-supplemented seedlings. Activities of catalase (CAT) and superoxide dismutase (SOD) were signifi-cantly (p � 0.05) high after 24 and 48 h of AsV treatment as compared to Fe-supplemented seedlings. Thegene expression analysis revealed up-regulation of metallothionein (MT1, MT2) and nodulin 26-likeintrinsic protein (NIP2;1) genes after 5d of As treatment, while their expressions were repressed un-der Fe-supplementation. Our results indicate that Fe regulates oxidative stress and promotes growthunder As stress.

� 2014 Published by Elsevier Masson SAS.

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1. Introduction

Arsenic (As) is toxic metalloid which is widely distributed inaquatic and terrestrial ecosystem (Phillips, 1990). It is considered asa class I carcinogen and food chain contaminant (Zhao et al., 2010).It exhibits four different valencies (-III, 0, III and V) with severalchemical forms. In soils, As is usually present as pentavalent arse-nate (AsV) and trivalent arsenite (AsIII), later being more toxic thanits pentavalent form (Zhao et al., 2010). Both of these forms areinter-convertible depending upon the redox sate of the soil(Tripathi et al., 2007). Ground water contamination with As is a

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serious issue in many South East Asian countries including India,Bangladesh, China and Vietman. Rice being the major staple cropgrown in these areas is affected by As toxicity, mainly due to irri-gation with As-contaminated water (Zhao et al., 2010; Williamset al., 2007; Brammer and Ravenscroft, 2009). Rice is highly effi-cient in accumulating As in comparison to other crops like wheatand barley (Williams et al., 2007). High As accumulation capacity ofrice poses immense health hazards to almost 50% of world popu-lation who are dependent on rice as their staple food (Norton et al.,2009; Tripathi et al., 2012). Studies have also shown that rice andrice-related products are also major source of As exposure inhumans who are not directly exposed to As contaminated drinkingwater (Mondol and Polya, 2008; Rahman et al., 2009). Being a redoxactive metalloid, it stimulates the production of reactive oxygenspecies (ROS) by the inter-converting one form to other, causingoxidative stress (Tripathi et al., 2012; Hartley-Whitaket et al., 2001;

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Choudhury and Panda, 2004; Mylona et al., 1998; Shaibur et al.,2006; Singh et al., 2007; Shri et al., 2009). As interacts with sulf-hydryl (-SH) groups of enzymes and proteins, leading to inhibitionof several important cellular functions (Mehrag and Hartley-Whitaker, 2002). Studies have also shown that As (AsV) acts asphosphate analog and hinder ATP production and oxidative pho-phorylation (Tripathi et al., 2007).

Evidences from physiological studies suggest that As transportoccurs via aquaporins in rice (Meharg and Jardine, 2003). Though,the exact mechanism of As transport in plants remain unclear,studies have revealed the possible role of nodulin26-like intrinsicproteins (NIPs) (Ali et al., 2009). In Arabidopsis, nodulin 26-likeintrinsic proteins (NIPs) like NIP5; 1, 6; 1 and 7; 1 are involved inAs transport and loss of NIP 7; 1 function leads to As tolerance(Isayenkov and Maathuis, 2008). Plants have also evolved severalmechanisms to combat the increasing stress load. Such intrinsicmechanisms for counteracting As toxicity in plants include phy-tochelatin (PC) dependant detoxification vis-à-vis induction of sul-fate uptake and reduction pathways (Rausch and Wachter, 2005).Higher level of PCs and PC-synthase activity alongwith coordinatedthiol metabolism were reported in rice, which induce As tolerance(Tripathi et al., 2012). Further, PC-arsenite complexion in rice leavesreduces translocation of As from leaves to grains (Duan et al., 2011).In addition, the potential role of various metallothioniens (MTs) inarsenic detoxification in ricewas also reported (Gautamet al., 2012).

Iron (Fe) is one of the major elements required by plants fornormal growth and metabolism. It is an essential element forphotosynthesis, respiration, DNA synthesis and co-factors forseveral enzymes (Hell and Stephan, 2003; Jeong and Connolly,2009). In soil, it is mainly present in insoluble oxidized (FeIII)form. In flooded rice a field, Fe is converted from FeIII to ferrous(FeII) form and quickly released from the soil and sequester As(Takahashi, 2004). Studies have also shown that Fe plaque forma-tion over root surface reduces As accumulation (Meharg, 2004). Therole of Fe in controlling As toxicity in plants were reported inseveral plant species, most of which concerns As accumulation andspeciation patterns (Meharg, 2004; Juskelis et al., 2013; Stone,2008). The role of Fe in mediating As induced oxidative stress asa strategy for As tolerance have been poorly reported. In the presentstudy, we investigate the role of Fe supplementation on growth andoxidative stress responses under As stress. To elucidate theameliorative role of Fe, we evaluate growth responses, As-accumulation, ROS production, lipid peroxidation, antioxidantlevel and expression pattern of some important genes in rice.

2. Methods

2.1. Plant material and treatments

Viable rice seeds (cv: Ranjit) were procured from RegionalAgricultural Research Station, Karimganj, India, surfaced sterilizedwith 0.1% mercuric chloride (HgCl2) and washed thoroughly withdeionised water. Sterile seeds were germinated over moistenedfilter papers in dark for 48 h. Uniformly germinated seeds weretransferred to plastic cups containing 350 ml Hoagland nutrientsolution at pH 6.2 (Hoagland and Arnon, 1950) and grown for 5dunder white light (52 mEm�2 s�1 PAR) at 25 �C with 16 h photo-period. Lethal dose (LD50) was measured for determining theconcentration of As [sodium arsenate (Na2AsO4)] and Fe [ferricchloride (FeCl3)] (see Supplementary Fig. 1 aec). Based on LD50results (LD50As ¼ 200 mM; LD50Fe ¼ 3.5 mM) final concentration ofAs (0 and 100 mM) and Fe (2.5 mM) were selected. Two differentsets, either in presence or absence of Fe were prepared and As(100 mM)was added to the nutrient solution. After 24 and 48 h, rootand shoot were used for analysis.

Please cite this article in press as: Nath, S., et al., Arsenic stress in riceBiochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.04.013

2.2. Growth, uptake and SEM analysis

The root and shoot length were measured for five plants pertreatment after 24 and 48 h using a centimeter scale. To measurethe dry biomass,1 g of plantmaterial was dried at 80 �C for 48 h andweighed. For measurement of As uptake, tissue samples werebriefly rinsed with deionised water and the excess water wassoaked out with a paper towel. Sample was dried as mentionedabove. 100 mg of dried sample was digested with 5 ml acidicmixture of nitric acid (HNO3) and hydrochloric acid (HCl) in theratio of 3:1. After complete digestion, the final volumewas adjustedto 20 ml with deionised water. The total As and Fe content wasrecorded using Atomic Absorption Spectrometer (3110/AAnalysi,Perkin Elmer, USA). For scanning electron microscopy (SEM) anal-ysis, root samples were fixed in 2.5% glutaraldehyde in 0.2 M so-dium phosphate buffer for 2 h at 4 �C. The post fixation, stainingand SEM analysis were carried out at Electron Microscopy Division,Sophisticated Analytical Instrumentation Facility at NEHU, Shillong.

2.3. Hydrogen peroxide and lipid peroxidation

Hydrogen peroxide (H2O2) content was determined as per themethod of Sagisaka (1976). Briefly, 200 mg of tissue sample washomogenized with 10% (w/v) trichloroacetic acid (TCA) andcentrifuged at 17, 000 g for 15min at 4 �C. The assay mixture con-tained 1.6 ml supernatant tissue extract, 0.4 ml 50% (w/v) TCA,0.4 ml ferrous ammonium sulfate (FeNHSO4) and 2.5 mM potas-sium thiocyanate (KSCN). The absorbance was recorded at 480 nm.

Lipid peroxidation was measured as per the method ofZhangZhang (1992) in terms of malondialdehyde (MDA) content.200 mg of tissue sample was homogenized in 0.25% (w/v) thio-barbituric acid (TBA) prepared in 10% (w/v) TCA. The extract washeated at 95 �C for 30min and cooled in ice. The mixture wascentrifuged at 10, 000 g for 10min at 4 �C and absorbance of thesupernatant was recorded at 532 nm. The non-specific turbiditywas corrected by subtracting the absorbance value at 600 nm.

2.4. Catalase (CAT) and superoxide dismutase (SOD) activities

The enzymes from plant tissues were extracted with sodiumphosphate buffer (0.1 M, pH 6.8) and centrifuged at 17, 000 g at 4 �Cfor 15min. The supernatant obtained was used for assay of en-zymes. The reaction mixture of CAT (EC 1.11.1.6) contained 2 mlsodium phosphate buffer (0.1 M), 0.5 ml H2O2 (30 mM) and 0.5 mlsterile distilled water. The reaction mixturewas incubated for 1minand the absorbance was recorded at 240 nm. The CAT activity wasexpressed as U min�1 g�1 f.w. using an extinction coefficient of43.6 mM�1 cm�1 (Chance and Maehly, 1955). The reaction mixturefor SOD (EC 1.15.1.1) contained triseHCl buffer (79.2 mM, pH 6.8),EDTA (0.12 mM), tetraethylenediamine (10.8 mM), bovine serumalbumin (0.0033%) nitroblue tetrazolium (600 mM in 5 mM KOH)and 0.2 ml supernatant enzyme extract. The reaction was initiatedby placing the glass vials under fluorescent light (20 W Phillips,India). By switching on the light on and off, the reaction wasinitiated and terminated. The absorbance for formazan formationwas recorded at 560 nM. The activity was expressed as DA560 g�1

(fr.wt.)10 min�1 (Giannopolitis and Ries, 1980).

2.5. Ascorbate (AsA) and glutathione (GSH) content

For the estimation of ascorbate and glutathione, 200 mg of theplant tissue was homogenized in sterile distilled water andcentrifuged at 17,000 g for 15min at 4 �C. For the estimation of AsAcontent, the reaction mixture contained sodium molybdate (2%w/v), sulfuric acid (0.1N H2SO4), sodium phosphate (1.5N Na3PO4) and


Table 1Primer sequences for amplification of genes in rice using reverse transcriptase po-lymerase chain reaction (PCR).

Primer name Forward (50e30) Reverse (30e50)

OsMT1 50ATGTCTTGCAGCTGTGGA30 30AGTTGCAAGGGTTGCACC50

OsMT2 50ATGTCGTGCTGCGGAGGA30 30GCAGTTGCAGGGGTTGCA50

OsNIP2; 1 50ACCATGTACTACGGCGAG30 30CGCGCATATCGCTCCGGT50

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supernatant plant extract. The mixture was incubated at 60 �C for40min and centrifuged at 3, 000 g for 10min. The absorbance wasrecorded at 660 nm (Oser, 1979). For GSH estimation, the assaymixture contained 0.5 ml potassium phosphate buffer (0.5N at pH7.5) with ethylenediamine tetra acetic acid (EDTA), 0.2 ml 5,50-dithiobisnitrobenzoic acid (6 mM DTNB), 0.1 ml reduced nicotin-amide adenine dinucleotide phosphate (2 mM NADPH) and 1 ml of1-U yeast GR type III. The change in absorbance was recorded at412 nm (Grifith, 1980).

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2.6. Gene expression analysis

100 mg of frozen root tissue was thoroughly grounded in liquidnitrogen using a pre-chilled mortar and pestle. RNA was extractedusing 5 ml DB homogenizing buffer (Bilgin et al., 2009). The con-centration of RNA was determined spectrophotometrically at260 nm. The RNA purity was also checked spectrophotometricallyby means of the 260/280 ratio and also electrophoretically usingagarose (1.2%). Total RNA (8 ml) was reverse transcribed to firststrand cDNA by using cDNA synthesis kit (Fermentas Life Sciences)following the manufacturer’s instructions. Four pairs of PCRprimers (Table 1) were designed to amplify the expression of MT1,MT2 and NIP2;1 along with actin. The PCR reactions were per-formed in a final volume containing 2.5 ml PCR buffer, dNTP(10 mM), 20 mM primers, 1U taq polymerase, 1U template DNA

Fig. 1. Effect of arsenic on root and shoot elongation (aeb) and dry biomass (ced) after 24then treated with 100 mM of AsV either in presence or absence of 2.5 mM iron (Fe). The datasignificance differences at p � 0.05 as compared to controls and treatments. The growth prepresents the scanning electron micrographs of roots after 48 h of AsV treatment.


(cDNA) for 25, 27 and 30 cycles (95 �C e 30S, 60 �C e 30S, 72 �C e

30S) followed by final extension of 5 min at 72 �C. Finally, 10 ml ofreaction volume was separated on agarose (1.5% w/v) gel.

2.7. Statistical analysis

Each experiment was repeated three times and the data pre-sented are mean � SE. The results were analyzed statistically todetermine the significant difference between each treatmentgroups and control.

3. Results

3.1. Effect of Fe-supplementation of growth of rice under As stress

Exposure of AsV for 24 and 48 h significantly (p� 0.05) inhibitedthe root and shoot growth (Fig. 1aeb). The root length of As treatedseedlings after 24 and 48 h reduced by 1.74 and 2 folds respectivelyas compared to control (Fig. 1a). Further, we observed significant(p � 0.05) inhibition of shoot growth (Fig. 1b). In Fe-supplementedseedlings, no variation was observed in shoot length after 24 h ascompared to AsV alone, but increased significantly (p � 0.05) after48 h. Comparing the effect of Fe-supplementation on root growthduring AsV stress, we observe significant (p � 0.05) increase in rootlength in Fe supplemented seedlings as compared to AsV alone. Theroot length increased approximately by 1.37 fold in Fe e supple-mented seedlings in comparison to AsV alone after 48 h oftreatment.

The root and shoot dry biomass (Fig. 1ced) showed strong effectof AsV stress. AsV treatment for 24 and 48 h reduced the root drybiomass either in presence or absence of Fe significantly (p � 0.05).In absence of Fe supplementation, AsV inhibited root growth by 1.81and 1.89 folds as compared to control. In presence of Fe, AsV

treatment resulted in significant (p � 0.05) decline of root dry

and 48 h of AsV treatment. Rice seedlings were grown in Hoagland solution for 5d andpresented are means of three replicates �SE. Legends indicated as a and b representsattern of rice seedlings (e) under AsV stress after 48 h of treatment is presented. (f)

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Fig. 2. Arsenic uptake in root and shoot of rice seedlings after 24 and 48 h of AsV treatment in presence or absence of Fe. The data presented are means of three replicates �SE.Legends indicated as a and b represents significance differences at p � 0.05 as compared to controls and treatments.


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biomass as well. The shoot dry biomass on the other hand did notchange after 24 h but it reduced significantly (p � 0.05) after 48 h.

The growth of rice seedlings after 48 h of AsV stress indicatesnoticeable changes as compared to control and Fe e supplemen-tation (Fig. 1e). Noticeable inhibition of root and shoot growthalong with visible symptoms of chlorosis were observed under AsV

stress. In Fe esupplemented seedlings, we observe strong root andshoot growth as compared to AsV alone as compared to control. Nomajor symptoms of AsV toxicity were visible in shoot as comparedto As alone. In order to evaluate the effect of AsVstress on rootgrowth, we performed SEM analysis of root after 48 h of stress(Fig. 1f). In As treated seedling, root e cracks were visible, whichindicated possible loss of cellular integrity. In Fe-supplementedseedlings, root surface appears smooth and no major alterationscould be observed.

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3.2. As uptake

The accumulation of AsV in rice root and shoot after 24 and 48 hof treatment either in presence or absence of Fe is shown in Fig. 2.We observe high accumulation of AsV in root and shoot after 24 and48 h of treatment. In Fe e supplemented seedlings, AsV accumu-lation was considerably low as compared to AsV alone. AsV stress

Fig. 3. Effect of AsV on hydrogen peroxide (aeb) and malondialdehyde (ced) content in rpresence or absence of Fe. The data presented are means of three replicates �SE. Legends indand treatments.


showed 6 fold increase in AsV accumulation in root following 24and 48 h of treatment as compared to Fee supplemented seedlings.In shoots, 2.3 fold increase in accumulation was observed, while itincreased by 4.65 fold after 48 h as compared to Fee supplementedseedlings. AsV was undetectable in control plants.

3.3. Effect of Fe e supplementation on hydrogen peroxide (H2O2)content and lipid peroxidation under As stress

The hydrogen peroxide (H2O2) content increased significantly(p� 0.05) after 24 and 48 h of AsV treatment in presence or absenceof Fe as compared to controls (Fig. 3aeb). As treatment in absenceof Fe enhanced H2O2 production in roots by 1.9 and 3 foldsrespectively after 24 and 48 h as compared to control. In shoots,H2O2 level increased by 1.8 and 2.7 folds after 24 and 48 hrespectively as compared to control. For Fe e supplemented seed-lings, H2O2 level also increased significantly (p � 0.05), though noconsiderable (p � 0.05) change was observed in shoot after 24 h ascompared to controls. Comparing H2O2 levels in AsV treated groupsin presence or absence of Fe, we observe high concentration ofH2O2 in absence of Fe. AsV alone increased the H2O2 content by 1.5and 1.6 folds in roots after 24 and 48 h respectively as compared toFe e supplemented seedlings. In shoots, H2O2 content was also

oot and shoot of rice seedlings respectively after 24 and 48 h of treatment either inicated as a and b represents significance differences at p � 0.05 as compared to controls

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Fig. 4. Effect of AsV on catalase (aeb) and superoxide dismutase (ced) activities in root and shoot of rice seedlings respectively after 24 and 48 h of treatment either in presence orabsence of Fe. The data presented are means of three replicates �SE. Legends indicated as a and b represents significance differences at p � 0.05 as compared to controls andtreatments.


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significantly (p � 0.05) high after 24 and 48 h as compared tocontrols.

The lipid peroxidation was measured in terms of malondialde-hyde (MDA) produced under AsV stress in root and shoot of riceseedlings (Fig. 3ced). Upon AsV exposure, the MDA contentincreased significantly (p � 0.05) as compared to control. In pres-ence of Fe, theMDA content in root did not showmajor change after24 h of treatment but it increased (p� 0.05) after 48 h of treatmentas compared to controls. In comparison to Fe e supplementedseedlings, the MDA content was significantly (p� 0.05) high in rootand shoot, where almost 4 and 2 fold increase in MDA content wasobserved after 24 and 48 h respectively in roots under AsV treat-ment alone. The shoot MDA content also increased significantly(p � 0.05) after 24 and 48 h under AsV stress as compared to Fe e

supplemented seedlings.The glutathione (GSH) levels (Fig. 4ced) were also significantly

(p� 0.05) hih in root and shoot under AsV stress. The GSH level was

Fig. 5. Effect of AsV on ascorbate (aeb) and glutathione (ced) content in root and shoot of ricFe. The data presented are means of three replicates �SE. Legends indicated as a and b rep


increased by 4.6 and 3.86 folds respectively after 24 and 48 h of AsV

treatment as compared to control. In Fe e supplemented seedlings,GSH level was also high in both root and shoot as compared tocontrol. GSH levels for AsV treated seedlings in absence of Fe e

supplementation were significantly (p � 0.05) high as compared toseedlings under As treatment in presence of Fe e supplementation.

3.4. Effect of Fe e supplementation on antioxidant enzymes underAs stress

The effect of AsV on catalase (CAT) and superoxide dismutase(SOD) activities in presence or absence of Fewas evaluated (Fig. 4aed). The total CAT and SOD activity increased significantly (p � 0.05)in root and shoot under AsV treatment both in presence or absenceof Fe. Maximum CAT activity was observed in root of AsV treatedseedlings in absence of Fe e supplementation. For roots of Fe e

supplemented seedlings, the CAT activity was almost close to

e seedlings respectively after 24 and 48 h of treatment either in presence or absence ofresents significance differences at p � 0.05 as compared to controls and treatments.

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Fig. 6. Expression pattern of metallothionein (MT1 and MT2) and nodulin 26-likeintrinsic (NIP2;1) genes in rice after 5d of AsV treatment in presence or absence ofFe and 2.5 mM Fe alone. The PCR primers (see Table 1) were designed to amplify theexpression of the these genes along with actin and the reaction volume was separatedon 1.5% (w/v) agarose gel.


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control levels after 24 h. Moreover, the CAT activity in shoots of Fesupplemented seedlings was significantly (p� 0.05) enhanced after48 h of AsV treatment, whereas it was relatively low after 24 h withrespect to controls. Compared to Fe e supplemented seedlings, theCAT activity enhanced significantly in root and shoot under AsV

stress in absence of Fe.

3.5. Effect of Fe e supplementation on ascorbate (AsA) andglutathione (GSH) content under As stress

We evaluated the ascorbate (AsA) and glutathione (GSH) con-tent in rice seedlings exposed to AsV stress for 24 and 48 h (Fig. 5aed). After 24 and 48 h of AsV treatment, there was significant in-crease (p � 0.05) in AsA and GSH content in root and shoot ascompared to controls. AsV stress for 24 and 48 h in absence of Feincreased the AsA level in root by 6 and 8 folds respectively ascompared to control. In shoot, significantly high levels (p � 0.05) ofAsA were observed after 24 and 48 h. In Fe e supplementedseedlings, AsV stress did not alter the AsA levels for 24 and 48 h inroot and shoot; however, significant increase (p � 0.05) wasobserved as compared to control. AsA levels were significantly highin under AsV alone as compared to Fe e supplemented seedlings(p � 0.05).

3.6. Gene expression analysis

We evaluated the expression of threemajor genesMT1,MT2 andNIP2;1 in rice roots after 5d of AsV stress (Fig. 6). These three geneswere highly expressed when exposed to low concentration (5 mM)of AsV stress.MT1,MT2 and NIP2;1were highly expressed as well at100 mM of AsV stress for 5d. In presence of 2.5 mM Fe, mildexpression of MT1 and NIP2;1 were observed, while no expressionof MT2 was noticed. Treatment of 2.5 mM Fe did not express thesegenes in roots after 5d.

4. Discussion

Arsenic is a carcinogenic metalloid, which is released into theenvironment by natural and anthropogenic sources. Rice cultiva-tion in South Asian countries like Bangladesh, China, India andVietman is severely affected by arsenic toxicity, largely due toirrigation of rice field with arsenic contaminated ground water(Mehrag and Hartley-Whitaker, 2002). Rice can accumulate highconcentration of arsenic in grains, which poses severe health haz-ards for rice consuming population worldwide. Several studieshave reported that arsenic induces oxidative stress in rice (Tripathi


et al., 2012; Shri et al., 2009; Rai et al., 2011; Dave et al., 2013),however, the ameliorative role of iron in modulating oxidativestress responses during arsenic toxicity have been poorly reported.In the present investigation, we studied the effect of iron (Fe)supplementation on arsenic (AsV) induced oxidative stress in rice.Our results demonstrate that AsV inhibits growth, while Fe sup-plementation resulted in improved growth response and low AsV

accumulation. Further, the hydrogen peroxide (H2O2) and malon-dialdehyde (MDA) levels were increased during AsV exposure for 24and 48 h, while in Fe e supplemented plants, both H2O2 and MDAlevels were low in comparison to AsV alone. Abiotic stresses,including heavy metals and metalloids are known to induce reac-tive oxygen species (ROS) and causes oxidative stress. Previousstudies have demonstrated that arsenic generates reactive oxygenspecies (ROS), which results in degradation of important bio-molecules like lipid, nucleic acids and proteins (Hartley et al.,2001; Tripathi et al., 2007). These ROS include H2O2, superoxideradical (O2

C�) and hydroxyl radical (OHC), that attacks importantmetabolic functions by degrading enzymes and other metabolites(Haliwell and Gutteridge,1989; Mittler, 2002). H2O2 production hasbeen implicated during many stress conditions. It is produced byreduction of oxygen (O2) to form O2

C� and subsequently to H2O2(Haliwell and Gutteridge, 1989). The high production of H2O2 dur-ing AsV stress in absence of Fe indicates that it impart greater affectby causing strong oxidative damage. Further, it possibly resulted ingrowth inhibition, leaf chlorosis and reduction of plant biomass,which are evident in the present investigation (Fig. 1aef). The in-crease in H2O2 content during AsV stress has possibly initiated lipidperoxidation, which resulted in oxidative stress. Though, we didnot observe any major difference in shoot MDA levels after 24 and48 h, there was a strong increase in MDA level in roots after 48 hduring AsV stress in Fe e supplemented seedlings as compared tocontrols. The elevated levels of MDA in AsV treated seedlings inabsence of Fe showed higher rate of lipid peroxidation and strongsusceptibility to oxidative stress. In mung bean, increase in ROSproduction and lipid peroxidation was reported under AsV stress(Singh et al., 2007).

We investigated responses of antioxidant enzymes viz., catalase(CAT) and superoxide dismutase (SOD) in rice seedlings during AsV

stress. The results indicated that CAT activity remained unaffectedin presence of Fe but significantly increased under AsV alone ascompared to controls. Superoxide dismutase (SOD) activity on theother hand showed elevated activity during AsV stress either inpresence or absence of Fe. For both CATand SOD, the activities werehigh in AsV treated seedlings in absence of Fe. CAT scavenges H2O2in cells and does not require a reluctant. Studies have shown thatCAT activity increases even if H2O2 levels reach high values(Mhamdi et al., 2010). High H2O2 levels and increase in CAT activityunder AsV stress indicated that CAT is probably a highly expressedenzyme in rice, which makes it an integral part of ROS detoxifica-tion system. SOD on the other hand converts O2

C� to H2O2 in cells.The increase in SOD activity during AsV stress was probably due tohigh conversion of O2

C� to H2O2 that resulted in increase of H2O2pool in absence of Fe e supplementation. Low SOD activity andH2O2 production in Fe e supplemented seedlings under AsV stresssignifies ameliorative role of Fe in controlling ROS production.

The non-enzymatic antioxidants viz., ascorbate (AsA) andglutathione (GSH) showed strong increase under AsV alone after 24and 48 h as compared to control and Fe e supplemented seedlings.In presence of Fe, both AsA and GSH levels were high as comparedcontrol, but did not show considerable difference during the stressperiod. AsA and GSH are two major non-enzymic antioxidants,which play an important role in scavenging ROS. AsA acts as asubstrate for ascorbate peroxidase (APX) to detoxify H2O2 (Smrinoffand Wheeler, 2000; Wheeler et al., 1998). Higher levels of AsA in


Q1


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AsV stressed plants indicated its possible role in H2O2 detoxifica-tion. GSH is widely used as a marker for oxidative stress in plants.During the ascorbateeglutathione cycle, GSH acts as electron donorto regenerate AsA from its oxidized form thereby playing a crucialrole in detoxification of O2

C� and H2O2 (Noctor and Foyer, 1998). Italso acts as a substrate for glutathione peroxidase (GPX), which isinvolved in ROS detoxification (Tausz et al., 2004).

In plants, metallothionines (MTs) are important components formaintaining homeostasis of essential metals and detoxification oftoxic elements like cadmium (Cd) and As (Robinson et al., 1993).Studies have shown that there are eleven class I MT genes in ricegenome that are expressed differentially during growth anddevelopment (Gautam et al., 2012). Further, the MTs confer toler-ance against copper (Cu) and Cd in plants (Zhou and Goldsbrough,1994). Our results indicate that both MT1 and MT2 were stronglyexpressed during AsV stress for 5d. When AsV stress was given inpresence of Fe, we observed mild expression of MT1 only. For MTs,the ability to bind and sequester metal ions including arsenic de-pends upon the distribution and organization of cysteine residuesand their regulated expression during stress is a major option formetal detoxification and homeostasis (Gautam et al., 2012; Ushaet al., 2009; Singh et al., 2011). NIPs play important role inarsenic transport in plants. In rice, OsNIP2;1 (Lsi1) was identified inroots as major pathway for arsenite uptake (Ma et al., 2006). Later,other NIPs like OsNIP1;1, OsNIP2;2 and OsNIP3;1 were identified forAsIII transport (Ma et al., 2008). Though, there are no major reportson the involvement of NIPs in transport of AsV, we report here it’spossible role in uptake. NIP2;1was highly expressed in roots duringAsV stress for 5d, however, no significant expression was observedin Fe e supplemented seedlings. This also account for the fact thatunder Fe e supplementation AsV uptake was limited, which lead tostrong growth and development as compared to AsV alone.

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100101102103104105106107108109110111112113

5. Conclusion

The overall results reported in this investigation reveal thattreatment of AsV caused oxidative stress in rice seedlings. Weobserved major decline in plant biomass and growth along withsignificantly high uptake of arsenic. Elevated levels of H2O2,enhanced rate of lipid peroxidation and changes in antioxidantmachinery signifies AsV as a potential inducer of oxidative stress inrice. We have also reported that AsV uptake possibly occurs via NIPs(NIP2;1) and MTs are important in As detoxification. Under Fe e

supplementation, strong growth responses and low arsenic uptakewere observed as compared to AsV alone. The extent of oxidativeload was significantly reduced in Fe - supplemented seedlingsduring AsV stress.

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Acknowledgments

We acknowledge Regional Agricultural Research Station(Akbarpur, Karimganj) India for providing rice seeds. We arethankful to Central Instrumentation Laboratory, Assam Universityfor providing atomic absorption spectrometry facility and SAIF(North Eastern Hill University, Shillong) for scanning electron mi-croscopy facility.

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Authors contribution

The research work is carried out with equal contribution fromall the authors. SN, PP, SM and MD did all the experiments. SKPdesigned the experiments and gave valuable suggestions for theresearch work. SC, LS and SKP wrote the paper analyzed the data.


Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2014.04.013.

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arsenic stress in rice: redox consequences and regulation by iron

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