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Comparative transcriptome analysis of arsenate and arsenitestresses in rice seedlings

Debasis Chakrabarty 1, Prabodh Kumar Trivedi 1, Prashant Misra, Manish Tiwari, Manju Shri, Devesh Shukla,Smita Kumar, Arti Rai, Ashutosh Pandey, Deepti Nigam, Rudra Dev Tripathi, Rakesh Tuli *

National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, UP, India

a r t i c l e i n f o

Article history:Received 30 June 2008Received in revised form 8 September 2008Accepted 16 September 2008Available online 8 November 2008

Keywords:ArsenicGenome-wide expressionGlutathione metabolismMicroarrayOryza sativaMetabolic network

a b s t r a c t

The effect of arsenic (As) exposure on genome-wide expression was examined in rice (Oryza sativa L., ssp.Indica). A group of defense and stress-responsive genes, transporters, heat-shock proteins, metallothione-ins, sulfate-metabolizing proteins, and regulatory genes showed differential expression in rice seedlingschallenged with arsenate (AsV) and arsenite (AsIII). AsV stress led to upregulation or downregulation ofan additional set of genes in comparison to AsIII. Differential expression of several genes that showed thehighest contrast in a microarray analysis was validated by following the quantitative changes in the lev-els of individual transcripts following challenge with AsV, AsIII, Cd, Cr, and Pb. Most of the selected genesresponded to challenge by heavy metals such as arsenic. However, expression of one of the cytochromeP450 genes (Os01g43740) in rice root was induced by AsV but not by other heavy metals. Similarly, oneglutaredoxin (Os01g26912) is expressed specifically in the AsIII-treated shoot.

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1. Introduction

The presence of arsenic (As) in groundwater and food poses aserious health risk to people worldwide. The inorganic forms ofthe element, arsenite (AsIII) and arsenate (AsV), are more abundantin soils compared to the organic As species such as monomethylar-sonic acid and dimethylarsonic acid (Tripathi et al., 2007). Themechanism by which arsenic is taken up by plants differs for AsIIIand AsV. AsV is taken up by the high-affinity phosphate uptakesystem (Ullrich-Eberius et al., 1989), whereas AsIII uptake isthought to be accomplished through the aquaporins of the roots(Meharg and Jardine, 2003). Both inorganic forms of arsenic arehighly toxic because they interfere with various metabolic path-ways in cells such as the interaction of substrates/enzymes withthe sulfhydryl groups of proteins and the replacement of phos-phate in ATP for energy (Tripathi et al., 2007). Hence, plants ex-posed to arsenic show symptoms including decreases in bothplant growth and crop yield (Carbonell Barrachina et al., 1995).In addition, arsenic stimulates the formation of free radicals andreactive oxygen species, resulting in oxidative stress (Requejoand Tena, 2005). Plants respond to AsV and AsIII stresses differen-tially by stimulating the antioxidant system and thiol metabolism,respectively (Mishra et al., 2008). In spite of many previous studies

having been conducted on the effects of arsenic stress, the precisemolecular mechanisms related to both the effects of arsenic phyto-toxicity and the defense reactions of plants against arsenic expo-sure remain poorly understood at present. Recently, Dasguptaet al. (2004) have reported the association of As tolerance withthe chromosome 6 in rice through quantitative trait loci (QTL)analysis. The study has been conducted using a doubled haploidpopulation established by the anther culture of F1 plants obtainedfrom a cross between a Japonica cultivar, CJ06, and an Indica culti-var, TN1. Four QTLs present in rice chromosomes have been thusreported to be responsible for the uptake of As in different tissuesand at various developmental stages (Zhang et al., 2008).

To characterize the arsenic-responsive genes, an analysis of thegenome-wide expression of the rice transcriptome was carried out.A DNA microarray chip was used to identify the differentially ex-pressed genes in rice following exposure to AsIII and AsV duringgermination and growth of the seedling. New genes identified inthis study may provide more clues to understanding the molecularmechanism of response to As-induced stress in rice.

2. Materials and methods

2.1. Plant material, growth conditions, and treatments

Mature seeds of the Indica cultivar, IR64 (Oryza sativa L.) weremanually dehusked, sterilized, and cultured on 1/2 Murashigeand Skoog (MS) medium, containing either 25 lM AsIII (NaAsO2,

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* Corresponding author. Tel.: +91 522 2205841; fax: +91 522 205836.E-mail address: rakeshtuli@hotmail.com (R. Tuli).

1 Contributed equally to the results of this study.

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ICN, USA) or 250 lM AsV (Na2HAsO4, J.T. Baker, UK). Twenty seedswere placed in each plate, covered by a lid, and incubated under a16-h light (110—130 lE m�2 s�1)/8-h dark photoperiod at 26 �C.For treatment with Pb [Pb(NO3)2; HiMedia, India), CrVI (K2Cr2O7;SRL, India), and Cd (CdCl2; Rankem, India), 100 lM of each elementwas used independently on the germinating seeds, as in the casesof AsV and AsIII. Shoot length, root length, root weight, and shootweight were measured 10 days after sowing.

2.2. Total RNA extraction and transcriptome analyses

Total RNA from 10-d-old seedlings, grown with or without AsVor AsIII, was extracted using the QIAGEN RNeasy Plant Maxi Kit(QIAGEN, MD) for microarray analysis. Affymetrix GeneChip� RiceGenome Arrays (Gene Expression Omnibus platform Accession No.GPL2025) were used for microarray analyses. Target preparation,hybridization to arrays, washing, staining, and scanning were car-ried out according to the instructions of the manufacturer (Affyme-trix, USA). Three independent replicated experiments were carriedout for all the treatments. The hybridization data were analyzedusing the Affymetrix GeneChip Operating Software (GCOS version1.3). Satisfactory image files were analyzed to generate the probeintensity (.cel) files using the default settings of GCOS. Normaliza-tion of all the arrays was carried out using three independentmethods: namely, probe logarithmic intensity error (PLIER), robustmultiarray average (RMA), and GeneChip robust multiarray(GCRMA), using the ArrayAssist software (Stratagene, La Jolla, CA,USA). To identify statistically significant differentially expressedgenes, a combined criterion of 2-fold or greater change and a P va-lue <0.05 in the t-tests was adopted. Only common probe sets thatwere present in all the three analyses were considered significant.To obtain annotations for the arsenic-regulated probe sets, targetsequences from the sequence information file of the rice genomearray were searched using BLASTn against the TIGR rice pseudo-molecules, release 5 (http://www.tigr.org/tdb/e2k1/osa1).

2.3. Pathway analysis

All the genes identified in the microarray analysis using thecombined criterion of 2-fold or greater change and a P value<0.05 in the t-tests were used to generate the cellular-functionpathways and interactomes using the software Pathway Studio(Ariadne Genomics, MD, USA). Pathways were generated for thegenes modulated by AsV and AsIII independently. Another com-mon pathway was generated for genes whose expression wasmodulated by both AsV and AsIII treatments.

2.4. Expression analysis using semiquantitative RTPCR

Rice plants were grown on ½MS medium containing AsV(250 lM), AsIII (25 lM), or other metals (Pb, Cr, and Cd;100 lM), as described above. Total RNA from the root and theshoot of 10-d-old seedlings was extracted using the QIAGENRNeasy Plant Maxi Kit (QIAGEN, MD), followed by treatment withRNAase-free DNase (Fermantas, Life Sciences, Ontario, Canada).RTPCR was carried out using 20 lL of the cDNA correspondingto the set of selected genes in a reaction containing 2X PCR Mas-ter mix (Fermantas, Life Sciences, Ontario, Canada). The list of se-lected genes and oligonucleotide primers (MWG, India) used inthe study is provided as Supplementary Table S1. The primersfor rice actin gene were used as loading control to ensure thatequal amounts of cDNA were used in all the reactions. The PCRreaction was carried out using the following cycle conditions:an initial denaturation at 94 �C for 2 min, 30 cycles at 94 �C for30 s, 55 �C for 30 s, and 72 �C for 30 s, followed by a final 5-minextension at 72 �C.

3. Results and discussion

3.1. Growth of rice seedlings exposed to arsenic

Seeds germinated on media containing different concentrationsof AsV and AsIII show a decline in germination and retardation inthe growth of the seedlings during the 10 days of observation.The toxic effect was much higher in AsIII in comparison to AsV(data not shown). In our study, we observed similar toxic effectsin AsV (250 lM) or ASIII (25 lM) with a significant decline (40–50%) in the length and weight of roots (Fig. 1A and B) demonstrat-ing iso-toxic effect. Our study as well as results from other groupsclearly suggest that AsIII is more toxic in comparison to AsV (Fitzand Wenzel 2002; Abedin and Meharg, 2002; Meharg and Hart-ley-Whitaker 2002; Dembitsky and Rezanka 2003; Liu et al.,2004; Mahimairaja et al., 2005). Reduced plant growth in responseto arsenic exposure has also been reported by numerous investiga-tors in other plants (Liu et al., 2005).

Fig. 1. Root (j) and shoot (h) length (A), root and shoot weight (B) of rice IR64seedlings grown on AsV (250 lM) and AsIII (25 lM). Bars represent SD of means. ***

indicate values that differ significantly from control at P < 0.001 according toStudent’s paired t-test. Venn diagram of the number of probe sets up- anddownregulated in response to AsV and AsIII stress (C). The number of probe setswhich were upregulated and the number of probe sets which were downregulated(italic) with an adjusted P value of <0.05 and a >2-fold change in gene expression.

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3.2. Transcriptome analysis of gene expression

Affymetrix GeneChip Rice Genome Arrays containing 51,279probes to query the transcripts are hybridized with the cRNA pre-pared from RNA that is isolated from seedlings grown on ½MSmedium containing AsV or AsIII. In the control and in the AsV- orAsIII-treated seedlings, an average of 47% probe sets hybridize tothe transcripts, as analyzed by GCOS. In seedlings exposed to AsVtreatment, 618, 240, and 386 probe sets are found to be upregu-lated using PLIER, RMA, and GCRMA normalizations, respectively,as per the determined criteria. Only 223 probe sets are commonto all the three normalizations. Similarly, although many probesets are upregulated in AsIII-treated plants in comparison to thecontrol in all the three normalization procedures, only 85 probesets are common. Analyses using similar criteria identify 205and 86 probe sets as being downregulated in seedlings treatedwith AsV and AsIII, respectively. Thirty-one upregulated and 61downregulated probe sets are common between the AsV- andAsIII-treated rice seedlings (Fig. 1C). A set of common up- anddownregulated genes belong to various gene families related totransporters, stress-related genes, regulatory proteins, growthand developments and secondary metabolisms ( SupplementaryTables S2 and S3). A list of the up-/downregulated probe sets underAsIII and AsV treated rice seedlings with their annotations areprovided in Table 1 and Table 2, respectively.

AsV stress produces a large additional set of probe sets that areeither upregulated or downregulated, in comparison to AsIII. Theoccurrence of responses specific for AsV appears reasonable be-cause AsV differs from AsIII in its mechanism of action as well asdifferential uptake machinery (Ullrich-Eberius et al., 1989; Tripathiet al., 2007). In total, 507 probe sets are differentially expressedduring arsenic stress, which corresponds to approximately 1% ofthe total Affymetrix GeneChip Rice Genome Array; thus arseniccan be concluded to cause a profound effect on basal cellularmetabolism.

3.3. Gene annotation

Different probe sets have been classified according to their bio-logical functions, namely, their involvement in defense and stress,metabolism, growth and development, secondary metabolism,transcription, signal transduction, and transport. Many genes pres-ent are classified as ‘‘unknown” because the annotation of the RiceGenome does not assign any putative functions and many largesets of genes are stated as ‘‘hypothetical” or ‘‘expressed”. A largeset of differentially expressed genes belongs to the defense andstress response in plants, in addition to being involved in generalmetabolism (Fig. 2).

The current analyses show that 72 and 27 genes are unique todifferential regulation by AsV and AsIII stresses, respectively.Among them, genes coding for proteins such as glutathione S-transferase (GSTs; seven from the TAU class; Os01g49710,Os10g34020, Os10g38350, Os10g38720, Os10g38189,Os10g38610, Os01g49720; and three from an unknown class;Os09g20220, Os01g72150 and Os01g72140), which are involvedin xenobiotic metabolism, are found to be upregulated in AsVstress. Among these, only 1 gene is upregulated in AsIII stress.Three other GSTs are also upregulated in AsIII stress, but the induc-tion is less than 2-fold. Moreover, 2 GSTs (Os01g27630 andOs01g27390) are downregulated by AsV.

GSTs are known to be induced by a number of intracellular andenvironmental factors that include heavy-metal stress. Normally,GSTs catalyze the conjugation of toxic molecules with reduced glu-tathione (GSH) and target them for ATP-dependent transport intothe vacuole. Some isoforms of GST are reported to show dual activ-ity, additionally functioning as a glutathione peroxidase in the

presence of H2O2 or organic/lipid hydroperoxides (Marrs, 1996).This phenomenon provides further evidence for the role of GSTsin antioxidant metabolism. Furthermore, a considerable numberof TAU-class GSTs, which are known to be induced by osmoticstress, extreme temperatures, and treatments that invoke plant de-fense reactions, are found to be differentially expressed in thisstudy (Marrs, 1996).

Another class of stress-related genes involved in glutathionebiochemistry is the family of glutaredoxins. Two glutaredoxins(Os02g40500 and Os01g26912) are upregulated in AsIII stress,whereas only one (Os02g40500) is upregulated in AsV stress. Oneof the glutaredoxins, PvGrx5, has been previously reported to havea role in regulating the intracellular AsIII levels in the arsenichyperaccumulator fern, Pteris vittata L. (Sundaram et al., 2008).

Cytochrome P450-related genes are also upregulated in AsV andAsIII stresses. Cytochrome P450s constitute a large superfamily ofenzymes, with 356 members in rice that are involved in the metab-olism of toxic compounds. Ten members are commonly upregu-lated in AsV. Out of these 10, some of the members are alsoupregulated in AsIII but their fold change in expression is below2. Numerous genes related to the peroxidase family are also down-regulated in both AsV (20 nos.) and AsIII (10 nos.) stresses. It mightbe possible that the thylakoid membranes are susceptible to lipidperoxidation caused by arsenic stress. It is presumed herein thatthe downregulation of peroxidases may be due to the arsenicstress-induced damage to thylakoid membranes caused by lipoxy-genases. This can be correlated with the 4-fold upregulation of achloroplast-targeted lipoxygenase (Os08g39850) that has been de-tected in this study.

Four (Os12g38300; Os12g38290; Os12g38051 and Os12g38064) and two (Os12g38290 and Os12g38051) metallothioneins(MTs) are upregulated in AsV and AsIII stresses, respectively. Re-cent studies in animals and plants have shown that MTs are in-volved in maintaining the homeostasis of essential metals andfunction in metal detoxification (Hall, 2002).

A total of 11 shock proteins (HSPs: Os02g54140, Os03g16030,Os03g16920, Os02g52150, Os04g01740, Os04g36750, Os03g16020, Os01g08860, Os04g45480, Os03g14180, and Os04g01740)are upregulated in AsV stress. Of these HSPs, only 3 (Os02g54140, Os03g16030, and Os03g16920) are observed to be upregu-lated in AsIII stress. HSPs are produced by all organisms in re-sponse to increased temperature and certain other stresses. PlantHSPs consist of a small family of high-molecular-weight and a lar-ger group of low-molecular-weight proteins, ranging from 15 to20 kDa (Dafny-Yelin et al., 2008). Of the 11 HSPs that are upregu-lated, 7 belong to the low-molecular-weight category. One of themembers of the germin gene family (Os12g05840), which has beenshown to have both super oxide dismutase (SOD) and oxalate oxi-dase (Godfrey et al., 2007) activities, is upregulated in both AsVand AsIII stresses.

A large number of transporters are differentially expressed afterchallenge with AsV and AsIII. One of the members of the sulfate-transporter gene family (Os03g09970) is upregulated more than10-fold in both AsV and AsIII stresses. There are many different sul-fate transporters in plants that differ in their intracellular locations,expression patterns, and kinetic properties. Sulfate can enter plantsthrough a high-affinity sulfate transporter present in the plasmamembrane (Nocito et al., 2006), which may lead to the enhancedproduction of S-rich metal-binding peptides (such as GSH, phyto-chelatins), thus providing metal tolerance and resulting in metalaccumulation. These sulfate transporters are expressed mainly inthe roots of plants and are upregulated under sulfur-starved condi-tions and heavy-metal stress. Transgenic mustard plants, devel-oped using a high-affinity sulfate-transporter construct, has beenobserved to show altered metal tolerance and accumulation(Lindblom et al., 2006). The metal transporter genes, OsNrmp1

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Table 1List of probe sets up- and downregulated by AsV stress in rice.

Probe ID Locus Description P value Fold change

UpregulatedOs.1000.1.S1_at Os01g01660 Isoflavone reductase homolog IRL, putative, expressed 0.005749223 3.1839051Os.10018.S1_at Os02g43410 Transposon protein, putative, unclassified, expressed 0.003690533 2.922332Os.10251.S1_at Os10g33370 Acyltransferase, putative, expressed 9.71E�04 2.4006672Os.10348.S1_at Os01g41430 Flavonol-3-O-glycoside-7-O-glucosyltransferase 1, putative, expressed 1.08E-04 2.5598378Os.10682.S1_at AK109215 Unknown 1.72E�04 6.9054685Os.103.S1_s_at Os04g01740 Heat-shock protein 82, putative, expressed 5.63E�05 35.784443Os.1039.3.3.S1_at Os04g01740 Heat-shock protein 82, putative, expressed 2.91E�06 15.27499Os.1046.S1_at Os06g39240 Endothelial differentiation-related factor 1, putative, expressed 7.21E�05 57.761948Os. 1193.S1_at Os03g55240 Cytochrome P450 81 E1, putative, expressed 3.52E�05 3.0716093Os. 1266.S1_at Os04g27060 Auxin-induced protein PCNT115, putative, expressed 2.16E�06 6.444453Os. 1341.S1_a_at Os10g01134 Serine carboxypeptidase 1 precursor, putative, expressed 7.66E�04 2.565818Os. 1376.S1_at Os06g06490 U-box domain containing protein, expressed 1.10E�04 3.3352544Os. 1417.S1_at Os02g26810 Trans-cinnamate 4-monooxygenase, putative, expressed 0.037341837 3.8989396Os. 1775.S2 at Os01g53920 Receptor-like protein kinase 5 precursor, putative, expressed 0.00703215 2.553484Os. 1800.S1_at Os01g50100 Multidrug resistance protein 4, putative, expressed 0.003496796 3.626145Os. 1800.S1_s_at Os01g50100 Multidrug resistance protein 4, putative, expressed 0.014067366 3.0765574Os. 1816.S1_at Os04g37490 NAD(P)H-dependent oxidoreductase, putative, expressed 2.46E�04 2.9930441Os. 191.1S1_at Os03g04060 Basic endochitinase C precursor, putative, expressed 3.22E�04 2.7317624Os.12038.S1_at Os01g65700 Expressed protein 0.003528639 4.8402133Os.12106.S1_at Os03g48780 Oxalate oxidase 2 precursor, putative, expressed 0.001429441 3.2162275Os.12253.S1_at Os01g45110 Cytokinin-O-glucosyltransferase 1, putative, expressed 2.68E�06 3.5605989Os.12257.S1_at Os02g52150 Heat-shock 22 kDa protein, mitochondrial precursor, putative, expressed 8.93E�05 3.6164477Os.12629.S2_at AK068661 Unknown 0.001773725 3.417835Os.12732.S1_a_at Os10g42320 Nonsense-mediated mRNA decay protein 3, putative, expressed 2.83E�04 2.4345098Os.13008.S1_at Os08g39850 Lipoxygenase 8, chloroplast precursor, putative, expressed 0.041982774 4.21144Os.14079.S1_at Os03g58300 Indole-3-glycerol phosphate lyase, chloroplast precursor, putative, expressed 0.002392372 2.709829Os.14616.S1_at Os07g34260 Chalcone synthase DII, putative, expressed 0.004396281 3.7405457Os.14951.S1_at Os08g39730 Cytochrome P450 76C2, putative, expressed 0.006891173 6.0923824Os.14955.S1_at Os03g31730 Expressed protein 8.33E�05 2.5508919Os.15112.S1_ at Os07g23410 Omega-6 fatty acid desaturase, endoplasmic reticulum isozyme 2, putative, expressed 0.002479626 3.014625Os.15471.S1_at Os07g23410 Omega-6 fatty acid desaturase, endoplasmic reticulum isozyme 2, putative, expressed 4.06E�05 3.3994958Os.15838.A1_at Os07g44410 WD40-like Beta Propeller Repeat family protein, expressed 1.45E�04 5.1368575Os.16317.S1_at Os04g45480 Heat-shock protein STI, putative, expressed 9.98E�04 5.059758Os.16435.S1_at Os02g53410 Expressed protein 0.003449988 2.8375697Os.16744.S1_at Os01g58670 Expressed protein 1.09E�04 4.0464234Os.16879.S1_at Os06g20820 Expressed protein 0.001051539 4.014702Os. 7271.S1_at Os12g07310 Citrate-binding protein precursor, putative, expressed 3.98E�04 4.389757Os.17316.S1_at Os04g49194 Naringenin, 2-oxoglutarate 3-dioxygenase, putative, expressed 2.18E�04 6.1484003Os.17659.S1_ at Os12g15680 L-Ascorbate oxidase precursor, putative, expressed 2.79E�04 3.2314115Os.18064.S1_at CF337042 Unknown 0.00127395 29.290302Os.18707.S1_at Os11g04020 Major facilitator superfamily antiporter, putative, expressed 0.001045214 5.0044637Os.19547.S1_at Os08g02030 Expressed protein 0.00262016 4.667184Os.19547.S1_x_at Os08g02030 expressed protein 0.001715894 4.805008Os.19632.S1_ at Os03g46470 Zinc transporter 10 precursor, putative, expressed 4.65E�05 4.688128Os.19740.S2_at Os03g02874 Retrotransposon protein, putative, Ty3-gypsy subclass, expressed 3.82E�04 19.085537Os.19822.S1_ at Os03g09970 Sulfate transporter 1.2, putative, expressed 1.20E�06 48.225983Os.21103.S1_x_at Os03g42130 Gibberellin 20 oxidase 2, putative, expressed 3.76E�05 2.7421708Os.21103.2. 2.S1_at Os03g42130 Gibberellin 20 oxidase 2, putative, expressed 0.001805404 4.9919105Os.21318.S1_at Os06g04510 Enolase 1, putative, expressed 2.53E�05 2.335791Os.22731.S1_at Os03g 16020 17.4 kDa class I heat-shock protein 2, putative, expressed 5.01E�05 8.400491Os.22957.S1_ at Os01g72140 Glutathione S-transferase, putative, expressed 0.00323777 2.5255013Os.23021.S1_at Os03g62480 Anthocyanidin 5,3-O-glucosyltransferase, putative, expressed 0.001397501 3.1563632Os.23183.S1_at Os09g28160 Phosphate carrier protein, mitochondrial precursor, putative, expressed 7.04E�04 4.0863028Os.23207.S1_at Os08g04340 Blue copper protein precursor, putative, expressed 0.017692199 3.5108747Os.23233.S1_at Os04g46940 Copper-transporting ATPase 3, putative, expressed 7.65E�04 2.4922578Os.23327.2. 2.S1_a_at Os10g10130 OsWAK112d - OsWAK receptor-like protein kinase, expressed 6.25E�04 3.2063248Os.23397.S1_at Os08g37874 Oxidoreductase, putative, expressed 2.81E�04 3.2775242Os.23397.S2_at Os08g37874 Oxidoreductase, putative, expressed 8.71E�05 4.5989285Os.23518.A1_at Os02g36190 Cytochrome P450 71 D7, putative, expressed 0.019189073 2.946142Os.23578.S1_at AK070059 Unknown 5.19E�05 11.056473Os.2376.1S1_at Os03g17480 IN2-1 protein, putative, expressed 4.58E�05 3.824379Os.24314.A1_s_at CA761625 Unknown 3.89E�04 4.217265Os.24906.S1_at Os01 g72460 NADPH quinone oxidoreductase 2, putative, expressed 8.53E�04 3.352058Os.25409.S1_ at Os06g45184 Retrotransposon protein, putative, Ty3-gypsy subclass, expressed 2.35E�04 10.347247Os.25409.S1_x_at Os06g45184 Retrotransposon protein, putative, Ty3-gypsy subclass, expressed 3.92E�05 15.84622Os.25497.S1_at Os11g47500 Xylanase inhibitor protein 1 precursor, putative, expressed 0.001582074 2.5107698Os.25497.S1_x_at Os11g47500 Xylanase inhibitor protein 1 precursor, putative, expressed 7.71E�04 2.604424Os.25639.S1_at Os04g28420 Peptidyl-prolyl isomerase, putative, expressed 1.18E�05 18.7189Os.26063.S1_at Os11g15624 Expressed protein 3.60E�04 2.5617964Os.27271.S1_a_at Os04g25060 Protein kinase, putative, expressed 0.004088015 2.296572Os.27671 S1_at Os04g25650 Protein kinase, putative, expressed 6.5 6E�04 2.5874338Os.27759.S1_at Os01g46720 Protein kinase domain containing protein, expressed 9.52E�05 2.5156193Os.27876.S1_x_at Os08g04540 Aromatic-L-amino-acid decarboxylase, putative, expressed 0.009065542 12.716974Os.28593.S1_ at Os06g31090 GAMYB-binding protein, putative, expressed 8.56E�05 4.8248067Os.28964.S1_at Os01 g42790 Xylem cysteine proteinase 2 precursor, putative, expressed 0.002432766 2.6236691

(continued on next page)

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Table 1 (continued)

Probe ID Locus Description P value Fold change

Os.30670.S1_at Os07g22730 Ethylene-responsive transcription factor 15, putative, expressed 6.36E�04 2.481241Os.30886.S1_at Os03g61360 Epoxide hydrolase 2, putative, expressed 3.16E�04 3.1904807Os.30886.S1_x_at Os03g61360 Epoxide hydrolase 2, putative, expressed 1.51E�04 3.3876748Os.30886.2.S1_x_at Os03g61360 Epoxide hydrolase 2, putative, expressed 1.52E�04 2.9442291Os.33102.1.S1_at Os01g43740 Cytochrome P450 72A1, putative, expressed 1.63E�04 8.505603Os.33625.4.S1_ at Os01 g70850 Esterase PIR7B, putative, expressed 1.74E�05 7.1135902Os.3406.1.S1_at Os04g51160 Transposon protein, putative, unclassified, expressed 4.07E�04 7.426893Os.34496.1.S1_at Os03g61500 PGPS/D12, putative, expressed 0.017823705 3.5318103Os.34501.1.S1_ x at Os01g43890 SCPL50, putative, expressed 0.013815586 2.340366Os.34962.1.S1_at Os01g38110 Cytochrome P450 76C4, putative, expressed 0.012022875 7.763336Os.35642.2.S1_x_at Os01g58280 Cucumisin precursor, putative, expressed 0.026283357 3.703265Os.36496.1.S1_at AK063249 Unknown 1.86E�06 10.7123Os.37644.1.S1_at AK107857 Unknown 0.016586656 2.5243096Os.37773.1.S1_ at Os03g 16030 17.4 kDa class I heat-shock protein 3, putative, expressed 1.06E�05 194.37846Os.37773.1.S1_x_at Os03g 16030 17.4 kDa class I heat-shock protein 3, putative, expressed 4.45E�05 153.70242Os.37783.1.S1_a_at Os12g38064 Metallothionein-like protein 1, putative, expressed 8.45E�05 2.6023262Os.37783.2.S1_x_at Os12g38300 Metallothionein-like protein 1, putative 2.08E�04 2.4392877Os.38022.1.S1_at Os01g49710 Glutathione S-transferase GSTU6, putative, expressed 3.58E�04 3.402858Os.38992.1.A1_x_at Os08g45120 Hypothetical protein 6.20E�06 5.7553525Os.39228.1.S1_at Os10g34020 Glutathione S-transferase GSTU6, putative, expressed 9.53E�05 12.10607Os.39725.1.S1_ at Os12g30680 Retrotransposon protein, putative, unclassified 0.014868993 3.1137257Os.40007.1.S1_x_at CF318727 Unknown 0.003217326 2.5227277Os.40018.1.S1_at Os05g45410 Heat-shock factor, putative, expressed 0.015285763 2.6589158Os.409.1.S1_at Os07g15460 Metal transporter Nramp6, putative, expressed 2.17E�04 2.9199607Os.41109.1.S1_at Os07g43740 Ubiquitin-protein ligase CIP8, putative, expressed 2.60E�04 3.688651Os.41109.1.S1_x_at Os07g43740 Ubiquitin-protein ligase CIP8, putative, expressed 4.19E�04 4.1337633Os.41199.1.A1_at Os01 g43774 Cytochrome P450 72A1, putative, expressed 6.13E�05 30.16366Os.41468.1.S1_at Os01 g47050 Kelch motif family protein, expressed 0.007886614 2.7222812Os.43896.1.S1_at Os03g08900 Transparent testa 12 protein, putative, expressed 1.92E�04 5.9710608Os.44475.1.S1_at Os07g37730 Rotenone-insensitive NADH-ubiquinone oxidoreductase, mitochondrial precursor, putative, expressed 0.013024659 4.503295Os.44475.1.S1_x_at Os07g37730 0.021737227 3.4588404Os.45955.1.S1_at Os07g43260 Rotenone-insensitive NADH-ubiquinone oxidoreductase, mitochondrial precursor, putative, expressed 6.74E�05 4.5857215Os.46050.1.A1_x_at Os07g43604 0.005402513 3.8765907Os.4618.1.S1_at Os01g32670 SKP1-like protein 1 A, putative, expressed 0.00148309 2.9750328Os.46635.1.S1_x_at Os10g38350 Expressed protein 0.006748201 3.6931367Os.46670.1.S1_at Os10g31560 Expressed protein 0.026716316 5.369744Os.46698.2.S1_x_at Os10g20350 Glutathione S-transferase GSTU6, putative, expressed 0.002173139 3.308409Os.46825.1.S1_at Os10g38720 Glycine-rich cell wall structural protein 2 precursor, putative, expressed 2.31E�04 4.2465205Os.4759.1.S1_at Os09g20220 Transparent testa 12 protein, putative, expressed 1.20E�04 3.1307435Os.48053.1.A1_at AA753122 Glutathione S-transferase GSTU6, putative, expressed 0.004260087 2.6765141Os.48057.1.S1_at Os03g 13390 Glutathione S-transferase, putative, expressed 8.99E�05 4.0128818Os.4914.1.S1_s_at Os03g51459 Unknown 2.58E�04 3.0550814Os.49198.1.S1_at Os04g58090 NAD(P)H-dependent oxidoreductase, putative, expressed 0.006171342 3.0438855Os.49607.1.S1_at Os03g09880 Expressed protein 1.84E�04 5.820457Os.49648.1.S1_at Os02g54140 NHL25, putative, expressed 0.019931039 5.6354346Os.49648.1.S1_s_at Os02g54140 AIR12, putative, expressed 0.013005853 4.707556Os.49755.1.S1_at Os10g38189 17.5 kDa class II heat-shock protein, putative, expressed 9.33E�05 2.4326582Os.4978.1.S1_at Os03g54000 17.5 kDa class II heat-shock protein, putative, expressed 0.03231236 3.8442008Os.50015.1.S1_at Os06g44010 Glutathione S-transferase GSTU6, putative, expressed 0.03551279 2.4785903Os.50798.1.S1_at Os08g01480 Oligopeptide transporter 3, putative, expressed 0.006523595 8.085375Os.50903.1.S1_ at Os09g08130 OsWRKY28 - Superfamily of rice TFs having WRKY and zinc finger domains, expressed 0.011397252 7.3308306Os.50995.1.S1_at Os01 g50170 Cytochrome P450 71C4, putative, expressed 1.01E�04 2.7992146Os.51043.S1_at Os01 g07730 Indole-3-glycerol phosphate synthase, chloroplast precursor, putative, expressed 2.96E�05 2.843946Os.51147.S1_ at Os03g16960 Aspartic proteinase nepenthesin-1 precursor, putative, expressed 0.016662726 2.8433242Os.51343.S1_at Os03g 10740 Triose phosphate/phosphate translocator, non-green plastid,chloroplast precursor, putative, expressed 8.76E�04 2.653762Os.51401.S1_at AK062714 2.00E�04 3.3666098Os.51491.S1_at AK062942 CRK6, putative, expressed 6.75E�04 2.845673Os.51532.S1_at Os12g05840 Expressed protein 4.72E�04 11.565012Os.51778.S1_ at Os02g15930 Unknown 6.86E�04 26.819876Os.51787.S1_at Os12g24320 Unknown 0.001303767 5.4468565Os.51791.S1_at Os03g06970 Germin-like protein subfamily 1 member 8 precursor, putative, expressed 5.64E�05 7.1877704Os.519.1.S1_at Os01g08860 Expressed protein 0.001091599 21.840212Os.52845.S1_at AK069569 Cell Division Protein AAA ATPase family, putative, expressed 0.0391555 2.1044433Os.53150.S1_at Os08g41730 Pollenless3, putative, expressed 0.001159806 2.4826586Os.53614.S1_at Os02g51930 17.5 kDa class II heat-shock protein, putative, expressed 5.06E�04 2.6994185Os.53852.S1_ at Os04g 13210 Unknown 3.80E�04 3.460439Os.54157.S1_at Os03g55050 Proteasome subunit beta type 1, putative, expressed 2.34E�05 19.642946Os.54195.S1_at Os11g05390 Cytokinin-O-glucosyltransferase 2, putative, expressed 1.95E�04 4.267024Os.54291.S1_at Os11g42200 Multidrug resistance-associated protein 4, putative, expressed 0.014253677 3.8704424Os.54318.S1_at Os11g04290 Cytokinin-O-glucosyltransferase 2, putative, expressed 0.01285922 3.0681381Os.54615.S1_at Os06g18140 Tetracycline transporter protein, putative, expressed 0.005872716 2.8560975Os.54930.S1_at Os07g42994 Laccase LAC2-1, putative, expressed 1.65E�04 2.7788708Os.54936.S1_at Os03g57640 Cytochrome P450 94A1, putative, expressed 0.001130999 3.0025969Os.55193.S1_at Os12g42280 Indole-3-acetate beta-glucosyltransferase, putative, expressed 0.001657099 5.685966Os.55250.S1_at Os09g32000 Hydrolase, putative, expressed 9.88E�04 3.1811702Os.55429.S1_at Os06g11200 Gibberellin receptor GID1 L2, putative, expressed 0.001310258 18.189714

692 D. Chakrabarty et al. / Chemosphere 74 (2009) 688–702

Author's personal copy

Table 1 (continued)

Probe ID Locus Description P value Fold change

Os.55436.S1_at AK108093 Viviparous-14, putative, expressed 2.17E�04 9.028138Os.5562.1S1_at Os04g51150 TMV response-related gene product, putative, expressed 8.50E�05 3.1143813Os.5581.1S1_at Os03g56540 12-Oxophytodienoate reductase 2, putative, expressed 1.03E�04 4.0369015Os.55827.S1_at Os11g29870 Unknown 0.007501041 9.480225Os.55961.S1_at Os07g34280 Transposon protein, putative, unclassified, expressed 0.036349285 29.284523Os.56004.S1_at Os03g22590 Mitochondrial import inner membrane translocase subunit TIM14, putative, expressed 0.00217845 3.6265452Os.56074.S1_at Os02g03710 OsWRKY72 - Superfamily of rice TFs having WRKY and zinc finger domains, expressed 0.001699779 3.3127103Os.56349.S1_at Os07g43160 2-Hydroxyisoflavanone dehydratase, putative, expressed 0.001019277 4.2673535Os.56946.S1_at Os11g14910 MTN3, putative, expressed 1.70E�04 4.8861213Os.57142.S1_at AK111011 Expressed protein 0.027405836 5.141121Os.6157.1.S1_at Os01 g37630 Expressed protein 3.98E�04 3.4103649Os.6244.1.S1_at Os03g 13740 NADP-dependent oxidoreductase P1, putative, expressed 0.027029183 3.296992Os.6244.1.S1_ x at Os03g 13740 Unknown 0.02528493 3.044923Os.68.1.S1_at Os01 g16310 Esterase PIR7B, putative, expressed 3.59E�04 6.613592Os.6901.1.S1_at AK102892 Immediate-early fungal elicitor protein CMPG1, putative, expressed 2.13E�05 9.062847Os.7537.1.S1_at Os04g36750 Immediate-early fungal elicitor protein CMPG1, putative, expressed 0.004720983 8.80069Os.7989.1.S1_at Os02g20360 Glycosyl hydrolases family 31 protein, expressed 7.27E�04 2.6213818Os.8503.1.S1_at Os06g39110 Unknown 0.002213319 2.8095355Os.8741.1.S1_at Os03g 12820 22.0 kDa class IV heat-shock protein precursor, putative, expressed 5.06E�05 4.0049977Os.8778.1.S1_ a at Os06g11290 Tyrosine aminotransferase, putative, expressed 3.48E�04 4.160767Os.8868.1.S1_at Os10g38610 Expressed protein 1.46E�06 6.475877Os.8926.1.S1_at Os03g14180 ATP8, putative, expressed 0.038839553 3.3957715Os.8987.1.S1_ at Os03g51459 12-oxophytodienoate reductase 2, putative, expressed 3.80E�04 3.3286092Os.9017.1.S1_x_at Os07g23570 Glutathione S-transferase GSTU6, putative, expressed 0.004857687 3.2924836Os.9067.1.S1_at Os07g44140 Small heat-shock protein, chloroplast precursor, putative, expressed 0.001907748 2.374002Os.9107.1 .S1 _a_at Os09g27260 Expressed protein 0.002723023 8.033096Os.9107.2.S1_x_at Os09g27260 Cytochrome P450 72A1, putative, expressed 3.26E�05 8.08841Os.9216.1.S1_ at Os03g26210 Cytochrome P450 72A1, putative, expressed 9.98E�05 2.493362Os.9311.1.S1_at Os03g19420 TMV response-related gene product, putative, expressed 8.39E�05 2.9797976Os.9427.1.S1_at Os10g05020 TMV response-related gene product, putative, expressed 3.02E�04 2.9120028Os.9805.1.S1_at Os12g18410 Amelogenin precursor like protein, putative, expressed 0.00646947 2.5489602Os.9827.1.S1_at Os01 g37000 Nicotianamine synthase 2, putative, expressed 0.010360736 3.7558618OsAffx.11535.1. S1 _at Os01g49720 Transposon protein, putative, unclassified, expressed 9.99E�04 6.9896817OsAffx.12887.2.S1_at Os03g 16920 Expressed protein 3.12E�04 13.968477OsAffx.12887.2.S1 s at Os03g 16920 Carboxyl-terminal peptidase, putative, expressed 0.003261165 9.018633OsAffx.13376.1 .S1 _s_at Os03g46110 Glutathione S-transferase GSTU6, putative, expressed 7.98E�05 4.9117975OsAffx.14069.1 .S1 _at Os04g31290 Heat-shock cognate 70 kDa protein, putative, expressed 5.37E�05 5.828531OsAffx.14069.1 .S1 _x_at Os04g31290 Heat-shock cognate 70 kDa protein, putative, expressed 0.002657257 7.6790915OsAffx.15530.1 .S1 _at Os06g20960 expressed protein 0.001360526 4.939891OsAffx.16082.1 .S1_at Os07g04560 Helix-loop-helix DNA-binding domain containing protein 0.028775755 3.9059362OsAffx.16847.1 .S1_s_at Os08g05970 Helix-loop-helix DNA-binding domain containing protein 2.07E�05 11.13056OsAffx.17942.1 .S1_at Os09g28160 Jasmonate O-methyltransferase, putative 0.013777263 3.5441225OsAffx.18335.1 .S1 _at Os10g21630 NAC domain-containing protein 94, putative 6.17E�04 5.8136163OsAffx.18681.1 .S1 _s_at Os11g05170 Expressed protein 0.001444345 3.4954681OsAffx.18687.1 .S1 _x_at Os11g05390 Phosphate carrier protein, mitochondrial precursor, putative, expressed 8.79E�05 3.7125852OsAffx.19095.1 .S1 _at Os11g31060 Expressed protein 0.030268421 3.8989778OsAffx.20211.1.S1_at Os10g36580 Expressed protein 0.001440553 3.6964302OsAffx.23327.2.S1_at Os01 g21620 Tetracycline transporter protein, putative, expressed 3.89E�04 9.118891OsAffx.23652.1.S1_at CT832378 BAG domain containing protein, expressed 3.97E�04 12.046585OsAffx.24011.1.S1_at Os01g72150 Expressed protein 1.79E�04 4.5925746OsAffx.25008.1.S1_s_at Os03g07510 conserved hypothetical protein 1.76E�05 3.1359076OsAffx.26357.1.S1_at Os04g36830 Unknown 0.011450969 3.9587166OsAffx.27605.1.S1_s_at Os06g13190 Glutathione S-transferase, putative, expressed 0.00643303 2.4058986OsAffx.27606.1.S1_at Os06g13220 Expressed protein 0.002235957 2.136483OsAffx.27741.1.S1_s_at Os06g20820 Expressed protein 0.021339474 3.0853343OsAf fx.28152.1 .S1 _s_at Os06g47530 Expressed protein 1.74E�04 2.5651634OsAffx.28276.1.S1_x_at Os07g04970 Growth regulator, putative, expressed 0.001439992 3.1908412OsAffx.2928.1.S1_ at Os02g40500 Expressed protein 7.54E�04 6.553097OsAffx.2947.1.S1_at Os02g43370 Serine/threonine-protein kinase receptor precursor, putative, expressed 2.17E�05 3.3124826OsAffx.31223.1 .S1 _at Os11g30290 Transferase, putative, expressed 0.002563707 7.806357OsAffx.32080.1.S1_at Os12g38290 OsGrx_C2.1 - glutaredoxin subgroup I, expressed 1.33E�04 13.293832OsAffx.32233.1.S1_x_at Os04g16774 Transposon protein, putative, unclassified, expressed 0.032152306 6.1103687OsAffx.3313.1.S1_at Os03g24690 PHD finger-like domain-containing protein 5A, putative 0.01615539 4.6233735OsAffx.4084.1.S1_at Os04g44250 Metallothionein-like protein 1, putative, expressed 3.71E�05 3.4215672Os Affx.5 702.1 .S1 _s_at Os08g04560 Conserved hypothetical protein 0.001414529 3.7362373OsAffx.6464.1.S1_at Os09g31990 Terpene synthase 7, putative, expressed 2.01E�06 6.4562354OsAffx.6703.1.S1_s_at Cytokinin-O-glucosyltransferase 3, putative, expressed 1.41E�04 4.9304276OsAffx.7332.1.S1_ at Os11g37940 Aromatic-L-amino-acid decarboxylase, putative, expressed 0.002609524 3.8680687OsAffx.7530.1.S1_s_at Os12g08930 Expressed protein 0.002049745 4.0377316OsAffx.7606.1.S1_at Os12g15680 Unknown 0.003847878 3.3820632OsAffx.7690.1.S1_at Os12g24650 Win2 precursor, putative, expressed 0.022749351 3.2710202OsAffx.7826.1.S1_at Os12g38051 Expressed protein 1.34E�04 28.024647

L-Ascorbate oxidase precursor, putative, expressedLeucine aminopeptidase 1, chloroplast precursor, putative, expressedMetallothionein-like protein 1, putative, expressed

(continued on next page)

D. Chakrabarty et al. / Chemosphere 74 (2009) 688–702 693

Author's personal copy

Table 1 (continued)

Probe ID Locus Description P value Fold change

DownregulatedOs.10075.1 .S1_at Os07g30090 Actin-depolymerizing factor 1, putative, expressed 1.91E�04 7.811657Os.10394.2.S1_at Os08g42030 Peroxidase 73 precursor, putative, expressed 3.16E�04 2.4578886Os.10410. .S1_at Os08g02210 Expressed protein 0.001710042 2.8268347Os.10556. .S1_ at Os02g15860 Expressed protein 0.002106316 2.4025197Os.10583. S1_at Os06g04920 Zinc finger protein, putative, expressed 0.001844075 2.7217982Os.11129. S1_at Os10g35840 Glutamyl-tRNA reductase, chloroplast precursor, putative, expressed 3.90E�05 2.5336297Os.11551. .S1_at Os05g04410 Peroxidase 2 precursor, putative, expressed 6.83E�05 4.135939Os.11555. .S1_at Os05g04450 Peroxidase 12 precursor, putative, expressed 1.64E�04 5.942772Os.11920. S1_s_at Os04g39320 Expressed protein 1.74E�04 2.6711402Os.12096.3.S1_a_at Os12g01530 Ferritin-1, chloroplast precursor, putative, expressed 7.02E�04 3.44587Os.12096.3.S1_x_at Os12g01530 Ferritin-1, chloroplast precursor, putative, expressed 8.87E�05 3.2939792Os.12096.4.S1_s_at Os11g01530 Ferritin-1, chloroplast precursor, putative, expressed 3.63E�04 4.58151Os.12313.1. S1_at Os12g08770 Photosystem I reaction center subunit N, chloroplast precursor, putative, expressed 0.004422839 2.926885Os.12340.1.S1_at Os01g11730 Esterase precursor, putative, expressed 0.04046856 2.813869Os.12393.2.S1_at Os08g01380 Ferredoxin-1, chloroplast precursor, putative, expressed 3.30E�04 3.4880705Os.12393.2.S1_s_at Os08g01380 Ferredoxin-1, chloroplast precursor, putative, expressed 6.10E�04 3.6261017Os.12393.2.S1_x_at Os08g01380 Ferredoxin-1, chloroplast precursor, putative, expressed 7.08E�04 2.7496037Os.12501.1.S1_at Os01g55940 Indole-3-acetic acid-amido synthetase GH3.2, putative, expressed 8.30E�04 5.3497086Os.12593.1.S1_s_at Os08g36900 Alpha-amylase isozyme 3E precursor, putative, expressed 0.006217466 5.007596Os.13392.1.S1_at Os03g36560 Peroxidase 27 precursor, putative, expressed 1.91E�04 3.6938155Os.142.2.A1_a_at Os01g25484 Ferredoxin-nitrite reductase, chloroplast precursor, putative, expressed 2.94E�04 3.6687436Os.15798.1.S1_at Os06g07040 OsIAA20 – auxin-responsive Aux/IAA gene family member, expressed 1.51E�05 8.283555Os.15854.1.S1_ at BM418911 Unknown 0.02844432 6.381501Os.16010.1.S1_at Os05g42150 Indole-3-acetic acid-amido synthetase GH3.4, putative, expressed 4.34E�08 6.8799257Os.1606.1 .S1_at Os04g56430 CRK5, putative, expressed 0.026852988 2.7703824Os.16500.1.S1_at Os01 g27630 Glutathione S-transferase GSTF2, putative, expressed 0.013016445 6.2901974Os.170.3.S1_at Os01g64120 Ferredoxin-6, chloroplast precursor, putative, expressed 5.20E�04 4.4330897Os.17076.1.S1_at Os09g 10340 Cytochrome P450 71 D7, putative, expressed 0.001724992 2.7402346Os.17108.1.S1_at Os12g38170 Pathogenesis-related protein 5 precursor, putative, expressed 0.032476246 2.454789Os.17921.1.S1_at Os05g47540 Phosphoethanolamine N-methyltransferase, putative, expressed 8.46E�04 2.3824267Os.18006.1.S1_a_at Os01g72810 Secreted glycoprotein, putative, expressed 0.022367688 3.1849325Os.18598.1.S1_at Os03g49260 Lipoxygenase 1, putative, expressed 0.030886626 2.6591372Os.18818.1.S1_a_at Os01 g12710 Oxidoreductase, putative, expressed 0.013631964 2.7899199Os.18981.1.S1_x_at Os07g18120 Abscisic-aldehyde oxidase, putative, expressed 0.001979924 2.5359845Os.19761. .S1_at Os01 g03950 Alpha-D-xylosidase, putative, expressed 1.40E�04 2.8862777Os.19863.1.S1_a_at AB069966 Unknown 0.00279149 2.4677284Os.20290.1.S1_ at Os07g01410 Peroxidase 1 precursor, putative, expressed 0.00366541 5.7392135Os.20572.2.S1_at Os03g22610 Peptidyl-tRNA hydrolase, putative, expressed 0.040368628 2.8947356Os.2123.1.S1_at Os01g54300 Hydrolase, hydrolyzing O-glycosyl compounds, putative, expressed 1.69E�04 3.5025482Os.21893.1.S1_x_at Os04g59160 Peroxidase 12 precursor, putative, expressed 8.74E�04 12.596871Os.21893.2.S1_at Os04g59160 Peroxidase 12 precursor, putative, expressed 5.50E�05 8.326304Os.21894.1.S1_ at Os10g31720 Glycine-rich cell wall structural protein 2 precursor, putative, expressed 0.004958325 2.7112608Os.23059.1.S1_s_at CA754790 Unknown 6.37E�04 3.940176Os.23256.1.S1_at Os02g56120 OsIAA9 - Auxin-responsive Aux/IAA gene family member, expressed 5.40E�05 4.83383Os.23440.1.A1_at Os04g46910 Actin-depolymerizing factor, putative, expressed 2.70E�05 6.1264358Os.23471.2.S1_at Os11g18366 Cycloartenol synthase, putative, expressed 0.010878207 5.003564Os.24041.1.A1_at CB622792 Unknown 0.002861068 7.6912813Os.24865.1.A1_at Os05g09724 Stem 28 kDa glycoprotein precursor, putative, expressed 4.01E�05 3.013568Os.24972.1.S1_ at Os02g09930 CSLA1 – cellulose synthase-like family A; mannan synthase, expressed 1.10E�04 2.4206884Os.264.1.S1_at Os04g59260 Peroxidase 12 precursor, putative, expressed 0.001089746 3.6702936Os.26983.1.S1_s_at Os01g04570 Ser/Thr protein kinase, putative, expressed 0.010057966 2.482684Os.27417.1.S1_at Os01 g52170 HCF101, putative, expressed 0.001677459 2.929378Os.27961.1.S1_at Os02g21890 Expressed protein 3.01E�06 2.4014084Os.28098.1.S1_at Os10g32810 Beta-amylase, putative, expressed 0.001869579 3.386423Os.2957.1.S1_at Os03g25330 Peroxidase 66 precursor, putative, expressed 8.24E�05 3.7252936Os.2961.1.S1_at Os03g25340 Peroxidase 47 precursor, putative, expressed 0.001113532 4.6347456Os.30269.1.S1_at Os07g01340 Gibberellin 2-beta-dioxygenase 7, putative, expressed 4.79E�04 4.150904Os.31056.1.S1_at Os01 g03900 Expressed protein 3.52E�04 2.467375Os.31778.2.S1_at Os01g61610 Gibberellin 20 oxidase 2, putative, expressed 0.004479461 2.8396807Os.31778.2.S1_x_at Os01g61610 Gibberellin 20 oxidase 2, putative, expressed 0.008484233 2.688959Os.31893.1.S1_at Os01 g19290 Nodulin-like protein, putative, expressed 3.84E�04 3.9868486Os.32459.1.S1_at Os01g03310 Bowman-Birk type bran trypsin inhibitor precursor, putative, expressed 0.010813518 2.8557549Os.32471.1.S1_at Os03g55410 Peroxidase 51 precursor, putative, expressed 0.02173449 2.330059Os.32630.1.S1_at Os10g32700 Hypersensitive-induced response protein, putative, expressed 0.002833107 3.7819426Os.32667.1.S1_at Os01 g12070 Endoglucanase 1 precursor, putative, expressed 1.09E�04 27.994522Os.32667.2.S1_x_at Os01 g12070 Endoglucanase 1 precursor, putative, expressed 1.67E�05 34.68029Os.32727.1.S1_at Os01g43480 ATP binding protein, putative, expressed 5.37E�05 5.599682Os.34118.1.S1_at AK072776 Unknown 2.49E�04 2.5143225Os.34488.1.S1_at Os03g61590 Expressed protein 5.16E�06 2.6295664Os.35921.1.S1_at Os01g60730 RING-H2 finger protein ATL5A, putative, expressed 2.41E�05 3.2795386Os.36278.1.S1_x_at Os03g03724 Expressed protein 0.011838879 3.4040573Os.36346.2.S1_at Os01g67030 Dopamine beta-monooxygenase, putative, expressed 2.36E�04 10.375564Os.36663.1.S1_at Os01 g27390 Glutathione S-transferase GSTF2, putative, expressed 8.19E�05 2.4601579Os.37622.1.S1_at Os01g67010 Expressed protein 0.001369375 16.68239

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Table 1 (continued)

Probe ID Locus Description P value Fold change

Os.37876.1.S1_at Os10g39920 Expressed protein 0.001203166 2.5344646Os.39991.1.A1_at Os09g28420 Alpha-amylase isozyme 3B precursor, putative, expressed 0.03368021 5.6550894Os.42521.1.S1_at Os01 g61230 Dihydroflavonol-4-reductase, putative, expressed 7.91E�05 3.6915357Os.46052.1.S1_at Os10g40440 Cortical cell-delineating protein precursor, putative, expressed 0.001701215 4.468893Os.46538.1.S1_ at Os10g11270 Flavonol 3-sulfotransferase, putative, expressed 0.007517009 2.8623204Os.46551.1.S1_at Os10g17260 Flavonoid 3-monooxygenase, putative, expressed 0.001267691 5.2067237Os.48074.1.A1_at Os05g19910 Taxadien-5-alpha-ol O-acetyltransferase, putative, expressed 9.95E�04 2.5591729Os.4844.1.S1_ at Os05g06970 Peroxidase 1 precursor, putative, expressed 0.002160482 2.9833415Os.49116.S1_at Os02g45320 ATPP2-A13, putative, expressed 1.90E�05 3.5894277Os.49545.S1_at Os11g32030 Sex determination protein tasselseed-2, putative, expressed 8.93E�05 3.0498958Os.49608.S1_at Os03g15340 Chemocyanin precursor, putative, expressed 1.50E�04 3.151068Os.4965.1S1_at Os03g59440 Disease resistance response like protein, putative, expressed 2.51E�04 11.652224Os.49663.S1_ at Os02g39000 DNA binding protein, putative, expressed 0.001065043 5.482216Os.50125.S1_at Os04g57860 Endoglucanase precursor, putative, expressed 2.42E�04 10.362979Os.5037.1S1_s_at Os12g16010 Sex determination protein tasselseed-2, putative, expressed 2.88E�05 3.2635279Os.50383.S1_at Os06g05430 Expressed protein 0.002191499 3.8365304Os.50481.S1_at Os06g50140 Endoglucanase 1 precursor, putative, expressed 9.05E�04 2.579006Os.50555.S2_at Os11g03860 Ser/Thr protein kinase, putative, expressed 5.90E�04 3.6676476Os.50606.S1_at Os12g05394 Serine/threonine-protein kinase PK7, putative, expressed 1.00E�04 3.2668247Os.5066.1S1_ at Os01g12000 Expressed protein 0.002295664 3.3522031Os.5086.1S1_at Os03g05640 Inorganic phosphate transporter 1–7, putative, expressed 9.07E�05 5.68602Os.51266.S1_at Os08g02200 Expressed protein 0.025316738 3.6353467Os.51473.2.2.S1_x_at Os05g48160 Electron carrier/electron transporter/iron ion binding protein, putative, expressed 9.03E�04 4.1588793Os.51753.A1_at Os12g35340 OsGrx_C11 – glutaredoxin subgroup III, expressed 0.044551436 3.8831353Os.51831.S1_at Os02g15340 NAC domain-containing protein 76, putative, expressed 0.007797421 3.713341Os.51835.S1_a_at Os05g48700 Gibberellin 2-beta-dioxygenase, putative, expressed 2.71E�05 6.2404566Os.52112.S1_at Os07g48320 Acid phosphatase 1 precursor, putative, expressed 1.76E�04 3.147764Os.52171.S1_at Os06g38950 ATATH6, putative, expressed 1.71E�04 5.6394796Os.5242.1S1_at Os01g58260 Peptidas/subtilase, putative, expressed 7.42E�04 3.9128084Os.52449.S1_at Os09g19830 YDG/SRA domain containing protein, expressed 0.01085348 2.582505Os.52536.S1_at Os04g43800 Phenylalanine ammonia-lyase, putative, expressed 6.28E�04 2.6888363Os.53348.2.2.S1_at Os11g17014 NB-ARC domain containing protein, expressed 3.14E�04 7.886472Os.53639.S1_at Os04g37650 Fucosyltransferase 7, putative, expressed 0.040104236 2.328965Os.54019.S1_at Os05g15690 Beta-expansin 2 precursor, putative, expressed 3.84E�04 4.779497Os.54066.S1_at Os02g33670 Expressed protein 0.004811036 3.3618808Os.54109.S1_at Os03g38800 Mitochondrial protein, putative, expressed 9.35E�04 2.64233Os.54148.S1_at Os02g36350 Expressed protein 4.25E�04 4.6888766Os.54203.S1_at Os09g32260 ANAC079/ANAC080, putative, expressed 0.003454529 2.765276Os.54351.S1_at Os08g31250 Protein binding protein, putative, expressed 0.006244707 2.9664717Os.54354.S1_at Os03g27480 Retinoid-inducible serine carboxypeptidase precursor, putative, expressed 5.86E�05 4.1880245Os.54453.S1_at Os08g44220 Expressed protein 0.002151351 10.64751Os.54472.S1_at Os07g32060 Anthocyanidin 5,3-O-glucosyltransferase, putative, expressed 7.67E�04 2.9080863Os.54653.S1_ at Os06g09870 Glycine-rich cell wall structural protein 2 precursor, putative, expressed 3.58E�09 146.52873Os.54770.S1_at Os02g03120 Endoglucanase 1 precursor, putative, expressed 4.85E�04 3.5744443Os.55065.S1_at Os06g04540 DNA-binding protein, putative, expressed 9.07E�05 2.7976108Os.55284.S1_at Os05g07140 RING-H2 finger protein ATL3F, putative, expressed 1.41E�04 3.0170562Os.55488.S1_at Os08g28790 Dirigent-like protein pDIR3, putative, expressed 0.002438948 3.4370375Os.55511.S1_at Os04g45520 Integral membrane protein, expressed 4.87E�06 18.335352Os.55696.S1_at Os12g39100 Expressed protein 9.66E�04 3.0989177Os.55785.S1_ at Os07g31610 Peroxidase 7 precursor, putative, expressed 2.00E�04 10.536312Os.55837.S1_at Os01g10370 Transcriptional factor TINY, putative, expressed 0.00841639 5.58046Os.56824.1.S1_at Os06g47750 Phytosulfokine receptor precursor, putative, expressed 0.002613655 3.2945552Os.57112.1.S1 at Os04g56950 Jasmonate O-methyltransferase, putative, expressed 0.001286196 3.4668465Os.57170.1.S1_at Os03g41130 Expressed protein 2.01E�04 25.739874Os.5 7289.1 .S1 _at Os12g26960 Expressed protein 5.20E�06 5.7003336Os.57309.1.S1_at Os04g19960 Expressed protein 3.00E�04 5.668042Os.5963.1.S1_at Os04g39600 Fasciclin-like arabinogalactan protein 7 precursor, putative, expressed 6.91E�05 9.22947Os.6271.1.S1_ at Os07g39320 DNA binding protein, putative, expressed 6.56E�05 6.1384125Os.6610.1.S1_at Os07g28400 Kinesin like protein, putative, expressed 1.09E�04 4.1943994Os.6649.1.S1_at Os01g74110 Zinc transporter 2 precursor, putative, expressed 0.003314698 2.8316412Os.6651.1.S1_at Os07g43670 Ribonuclease 1 precursor, putative, expressed 2.72E�05 4.2721634Os.7112.1 .S1_at Os03g05500 LOB domain protein 16, putative, expressed 0.001200973 10.858128Os.7342.1.S1_at Os02g57490 LOB domain protein 16, putative, expressed 0.001922563 9.4122305Os.7445.1.S1_at Os04g39489 AAP7, putative, expressed 0.009140713 2.6275334Os.7826.1 .S1_ a at Os05g10730 Multidrug resistance-associated protein 6 precursor, putative, expressed 0.004830555 2.8675768Os.7938.1.S1_at Os04g59190 Peroxidase 2 precursor, putative, expressed 4.56E�06 34.17653Os.7986.1.S1_at Os03g03910 Catalase-1, putative, expressed 0.001756811 4.5199585Os.8089.1.S1_at Os02g17240 Pollen-specific kinase partner protein, putative, expressed 0.04189629 2.532701Os.8356.1.S1_at AU095460 Unknown 0.005462986 2.9954367Os.8648.1.S1_s_at Os08g37250 Patatin class 1 precursor, putative, expressed 2.45E�04 3.4730833Os.8673.1.S1_at Os11g20090 O-Methyltransferase ZRP4, putative, expressed 0.021641677 3.0292666Os.8682.1.S1_a_at Os10g08780 Expressed protein 0.004425649 2.5078702Os.8682.2.S1_x_at Os10g08780 Expressed protein 0.005245081 2.3077297Os.8833.1.S1_at Os04g48070 Homeobox protein OCL1, putative, expressed 1.47E�04 3.2882292Os.8855.1.S1_at Os02g31840 Expressed protein 0.001008545 3.0511832Os.9086.1.S1_at Os03g 10210 DNA binding protein, putative, expressed 3.99E�05 3.0027695

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and Os07g15460, which complement the fet3fet4 yeast mutantdefective both in low- and high-affinity iron transports (Curieet al., 2000), are also upregulated on challenge with both AsVand AsIII. Two glutathione-related transporters (Os03g54000 andOs04g13210), two MATE-efflux family proteins (Os10g20350 andOs03g08900), and one zinc/iron-transport family protein(Os03g46470) are upregulated specifically in AsV stress. Concomi-tantly, one multidrug-resistance protein (MRP; Os01g50100) isupregulated in both AsV and AsIII stresses. It has recently been re-ported that AsIII interacts with MRPs, and GSH appears to play amajor role in this process (Liu et al., 2001). In vitro studies suggestthat the arsenic species are either cotransported with GSH or as tri-GSH conjugates (Rea, 1999). Moreover, one MATE-efflux protein(Os03g62270), one phosphate:H+-symporter (Os03g05640), twoamino acid transporters (Os04g39489 and Os04g56470), oneATP-binding cassette transporter (Os06g38950), and one zinc

transporter (Os01g74110) are downregulated specifically in AsVstress, whereas one aquaporin (Os10g36924) is downregulated inAsIII stress only.

Twenty-nine transcription factors are differentially expressed inarsenic stress. Among these, one zinc-finger C3HC4-type protein(Os07g43740) and one helix-turn-helix protein (Os06g39240) areupregulated in both AsV and AsIII stresses. Two helix-loop-helix(HLH) DNA-binding-domain containing proteins (Os04g31290and Os03g26210) and two WRKY proteins (Os06g44010 andOs11g29870) are specifically upregulated in AsV stress. Databasesearch has resulted in the identification of one nearest homologof Os04g31290 in tomato which has been shown to be the masterregulator of iron uptake (Ling et al., 2002). In rice, a genome-wideanalysis of the basic HLH (bHLH) gene family indicates that thisgene is not expressed in any tissue, as reported by Li et al.(2006). This suggests that the bHLH might be an arsenate-regu-

Table 1 (continued)

Probe ID Locus Description P value Fold change

Os.9328.1.S1_at Os02g40730 Ammonium transporter 1, member 2, putative, expressed 8.25E�05 8.007161Os.9637.1.S1_at Os06g48050 Expressed protein 5.47E�04 4.9033127Os.9660.1.S1_at Os09g23300 Protein CCC1, putative, expressed 0.00191456 3.528239Os.9885.1.S1_x_at Os03g61090 Expressed protein 1.71E�04 3.586686Os.9885.2.S1_at Os03g61090 Expressed protein 0.001386923 5.313668Os.9885.2.S1_x_at Os03g61090 Expressed protein 2.26E�04 3.9017758OsAffx.12171.1 .S1_at Os10g03400 CRAL/TRIO, N-terminus family protein, expressed 0.001010625 3.475365OsAffx.12569.1 .S1 _at Os02g49850 Chemocyanin precursor, putative, expressed 8.05E�04 2.9766252OsAffx.14278.1 .S1 _at Os02g0731400 Unknown 0.005550358 3.5287218OsAffx.14436.1 .S1 _at Os04g56470 AAP7, putative, expressed 5.25E�05 2.8300571OsAffx.14448.1 .S1 _at Os04g56930 Beta-fructofuranosidase, insoluble isoenzyme 5, putative, expressed 4.83E�06 4.1287985OsAffx.15474.1 .S1_ s at Os06g17450 Expressed protein 5.70E�04 3.080881OsAffx.17374.1 .S1_at Os08g37250 Patatin class 1 precursor, putative, expressed 0.001013363 4.578216OsAffx.17516.1.S1_at Os08g44940 LOB domain protein 16, putative 0.028639052 3.4591627OsAffx.18126.1 .S1 _at Os10g04450 Expressed protein 1.73E�04 2.7185562OsAffx.18919.1.S1_s_at Os11g18366 Cycloartenol synthase, putative, expressed 0.008048005 3.6889255OsAffx.19559.1 .S1 _at Os12g08790 Expressed protein 0.001985192 2.8134394OsAffx.19632.1 .S1 _at Os12g12590 NADP-dependent oxidoreductase P1, putative, expressed 0.001160866 5.5819883OsAffx.20155.1.S1_ at Os10g39890 Pollen proteins Ole e I family protein, expressed 3.32E�04 6.8564773OsAffx.20168.1.S1_at Os10g39160 Peroxidase 2 precursor, putative 0.002169282 7.708713OsAffx.20242.2.S1_at Os10g34040 Nodulin, putative, expressed 4.04E�04 3.448851OsAffx.22888.1 .S1_ at NM_183766.1 Unknown 0.004985553 3.5152092OsAffx.22888.1.S1_x_at NM_183766.1 Unknown 0.004590788 3.2317755OsAffx.22919.1.S1_x_at Os01g18950 Cationic peroxidase 1 precursor, putative 0.002343593 20.773216OsAffx.22919.2.S1_x_at Os01g18970 Peroxidase 28 precursor, putative 5.23E�04 10.62063OsAf fx.23104.1 .S1_at Os01g03914 Metal tolerance protein C3, putative, expressed 0.001166996 9.285023OsAffx.24297.1 .S1_ at Os02g17900 Xyloglucan endotransglucosylase/hydrolase protein 26 precursor, putative, expressed 1.33E�05 17.687937OsAffx.24928.1.S1_at Os02g17880 Xyloglucan endotransglucosylase/hydrolase protein 26 precursor, putative 4.41E�04 10.852989OsAffx.25840.1.S1_at Os04g02560 Expressed protein 1.01E�05 6.3156834OsAffx.27098.1.S1_at Os05g31160 Peptide chain release factor 2, putative, expressed 1.36E�04 8.349067OsAffx.27508.1.S1_at Os07g44480 Peroxidase, putative, expressed 7.81E�04 24.422766OsAffx.27946.1.S1_at Os06g35060 Heavy metal-associated domain containing protein, expressed 2.45E�04 4.120428OsAffx.28364.1.S1_x_at Os07g09520 Expressed protein 0.001991753 3.2774386OsAffx.28409.2.S1_s at Os07g12240 Calmodulin TaCaM2-1, putative 1.25E�04 7.1663985OsAffx.28501.1.S1_x_at Os07g18120 Abscisic-aldehyde oxidase, putative, expressed 0.002997365 3.0399554OsAffx.28771.1.S1_at Os07g35940 Beta-amylase, putative 0.0356296 6.729333OsAffx.28900.1.S1_at Os07g44460 Peroxidase 66 precursor, putative 2.65E�04 7.03718OsAffx.28900.1.S1_x_at Os07g44460 Peroxidase 66 precursor, putative 2.47E�05 12.113911OsAffx.29448.1.S1_at Os08g31170 Protein binding protein, putative 3.71E�04 4.7105365OsAffx.30613.1.S1_x_at Os02g 14250 Transposon protein, putative, unclassified 3.90E�04 16.046747OsAffx.30613.3.S1_at Os02g32170 Transposon protein, putative, unclassified 0.001301376 15.146397OsAffx.30613.3.S1_x_at Os10g30680 Transposon protein, putative, unclassified 1.20E�04 14.035327OsAffx.31010.1.S1_at Os11g16970 Carnitine racemase like protein, putative, expressed 3.68E�05 2.3245091OsAffx.31011.1 .S1_at Os11g16990 Hypothetical protein 2.79E�05 7.552901OsAffx.31072.1 .S1 _at Os11g20160 O-Methyltransferase ZRP4, putative, expressed 1.9 6E�04 11.387556OsAffx.3672.1.S1_at Os03g62270 Antiporter/drug transporter/transporter, putative, expressed 8.93E�05 2.6762497OsAffx.4107.1.S1_s_at Os04g46350 Homeobox-leucine zipper protein ATHB-4, putative, expressed 5.08E�05 3.873269OsAffx.4172.1.S1_at Os04g53710 Early nodulin-like protein 1 precursor, putative, expressed 6.27E�04 7.9086924OsAffx.4286.1.S1_at Os05g09410 Histidine-containing phosphotransfer protein 4, putative, expressed 0.001632553 3.445665OsAffx.4731.1.S1_s_at Os06g05430 Expressed protein 0.001648334 4.413975OsAffx.4907.1.S1_x_at Os06g22919 Xyloglucan endotransglucosylase/hydrolase protein 21 precursor, putative, expressed 1.42E�05 10.285092OsAffx.5450.1.S1_at Os07g26460 Pleckstrin homology domain containing, family A, putative 3.51E�05 6.5833235OsAffx.5542.1.S1_at Os07g35860 Uclacyanin-2 precursor, putative, expressed 5.79E�06 10.528939Os Affx.5 729.1 .S1 _at Os08g07070 Expressed protein 0.002952245 16.71244OsAffx.6248.1.S1_at Os09g 12240 serine/threonine-protein kinase BRI1-like 1 precursor, putative, expressed 0.006442346 3.4852114

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Table 2List of probe sets up- and downregulated by AsIII stress in rice.

Probe ID Locus Description P value Fold change

UpregulatedOs.10171.1 .S1_at Os03g01300 Cortical cell-delineating protein precursor, putative, expressed 1.25E�04 2.5363476Os.10933.1.S1_at Os01g50910 Late embryogenesis abundant protein, group 3, putative, expressed 0.012543627 2.8060963Os.11046.1 .S1_at Os06g39240 Endothelial differentiation-related factor 1, putative, expressed 0.03825447 3.8745317Os.11164.1.S1_at Os07g03279 Pathogenesis-related protein 1 precursor, putative, expressed 0.003292917 2.0368955Os.11266.1 .S1_at Os04g27060 Auxin-induced protein PCNT115, putative, expressed 0.006280016 2.4047935Os.12112.1 .S1_at Os01g60830 Expressed protein 0.001574702 2.8673124Os.12293.2.S1_at Os01g25510 Alpha-amylase isozyme C, putative, expressed 0.017797368 2.042217Os.12452.1.S1_s_at Os08g36910 Alpha-amylase isozyme 3D precursor, putative, expressed 0.003928359 3.4433072Os.12551.1.S1_s_at Os05g46480 Late embryogenesis abundant protein, group 3, putative, expressed 0.03010704 2.1109178Os.12593.1.S1_s_at Os08g36900 Alpha-amylase isozyme 3E precursor, putative, expressed 0.006452816 2.4677641Os.12812.1.S1_at Os05g28210 Embryonic abundant protein 1, putative, expressed 2.28E�04 2.2030737Os.1316.1.S2_at Os02g04780 Expressed protein 0.011185423 3.6864135Os.13715.2.S1_x_at Os12g 16880 Prolamin PPROL 17 precursor, putative, expressed 0.003449937 7.3691444Os.13972.1.S1_at Os07g44430 Peroxiredoxin, putative, expressed 0.02493674 2.9288552Os.14381.1.S1_at Os03g28300 ATP binding protein, putative, expressed 2.68E�06 2.0714645Os.14921.1.S1_ at Os03g56930 Protein app1, putative, expressed 0.001333023 5.3034315Os.16041.1.S1_at Os03g 16940 Glyoxalase family protein superfamily, putative, expressed 0.034059606 3.3052177Os.18939.1 .S1_s_at AK066114 Unknown 0.00325386 7.7354345Os.19822.1.S1_at Os03g09970 Sulfate transporter 1.2, putative, expressed 8.81E�04 10.259973Os.19895.1 .S1_at Os11g46860 Protein kinase, putative, expressed 1.35E�04 2.1099746Os.2376.1.S1_at Os03g17480 IN2-1 protein, putative, expressed 0.005813882 2.053895Os.25955.1.S1_at Os11g34940 Plastid-specific ribosomal protein 6 precursor, putative, expressed 5.67E�04 50.003994Os.30366.1.S1_at Os07g45080 Expressed protein 6.45E�04 2.8038397Os.30886.1.S1_at Os03g61360 Epoxide hydrolase 2, putative, expressed 5.79E�05 2.0787106Os.30886.1.S1_x_at Os03g61360 Epoxide hydrolase 2, putative, expressed 6.37E�04 2.0529706Os.31022.1.S1_at Os06g02040 Seed maturation protein, putative, expressed 0.04766129 4.626753Os.31088.1.S1_at Os01g26912 OsGrx_C7.2 – glutaredoxin subgroup III, expressed 0.002260646 2.7883646Os.323.1.S1_at Os07g03710 Pathogenesis-related protein PRB1-3 precursor, putative, expressed 3.46E�04 2.157379Os.32360.1.A1_at Os04g58760 Plant integral membrane protein TIGR01569 containing protein, expressed 6.89E�04 2.1511366Os.33625.4.S1_at Os01 g70850 Esterase PIR7B, putative, expressed 0.007290491 2.4272804Os.3406.1.S1_at Os04g51160 Transposon protein, putative, unclassified, expressed 0.002194945 2.8553603Os.3419.1.S1_ a at Os01g50700 Dehydrin family protein, expressed 0.04274455 2.960579Os.34496.1.S1_at Os03g61500 PGPS/D12, putative, expressed 5.05E�05 2.378092Os.36307.1.S1_at Os04g35490 Expressed protein 0.01632564 4.949016Os.36496.1.S1_at AK063249 Unknown 0.008845395 2.5448942Os.37558.1.S1_at Os01g06630 Small hydrophilic plant seed protein, expressed 0.013775184 2.9894Os.37773.S1_ at Os03g 16030 17.4 kDa class I heat-shock protein 3, putative, expressed 0.014657695 10.801911Os.37773.S1_x_at Os03g 16030 17.4 kDa class I heat-shock protein 3, putative, expressed 0.007473562 12.23972Os.41109.S1_at Os07g43740 Ubiquitin-protein ligase CIP8, putative, expressed 6.94E�04 2.50485Os.41109.S1_x_at Os07g43740 Ubiquitin-protein ligase CIP8, putative, expressed 0.001105405 2.811713Os.43896.S1_at Os03g08900 Transparent testa 12 protein, putative, expressed 9.72E�04 2.679793Os.44475.S1_at Os07g37730 Rotenone-insensitive NADH-ubiquinone oxidoreductase, mitochondrial precursor, putative, expressed 7.85E�04 2.2664042Os.46021.S1_ at Os01 g46580 0.010744967 2.3006623Os.46582.S1_at Os10g36180 ARP2/3 complex 34 kDa subunit, putative, expressed 0.027952235 2.7812102Os.47380.S1_at Os04g46810 Expressed protein 2.63E�04 2.6673694Os.49648.S1_at Os02g54140 Cortical cell-delineating protein precursor, putative, expressed 0.004046949 2.7051115Os.49648.S1_s_at Os02g54140 17.5 kDa class II heat-shock protein, putative, expressed 0.007435185 3.1128614Os.50019.S1_at Os03g04080 17.5 kDa class II heat-shock protein, putative, expressed 0.009377864 5.8694215Os.50053.A1_at Os07g05940 Expressed protein 0.008708942 3.901959Os.5130.1S1_ at Os04g34630 Viviparous-14, putative, expressed 0.001003445 2.241471Os.51532.S1_at Os12g05840 Peroxidase 72 precursor, putative, expressed 0.008077763 2.4465659Os.51641.S1_at Os07g03590 Germin-like protein subfamily 1 member 8 precursor, putative, expressed 0.009354321 2.8903933Os.51718.S1_at Os11g26780 Pathogenesis-related protein PRB1-3 precursor, putative, expressed 0.015439005 2.8541846Os.51789.S1_at Os03g42520 Dehydrin Rab16B, putative, expressed 0.020020213 3.0236418Os.51813.S1_at AK063821 Expressed protein 3.33E�05 17.965927Os.52845.S1_at AK069569 Unknown 4.79E�04 2.4803464Os.53210.S1_at Os11g26760 Unknown 0.045720574 2.4495614Os.5325.1S1_at Os08g23870 Dehydrin Rab16C, putative, expressed 0.039178148 3.7990863Os.53438.S1_at AK072622 Seed maturation protein, putative, expressed 1.65E�04 2.6748827Os.53526.S1_at Os04g52110 Unknown 0.024053326 4.721352Os.54997.S1_at Os03g51350 Embryonic protein DC-8, putative, expressed 0.020868113 3.783074Os.55259.S1_at Os04g32480 Expressed protein 1.04E�04 4.792576Os.55907.S1_at AK108976 ZIM motif family protein, expressed 0.00168589 3.8223014Os.55961.S1_at Os07g34280 Unknown 1.86E�04 9.980069Os.5717.1.S1_at Os04g43170 2-Hydroxyisoflavanone dehydratase, putative, expressed 0.03412682 5.145434Os.6330.1.S1_at Os03g07170 Caleosin 1, putative, expressed 0.035861187 4.5989513Os.6618.1.S1_at Os04g49980 Expressed protein 0.02348928 5.5303783Os.8177.1.S1_a_at Os07g10580 Seed maturation protein, putative, expressed 0.004225229 2.519963Os.9107.2.S1_x_at Os09g27260 Prolamin precursor, putative, expressed 0.002280593 2.629391Os.941.1.S1_at Os01g52830 TMV response-related gene product, putative, expressed 0.021091063 4.4019175Os.9827.1.S1_at Os01 g37000 Secreted protein, putative, expressed 4.07E�04 3.6237378Os.9988.1.S1_at Os11g32890 Carboxyl-terminal peptidase, putative, expressed 0.026153458 5.188101OsAffx.11535.1 .S1_ at Os01g49720 Expressed protein 0.005038577 2.3794348OsAffx.12887.2.S1_at Os03g 16920 Glutathione S-transferase GSTU6, putative, expressed 0.013972967 2.4868436

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Table 2 (continued)

Probe ID Locus Description P value Fold change

OsAffx.12887.2.S1_s_at Os03g 16920 Heat-shock cognate 70 kDa protein, putative, expressed 0.042550996 2.4696164OsAffx.12954.1 .S1 _at Os03g20680 Heat-shock cognate 70 kDa protein, putative, expressed 0.005202627 2.1823668OsAffx.14523.1 .S1 _s_at Os05g03130 Embryonic protein DC-8, putative, expressed 0.029849583 3.0090632OsAffx.16847.1 .S1_s_at Os08g05970 Expressed protein 0.005745885 3.3219216OsAffx.21963.1 .S1 _s_at Os01 g19770 Expressed protein 0.045467142 2.9706032OsAffx.24280.2.S1_ at Os07g08150 Stress-inducible membrane pore protein, putative, expressed 1.63E�04 3.0219204OsAffx.25008.1.S1_s_at Os03g07510 Early light-induced protein, chloroplast precursor, putative, expressed 0.00350515 3.1984828OsAffx.2928.1.S1_at Os02g40500 Expressed protein 0.003097327 2.3900723OsAffx.32080.1 .S1_ at Os12g38290 OsGrx_C2.1 – glutaredoxin subgroup I, expressed 1.11E�04 6.521656OsAffx.4557.1.S1_s_at Os05g39250 Metallothionein-like protein 1, putative, expressed 0.025276499 2.3402073OsAffx.7826.1.S1_at Os12g38051 Expressed protein 3.93E�05 16.10698

Metallothionein-like protein 1, putative, expressed

DownregulatedOs.10444.1. S1_at Os07g35280 Receptor-like protein kinase homolog RK20-1, putative, expressed 1.45E�05 2.4533386Os.11551.1.S1_at Os05g04410 Peroxidase 2 precursor, putative, expressed 8.67E�05 2.8729682Os.11920.1.S1_s_at Os04g39320 expressed protein 8.63E�05 2.6402802Os.13392.1.S1_at Os03g36560 Peroxidase 27 precursor, putative, expressed 4.48E�06 2.43654Os.15798.1.S1_at Os06g07040 OsIAA20 – Auxin-responsive Aux/IAA gene family member, expressed 1.48E�05 5.434033Os.15854.1.S1_ at BM418911 Unknown 0.002662502 2.308534Os.16010.1.S1_at Os05g42150 Indole-3-acetic acid-amido synthetase GH3.4, putative, expressed 3.54E�05 3.4885685Os.16500.1.S1_at Os01 g27630 Glutathione S-transferase GSTF2, putative, expressed 0.00124726 4.2301393Os.18006.1.S1_a_at Os01g72810 Secreted glycoprotein, putative, expressed 1.77E�04 2.2248764Os.18462.1.S1_at Os03g30950 Acyl-desaturase, chloroplast precursor, putative, expressed 1.22E�04 2.8622296Os.2123.1.S1_at Os01g54300 Hydrolase, hydrolyzing O-glycosyl compounds, putative, expressed 4.70E�06 3.0026116Os.21893.1.S1_x_at Os04g59160 Peroxidase 12 precursor, putative, expressed 1.05E�05 2.970623Os.21893.2.S1_at Os04g59160 Peroxidase 12 precursor, putative, expressed 2.43E�06 2.5284595Os.22577.2.S1_x_at Os01 g32770 Seed specific protein Bn15D17A, putative, expressed 0.024708059 2.5645049Os.23256.1.S1_at Os02g56120 OsIAA9 – Auxin-responsive Aux/IAA gene family member, expressed 5.26E�04 2.884093Os.24041.1.A1_at CB622792 Unknown 0.001500505 2.2300873Os.24972.1.S1_at Os02g09930 CSLA1 – cellulose synthase-like family A; mannan synthase, expressed 4.66E�06 2.1288786Os.25578.1.S1_at Os07g35350 Hydrolase, hydrolyzing O-glycosyl compounds, putative, expressed 0.001460651 2.2332582Os.26983.1.S1_s_at Os01g04570 Ser/Thr protein kinase, putative, expressed 1.52E�04 2.623972Os.27483.1.S1_at Os08g 13440 Germin-like protein subfamily 1 member 17 precursor, putative, expressed 1.91E�04 4.0395966Os.28433.4.A1_at Os01g58290 Cucumisin precursor, putative, expressed 6.65E�04 2.55282Os.28433.5.S1_x_at Os01g58290 Cucumisin precursor, putative, expressed 7.02E�04 3.1006265Os.2961.1.S1_at Os03g25340 Peroxidase 47 precursor, putative, expressed 5.41E�09 5.9793158Os.31056.1.S1_at Os01 g03900 Expressed protein 2.97E�05 2.9322095Os.31171.1.S1_at Os01g40290 Expressed protein 0.004975374 3.6446333Os.31893.1.S1_at Os01 g19290 Nodulin-like protein, putative, expressed 4.37E�05 2.7275617Os.32667.1.S1_ at Os01 g12070 Endoglucanase 1 precursor, putative, expressed 2.82E�07 8.846673Os.32667.2.S1_x_at Os01 g12070 Endoglucanase 1 precursor, putative, expressed 2.21E�06 7.459667Os.32727.1.S1_at Os01g43480 ATP binding protein, putative, expressed 1.60E�05 5.846405Os.36346.2.S1_at Os01g67030 Dopamine beta-monooxygenase, putative, expressed 0.034313455 2.2013311Os.37622.1.S1_at Os01g67010 Expressed protein 0.007980204 3.0087888Os.46052.1.S1_at Os10g40440 Cortical cell-delineating protein precursor, putative, expressed 0.002692644 2.808154Os.4844.1.S1_at Os05g06970 Peroxidase 1 precursor, putative, expressed 6.70E�05 2.7344198Os.49116.1.S1_at Os02g45320 ATPP2-A13, putative, expressed 1.04E�06 2.6828265Os.49545.1.S1_at Os11g32030 Sex determination protein tasselseed-2, putative, expressed 2.40E�05 2.3033772Os.49608.1.S1_at Os03g15340 Chemocyanin precursor, putative, expressed 1.52E�05 2.09399Os.49663.1.S1_at Os02g39000 DNA binding protein, putative, expressed 1.62E�05 3.8656454Os.50383.1.S1_at Os06g05430 Expressed protein 2.65E�04 2.1732059Os.50555.1.S2_at Os11g03860 Ser/Thr protein kinase, putative, expressed 7.24E�05 2.2175531Os.50606.1.S1_at Os12g05394 Serine/threonine-protein kinase PK7, putative, expressed 3.37E�04 2.519327Os.5066.1.S1_ at Os01g12000 Expressed protein 7.01E�04 2.5683227Os.51285.S1_at Os01g04390 Expressed protein 2.99E�05 2.1595392Os.51427.S1_at Os04g55980 GRound-Like protein, putative, expressed 0.017401934 3.1965065Os.51831.S1_at Os02g15340 NAC domain-containing protein 76, putative, expressed 7.57E�04 3.2455897Os.51835.S1_a_at Os05g48700 Gibberellin 2-beta-dioxygenase, putative, expressed 1.22E�05 4.5942597Os.5242.1S1_ at Os01g58260 Peptidase/ subtilase, putative, expressed 3.11E�04 2.6854098Os.52570.S1_at Os12g44110 ligA, putative, expressed 7.80E�04 2.5082781Os.53348.2. 2.S1_at Os11 g17014 NB-ARC domain containing protein, expressed 0.001117023 2.5455925Os.54109.S1_at Os03g38800 Mitochondrial protein, putative, expressed 4.59E�05 3.3835647Os.54316.S1_at Os03g25060 Expressed protein 2.36E�05 2.7310393Os.54354.S1_at Os03g27480 Retinoid-inducible serine carboxypeptidase precursor, putative, expressed 1.72E�05 3.3126776Os.54653.S1_at Os06g09870 Glycine-rich cell wall structural protein 2 precursor, putative, expressed 2.49E�04 3.1875672Os.54770.S1_ at Os02g03120 Endoglucanase 1 precursor, putative, expressed 7.97E�05 3.6305342Os.55284.S1_at Os05g07140 RING-H2 finger protein ATL3F, putative, expressed 4.46E�05 2.6042767Os.55696.S1_at Os12g39100 Expressed protein 2.92E�05 2.4041243Os.56365.S1_at Os03g19370 Ocs element-binding factor 1, putative, expressed 1.35E�04 2.4775271Os.56899.S1_at Os07g44590 Peroxidase 66 precursor, putative, expressed 0.003683018 2.1222537Os.57112.S1_at Os04g56950 Jasmonate O-methyltransferase, putative, expressed 4.09E�04 2.6883378Os.57170.S1_at Os03g41130 Expressed protein 0.002831145 2.7146943Os.57309.S1_at Os04g19960 Expressed protein 1.45E�05 3.1186292Os.6237.1.S1_at Os05g02480 Expressed protein 4.19E�04 2.636499Os.6271.1.S1_at Os07g39320 DNA binding protein, putative, expressed 3.85E�04 2.7242424

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lated transcription factor in rice. One zinc-finger C3HC4-type pro-tein (Os05g07140) is downregulated in both AsV and AsIII stresses.In addition to this gene, three proteins of the zinc-finger family(Os06g04920, Os01g60730, and Os07g34120) are specificallydownregulated in AsV stress. In addition to the transcription fac-tors, F-box, U-box, and protein kinases are up- and downregulatedin both the cases. Among the three upregulated protein kinases,two (Os3g28300 and Os11g46860) are specific to AsIII and six(Os1g46720, Os01g53920, Os10g10130, Os04g25060, Os04g25650,Os06g47530) are specific to AsV. Among the 6 downregulated pro-tein kinases, two (Os9g12240 and Os2g17240) are specific to AsVand one (Os7g35280) is specific to AsIII.

3.4. Metabolic networks from transcriptome expression profiles

Analysis of potential metabolic networks from microarray stud-ies is a very useful method for maximizing information. To identifythe potential changes in cellular functions, transcriptome interac-tion networks of all the up- and downregulated data sets in the ar-ray have been analyzed using the software Pathway Studio (AriadneGenomics). The pathway analysis shows that arsenic-stress re-sponse influences the pathways related to photosynthesis, plant de-fense, and signal transduction in both cases. The pathway analysismoreover indicates that abscisic acid metabolism and germinationof the seedlings are affected much more in AsIII stress compared to

Table 2 (continued)

Probe ID Locus Description P value Fold change

Os.6653.1.S1_at AU164614 Unknown 4.06E�04 4.4604163Os.6764.1.S1_at AU096803 Probable wound-induced protein (Arabidopsis thaliana) 0.001060405 2.4548953Os.7342.1.S1_at Os02g57490 LOB domain protein 16, putative, expressed 1.06E�04 3.1710799Os.7428.1.S1_at Os06g44470 Pollen allergen Phl p 2 precursor, putative, expressed 4.20E�04 2.4012487Os.7938.1.S1_at Os04g59190 Peroxidase 2 precursor, putative, expressed 0.009827014 2.781223Os.8855.1.S1_at Os02g31840 Expressed protein 2.91E�04 2.991697Os.9086.1.S1_at Os03g 10210 DNA binding protein, putative, expressed 5.34E�05 3.0671852OsAffx.14278.1 .S1 _at Os04g45590 Lactoylglutathione lyase, putative, expressed 6.5 6E�04 3.8197377OsAffx.14436.1 .S1 _at Os04g56470 AAP7, putative, expressed 6.25E�05 2.5434856OsAffx.14448.1 .S1 _at Os04g56930 Beta-fructofuranosidase, insoluble isoenzyme 5, putative, expressed 3.52E�05 2.2755945OsAffx.17516.1.S1_at Os08g44940 LOB domain protein 16, putative 0.001317835 3.6695514OsAffx.19068.1 .S1 _x_at Os11g29420 Lipid binding protein, putative 0.001849828 5.1331053OsAffx.24297.1.S1_at Os02g17900 Xyloglucan endotransglucosylase/hydrolase protein 26 precursor, putative, expressed 2.99E�04 2.471011OsAffx.24928.1 .S1_ at Os02g17880 Xyloglucan endotransglucosylase/hydrolase protein 26 precursor, putative 8.29E�04 2.4203243OsAffx.25840.1.S1_at Os04g02560 Expressed protein 1.33E�04 2.6880703OsAffx.27508.100.S1_s_at Os10g36924 Aquaporin NIP5.1, putative, expressed 0.025144592 2.0350194OsAffx.27946.1.S1_at Os06g35060 Heavy metal-associated domain containing protein, expressed 2.49E�04 3.7222154OsAffx.30613.1.S1_x_at Os02g 14250 Transposon protein, putative, unclassified 4.40E�05 5.013218OsAffx.30613.3.S1_at Os07g34120 Transposon protein, putative, unclassified 2.15E�04 4.541455OsAffx.30613.3.S1_x_at Os07g34120 Transposon protein, putative, unclassified 1.39E�04 5.373925OsAffx.31011.1 .S1_ at Os11g16990 Hypothetical protein 5.86E�05 2.9293015OsAffx.4107.1.S1_s_at Os04g46350 Homeobox-leucine zipper protein ATHB-4, putative, expressed 2.58E�06 2.7595274OsAffx.4731.1 .S1_s_at Os06g05430 Expressed protein 0.001669405 2.2782338Os Affx.5 729.1 .S1 _at Os08g07070 Expressed protein 2.33E�05 5.870749

Fig. 2. The classification of differentially expressed probe sets during arsenic stress in rice. The upregulated (A and C) and downregulated (B and D) sets of genes in AsV andAsIII stress, respectively, are shown. The size of each set is proportional to the relative abundance of genes in the set of biological functions. Different probe sets were dividedin to defence and stress (DS), transporters (TR), Transcription factors (TF), growth and development (GD), signal transduction (ST), metabolism (MB), secondary metabolism(SM), transposon (TP) and unknown (Uk) categories for their classification.

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AsV challenge (Fig. 3 and Supplementary Fig. S1). By clicking on anyof the biological objects within the Pathway, more detailed infor-mation for each object can be obtained (for further information

and detailed pathway files, please contact authors). The nodes showthe metabolomics that are predicted to modulate molecular func-tions. Our analysis suggests that AsV affects mainly cell wall, pri-

Fig. 3. Metabolic pathway network constructed from differentially expressed transcriptome under AsV and AsIII stress.

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mary and secondary metabolism whereas AsIII mainly affects hor-monal and signaling process. This approach has created a vast res-ervoir of information which can be utilized systematically forunderstanding detailed mechanisms involved in As stress in rice.

3.5. Validation of microarray data by RTPCR

Oligonucleotide primers for the 16 genes related to transport,stress, defense, and one gene of unknown function have been usedfor validation of the microarray data through semiquantitativeRTPCR (Fig. 4A). Though the microarray analysis is carried out onRNA from entire seedlings, the validation has been carried out sep-arately for the root and shoot tissues, to resolve tissue-specific dif-ferences in the expression of selected genes. The results obtainedfrom all the 16 genes tested by RTPCR agree with the trend of reg-ulation identified by microarray testing. The sulfate-transportergene (Os03g09970) is more responsive to AsV than to AsIII in bothroot and shoot, although the induction in the root is more pro-nounced in AsV stress in comparison to AsIII. The oligopeptide

transporter gene (Os03g54000) is upregulated in both root andshoot on treatment with AsV, but it is only upregulated in shootswhen tested with AsIII. The metal transporter, Nramp1(Os07g15460), is expressed at a higher level in shoots as concludedfrom the microarray results of AsV-grown seedlings. Anothertransporter, glutathione-conjugate transporter (Os03g13210), isspecifically upregulated in AsV-stressed roots, which further vali-dates the microarray analysis. The expression of five differentHSPs, which show differential expression in AsV and AsIII stresses,is also analyzed. Most of the HSPs are upregulated only in the rootof AsV-treated seedlings, similar to that observed in the microarrayresults. All the metallothioneins analyzed show larger degree ofupregulation in case of AsV stress in comparison to AsIII. Of thetwo glutaredoxins analyzed, one (Os01g26912) is expressed specif-ically in the AsIII-treated shoot. The two cytochrome P450 genesanalyzed show a differential expression pattern: Os01g43740 isexpressed only in the roots of AsV-treated seedlings. An unknownprotein (Os02g15930) shows greater upregulation in AsV-treatedseedlings, similar to the data obtained in the microarray.

Fig. 4. Confirmation of the array results for selected genes by semi-quantities RTPCR. Results of the expression analysis done on the seedlings grown in AsV and AsIII (A) andCd, Cr and Pb (B) are shown.

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3.6. Expression of the above-selected genes under Cd, Cr, and Pbstresses

Expression analyses of the above-selected genes that have beenused to validate microarray data has been carried out for a fewother heavy metals (Fig. 4B). Expression of most of the genes underthese stresses was similar to that under arsenic stress, except inthe case of a cytochrome P450 gene (Os01g43740). Of the 5 differ-ent metals tested in this study, this specific cytochrome P450 isoverexpressed only in AsV-treated roots. This indicates thatOs01g43740 can be used as an important biomarker for AsV stressin rice. Most of the genes analyzed herein are related to defenseand stress, which, in addition, show a common expression patternunder heavy-metal stress. Analysis of a few more genes identifiedin the microarray data might provide further information relatedto metal-specific responses in plants.

In conclusion, genome-wide transcriptome analysis was carriedout in rice seedlings treated with different inorganic forms ofarsenic. The microarrays offered reproducible detection of arsenic-regulated gene expression. Most of the genes differentiallyexpressed under both AsIII and AsV stresses were related to glutathi-one metabolism, transport, and signal-transduction pathways.However, AsV stress led to the induction of a larger set of responsivegenes in comparison to AsIII, indicating the occurrence of AsV-spe-cific response in rice. However, somewhat unexpectedly, upregula-tion of phytochelatin synthase or arsenate reductase was notdetected by microarray analysis. This might be due to their nonre-sponsive behavior to arsenic stress, which is in agreement withprevious report (Ouziad et al., 2005). During preparation of our man-uscript, Norton et al., 2008 reported transcriptome changes AsVstress in rice varieties that are arsenate-tolerant (Bala) and -sensi-tive (Azucena). A number of genes that are up-/downregulated un-der AsV stress similar to our study were also reported by the samegroup. However, in our study we have compared the transcriptomicsof AsV and AsIII stress and generate a comprehensive network mapwhich created a vast reservoir of information and can be utilizedsystematically for understanding detailed mechanisms involved inmetabolism of AsV and AsIII, differentially.

Acknowledgments

The research was supported by Council of Scientific and Indus-trial Research, New Delhi. R.T. acknowledges the DST, Govt. of Indiafor providing financial assistance in the form of ‘‘J.C. Bose Fellow-ship”. P.M. and M.T. acknowledge Council of Scientific and Indus-trial Research and Indian Council of Medical Research, Govt. ofIndia for Junior Research Fellowships.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.chemosphere.2008.09.082.

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