Research ArticleGenome-Wide Transcriptome Analysis ofCadmium Stress in Rice

Youko Oono Takayuki Yazawa Hiroyuki Kanamori Harumi Sasaki Satomi MoriHirokazu Handa and Takashi Matsumoto

Agrogenomics Research Center National Institute of Agrobiological Sciences Tsukuba Ibaraki 305-8602 Japan

Correspondence should be addressed to Youko Oono yoonoaffrcgojp

Received 26 November 2015 Revised 26 January 2016 Accepted 28 January 2016

Academic Editor Yan-Bo Hu

Copyright copy 2016 Youko Oono et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Rice growth is severely affected by toxic concentrations of the nonessential heavy metal cadmium (Cd) To elucidate the molecularbasis of the response to Cd stress we performed mRNA sequencing of rice following our previous study on exposure to highconcentrations of Cd (Oono et al 2014) In this study rice plants were hydroponically treated with low concentrations of Cd andapproximately 211 million sequence reads were mapped onto the IRGSP-10 reference rice genome sequence Many genes includingsome identified under high Cd concentration exposure in our previous study were found to be responsive to low Cd exposurewith an average of about 11000 transcripts from each condition However genes expressed constitutively across the developmentalcourse responded only slightly to low Cd concentrations in contrast to their clear response to high Cd concentration which causesfatal damage to rice seedlings according to phenotypic changes The expression of metal ion transporter genes tended to correlatewith Cd concentration suggesting the potential of the RNA-Seq strategy to reveal novel Cd-responsive transporters by analyzinggene expression under different Cd concentrationsThis study could help to develop novel strategies for improving tolerance to Cdexposure in rice and other cereal crops

1 Introduction

Cadmium (Cd) is a widespread heavy metal pollutant thatis highly toxic to living cells Accumulation of the nonessen-tial metal Cd in plants is a major agricultural problemSpecifically Cd is absorbed by the roots from the soil andtransported to the shoot negatively affecting nutrient uptakeand homeostasis in plants even in very small amountsMany agricultural soils have become contaminated withCd through the use of phosphate fertilizers sludge andirrigation water containing Cd Cd exposure inhibits rootand shoot growth and ultimately reduces yield FurthermoreCd accumulation in the edible parts of plants such as seedgrains places humans at a risk because of its highly toxiceffects on human health Reducing the Cd concentration inplants below the maximum level indicated by the CodexAlimentarius Commission of FAOWHO [1] is necessary toavoid negative impacts on humanhealthThus it is importantto study the mechanisms of plant responses and defenses toCd exposure to overcome this problem

Cd causes oxidative stress and generates reactive oxygenspecies which can cause damage in various ways such asreacting with DNA causing mutation modifying proteinside chains and destroying phospholipids [2] Various bio-chemical and physiological processes associated with defensesystems are active in plants under Cd exposure Many genessuch as glutathione S-transferase (GST) for detoxificationand cysteine-rich metallothioneins (MT) for defense againstCd toxicity respond to Cd stress in plants and might conferCd tolerance in rice Transporters with heavy metal bindingdomains are key factors for root uptake of Cd from soil andefflux pumping of Cd at the plasma membrane however themanner in which these genes respond to low Cd concentra-tions has not been well investigated in rice

In a previous study we investigated the gene expressionof rice plants (Oryza sativa L cv Nipponbare) under a highCd concentration using the RNA-Seq platform A clear anddetailed view of the transcriptomic changes triggered by Cdexposure is important to understand the gene expressionnetwork of the basal response to Cd stress This could not be

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9739505 9 pageshttpdxdoiorg10115520169739505

2 BioMed Research International

obtained frompast studies using themicroarray platform butRNA-Seq can accurately quantify gene expression levels overa broad dynamic range with high resolution and sensitivity[3] We found that drought stress signaling pathways wereactivated under Cd exposure through the responses of manydrought-related genes [4] Thus the recently elucidated scaf-folding mechanisms for Cd signaling pathways are complexbut of great importance In this study we performed ricetranscriptome analysis under different lowCd concentrationsusing the RNA-Seq platform to deepen our understanding ofCd responses

2 Materials and Methods

21 Sample Preparation Rice (Oryza sativa ssp japonica cvNipponbare) seeds were germinated and grown by hydro-ponic culture in Yoshidarsquos solution [1425mM NH

4NO3

0323mM NaH2PO4 0513mM K

2SO4 0998mM CaCl

2

1643mM MgSO4 0009mM MnCl

2 0075mM (NH

4)6

Mo7O24 0019mM H

3BO3 0155mM CuSO

4 0036mM

FeCl3 0070mM citric acid and 0152mM ZnSO

4] [5] After

10 days seedlings of uniform size and growth were subjectedto Cd stress treatment by transferring them to a similarmedium with 02 1 or 50120583M Cd These values were chosenbased on a report that the total dissolved Cd in 64 fieldswith Cd-contaminated soils ranged from 003 to 182 120583gL[6] in previous experiences The plants were maintainedunder Cd stress conditions for 14 d Root and shoot sampleswere collected at approximately 900AM frozen in liquidnitrogen and stored at minus80∘C until subsequent analysesTotal RNA was extracted from both root and shoot samplesusing an RNeasy Plant Kit (Qiagen Hilden Germany)according to the manufacturerrsquos instructions Constructionof 34 cDNA libraries (2 tissues 4 conditions 2 treatmentsand 2-3 replicates) from total RNA using a TruSeq RNAsample preparation kit and sequencing with the IlluminaGenome Analyzer IIx (Illumina Inc San Diego CA USA)was performed according to the manufacturerrsquos protocols asa part of establishing TENOR (Transcriptome Encyclopediaof Rice httptenordnaaffrcgojp) [7] The resulting RNA-Seq data were deposited in the DDBJ Sequence Read Archive(Accession number DRA000959)

22 Identification of Differentially Expressed Transcripts Thebiological replicates (2-3) for each set of conditions werehighly correlated (coefficient gt 095) so reads from the sametreatment were merged for subsequent analysis Trimming ofIllumina adaptor sequences and low-quality bases (119876 lt 20)by Cutadapt [8] and mapping of preprocessed reads to theIRGSP-10 genome assembly (httprapdbdnaaffrcgojp)were performed as described previously [9] To estimate theexpression levels of each transcript all preprocessed readswere mapped to the IRGSP-10 genome assembly by Bowtiewith default parameters [10] The expression level for eachtranscript was calculated as the RPKM- (Reads per KilobaseExon Model per million mapped reads-) derived read count[11] based on the number of uniquely mapped reads thatoverlapped with exonic regions A 119866-test was performed to

detect differentially expressed transcripts in the control andCd treatments based on the statistical null hypothesis that theproportions of mapped reads to the transcripts were the samebetween the two conditions A false discovery rate (FDR lt001) was used in multiple-hypothesis testing to correct formultiple comparisons When calculating fold changes 1 wasadded to avoid division by 0

23 Hierarchical Clustering and Gene Ontology EnrichmentAnalysis The Cd-responsive transcripts in root and shootwere used for hierarchical clustering analysis We used theheatmap2 in the R package gplots (version 2110) to performclustering analyses of transcripts The 119885 scores were usedto compare significant changes in gene expression A GeneOntology (GO) termwas assigned to each transcript based onthe GO annotations for biological process molecular func-tion and cellular component in RAP-DB GO enrichmentwas evaluated by Fisherrsquos exact test with a FDR thresholdof 5 for responsive transcripts in the biological processcategory of each cluster The results were plotted as minus log 10of FDR values in a heatmap

24 qRT-PCR Analysis The expression of Cd upregulatedgenes in root sample was confirmed by qRT-PCR analysisRice seeds were germinated and grown in water in a growthchamber After 10 days the seedlings were subjected to differ-ent stress treatments by transferring them towater containingdifferent reagents RNA was extracted from them and thecDNA was synthesized according to the manufacturerrsquos pro-tocol and it is used for the further analysis as described previ-ously [4]The resulting cDNAwas used for PCR amplificationin the LightCycler 480 system (Roche Basel Switzerland)with each primer set (Os04g0600300 51015840-GGCGCTCTG-AGAATCATCAC-31015840 51015840-CATTCGGGAGCTCATCTCG-31015840 Os01g0692100 51015840-ATTCACGAGTCCGCGATG-31015840 51015840-CTCTCACCCGGATCACCC-31015840 Os12g0570700 51015840-GCA-CTCATCTCAAGCTTTTC-31015840 51015840-GCAAGACATCTTCTT-GG-31015840 Os12g0571000 51015840-ATTTCCTGAAGAGTTAAA-3101584051015840-TTCCGCAGCCGCAGCTTA-31015840) The detection thresh-old cycle for each reaction was normalized using Ubiq-uitin1 primers (51015840-CCAGGACAAGATGATCTGCC-31015840 51015840-AAGAAGCTGAAGCATCCAGC-31015840)

3 Results and Discussion

31 LowCdConcentration Exposure of Rice Plants and GrowthRetardation during the Treatment Weused rice plants grownin hydroponic culture which enabled us to control the Cdexposure easily High Cd concentration exposure has beenpreviously shown to elicit robust physiological responses andgene expression as acute toxic responses in rice seedlings [12ndash14] Growth retardation of the shoot was slightly visible after1 d (data not shown) the leaves turned yellow and the leaf tipsof the seedlings began to wilt after 4 d and all leaf blades werecurled completely and the seedlings were wilting after 10 dunder high Cd concentration (50120583M) exposure (Figure 1)While no visible symptoms were observed in shoots underlow Cd concentration exposure (02 and 1 120583M Cd) after

BioMed Research International 3

14d

Con

trol

02120583

M

1120583

M

50120583

M

10d

4d

0d

Figure 1 Phenotypic changes in rice plants grown in culturemedium with low concentrations of Cd (02 1120583M) and a highconcentration of Cd (50120583M) from 0 to 14 d

1 d growth retardation occurred gradually compared withthe control with symptoms starting to appear after 7 dPlants in the same growth chamber exposed to differentCd concentrations showed clear growth differences after10 d (Figure 1) Even after 28 d the seedlings under lowCd concentration exposure did not show yellow leaves orwilting (data not shown)These results suggested that highCdconcentration exposure causes fatal damage to plants whilelow Cd concentrations lead to growth retardation (Figure 1)which is supported by the fact that plant detoxificationprocesses are insufficient to copewith this toxicmetal beyonda 10 120583M dose [15]

32 Gene Expression Profiles under Low Cd Concentra-tion Exposure in Rice We next analyzed the transcriptomeprofiles of the response to Cd exposure using RNA-Seqduring plant growth at 1 4 and 10 d after Cd treat-ment and before treatment (0 d) For each set of con-ditions an average of approximately 151 million (922)quality-evaluated reads (total 211 million) were mappedto the rice genome sequence and used for further anal-ysis (Table S1 in Supplementary Material available onlineat httpdxdoiorg10115520169739505) The number ofupregulated transcripts ranged from 4529 to 6515 whereas

Root Shoot Root Shoot

1120583M Cd02 120583M Cd

1d 4d 10d 1d 4d 10d 1d 4d 10d 1d 4d 10d11000

9000

7000

5000

3000

1000

3000

5000

7000

9000

1000

Num

ber o

f up-

or d

ownr

egul

ated

tran

scrip

ts

Figure 2 Distribution of upregulated and downregulated tran-scripts in roots and shoots in response to Cd exposure RPKM foldchanges at 1 4 and 10 d were calculated for Cd-treated samplescompared with nontreated samples (0 d) The total numbers ofupregulated (upper) and downregulated (lower) transcripts in rootsand shoots identified by RNA-Seq were determined by119866-tests (FDRlt 001) at each stress time point (1 4 and 10 d) under 02 120583M (left)and 1 120583M (right) Cd exposureThe 119909-axis shows the time course andthe 119910-axis shows the number of transcripts

the number of downregulated transcripts ranged from 2359to 8734 under 02 120583M Cd (Figure 2) Twelve transcriptsincluding GST MT and DREB (drought responsive elementbinding protein) 1E were upregulated more than 20-foldamong the upregulated transcripts in roots at 02 120583M CdThe number of upregulated transcripts ranged from 5830to 7271 whereas the number of downregulated transcriptsranged from 2965 to 10020 under 1120583M Cd (Figure 2)Fifty-one transcripts including GST MT Prx (peroxidase)and heat shock proteins were upregulated more than 20-fold among the upregulated transcripts in roots at 1 120583MCd (Table 1) Induction of detoxification enzymes againstoxidation stress such as GST and Prx under Cd exposuremight be associated with the defense system that confers Cdtolerance to plants [16ndash18] even at lowCd concentrationsThecysteine-rich MT might function as a ligand for chelation ofmetal ions to defend against Cd toxicity [19] The DREBC-repeat binding factor (CBF) specifically interacts with theDRECRT cis-acting element and controls the expression ofmany stress-inducible genes in plants [20] The activationof gene expression in several drought stress signal pathwaysunder Cd exposure has been reported [4] Five heat shock

4 BioMed Research International

Table 1 Cadmium-upregulated transcripts identified in roots by RNA-Seq analysis

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

02 120583MCdOs10t0527400-01 Tau class GST protein 3 278 214 275 12 20 17Os03t0283000-00 In2-1 protein 275 28 10 13 11 15Os08t0156000-01 Conserved hypothetical protein 264 214 253 13 16 17Os01t0627967-00 Hypothetical protein 261 165 241 15 19 14Os04t0178300-02 Syn-copalyl diphosphate synthase 201 80 203 06 42 14

Os04t0301500-01 HLH (helix-loop-helix) DNA-bindingdomain containing protein 04 331 05 10 475 92

Os02t0676800-01 DREB1E (drought responsive elementbinding protein 1E) 09 287 09 12 109 20

Os02t0179200-01 Glutamine amidotransferase class-I domaincontaining protein 08 281 17 09 32 11

Os12t0154800-00 RmlC-like jelly roll fold domain containingprotein 40 214 57 10 14 12

Os12t0570700-01 MT (metallothionein)-like protein type 1 186 203 158 09 10 09Os03t0836800-01 IAA-amino acid hydrolase 1 43 65 336 10 10 10

Os10t0333700-00 Plant disease resistance response proteindomain containing protein 97 60 216 10 10 10

1 120583MCdOs04t0178300-02 Syn-copalyl diphosphate synthase 1220 321 255 05 10 36

Os04t0178300-01 Isoform 3 of Syn-copalyl diphosphatesynthase 1098 278 215 05 09 31

Os04t0178400-01 Cytochrome P450 CYP99A1 698 211 160 08 10 28Os03t0267000-00 Heat shock protein 180 575 77 109 12 07 07Os03t0266900-01 Heat shock protein 173 470 49 53 10 04 06Os01t0136200-01 Heat shock protein 1 437 39 13 10 10 10

Os07t0190000-01 1-Deoxy-D-xylulose 5-phosphate synthase 2precursor 424 115 86 07 11 39

Os07t0127500-01 PR-1a pathogenesis related protein precursor 400 56 50 08 08 21Os07t0154100-01 Viviparous-14 388 52 15 11 14 23Os07t0154201-00 Hypothetical gene 377 47 13 10 13 21Os12t0555200-01 Probenazole-inducible protein PBZ1 377 135 109 03 05 22Os06t0586000-01 Conserved hypothetical protein 376 93 65 06 09 14Os10t0527400-01 Tau class GST protein 3 343 180 324 11 14 20Os12t0555000-01 Probenazole-inducible protein PBZ1 332 135 110 06 07 25

Os03t0277700-01 Protein of unknown function DUF26domain containing protein 328 76 34 10 06 10

Os11t0687100-01 von Willebrand factor (type A domain) 325 41 138 07 07 23Os05t0211700-00 mdash 288 14 12 10 10 10Os06t0662550-01 Conserved hypothetical protein 285 78 88 08 08 16Os01t0944100-02 Conserved hypothetical protein 284 63 98 05 06 17Os06t0568600-01 Ent-kaurene oxidase 1 271 281 110 06 14 47Os12t0418600-01 Hypothetical conserved gene 267 20 13 10 10 10Os12t0258700-01 Cupredoxin domain containing protein 262 147 106 07 11 71Os01t0615100-01 Substilinchymotrypsin-like inhibitor 256 95 79 07 10 18Os04t0107900-02 Heat shock protein 81-1 256 25 16 10 10 09

BioMed Research International 5

Table 1 Continued

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

Os09t0493000-01 Conserved hypothetical protein 253 26 18 09 12 09Os01t0627967-00 Hypothetical protein 253 195 216 13 18 14Os01t0944100-03 Conserved hypothetical protein 252 46 63 06 06 18Os04t0180400-01 Cytochrome P450 99A2 244 43 60 05 05 31Os04t0108101-00 Hypothetical protein 244 23 14 10 10 10Os02t0269600-00 Subtilase 226 78 41 03 12 60Os01t0136000-00 Heat shock protein 175 225 31 12 10 14 12Os04t0180500-00 Hypothetical protein 222 40 54 05 06 31Os01t0946600-01 Conserved hypothetical protein 218 166 80 07 07 08Os09t0255400-02 Indole-3-glycerol phosphate synthase 214 51 38 07 09 23Os01t0348900-01 SalT gene product 212 65 89 01 01 02Os12t0491800-01 Ent-kaurene synthase 1A 211 15 17 04 08 55Os01t0132000-01 Wound-induced protease inhibitor 210 88 116 16 05 02Os11t0592200-01 Chitin-binding allergen Bra r 2 207 34 28 07 05 16Os01t0963000-01 Prx (Peroxidase) BP 1 precursor 206 38 44 07 11 13Os08t0189600-01 Oryza sativa germin-like protein 8-7 206 115 67 21 15 08Os07t0496250-01 Expansin-like B1 205 22 22 15 12 45Os01t0963000-04 Prx (Peroxidase) BP 1 precursor 203 37 44 07 11 13Os09t0255400-01 Indole-3-glycerol phosphate synthase 202 52 37 07 09 23Os11t0601950-01 cDNA clone002-114-B06 200 17 19 07 10 11Os03t0129400-01 Hypothetical protein 103 271 176 10 19 36Os01t0322700-01 Nonprotein coding transcript 122 255 157 09 13 25

Os03t0129400-02 EST AU078206 corresponds to a region ofthe predicted gene 94 243 163 11 14 28

Os12t0570700-01 MT (metallothionein)-like protein type 1 167 212 177 08 08 31Os12t0571000-01 MT (metallothionein)-like protein type 1 139 200 130 09 10 36Os08t0156000-01 Conserved hypothetical protein 154 179 260 11 15 16Os03t0836800-01 IAA-amino acid hydrolase 1 07 40 237 10 10 10

Reads were mapped to the rice genome and responsive genes were identified by 119866-tests Transcripts upregulated more than 20-fold in one or moretreatmentstime points in roots are shown Transcripts in bold were upregulated under both 1 and 02 120583MCd exposure

proteins (Hsps) were strongly upregulated in roots under 1120583MCd with the greatest relative expression at 1 d (Table 1)Thesegenes may contribute to cellular homeostasis by protectingmacromolecules such as enzymes protein complexes andmembranes under Cd exposureThis result suggested that theroots of hydroponically cultured rice might be affected moredirectly and earlier by Cd exposure There was a differencebetween the low Cd concentrations in that no Hsps werestrongly upregulated in roots at 02120583MCd (Table 1) suggest-ing that the effect of this condition might be small or showtime lag In shoots 15 and 11 transcripts were upregulatedmore than 20-fold among the upregulated transcripts under02 and 1 120583M Cd respectively (Table S2) Nine transcriptsincludingNramp1 (natural resistance-associatedmacrophageprotein) were upregulated under both 02 and 1120583M Cd(Table S2) In Arabidopsis Nramp1 localizes to the plasma

membrane and functions as a high-affinity transporter formanganese (Mn) uptake [21] while OsNramp5 uptakes Mnand Cd [22] Transporters with heavymetal binding domainsare often capable of transporting several metals such as FeZn Mn and Cd because of their low substrate specificity[23ndash26] We found that upregulation of a HLHDNA-bindingdomain containing transcription factor (Os04g0301500) inboth roots and shoots peaked at 4 d under 02120583M Cdthis protein may function as a regulatory factor under Cdexposure (Table 1 Table S2) The number of downregulatedtranscripts in roots peaked at 4 d after Cd exposure whilethe number in shoots gradually increased under low Cdconcentration exposure (Figure 2) A few dozen transcriptswere downregulated less than 005-fold among the down-regulated transcripts in roots and shoots under Cd exposure(Table S2)Therefore a small part of transcripts were strongly

6 BioMed Research International

up- or downregulated among several thousand responsivetranscripts under low Cd concentration exposure Large-scale changes in gene expression occurred in rice under Cdexposure even at low concentrations possibly because Cd isa nonessential metal for the plant

To obtain a functional annotation of responsive tran-scripts under Cd exposure we used GO biological processcategories The responsive transcripts in shoot and root wereclustered into several groups based on their expression pat-terns GO enrichment analysis was performed using clusteredtranscripts assigned by GO terms in RAP-DB (The RiceAnnotation Project Database [httprapdbdnaaffrcgojp])(Supplementary Figure S1) Enriched GO terms significantlyin each cluster may represent the functional categories inrice under Cd exposure Enriched GO terms of graduallyupregulated transcripts under Cd exposure include metalion transport (GO0030001) (cluster 3 in root under 02 120583MCd cluster 4 in root under 1120583M Cd) which may functionin Cd transport Response to oxidative stress (GO0006979)and responsive to oxidative stress (GO0006979) were alsoincluded in cluster 3 and cluster 4 respectivelyThis suggestedthat they might function in defense against Cd EnrichedGO terms of gradually downregulated transcripts underCd exposure include translation (GO0006412) translationelongation (GO0006414) DNA replication (GO0006260)andDNA repair (GO0006281) (cluster 1 in root under 02120583MCd cluster 2 in root under 1120583M Cd) Photosynthesis lightharvesting (GO0009765) and photosynthesis (GO0015979)were also included in both clusters These may function inplant growthThus these correspond to the observed changesin phenotype (Figure 1) which clearly validated the RNA-Seqexpression profiling data obtained from rice tissue under Cdstress condition However the pattern of gene expression isquite complex and would require more detailed analysis

33 Constitutively Expressed Genes Responded Differentlyunder Low Cd Concentration to High Cd Concentration Asmany genes responded to both low and high Cd concentra-tions [4] we assessed the effect of the stress degree on riceseedlings through the expression of constitutively expressedgenes We investigated the expression of 18 genes annotatedby the RAP that were expressed constitutively in 39 tissuescollected throughout the life cycle of the rice plant fromtwo varieties according to 190 Affymetrix GeneChip RiceGenome Arrays in addition to four genes annotated by theRAP that have frequently been used as internal controlsin expression analyses [27] The results showed that theexpression of more than half of them fluctuated drastically(gt2 orlt2) in roots or shoots after 1 d of highCd concentrationexposure (Figure 3) This drastic response may be partlybecause RNA-Seq can accurately quantify gene expressionlevels over a broad dynamic range with high resolution andsensitivity [10 28 29] However our results suggest that theirexpression is greatly affected by strong stress even thoughthey are expressed constitutively across the developmentalcourse Note that a high Cd concentration can cause fataldamage to rice seedlings such as by affecting homeostasiswhich corresponds to the observed changes in phenotype(Figures 1 and 3)

34 Comparative Gene Expression Analysis between Low andHigh Cd Concentrations Reveals Novel Cd-Responsive Trans-porters We investigated the expression of metal transportergenes containing metal ion binding Pfam domains [PF01554(MatE) PF08370 (PDR assoc) PF01545 (Cation efflux)PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)]that may function in Cd transport under Cd exposureThe expression of 183 transport transcripts was comparedbetween low and high Cd concentration treatments in rootsand shoots at 1 d because Cd uptake from the hydroponicculture and efflux pumping are initial responses to Cdexposure (Figure 4 Table S3) The transcripts tended tobe more responsive in roots and shoots under higher Cdconcentration exposure This result indicated the potentialof the RNA-Seq strategy to reveal novel Cd-responsivetransporters by analyzing gene expression under exposureto different Cd concentrations The responsive transcriptsmight function in roots at the early stage of Cd exposureNo transcripts were upregulated more than 3-fold in shootsunder low Cd exposure (Figure 4 Table S3) suggesting thatthe effect takes more time to appear in shootsOs03g0667500(Zip root) encoding iron-regulated transporter 1 (IRT1) wasupregulated more than 5-fold under low Cd concentrationsbut responded only slightly under the high Cd concentrationIRT1s often transport Cd because of their low substrate speci-ficity [24ndash26 30]Os02g0585200 (HMA root)Os03g0152000(HMA root) Os0g0584800 (HMA root) Os01g0609900(PDR assoc shoot) and Os01g0609300 (PDR assoc shoot)showed the highest (32-fold) upregulation under high Cdconcentration exposure and responded only slightly to lowCd concentrations (Table S3) The balance between Cd andvarious other metal ions in the hydroponic culture mightaffect the expression of these genes because specific systemsfor transporting Cd may have not developed in rice as it is anonessentialmetalThe effects of other ions on the expressionof transporters [4] and responsive genes associated withdefense systems against Cd (Supplementary Figure S2) havebeen indicated

4 Conclusions

We generated gene expression profiles for rice seedlingsgrown under low Cd concentrations Phenotypic observa-tions and constitutive gene expression indicated that low Cdconcentrations cause growth retardation but are far frombeing fatal in rice Several genes associated with defense sys-tems were strongly upregulated the expression of metal iontransporter genes tended to correlate with Cd concentrationand GO enrichment analysis of the clustered genes based ontheir expression patterns suggesting that our transcriptomeprofiles reflect responses to Cd in rice Our data also suggestthat it could be dangerous to eat plants that do not showspecificCd pollution symptoms growing in soil contaminatedby small amounts of Cd Establishing the exact compositionand organization of the transcriptional network underlyingthe response to Cd exposure will provide a robust tool forimproving crops in the future for example by creating lowCd uptake plants

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

obtained frompast studies using themicroarray platform butRNA-Seq can accurately quantify gene expression levels overa broad dynamic range with high resolution and sensitivity[3] We found that drought stress signaling pathways wereactivated under Cd exposure through the responses of manydrought-related genes [4] Thus the recently elucidated scaf-folding mechanisms for Cd signaling pathways are complexbut of great importance In this study we performed ricetranscriptome analysis under different lowCd concentrationsusing the RNA-Seq platform to deepen our understanding ofCd responses

2 Materials and Methods

21 Sample Preparation Rice (Oryza sativa ssp japonica cvNipponbare) seeds were germinated and grown by hydro-ponic culture in Yoshidarsquos solution [1425mM NH

4NO3

0323mM NaH2PO4 0513mM K

2SO4 0998mM CaCl

2

1643mM MgSO4 0009mM MnCl

2 0075mM (NH

4)6

Mo7O24 0019mM H

3BO3 0155mM CuSO

4 0036mM

FeCl3 0070mM citric acid and 0152mM ZnSO

4] [5] After

10 days seedlings of uniform size and growth were subjectedto Cd stress treatment by transferring them to a similarmedium with 02 1 or 50120583M Cd These values were chosenbased on a report that the total dissolved Cd in 64 fieldswith Cd-contaminated soils ranged from 003 to 182 120583gL[6] in previous experiences The plants were maintainedunder Cd stress conditions for 14 d Root and shoot sampleswere collected at approximately 900AM frozen in liquidnitrogen and stored at minus80∘C until subsequent analysesTotal RNA was extracted from both root and shoot samplesusing an RNeasy Plant Kit (Qiagen Hilden Germany)according to the manufacturerrsquos instructions Constructionof 34 cDNA libraries (2 tissues 4 conditions 2 treatmentsand 2-3 replicates) from total RNA using a TruSeq RNAsample preparation kit and sequencing with the IlluminaGenome Analyzer IIx (Illumina Inc San Diego CA USA)was performed according to the manufacturerrsquos protocols asa part of establishing TENOR (Transcriptome Encyclopediaof Rice httptenordnaaffrcgojp) [7] The resulting RNA-Seq data were deposited in the DDBJ Sequence Read Archive(Accession number DRA000959)

22 Identification of Differentially Expressed Transcripts Thebiological replicates (2-3) for each set of conditions werehighly correlated (coefficient gt 095) so reads from the sametreatment were merged for subsequent analysis Trimming ofIllumina adaptor sequences and low-quality bases (119876 lt 20)by Cutadapt [8] and mapping of preprocessed reads to theIRGSP-10 genome assembly (httprapdbdnaaffrcgojp)were performed as described previously [9] To estimate theexpression levels of each transcript all preprocessed readswere mapped to the IRGSP-10 genome assembly by Bowtiewith default parameters [10] The expression level for eachtranscript was calculated as the RPKM- (Reads per KilobaseExon Model per million mapped reads-) derived read count[11] based on the number of uniquely mapped reads thatoverlapped with exonic regions A 119866-test was performed to

detect differentially expressed transcripts in the control andCd treatments based on the statistical null hypothesis that theproportions of mapped reads to the transcripts were the samebetween the two conditions A false discovery rate (FDR lt001) was used in multiple-hypothesis testing to correct formultiple comparisons When calculating fold changes 1 wasadded to avoid division by 0

23 Hierarchical Clustering and Gene Ontology EnrichmentAnalysis The Cd-responsive transcripts in root and shootwere used for hierarchical clustering analysis We used theheatmap2 in the R package gplots (version 2110) to performclustering analyses of transcripts The 119885 scores were usedto compare significant changes in gene expression A GeneOntology (GO) termwas assigned to each transcript based onthe GO annotations for biological process molecular func-tion and cellular component in RAP-DB GO enrichmentwas evaluated by Fisherrsquos exact test with a FDR thresholdof 5 for responsive transcripts in the biological processcategory of each cluster The results were plotted as minus log 10of FDR values in a heatmap

24 qRT-PCR Analysis The expression of Cd upregulatedgenes in root sample was confirmed by qRT-PCR analysisRice seeds were germinated and grown in water in a growthchamber After 10 days the seedlings were subjected to differ-ent stress treatments by transferring them towater containingdifferent reagents RNA was extracted from them and thecDNA was synthesized according to the manufacturerrsquos pro-tocol and it is used for the further analysis as described previ-ously [4]The resulting cDNAwas used for PCR amplificationin the LightCycler 480 system (Roche Basel Switzerland)with each primer set (Os04g0600300 51015840-GGCGCTCTG-AGAATCATCAC-31015840 51015840-CATTCGGGAGCTCATCTCG-31015840 Os01g0692100 51015840-ATTCACGAGTCCGCGATG-31015840 51015840-CTCTCACCCGGATCACCC-31015840 Os12g0570700 51015840-GCA-CTCATCTCAAGCTTTTC-31015840 51015840-GCAAGACATCTTCTT-GG-31015840 Os12g0571000 51015840-ATTTCCTGAAGAGTTAAA-3101584051015840-TTCCGCAGCCGCAGCTTA-31015840) The detection thresh-old cycle for each reaction was normalized using Ubiq-uitin1 primers (51015840-CCAGGACAAGATGATCTGCC-31015840 51015840-AAGAAGCTGAAGCATCCAGC-31015840)

3 Results and Discussion

31 LowCdConcentration Exposure of Rice Plants and GrowthRetardation during the Treatment Weused rice plants grownin hydroponic culture which enabled us to control the Cdexposure easily High Cd concentration exposure has beenpreviously shown to elicit robust physiological responses andgene expression as acute toxic responses in rice seedlings [12ndash14] Growth retardation of the shoot was slightly visible after1 d (data not shown) the leaves turned yellow and the leaf tipsof the seedlings began to wilt after 4 d and all leaf blades werecurled completely and the seedlings were wilting after 10 dunder high Cd concentration (50120583M) exposure (Figure 1)While no visible symptoms were observed in shoots underlow Cd concentration exposure (02 and 1 120583M Cd) after

BioMed Research International 3

14d

Con

trol

02120583

M

1120583

M

50120583

M

10d

4d

0d

Figure 1 Phenotypic changes in rice plants grown in culturemedium with low concentrations of Cd (02 1120583M) and a highconcentration of Cd (50120583M) from 0 to 14 d

1 d growth retardation occurred gradually compared withthe control with symptoms starting to appear after 7 dPlants in the same growth chamber exposed to differentCd concentrations showed clear growth differences after10 d (Figure 1) Even after 28 d the seedlings under lowCd concentration exposure did not show yellow leaves orwilting (data not shown)These results suggested that highCdconcentration exposure causes fatal damage to plants whilelow Cd concentrations lead to growth retardation (Figure 1)which is supported by the fact that plant detoxificationprocesses are insufficient to copewith this toxicmetal beyonda 10 120583M dose [15]

32 Gene Expression Profiles under Low Cd Concentra-tion Exposure in Rice We next analyzed the transcriptomeprofiles of the response to Cd exposure using RNA-Seqduring plant growth at 1 4 and 10 d after Cd treat-ment and before treatment (0 d) For each set of con-ditions an average of approximately 151 million (922)quality-evaluated reads (total 211 million) were mappedto the rice genome sequence and used for further anal-ysis (Table S1 in Supplementary Material available onlineat httpdxdoiorg10115520169739505) The number ofupregulated transcripts ranged from 4529 to 6515 whereas

Root Shoot Root Shoot

1120583M Cd02 120583M Cd

1d 4d 10d 1d 4d 10d 1d 4d 10d 1d 4d 10d11000

9000

7000

5000

3000

1000

3000

5000

7000

9000

1000

Num

ber o

f up-

or d

ownr

egul

ated

tran

scrip

ts

Figure 2 Distribution of upregulated and downregulated tran-scripts in roots and shoots in response to Cd exposure RPKM foldchanges at 1 4 and 10 d were calculated for Cd-treated samplescompared with nontreated samples (0 d) The total numbers ofupregulated (upper) and downregulated (lower) transcripts in rootsand shoots identified by RNA-Seq were determined by119866-tests (FDRlt 001) at each stress time point (1 4 and 10 d) under 02 120583M (left)and 1 120583M (right) Cd exposureThe 119909-axis shows the time course andthe 119910-axis shows the number of transcripts

the number of downregulated transcripts ranged from 2359to 8734 under 02 120583M Cd (Figure 2) Twelve transcriptsincluding GST MT and DREB (drought responsive elementbinding protein) 1E were upregulated more than 20-foldamong the upregulated transcripts in roots at 02 120583M CdThe number of upregulated transcripts ranged from 5830to 7271 whereas the number of downregulated transcriptsranged from 2965 to 10020 under 1120583M Cd (Figure 2)Fifty-one transcripts including GST MT Prx (peroxidase)and heat shock proteins were upregulated more than 20-fold among the upregulated transcripts in roots at 1 120583MCd (Table 1) Induction of detoxification enzymes againstoxidation stress such as GST and Prx under Cd exposuremight be associated with the defense system that confers Cdtolerance to plants [16ndash18] even at lowCd concentrationsThecysteine-rich MT might function as a ligand for chelation ofmetal ions to defend against Cd toxicity [19] The DREBC-repeat binding factor (CBF) specifically interacts with theDRECRT cis-acting element and controls the expression ofmany stress-inducible genes in plants [20] The activationof gene expression in several drought stress signal pathwaysunder Cd exposure has been reported [4] Five heat shock

4 BioMed Research International

Table 1 Cadmium-upregulated transcripts identified in roots by RNA-Seq analysis

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

02 120583MCdOs10t0527400-01 Tau class GST protein 3 278 214 275 12 20 17Os03t0283000-00 In2-1 protein 275 28 10 13 11 15Os08t0156000-01 Conserved hypothetical protein 264 214 253 13 16 17Os01t0627967-00 Hypothetical protein 261 165 241 15 19 14Os04t0178300-02 Syn-copalyl diphosphate synthase 201 80 203 06 42 14

Os04t0301500-01 HLH (helix-loop-helix) DNA-bindingdomain containing protein 04 331 05 10 475 92

Os02t0676800-01 DREB1E (drought responsive elementbinding protein 1E) 09 287 09 12 109 20

Os02t0179200-01 Glutamine amidotransferase class-I domaincontaining protein 08 281 17 09 32 11

Os12t0154800-00 RmlC-like jelly roll fold domain containingprotein 40 214 57 10 14 12

Os12t0570700-01 MT (metallothionein)-like protein type 1 186 203 158 09 10 09Os03t0836800-01 IAA-amino acid hydrolase 1 43 65 336 10 10 10

Os10t0333700-00 Plant disease resistance response proteindomain containing protein 97 60 216 10 10 10

1 120583MCdOs04t0178300-02 Syn-copalyl diphosphate synthase 1220 321 255 05 10 36

Os04t0178300-01 Isoform 3 of Syn-copalyl diphosphatesynthase 1098 278 215 05 09 31

Os04t0178400-01 Cytochrome P450 CYP99A1 698 211 160 08 10 28Os03t0267000-00 Heat shock protein 180 575 77 109 12 07 07Os03t0266900-01 Heat shock protein 173 470 49 53 10 04 06Os01t0136200-01 Heat shock protein 1 437 39 13 10 10 10

Os07t0190000-01 1-Deoxy-D-xylulose 5-phosphate synthase 2precursor 424 115 86 07 11 39

Os07t0127500-01 PR-1a pathogenesis related protein precursor 400 56 50 08 08 21Os07t0154100-01 Viviparous-14 388 52 15 11 14 23Os07t0154201-00 Hypothetical gene 377 47 13 10 13 21Os12t0555200-01 Probenazole-inducible protein PBZ1 377 135 109 03 05 22Os06t0586000-01 Conserved hypothetical protein 376 93 65 06 09 14Os10t0527400-01 Tau class GST protein 3 343 180 324 11 14 20Os12t0555000-01 Probenazole-inducible protein PBZ1 332 135 110 06 07 25

Os03t0277700-01 Protein of unknown function DUF26domain containing protein 328 76 34 10 06 10

Os11t0687100-01 von Willebrand factor (type A domain) 325 41 138 07 07 23Os05t0211700-00 mdash 288 14 12 10 10 10Os06t0662550-01 Conserved hypothetical protein 285 78 88 08 08 16Os01t0944100-02 Conserved hypothetical protein 284 63 98 05 06 17Os06t0568600-01 Ent-kaurene oxidase 1 271 281 110 06 14 47Os12t0418600-01 Hypothetical conserved gene 267 20 13 10 10 10Os12t0258700-01 Cupredoxin domain containing protein 262 147 106 07 11 71Os01t0615100-01 Substilinchymotrypsin-like inhibitor 256 95 79 07 10 18Os04t0107900-02 Heat shock protein 81-1 256 25 16 10 10 09

BioMed Research International 5

Table 1 Continued

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

Os09t0493000-01 Conserved hypothetical protein 253 26 18 09 12 09Os01t0627967-00 Hypothetical protein 253 195 216 13 18 14Os01t0944100-03 Conserved hypothetical protein 252 46 63 06 06 18Os04t0180400-01 Cytochrome P450 99A2 244 43 60 05 05 31Os04t0108101-00 Hypothetical protein 244 23 14 10 10 10Os02t0269600-00 Subtilase 226 78 41 03 12 60Os01t0136000-00 Heat shock protein 175 225 31 12 10 14 12Os04t0180500-00 Hypothetical protein 222 40 54 05 06 31Os01t0946600-01 Conserved hypothetical protein 218 166 80 07 07 08Os09t0255400-02 Indole-3-glycerol phosphate synthase 214 51 38 07 09 23Os01t0348900-01 SalT gene product 212 65 89 01 01 02Os12t0491800-01 Ent-kaurene synthase 1A 211 15 17 04 08 55Os01t0132000-01 Wound-induced protease inhibitor 210 88 116 16 05 02Os11t0592200-01 Chitin-binding allergen Bra r 2 207 34 28 07 05 16Os01t0963000-01 Prx (Peroxidase) BP 1 precursor 206 38 44 07 11 13Os08t0189600-01 Oryza sativa germin-like protein 8-7 206 115 67 21 15 08Os07t0496250-01 Expansin-like B1 205 22 22 15 12 45Os01t0963000-04 Prx (Peroxidase) BP 1 precursor 203 37 44 07 11 13Os09t0255400-01 Indole-3-glycerol phosphate synthase 202 52 37 07 09 23Os11t0601950-01 cDNA clone002-114-B06 200 17 19 07 10 11Os03t0129400-01 Hypothetical protein 103 271 176 10 19 36Os01t0322700-01 Nonprotein coding transcript 122 255 157 09 13 25

Os03t0129400-02 EST AU078206 corresponds to a region ofthe predicted gene 94 243 163 11 14 28

Os12t0570700-01 MT (metallothionein)-like protein type 1 167 212 177 08 08 31Os12t0571000-01 MT (metallothionein)-like protein type 1 139 200 130 09 10 36Os08t0156000-01 Conserved hypothetical protein 154 179 260 11 15 16Os03t0836800-01 IAA-amino acid hydrolase 1 07 40 237 10 10 10

Reads were mapped to the rice genome and responsive genes were identified by 119866-tests Transcripts upregulated more than 20-fold in one or moretreatmentstime points in roots are shown Transcripts in bold were upregulated under both 1 and 02 120583MCd exposure

proteins (Hsps) were strongly upregulated in roots under 1120583MCd with the greatest relative expression at 1 d (Table 1)Thesegenes may contribute to cellular homeostasis by protectingmacromolecules such as enzymes protein complexes andmembranes under Cd exposureThis result suggested that theroots of hydroponically cultured rice might be affected moredirectly and earlier by Cd exposure There was a differencebetween the low Cd concentrations in that no Hsps werestrongly upregulated in roots at 02120583MCd (Table 1) suggest-ing that the effect of this condition might be small or showtime lag In shoots 15 and 11 transcripts were upregulatedmore than 20-fold among the upregulated transcripts under02 and 1 120583M Cd respectively (Table S2) Nine transcriptsincludingNramp1 (natural resistance-associatedmacrophageprotein) were upregulated under both 02 and 1120583M Cd(Table S2) In Arabidopsis Nramp1 localizes to the plasma

membrane and functions as a high-affinity transporter formanganese (Mn) uptake [21] while OsNramp5 uptakes Mnand Cd [22] Transporters with heavymetal binding domainsare often capable of transporting several metals such as FeZn Mn and Cd because of their low substrate specificity[23ndash26] We found that upregulation of a HLHDNA-bindingdomain containing transcription factor (Os04g0301500) inboth roots and shoots peaked at 4 d under 02120583M Cdthis protein may function as a regulatory factor under Cdexposure (Table 1 Table S2) The number of downregulatedtranscripts in roots peaked at 4 d after Cd exposure whilethe number in shoots gradually increased under low Cdconcentration exposure (Figure 2) A few dozen transcriptswere downregulated less than 005-fold among the down-regulated transcripts in roots and shoots under Cd exposure(Table S2)Therefore a small part of transcripts were strongly

6 BioMed Research International

up- or downregulated among several thousand responsivetranscripts under low Cd concentration exposure Large-scale changes in gene expression occurred in rice under Cdexposure even at low concentrations possibly because Cd isa nonessential metal for the plant

To obtain a functional annotation of responsive tran-scripts under Cd exposure we used GO biological processcategories The responsive transcripts in shoot and root wereclustered into several groups based on their expression pat-terns GO enrichment analysis was performed using clusteredtranscripts assigned by GO terms in RAP-DB (The RiceAnnotation Project Database [httprapdbdnaaffrcgojp])(Supplementary Figure S1) Enriched GO terms significantlyin each cluster may represent the functional categories inrice under Cd exposure Enriched GO terms of graduallyupregulated transcripts under Cd exposure include metalion transport (GO0030001) (cluster 3 in root under 02 120583MCd cluster 4 in root under 1120583M Cd) which may functionin Cd transport Response to oxidative stress (GO0006979)and responsive to oxidative stress (GO0006979) were alsoincluded in cluster 3 and cluster 4 respectivelyThis suggestedthat they might function in defense against Cd EnrichedGO terms of gradually downregulated transcripts underCd exposure include translation (GO0006412) translationelongation (GO0006414) DNA replication (GO0006260)andDNA repair (GO0006281) (cluster 1 in root under 02120583MCd cluster 2 in root under 1120583M Cd) Photosynthesis lightharvesting (GO0009765) and photosynthesis (GO0015979)were also included in both clusters These may function inplant growthThus these correspond to the observed changesin phenotype (Figure 1) which clearly validated the RNA-Seqexpression profiling data obtained from rice tissue under Cdstress condition However the pattern of gene expression isquite complex and would require more detailed analysis

33 Constitutively Expressed Genes Responded Differentlyunder Low Cd Concentration to High Cd Concentration Asmany genes responded to both low and high Cd concentra-tions [4] we assessed the effect of the stress degree on riceseedlings through the expression of constitutively expressedgenes We investigated the expression of 18 genes annotatedby the RAP that were expressed constitutively in 39 tissuescollected throughout the life cycle of the rice plant fromtwo varieties according to 190 Affymetrix GeneChip RiceGenome Arrays in addition to four genes annotated by theRAP that have frequently been used as internal controlsin expression analyses [27] The results showed that theexpression of more than half of them fluctuated drastically(gt2 orlt2) in roots or shoots after 1 d of highCd concentrationexposure (Figure 3) This drastic response may be partlybecause RNA-Seq can accurately quantify gene expressionlevels over a broad dynamic range with high resolution andsensitivity [10 28 29] However our results suggest that theirexpression is greatly affected by strong stress even thoughthey are expressed constitutively across the developmentalcourse Note that a high Cd concentration can cause fataldamage to rice seedlings such as by affecting homeostasiswhich corresponds to the observed changes in phenotype(Figures 1 and 3)

34 Comparative Gene Expression Analysis between Low andHigh Cd Concentrations Reveals Novel Cd-Responsive Trans-porters We investigated the expression of metal transportergenes containing metal ion binding Pfam domains [PF01554(MatE) PF08370 (PDR assoc) PF01545 (Cation efflux)PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)]that may function in Cd transport under Cd exposureThe expression of 183 transport transcripts was comparedbetween low and high Cd concentration treatments in rootsand shoots at 1 d because Cd uptake from the hydroponicculture and efflux pumping are initial responses to Cdexposure (Figure 4 Table S3) The transcripts tended tobe more responsive in roots and shoots under higher Cdconcentration exposure This result indicated the potentialof the RNA-Seq strategy to reveal novel Cd-responsivetransporters by analyzing gene expression under exposureto different Cd concentrations The responsive transcriptsmight function in roots at the early stage of Cd exposureNo transcripts were upregulated more than 3-fold in shootsunder low Cd exposure (Figure 4 Table S3) suggesting thatthe effect takes more time to appear in shootsOs03g0667500(Zip root) encoding iron-regulated transporter 1 (IRT1) wasupregulated more than 5-fold under low Cd concentrationsbut responded only slightly under the high Cd concentrationIRT1s often transport Cd because of their low substrate speci-ficity [24ndash26 30]Os02g0585200 (HMA root)Os03g0152000(HMA root) Os0g0584800 (HMA root) Os01g0609900(PDR assoc shoot) and Os01g0609300 (PDR assoc shoot)showed the highest (32-fold) upregulation under high Cdconcentration exposure and responded only slightly to lowCd concentrations (Table S3) The balance between Cd andvarious other metal ions in the hydroponic culture mightaffect the expression of these genes because specific systemsfor transporting Cd may have not developed in rice as it is anonessentialmetalThe effects of other ions on the expressionof transporters [4] and responsive genes associated withdefense systems against Cd (Supplementary Figure S2) havebeen indicated

4 Conclusions

We generated gene expression profiles for rice seedlingsgrown under low Cd concentrations Phenotypic observa-tions and constitutive gene expression indicated that low Cdconcentrations cause growth retardation but are far frombeing fatal in rice Several genes associated with defense sys-tems were strongly upregulated the expression of metal iontransporter genes tended to correlate with Cd concentrationand GO enrichment analysis of the clustered genes based ontheir expression patterns suggesting that our transcriptomeprofiles reflect responses to Cd in rice Our data also suggestthat it could be dangerous to eat plants that do not showspecificCd pollution symptoms growing in soil contaminatedby small amounts of Cd Establishing the exact compositionand organization of the transcriptional network underlyingthe response to Cd exposure will provide a robust tool forimproving crops in the future for example by creating lowCd uptake plants

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

14d

Con

trol

02120583

M

1120583

M

50120583

M

10d

4d

0d

Figure 1 Phenotypic changes in rice plants grown in culturemedium with low concentrations of Cd (02 1120583M) and a highconcentration of Cd (50120583M) from 0 to 14 d

1 d growth retardation occurred gradually compared withthe control with symptoms starting to appear after 7 dPlants in the same growth chamber exposed to differentCd concentrations showed clear growth differences after10 d (Figure 1) Even after 28 d the seedlings under lowCd concentration exposure did not show yellow leaves orwilting (data not shown)These results suggested that highCdconcentration exposure causes fatal damage to plants whilelow Cd concentrations lead to growth retardation (Figure 1)which is supported by the fact that plant detoxificationprocesses are insufficient to copewith this toxicmetal beyonda 10 120583M dose [15]

32 Gene Expression Profiles under Low Cd Concentra-tion Exposure in Rice We next analyzed the transcriptomeprofiles of the response to Cd exposure using RNA-Seqduring plant growth at 1 4 and 10 d after Cd treat-ment and before treatment (0 d) For each set of con-ditions an average of approximately 151 million (922)quality-evaluated reads (total 211 million) were mappedto the rice genome sequence and used for further anal-ysis (Table S1 in Supplementary Material available onlineat httpdxdoiorg10115520169739505) The number ofupregulated transcripts ranged from 4529 to 6515 whereas

Root Shoot Root Shoot

1120583M Cd02 120583M Cd

1d 4d 10d 1d 4d 10d 1d 4d 10d 1d 4d 10d11000

9000

7000

5000

3000

1000

3000

5000

7000

9000

1000

Num

ber o

f up-

or d

ownr

egul

ated

tran

scrip

ts

Figure 2 Distribution of upregulated and downregulated tran-scripts in roots and shoots in response to Cd exposure RPKM foldchanges at 1 4 and 10 d were calculated for Cd-treated samplescompared with nontreated samples (0 d) The total numbers ofupregulated (upper) and downregulated (lower) transcripts in rootsand shoots identified by RNA-Seq were determined by119866-tests (FDRlt 001) at each stress time point (1 4 and 10 d) under 02 120583M (left)and 1 120583M (right) Cd exposureThe 119909-axis shows the time course andthe 119910-axis shows the number of transcripts

the number of downregulated transcripts ranged from 2359to 8734 under 02 120583M Cd (Figure 2) Twelve transcriptsincluding GST MT and DREB (drought responsive elementbinding protein) 1E were upregulated more than 20-foldamong the upregulated transcripts in roots at 02 120583M CdThe number of upregulated transcripts ranged from 5830to 7271 whereas the number of downregulated transcriptsranged from 2965 to 10020 under 1120583M Cd (Figure 2)Fifty-one transcripts including GST MT Prx (peroxidase)and heat shock proteins were upregulated more than 20-fold among the upregulated transcripts in roots at 1 120583MCd (Table 1) Induction of detoxification enzymes againstoxidation stress such as GST and Prx under Cd exposuremight be associated with the defense system that confers Cdtolerance to plants [16ndash18] even at lowCd concentrationsThecysteine-rich MT might function as a ligand for chelation ofmetal ions to defend against Cd toxicity [19] The DREBC-repeat binding factor (CBF) specifically interacts with theDRECRT cis-acting element and controls the expression ofmany stress-inducible genes in plants [20] The activationof gene expression in several drought stress signal pathwaysunder Cd exposure has been reported [4] Five heat shock

4 BioMed Research International

Table 1 Cadmium-upregulated transcripts identified in roots by RNA-Seq analysis

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

02 120583MCdOs10t0527400-01 Tau class GST protein 3 278 214 275 12 20 17Os03t0283000-00 In2-1 protein 275 28 10 13 11 15Os08t0156000-01 Conserved hypothetical protein 264 214 253 13 16 17Os01t0627967-00 Hypothetical protein 261 165 241 15 19 14Os04t0178300-02 Syn-copalyl diphosphate synthase 201 80 203 06 42 14

Os04t0301500-01 HLH (helix-loop-helix) DNA-bindingdomain containing protein 04 331 05 10 475 92

Os02t0676800-01 DREB1E (drought responsive elementbinding protein 1E) 09 287 09 12 109 20

Os02t0179200-01 Glutamine amidotransferase class-I domaincontaining protein 08 281 17 09 32 11

Os12t0154800-00 RmlC-like jelly roll fold domain containingprotein 40 214 57 10 14 12

Os12t0570700-01 MT (metallothionein)-like protein type 1 186 203 158 09 10 09Os03t0836800-01 IAA-amino acid hydrolase 1 43 65 336 10 10 10

Os10t0333700-00 Plant disease resistance response proteindomain containing protein 97 60 216 10 10 10

1 120583MCdOs04t0178300-02 Syn-copalyl diphosphate synthase 1220 321 255 05 10 36

Os04t0178300-01 Isoform 3 of Syn-copalyl diphosphatesynthase 1098 278 215 05 09 31

Os04t0178400-01 Cytochrome P450 CYP99A1 698 211 160 08 10 28Os03t0267000-00 Heat shock protein 180 575 77 109 12 07 07Os03t0266900-01 Heat shock protein 173 470 49 53 10 04 06Os01t0136200-01 Heat shock protein 1 437 39 13 10 10 10

Os07t0190000-01 1-Deoxy-D-xylulose 5-phosphate synthase 2precursor 424 115 86 07 11 39

Os07t0127500-01 PR-1a pathogenesis related protein precursor 400 56 50 08 08 21Os07t0154100-01 Viviparous-14 388 52 15 11 14 23Os07t0154201-00 Hypothetical gene 377 47 13 10 13 21Os12t0555200-01 Probenazole-inducible protein PBZ1 377 135 109 03 05 22Os06t0586000-01 Conserved hypothetical protein 376 93 65 06 09 14Os10t0527400-01 Tau class GST protein 3 343 180 324 11 14 20Os12t0555000-01 Probenazole-inducible protein PBZ1 332 135 110 06 07 25

Os03t0277700-01 Protein of unknown function DUF26domain containing protein 328 76 34 10 06 10

Os11t0687100-01 von Willebrand factor (type A domain) 325 41 138 07 07 23Os05t0211700-00 mdash 288 14 12 10 10 10Os06t0662550-01 Conserved hypothetical protein 285 78 88 08 08 16Os01t0944100-02 Conserved hypothetical protein 284 63 98 05 06 17Os06t0568600-01 Ent-kaurene oxidase 1 271 281 110 06 14 47Os12t0418600-01 Hypothetical conserved gene 267 20 13 10 10 10Os12t0258700-01 Cupredoxin domain containing protein 262 147 106 07 11 71Os01t0615100-01 Substilinchymotrypsin-like inhibitor 256 95 79 07 10 18Os04t0107900-02 Heat shock protein 81-1 256 25 16 10 10 09

BioMed Research International 5

Table 1 Continued

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

Os09t0493000-01 Conserved hypothetical protein 253 26 18 09 12 09Os01t0627967-00 Hypothetical protein 253 195 216 13 18 14Os01t0944100-03 Conserved hypothetical protein 252 46 63 06 06 18Os04t0180400-01 Cytochrome P450 99A2 244 43 60 05 05 31Os04t0108101-00 Hypothetical protein 244 23 14 10 10 10Os02t0269600-00 Subtilase 226 78 41 03 12 60Os01t0136000-00 Heat shock protein 175 225 31 12 10 14 12Os04t0180500-00 Hypothetical protein 222 40 54 05 06 31Os01t0946600-01 Conserved hypothetical protein 218 166 80 07 07 08Os09t0255400-02 Indole-3-glycerol phosphate synthase 214 51 38 07 09 23Os01t0348900-01 SalT gene product 212 65 89 01 01 02Os12t0491800-01 Ent-kaurene synthase 1A 211 15 17 04 08 55Os01t0132000-01 Wound-induced protease inhibitor 210 88 116 16 05 02Os11t0592200-01 Chitin-binding allergen Bra r 2 207 34 28 07 05 16Os01t0963000-01 Prx (Peroxidase) BP 1 precursor 206 38 44 07 11 13Os08t0189600-01 Oryza sativa germin-like protein 8-7 206 115 67 21 15 08Os07t0496250-01 Expansin-like B1 205 22 22 15 12 45Os01t0963000-04 Prx (Peroxidase) BP 1 precursor 203 37 44 07 11 13Os09t0255400-01 Indole-3-glycerol phosphate synthase 202 52 37 07 09 23Os11t0601950-01 cDNA clone002-114-B06 200 17 19 07 10 11Os03t0129400-01 Hypothetical protein 103 271 176 10 19 36Os01t0322700-01 Nonprotein coding transcript 122 255 157 09 13 25

Os03t0129400-02 EST AU078206 corresponds to a region ofthe predicted gene 94 243 163 11 14 28

Os12t0570700-01 MT (metallothionein)-like protein type 1 167 212 177 08 08 31Os12t0571000-01 MT (metallothionein)-like protein type 1 139 200 130 09 10 36Os08t0156000-01 Conserved hypothetical protein 154 179 260 11 15 16Os03t0836800-01 IAA-amino acid hydrolase 1 07 40 237 10 10 10

Reads were mapped to the rice genome and responsive genes were identified by 119866-tests Transcripts upregulated more than 20-fold in one or moretreatmentstime points in roots are shown Transcripts in bold were upregulated under both 1 and 02 120583MCd exposure

proteins (Hsps) were strongly upregulated in roots under 1120583MCd with the greatest relative expression at 1 d (Table 1)Thesegenes may contribute to cellular homeostasis by protectingmacromolecules such as enzymes protein complexes andmembranes under Cd exposureThis result suggested that theroots of hydroponically cultured rice might be affected moredirectly and earlier by Cd exposure There was a differencebetween the low Cd concentrations in that no Hsps werestrongly upregulated in roots at 02120583MCd (Table 1) suggest-ing that the effect of this condition might be small or showtime lag In shoots 15 and 11 transcripts were upregulatedmore than 20-fold among the upregulated transcripts under02 and 1 120583M Cd respectively (Table S2) Nine transcriptsincludingNramp1 (natural resistance-associatedmacrophageprotein) were upregulated under both 02 and 1120583M Cd(Table S2) In Arabidopsis Nramp1 localizes to the plasma

membrane and functions as a high-affinity transporter formanganese (Mn) uptake [21] while OsNramp5 uptakes Mnand Cd [22] Transporters with heavymetal binding domainsare often capable of transporting several metals such as FeZn Mn and Cd because of their low substrate specificity[23ndash26] We found that upregulation of a HLHDNA-bindingdomain containing transcription factor (Os04g0301500) inboth roots and shoots peaked at 4 d under 02120583M Cdthis protein may function as a regulatory factor under Cdexposure (Table 1 Table S2) The number of downregulatedtranscripts in roots peaked at 4 d after Cd exposure whilethe number in shoots gradually increased under low Cdconcentration exposure (Figure 2) A few dozen transcriptswere downregulated less than 005-fold among the down-regulated transcripts in roots and shoots under Cd exposure(Table S2)Therefore a small part of transcripts were strongly

6 BioMed Research International

up- or downregulated among several thousand responsivetranscripts under low Cd concentration exposure Large-scale changes in gene expression occurred in rice under Cdexposure even at low concentrations possibly because Cd isa nonessential metal for the plant

To obtain a functional annotation of responsive tran-scripts under Cd exposure we used GO biological processcategories The responsive transcripts in shoot and root wereclustered into several groups based on their expression pat-terns GO enrichment analysis was performed using clusteredtranscripts assigned by GO terms in RAP-DB (The RiceAnnotation Project Database [httprapdbdnaaffrcgojp])(Supplementary Figure S1) Enriched GO terms significantlyin each cluster may represent the functional categories inrice under Cd exposure Enriched GO terms of graduallyupregulated transcripts under Cd exposure include metalion transport (GO0030001) (cluster 3 in root under 02 120583MCd cluster 4 in root under 1120583M Cd) which may functionin Cd transport Response to oxidative stress (GO0006979)and responsive to oxidative stress (GO0006979) were alsoincluded in cluster 3 and cluster 4 respectivelyThis suggestedthat they might function in defense against Cd EnrichedGO terms of gradually downregulated transcripts underCd exposure include translation (GO0006412) translationelongation (GO0006414) DNA replication (GO0006260)andDNA repair (GO0006281) (cluster 1 in root under 02120583MCd cluster 2 in root under 1120583M Cd) Photosynthesis lightharvesting (GO0009765) and photosynthesis (GO0015979)were also included in both clusters These may function inplant growthThus these correspond to the observed changesin phenotype (Figure 1) which clearly validated the RNA-Seqexpression profiling data obtained from rice tissue under Cdstress condition However the pattern of gene expression isquite complex and would require more detailed analysis

33 Constitutively Expressed Genes Responded Differentlyunder Low Cd Concentration to High Cd Concentration Asmany genes responded to both low and high Cd concentra-tions [4] we assessed the effect of the stress degree on riceseedlings through the expression of constitutively expressedgenes We investigated the expression of 18 genes annotatedby the RAP that were expressed constitutively in 39 tissuescollected throughout the life cycle of the rice plant fromtwo varieties according to 190 Affymetrix GeneChip RiceGenome Arrays in addition to four genes annotated by theRAP that have frequently been used as internal controlsin expression analyses [27] The results showed that theexpression of more than half of them fluctuated drastically(gt2 orlt2) in roots or shoots after 1 d of highCd concentrationexposure (Figure 3) This drastic response may be partlybecause RNA-Seq can accurately quantify gene expressionlevels over a broad dynamic range with high resolution andsensitivity [10 28 29] However our results suggest that theirexpression is greatly affected by strong stress even thoughthey are expressed constitutively across the developmentalcourse Note that a high Cd concentration can cause fataldamage to rice seedlings such as by affecting homeostasiswhich corresponds to the observed changes in phenotype(Figures 1 and 3)

34 Comparative Gene Expression Analysis between Low andHigh Cd Concentrations Reveals Novel Cd-Responsive Trans-porters We investigated the expression of metal transportergenes containing metal ion binding Pfam domains [PF01554(MatE) PF08370 (PDR assoc) PF01545 (Cation efflux)PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)]that may function in Cd transport under Cd exposureThe expression of 183 transport transcripts was comparedbetween low and high Cd concentration treatments in rootsand shoots at 1 d because Cd uptake from the hydroponicculture and efflux pumping are initial responses to Cdexposure (Figure 4 Table S3) The transcripts tended tobe more responsive in roots and shoots under higher Cdconcentration exposure This result indicated the potentialof the RNA-Seq strategy to reveal novel Cd-responsivetransporters by analyzing gene expression under exposureto different Cd concentrations The responsive transcriptsmight function in roots at the early stage of Cd exposureNo transcripts were upregulated more than 3-fold in shootsunder low Cd exposure (Figure 4 Table S3) suggesting thatthe effect takes more time to appear in shootsOs03g0667500(Zip root) encoding iron-regulated transporter 1 (IRT1) wasupregulated more than 5-fold under low Cd concentrationsbut responded only slightly under the high Cd concentrationIRT1s often transport Cd because of their low substrate speci-ficity [24ndash26 30]Os02g0585200 (HMA root)Os03g0152000(HMA root) Os0g0584800 (HMA root) Os01g0609900(PDR assoc shoot) and Os01g0609300 (PDR assoc shoot)showed the highest (32-fold) upregulation under high Cdconcentration exposure and responded only slightly to lowCd concentrations (Table S3) The balance between Cd andvarious other metal ions in the hydroponic culture mightaffect the expression of these genes because specific systemsfor transporting Cd may have not developed in rice as it is anonessentialmetalThe effects of other ions on the expressionof transporters [4] and responsive genes associated withdefense systems against Cd (Supplementary Figure S2) havebeen indicated

4 Conclusions

We generated gene expression profiles for rice seedlingsgrown under low Cd concentrations Phenotypic observa-tions and constitutive gene expression indicated that low Cdconcentrations cause growth retardation but are far frombeing fatal in rice Several genes associated with defense sys-tems were strongly upregulated the expression of metal iontransporter genes tended to correlate with Cd concentrationand GO enrichment analysis of the clustered genes based ontheir expression patterns suggesting that our transcriptomeprofiles reflect responses to Cd in rice Our data also suggestthat it could be dangerous to eat plants that do not showspecificCd pollution symptoms growing in soil contaminatedby small amounts of Cd Establishing the exact compositionand organization of the transcriptional network underlyingthe response to Cd exposure will provide a robust tool forimproving crops in the future for example by creating lowCd uptake plants

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table 1 Cadmium-upregulated transcripts identified in roots by RNA-Seq analysis

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

02 120583MCdOs10t0527400-01 Tau class GST protein 3 278 214 275 12 20 17Os03t0283000-00 In2-1 protein 275 28 10 13 11 15Os08t0156000-01 Conserved hypothetical protein 264 214 253 13 16 17Os01t0627967-00 Hypothetical protein 261 165 241 15 19 14Os04t0178300-02 Syn-copalyl diphosphate synthase 201 80 203 06 42 14

Os04t0301500-01 HLH (helix-loop-helix) DNA-bindingdomain containing protein 04 331 05 10 475 92

Os02t0676800-01 DREB1E (drought responsive elementbinding protein 1E) 09 287 09 12 109 20

Os02t0179200-01 Glutamine amidotransferase class-I domaincontaining protein 08 281 17 09 32 11

Os12t0154800-00 RmlC-like jelly roll fold domain containingprotein 40 214 57 10 14 12

Os12t0570700-01 MT (metallothionein)-like protein type 1 186 203 158 09 10 09Os03t0836800-01 IAA-amino acid hydrolase 1 43 65 336 10 10 10

Os10t0333700-00 Plant disease resistance response proteindomain containing protein 97 60 216 10 10 10

1 120583MCdOs04t0178300-02 Syn-copalyl diphosphate synthase 1220 321 255 05 10 36

Os04t0178300-01 Isoform 3 of Syn-copalyl diphosphatesynthase 1098 278 215 05 09 31

Os04t0178400-01 Cytochrome P450 CYP99A1 698 211 160 08 10 28Os03t0267000-00 Heat shock protein 180 575 77 109 12 07 07Os03t0266900-01 Heat shock protein 173 470 49 53 10 04 06Os01t0136200-01 Heat shock protein 1 437 39 13 10 10 10

Os07t0190000-01 1-Deoxy-D-xylulose 5-phosphate synthase 2precursor 424 115 86 07 11 39

Os07t0127500-01 PR-1a pathogenesis related protein precursor 400 56 50 08 08 21Os07t0154100-01 Viviparous-14 388 52 15 11 14 23Os07t0154201-00 Hypothetical gene 377 47 13 10 13 21Os12t0555200-01 Probenazole-inducible protein PBZ1 377 135 109 03 05 22Os06t0586000-01 Conserved hypothetical protein 376 93 65 06 09 14Os10t0527400-01 Tau class GST protein 3 343 180 324 11 14 20Os12t0555000-01 Probenazole-inducible protein PBZ1 332 135 110 06 07 25

Os03t0277700-01 Protein of unknown function DUF26domain containing protein 328 76 34 10 06 10

Os11t0687100-01 von Willebrand factor (type A domain) 325 41 138 07 07 23Os05t0211700-00 mdash 288 14 12 10 10 10Os06t0662550-01 Conserved hypothetical protein 285 78 88 08 08 16Os01t0944100-02 Conserved hypothetical protein 284 63 98 05 06 17Os06t0568600-01 Ent-kaurene oxidase 1 271 281 110 06 14 47Os12t0418600-01 Hypothetical conserved gene 267 20 13 10 10 10Os12t0258700-01 Cupredoxin domain containing protein 262 147 106 07 11 71Os01t0615100-01 Substilinchymotrypsin-like inhibitor 256 95 79 07 10 18Os04t0107900-02 Heat shock protein 81-1 256 25 16 10 10 09

BioMed Research International 5

Table 1 Continued

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

Os09t0493000-01 Conserved hypothetical protein 253 26 18 09 12 09Os01t0627967-00 Hypothetical protein 253 195 216 13 18 14Os01t0944100-03 Conserved hypothetical protein 252 46 63 06 06 18Os04t0180400-01 Cytochrome P450 99A2 244 43 60 05 05 31Os04t0108101-00 Hypothetical protein 244 23 14 10 10 10Os02t0269600-00 Subtilase 226 78 41 03 12 60Os01t0136000-00 Heat shock protein 175 225 31 12 10 14 12Os04t0180500-00 Hypothetical protein 222 40 54 05 06 31Os01t0946600-01 Conserved hypothetical protein 218 166 80 07 07 08Os09t0255400-02 Indole-3-glycerol phosphate synthase 214 51 38 07 09 23Os01t0348900-01 SalT gene product 212 65 89 01 01 02Os12t0491800-01 Ent-kaurene synthase 1A 211 15 17 04 08 55Os01t0132000-01 Wound-induced protease inhibitor 210 88 116 16 05 02Os11t0592200-01 Chitin-binding allergen Bra r 2 207 34 28 07 05 16Os01t0963000-01 Prx (Peroxidase) BP 1 precursor 206 38 44 07 11 13Os08t0189600-01 Oryza sativa germin-like protein 8-7 206 115 67 21 15 08Os07t0496250-01 Expansin-like B1 205 22 22 15 12 45Os01t0963000-04 Prx (Peroxidase) BP 1 precursor 203 37 44 07 11 13Os09t0255400-01 Indole-3-glycerol phosphate synthase 202 52 37 07 09 23Os11t0601950-01 cDNA clone002-114-B06 200 17 19 07 10 11Os03t0129400-01 Hypothetical protein 103 271 176 10 19 36Os01t0322700-01 Nonprotein coding transcript 122 255 157 09 13 25

Os03t0129400-02 EST AU078206 corresponds to a region ofthe predicted gene 94 243 163 11 14 28

Os12t0570700-01 MT (metallothionein)-like protein type 1 167 212 177 08 08 31Os12t0571000-01 MT (metallothionein)-like protein type 1 139 200 130 09 10 36Os08t0156000-01 Conserved hypothetical protein 154 179 260 11 15 16Os03t0836800-01 IAA-amino acid hydrolase 1 07 40 237 10 10 10

Reads were mapped to the rice genome and responsive genes were identified by 119866-tests Transcripts upregulated more than 20-fold in one or moretreatmentstime points in roots are shown Transcripts in bold were upregulated under both 1 and 02 120583MCd exposure

proteins (Hsps) were strongly upregulated in roots under 1120583MCd with the greatest relative expression at 1 d (Table 1)Thesegenes may contribute to cellular homeostasis by protectingmacromolecules such as enzymes protein complexes andmembranes under Cd exposureThis result suggested that theroots of hydroponically cultured rice might be affected moredirectly and earlier by Cd exposure There was a differencebetween the low Cd concentrations in that no Hsps werestrongly upregulated in roots at 02120583MCd (Table 1) suggest-ing that the effect of this condition might be small or showtime lag In shoots 15 and 11 transcripts were upregulatedmore than 20-fold among the upregulated transcripts under02 and 1 120583M Cd respectively (Table S2) Nine transcriptsincludingNramp1 (natural resistance-associatedmacrophageprotein) were upregulated under both 02 and 1120583M Cd(Table S2) In Arabidopsis Nramp1 localizes to the plasma

membrane and functions as a high-affinity transporter formanganese (Mn) uptake [21] while OsNramp5 uptakes Mnand Cd [22] Transporters with heavymetal binding domainsare often capable of transporting several metals such as FeZn Mn and Cd because of their low substrate specificity[23ndash26] We found that upregulation of a HLHDNA-bindingdomain containing transcription factor (Os04g0301500) inboth roots and shoots peaked at 4 d under 02120583M Cdthis protein may function as a regulatory factor under Cdexposure (Table 1 Table S2) The number of downregulatedtranscripts in roots peaked at 4 d after Cd exposure whilethe number in shoots gradually increased under low Cdconcentration exposure (Figure 2) A few dozen transcriptswere downregulated less than 005-fold among the down-regulated transcripts in roots and shoots under Cd exposure(Table S2)Therefore a small part of transcripts were strongly

6 BioMed Research International

up- or downregulated among several thousand responsivetranscripts under low Cd concentration exposure Large-scale changes in gene expression occurred in rice under Cdexposure even at low concentrations possibly because Cd isa nonessential metal for the plant

To obtain a functional annotation of responsive tran-scripts under Cd exposure we used GO biological processcategories The responsive transcripts in shoot and root wereclustered into several groups based on their expression pat-terns GO enrichment analysis was performed using clusteredtranscripts assigned by GO terms in RAP-DB (The RiceAnnotation Project Database [httprapdbdnaaffrcgojp])(Supplementary Figure S1) Enriched GO terms significantlyin each cluster may represent the functional categories inrice under Cd exposure Enriched GO terms of graduallyupregulated transcripts under Cd exposure include metalion transport (GO0030001) (cluster 3 in root under 02 120583MCd cluster 4 in root under 1120583M Cd) which may functionin Cd transport Response to oxidative stress (GO0006979)and responsive to oxidative stress (GO0006979) were alsoincluded in cluster 3 and cluster 4 respectivelyThis suggestedthat they might function in defense against Cd EnrichedGO terms of gradually downregulated transcripts underCd exposure include translation (GO0006412) translationelongation (GO0006414) DNA replication (GO0006260)andDNA repair (GO0006281) (cluster 1 in root under 02120583MCd cluster 2 in root under 1120583M Cd) Photosynthesis lightharvesting (GO0009765) and photosynthesis (GO0015979)were also included in both clusters These may function inplant growthThus these correspond to the observed changesin phenotype (Figure 1) which clearly validated the RNA-Seqexpression profiling data obtained from rice tissue under Cdstress condition However the pattern of gene expression isquite complex and would require more detailed analysis

33 Constitutively Expressed Genes Responded Differentlyunder Low Cd Concentration to High Cd Concentration Asmany genes responded to both low and high Cd concentra-tions [4] we assessed the effect of the stress degree on riceseedlings through the expression of constitutively expressedgenes We investigated the expression of 18 genes annotatedby the RAP that were expressed constitutively in 39 tissuescollected throughout the life cycle of the rice plant fromtwo varieties according to 190 Affymetrix GeneChip RiceGenome Arrays in addition to four genes annotated by theRAP that have frequently been used as internal controlsin expression analyses [27] The results showed that theexpression of more than half of them fluctuated drastically(gt2 orlt2) in roots or shoots after 1 d of highCd concentrationexposure (Figure 3) This drastic response may be partlybecause RNA-Seq can accurately quantify gene expressionlevels over a broad dynamic range with high resolution andsensitivity [10 28 29] However our results suggest that theirexpression is greatly affected by strong stress even thoughthey are expressed constitutively across the developmentalcourse Note that a high Cd concentration can cause fataldamage to rice seedlings such as by affecting homeostasiswhich corresponds to the observed changes in phenotype(Figures 1 and 3)

34 Comparative Gene Expression Analysis between Low andHigh Cd Concentrations Reveals Novel Cd-Responsive Trans-porters We investigated the expression of metal transportergenes containing metal ion binding Pfam domains [PF01554(MatE) PF08370 (PDR assoc) PF01545 (Cation efflux)PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)]that may function in Cd transport under Cd exposureThe expression of 183 transport transcripts was comparedbetween low and high Cd concentration treatments in rootsand shoots at 1 d because Cd uptake from the hydroponicculture and efflux pumping are initial responses to Cdexposure (Figure 4 Table S3) The transcripts tended tobe more responsive in roots and shoots under higher Cdconcentration exposure This result indicated the potentialof the RNA-Seq strategy to reveal novel Cd-responsivetransporters by analyzing gene expression under exposureto different Cd concentrations The responsive transcriptsmight function in roots at the early stage of Cd exposureNo transcripts were upregulated more than 3-fold in shootsunder low Cd exposure (Figure 4 Table S3) suggesting thatthe effect takes more time to appear in shootsOs03g0667500(Zip root) encoding iron-regulated transporter 1 (IRT1) wasupregulated more than 5-fold under low Cd concentrationsbut responded only slightly under the high Cd concentrationIRT1s often transport Cd because of their low substrate speci-ficity [24ndash26 30]Os02g0585200 (HMA root)Os03g0152000(HMA root) Os0g0584800 (HMA root) Os01g0609900(PDR assoc shoot) and Os01g0609300 (PDR assoc shoot)showed the highest (32-fold) upregulation under high Cdconcentration exposure and responded only slightly to lowCd concentrations (Table S3) The balance between Cd andvarious other metal ions in the hydroponic culture mightaffect the expression of these genes because specific systemsfor transporting Cd may have not developed in rice as it is anonessentialmetalThe effects of other ions on the expressionof transporters [4] and responsive genes associated withdefense systems against Cd (Supplementary Figure S2) havebeen indicated

4 Conclusions

We generated gene expression profiles for rice seedlingsgrown under low Cd concentrations Phenotypic observa-tions and constitutive gene expression indicated that low Cdconcentrations cause growth retardation but are far frombeing fatal in rice Several genes associated with defense sys-tems were strongly upregulated the expression of metal iontransporter genes tended to correlate with Cd concentrationand GO enrichment analysis of the clustered genes based ontheir expression patterns suggesting that our transcriptomeprofiles reflect responses to Cd in rice Our data also suggestthat it could be dangerous to eat plants that do not showspecificCd pollution symptoms growing in soil contaminatedby small amounts of Cd Establishing the exact compositionand organization of the transcriptional network underlyingthe response to Cd exposure will provide a robust tool forimproving crops in the future for example by creating lowCd uptake plants

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table 1 Continued

Transcript DescriptionFold change

Root Shoot1 d 4 d 10 d 1 d 4 d 10 d

Os09t0493000-01 Conserved hypothetical protein 253 26 18 09 12 09Os01t0627967-00 Hypothetical protein 253 195 216 13 18 14Os01t0944100-03 Conserved hypothetical protein 252 46 63 06 06 18Os04t0180400-01 Cytochrome P450 99A2 244 43 60 05 05 31Os04t0108101-00 Hypothetical protein 244 23 14 10 10 10Os02t0269600-00 Subtilase 226 78 41 03 12 60Os01t0136000-00 Heat shock protein 175 225 31 12 10 14 12Os04t0180500-00 Hypothetical protein 222 40 54 05 06 31Os01t0946600-01 Conserved hypothetical protein 218 166 80 07 07 08Os09t0255400-02 Indole-3-glycerol phosphate synthase 214 51 38 07 09 23Os01t0348900-01 SalT gene product 212 65 89 01 01 02Os12t0491800-01 Ent-kaurene synthase 1A 211 15 17 04 08 55Os01t0132000-01 Wound-induced protease inhibitor 210 88 116 16 05 02Os11t0592200-01 Chitin-binding allergen Bra r 2 207 34 28 07 05 16Os01t0963000-01 Prx (Peroxidase) BP 1 precursor 206 38 44 07 11 13Os08t0189600-01 Oryza sativa germin-like protein 8-7 206 115 67 21 15 08Os07t0496250-01 Expansin-like B1 205 22 22 15 12 45Os01t0963000-04 Prx (Peroxidase) BP 1 precursor 203 37 44 07 11 13Os09t0255400-01 Indole-3-glycerol phosphate synthase 202 52 37 07 09 23Os11t0601950-01 cDNA clone002-114-B06 200 17 19 07 10 11Os03t0129400-01 Hypothetical protein 103 271 176 10 19 36Os01t0322700-01 Nonprotein coding transcript 122 255 157 09 13 25

Os03t0129400-02 EST AU078206 corresponds to a region ofthe predicted gene 94 243 163 11 14 28

Os12t0570700-01 MT (metallothionein)-like protein type 1 167 212 177 08 08 31Os12t0571000-01 MT (metallothionein)-like protein type 1 139 200 130 09 10 36Os08t0156000-01 Conserved hypothetical protein 154 179 260 11 15 16Os03t0836800-01 IAA-amino acid hydrolase 1 07 40 237 10 10 10

Reads were mapped to the rice genome and responsive genes were identified by 119866-tests Transcripts upregulated more than 20-fold in one or moretreatmentstime points in roots are shown Transcripts in bold were upregulated under both 1 and 02 120583MCd exposure

proteins (Hsps) were strongly upregulated in roots under 1120583MCd with the greatest relative expression at 1 d (Table 1)Thesegenes may contribute to cellular homeostasis by protectingmacromolecules such as enzymes protein complexes andmembranes under Cd exposureThis result suggested that theroots of hydroponically cultured rice might be affected moredirectly and earlier by Cd exposure There was a differencebetween the low Cd concentrations in that no Hsps werestrongly upregulated in roots at 02120583MCd (Table 1) suggest-ing that the effect of this condition might be small or showtime lag In shoots 15 and 11 transcripts were upregulatedmore than 20-fold among the upregulated transcripts under02 and 1 120583M Cd respectively (Table S2) Nine transcriptsincludingNramp1 (natural resistance-associatedmacrophageprotein) were upregulated under both 02 and 1120583M Cd(Table S2) In Arabidopsis Nramp1 localizes to the plasma

membrane and functions as a high-affinity transporter formanganese (Mn) uptake [21] while OsNramp5 uptakes Mnand Cd [22] Transporters with heavymetal binding domainsare often capable of transporting several metals such as FeZn Mn and Cd because of their low substrate specificity[23ndash26] We found that upregulation of a HLHDNA-bindingdomain containing transcription factor (Os04g0301500) inboth roots and shoots peaked at 4 d under 02120583M Cdthis protein may function as a regulatory factor under Cdexposure (Table 1 Table S2) The number of downregulatedtranscripts in roots peaked at 4 d after Cd exposure whilethe number in shoots gradually increased under low Cdconcentration exposure (Figure 2) A few dozen transcriptswere downregulated less than 005-fold among the down-regulated transcripts in roots and shoots under Cd exposure(Table S2)Therefore a small part of transcripts were strongly

6 BioMed Research International

up- or downregulated among several thousand responsivetranscripts under low Cd concentration exposure Large-scale changes in gene expression occurred in rice under Cdexposure even at low concentrations possibly because Cd isa nonessential metal for the plant

To obtain a functional annotation of responsive tran-scripts under Cd exposure we used GO biological processcategories The responsive transcripts in shoot and root wereclustered into several groups based on their expression pat-terns GO enrichment analysis was performed using clusteredtranscripts assigned by GO terms in RAP-DB (The RiceAnnotation Project Database [httprapdbdnaaffrcgojp])(Supplementary Figure S1) Enriched GO terms significantlyin each cluster may represent the functional categories inrice under Cd exposure Enriched GO terms of graduallyupregulated transcripts under Cd exposure include metalion transport (GO0030001) (cluster 3 in root under 02 120583MCd cluster 4 in root under 1120583M Cd) which may functionin Cd transport Response to oxidative stress (GO0006979)and responsive to oxidative stress (GO0006979) were alsoincluded in cluster 3 and cluster 4 respectivelyThis suggestedthat they might function in defense against Cd EnrichedGO terms of gradually downregulated transcripts underCd exposure include translation (GO0006412) translationelongation (GO0006414) DNA replication (GO0006260)andDNA repair (GO0006281) (cluster 1 in root under 02120583MCd cluster 2 in root under 1120583M Cd) Photosynthesis lightharvesting (GO0009765) and photosynthesis (GO0015979)were also included in both clusters These may function inplant growthThus these correspond to the observed changesin phenotype (Figure 1) which clearly validated the RNA-Seqexpression profiling data obtained from rice tissue under Cdstress condition However the pattern of gene expression isquite complex and would require more detailed analysis

33 Constitutively Expressed Genes Responded Differentlyunder Low Cd Concentration to High Cd Concentration Asmany genes responded to both low and high Cd concentra-tions [4] we assessed the effect of the stress degree on riceseedlings through the expression of constitutively expressedgenes We investigated the expression of 18 genes annotatedby the RAP that were expressed constitutively in 39 tissuescollected throughout the life cycle of the rice plant fromtwo varieties according to 190 Affymetrix GeneChip RiceGenome Arrays in addition to four genes annotated by theRAP that have frequently been used as internal controlsin expression analyses [27] The results showed that theexpression of more than half of them fluctuated drastically(gt2 orlt2) in roots or shoots after 1 d of highCd concentrationexposure (Figure 3) This drastic response may be partlybecause RNA-Seq can accurately quantify gene expressionlevels over a broad dynamic range with high resolution andsensitivity [10 28 29] However our results suggest that theirexpression is greatly affected by strong stress even thoughthey are expressed constitutively across the developmentalcourse Note that a high Cd concentration can cause fataldamage to rice seedlings such as by affecting homeostasiswhich corresponds to the observed changes in phenotype(Figures 1 and 3)

34 Comparative Gene Expression Analysis between Low andHigh Cd Concentrations Reveals Novel Cd-Responsive Trans-porters We investigated the expression of metal transportergenes containing metal ion binding Pfam domains [PF01554(MatE) PF08370 (PDR assoc) PF01545 (Cation efflux)PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)]that may function in Cd transport under Cd exposureThe expression of 183 transport transcripts was comparedbetween low and high Cd concentration treatments in rootsand shoots at 1 d because Cd uptake from the hydroponicculture and efflux pumping are initial responses to Cdexposure (Figure 4 Table S3) The transcripts tended tobe more responsive in roots and shoots under higher Cdconcentration exposure This result indicated the potentialof the RNA-Seq strategy to reveal novel Cd-responsivetransporters by analyzing gene expression under exposureto different Cd concentrations The responsive transcriptsmight function in roots at the early stage of Cd exposureNo transcripts were upregulated more than 3-fold in shootsunder low Cd exposure (Figure 4 Table S3) suggesting thatthe effect takes more time to appear in shootsOs03g0667500(Zip root) encoding iron-regulated transporter 1 (IRT1) wasupregulated more than 5-fold under low Cd concentrationsbut responded only slightly under the high Cd concentrationIRT1s often transport Cd because of their low substrate speci-ficity [24ndash26 30]Os02g0585200 (HMA root)Os03g0152000(HMA root) Os0g0584800 (HMA root) Os01g0609900(PDR assoc shoot) and Os01g0609300 (PDR assoc shoot)showed the highest (32-fold) upregulation under high Cdconcentration exposure and responded only slightly to lowCd concentrations (Table S3) The balance between Cd andvarious other metal ions in the hydroponic culture mightaffect the expression of these genes because specific systemsfor transporting Cd may have not developed in rice as it is anonessentialmetalThe effects of other ions on the expressionof transporters [4] and responsive genes associated withdefense systems against Cd (Supplementary Figure S2) havebeen indicated

4 Conclusions

We generated gene expression profiles for rice seedlingsgrown under low Cd concentrations Phenotypic observa-tions and constitutive gene expression indicated that low Cdconcentrations cause growth retardation but are far frombeing fatal in rice Several genes associated with defense sys-tems were strongly upregulated the expression of metal iontransporter genes tended to correlate with Cd concentrationand GO enrichment analysis of the clustered genes based ontheir expression patterns suggesting that our transcriptomeprofiles reflect responses to Cd in rice Our data also suggestthat it could be dangerous to eat plants that do not showspecificCd pollution symptoms growing in soil contaminatedby small amounts of Cd Establishing the exact compositionand organization of the transcriptional network underlyingthe response to Cd exposure will provide a robust tool forimproving crops in the future for example by creating lowCd uptake plants

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

up- or downregulated among several thousand responsivetranscripts under low Cd concentration exposure Large-scale changes in gene expression occurred in rice under Cdexposure even at low concentrations possibly because Cd isa nonessential metal for the plant

To obtain a functional annotation of responsive tran-scripts under Cd exposure we used GO biological processcategories The responsive transcripts in shoot and root wereclustered into several groups based on their expression pat-terns GO enrichment analysis was performed using clusteredtranscripts assigned by GO terms in RAP-DB (The RiceAnnotation Project Database [httprapdbdnaaffrcgojp])(Supplementary Figure S1) Enriched GO terms significantlyin each cluster may represent the functional categories inrice under Cd exposure Enriched GO terms of graduallyupregulated transcripts under Cd exposure include metalion transport (GO0030001) (cluster 3 in root under 02 120583MCd cluster 4 in root under 1120583M Cd) which may functionin Cd transport Response to oxidative stress (GO0006979)and responsive to oxidative stress (GO0006979) were alsoincluded in cluster 3 and cluster 4 respectivelyThis suggestedthat they might function in defense against Cd EnrichedGO terms of gradually downregulated transcripts underCd exposure include translation (GO0006412) translationelongation (GO0006414) DNA replication (GO0006260)andDNA repair (GO0006281) (cluster 1 in root under 02120583MCd cluster 2 in root under 1120583M Cd) Photosynthesis lightharvesting (GO0009765) and photosynthesis (GO0015979)were also included in both clusters These may function inplant growthThus these correspond to the observed changesin phenotype (Figure 1) which clearly validated the RNA-Seqexpression profiling data obtained from rice tissue under Cdstress condition However the pattern of gene expression isquite complex and would require more detailed analysis

33 Constitutively Expressed Genes Responded Differentlyunder Low Cd Concentration to High Cd Concentration Asmany genes responded to both low and high Cd concentra-tions [4] we assessed the effect of the stress degree on riceseedlings through the expression of constitutively expressedgenes We investigated the expression of 18 genes annotatedby the RAP that were expressed constitutively in 39 tissuescollected throughout the life cycle of the rice plant fromtwo varieties according to 190 Affymetrix GeneChip RiceGenome Arrays in addition to four genes annotated by theRAP that have frequently been used as internal controlsin expression analyses [27] The results showed that theexpression of more than half of them fluctuated drastically(gt2 orlt2) in roots or shoots after 1 d of highCd concentrationexposure (Figure 3) This drastic response may be partlybecause RNA-Seq can accurately quantify gene expressionlevels over a broad dynamic range with high resolution andsensitivity [10 28 29] However our results suggest that theirexpression is greatly affected by strong stress even thoughthey are expressed constitutively across the developmentalcourse Note that a high Cd concentration can cause fataldamage to rice seedlings such as by affecting homeostasiswhich corresponds to the observed changes in phenotype(Figures 1 and 3)

34 Comparative Gene Expression Analysis between Low andHigh Cd Concentrations Reveals Novel Cd-Responsive Trans-porters We investigated the expression of metal transportergenes containing metal ion binding Pfam domains [PF01554(MatE) PF08370 (PDR assoc) PF01545 (Cation efflux)PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)]that may function in Cd transport under Cd exposureThe expression of 183 transport transcripts was comparedbetween low and high Cd concentration treatments in rootsand shoots at 1 d because Cd uptake from the hydroponicculture and efflux pumping are initial responses to Cdexposure (Figure 4 Table S3) The transcripts tended tobe more responsive in roots and shoots under higher Cdconcentration exposure This result indicated the potentialof the RNA-Seq strategy to reveal novel Cd-responsivetransporters by analyzing gene expression under exposureto different Cd concentrations The responsive transcriptsmight function in roots at the early stage of Cd exposureNo transcripts were upregulated more than 3-fold in shootsunder low Cd exposure (Figure 4 Table S3) suggesting thatthe effect takes more time to appear in shootsOs03g0667500(Zip root) encoding iron-regulated transporter 1 (IRT1) wasupregulated more than 5-fold under low Cd concentrationsbut responded only slightly under the high Cd concentrationIRT1s often transport Cd because of their low substrate speci-ficity [24ndash26 30]Os02g0585200 (HMA root)Os03g0152000(HMA root) Os0g0584800 (HMA root) Os01g0609900(PDR assoc shoot) and Os01g0609300 (PDR assoc shoot)showed the highest (32-fold) upregulation under high Cdconcentration exposure and responded only slightly to lowCd concentrations (Table S3) The balance between Cd andvarious other metal ions in the hydroponic culture mightaffect the expression of these genes because specific systemsfor transporting Cd may have not developed in rice as it is anonessentialmetalThe effects of other ions on the expressionof transporters [4] and responsive genes associated withdefense systems against Cd (Supplementary Figure S2) havebeen indicated

4 Conclusions

We generated gene expression profiles for rice seedlingsgrown under low Cd concentrations Phenotypic observa-tions and constitutive gene expression indicated that low Cdconcentrations cause growth retardation but are far frombeing fatal in rice Several genes associated with defense sys-tems were strongly upregulated the expression of metal iontransporter genes tended to correlate with Cd concentrationand GO enrichment analysis of the clustered genes based ontheir expression patterns suggesting that our transcriptomeprofiles reflect responses to Cd in rice Our data also suggestthat it could be dangerous to eat plants that do not showspecificCd pollution symptoms growing in soil contaminatedby small amounts of Cd Establishing the exact compositionand organization of the transcriptional network underlyingthe response to Cd exposure will provide a robust tool forimproving crops in the future for example by creating lowCd uptake plants

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

Root

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4 Re

lativ

e exp

ress

ion

valu

e (lo

g 2)

(a)

Tubu

lin b

eta-

6 ch

ain

(Os01g0805900)

Prot

ein

tran

slatio

n fa

ctor

SU

I1(O

s07g0529800)

Pept

idyl

-pro

lyl c

is-tr

ans i

som

eras

e(O

s02g0121300)

Gly

cine

-ric

h RN

A-bi

ng p

rote

in(O

s03g0670700)

GTP

-bin

ding

nuc

lear

pro

tein

(Os05g0574500)

Ubi

quiti

n fu

sion

prot

ein

(Os03g0234200)

Ubi

quiti

n-co

njug

atin

g en

zym

e(O

s01g0819500)

Tran

slatio

n in

itiat

ion

fact

or(O

s03g0758800)

Trio

seph

osph

ate i

som

eras

e(O

s01g0147900)

Gly

cine

-ric

h RN

A-bi

ndin

g pr

otei

n(O

s12g0632000)

Pept

idyl

-pro

lyl i

som

eras

e(O

s02g0760300)

Ubi

quiti

n m

onom

er(O

s06g0681400)

60S

ribos

omal

pro

tein

L31

(Os02g0717800)

Profi

lin(O

s06g0152100)

Elon

gatio

n fa

ctor

1-al

pha

(Os03g0177500)

Endo

thel

ial d

iffer

entia

tion

fact

or(O

s08g0366100)

GA

PDH

(Os08g0126300)

AD

P-rib

osyl

atio

n fa

ctor

(Os05g0489600)

Expr

esse

d pr

otei

n(O

s06g0686700)

GA

PDH

(Os02g0601300)

Prot

ein

elon

gatio

n fa

ctor

(Os02g0519900)

Poly

ubiq

uitin

(Os02g0161900)

Shoot

02 120583M Cd 1d02 120583M Cd 4d02 120583M Cd 10d1120583M Cd 1d

1120583M Cd 4d1120583M Cd 10d50120583M Cd 1d

minus4

minus3

minus2

minus1

0

1

2

3

4

Relat

ive e

xpre

ssio

n va

lue (

log 2

)

(b)

Figure 3 Response of constitutively expressed genes in roots and shoots to Cd exposure The relative expression of constitutively expressedgenes [27] in roots (a) and shoots (b) is shownunderCd exposure at each stress time point (1 4 and 10 d) during 02120583M(white grey and black)and 1 120583M (light blue light green and green) Cd exposure compared with nontreatment (0 d) The red bar shows the relative expression at 1 dunder 50 120583M Cd exposure The 119909-axis shows the genes and the 119910-axis shows relative expression Wang et al [27] suggested the followinggenes as candidates for constitutive expression glycine-rich RNA-binding protein (Os12g0632000) expressed protein (Os06g0686700)profilin (Os06g0152100) ADP-ribosylation factor (Os05g0489600) triosephosphate isomerase (Os01g0147900) glycine-rich RNA-bindingprotein (Os03g0670700) peptidyl-prolyl cis-trans isomerase (Os02g0121300) endothelial differentiation factor (Os08g0366100) ubiquitinmonomer (Os06g0681400) protein translation factor SUI1 (Os07g0529800) GAPDH (Os08g0126300) polyubiquitin (Os02g0161900) proteinelongation factor (Os02g0519900) translation initiation factor (Os03g0758800) ubiquitin-conjugating enzyme (Os01g0819500) GTP-bindingnuclear protein (Os05g0574500) peptidyl-prolyl isomerase (Os02g0760300) and 60S ribosomal protein L31 (Os02g0717800) Their paperalso introduced the following genes that have frequently been used as internal controls in expression analyses elongation factor1-alpha(Os03g0177500) ubiquitin fusion protein (Os03g0234200) GAPDH (Os02g0601300) and tubulin beta-6 chain (Os01g0805900)

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

Os0

1t06

0930

0-01

PD

R_as

soc

Os0

1t06

0990

0-02

PD

R_as

soc

Os1

0t03

4400

0-01

Mat

E O

s02t

0585

100-

00 H

MA

O

s02t

0585

200-

01 H

MA

O

s03t

0152

000-

01 H

MA

O

s02t

0584

700-

01 H

MA

O

s02t

0584

800-

01 H

MA

O

s10t

0344

900-

01 M

atE

Os0

5t04

7240

0-00

Zip

O

s08t

0405

700-

01 H

MA

O

s02t

0131

800-

01 N

ram

p O

s04t

0390

100-

01 H

MA

O

s03t

0861

400-

00 H

MA

O

s01t

0125

600-

01 H

MA

O

s06t

0495

500-

01 M

atE

Os1

1t01

4750

0-01

HM

A

Os1

2t01

4460

0-01

HM

A

Os1

1t01

4750

0-02

HM

A

Os0

1t06

7880

0-01

HM

A

Os0

7t01

0820

0-00

Mat

E O

s06t

0566

300-

00 Z

ip

Os0

2t05

1060

0-01

HM

A

Os0

4t02

9820

0-01

Cat

ion_

efflux

O

s03t

0226

400-

01 C

atio

n_effl

ux

Os0

3t02

2640

0-02

Cat

ion_

efflux

O

s08t

0512

200-

00 H

MA

O

s04t

0573

200-

01 H

MA

O

s04t

0573

200-

02 H

MA

O

s12t

0512

700-

01 P

DR_

asso

c O

s02t

0196

000-

01 Z

ip

Os0

1t01

9250

0-00

HM

A

Os0

8t05

5020

0-01

Mat

E O

s09t

0468

000-

01 M

atE

Os0

2t08

3270

0-01

Cat

ion_

efflux

O

s01t

0919

100-

00 M

atE

Os0

2t08

3270

0-02

Cat

ion_

efflux

O

s03t

0120

400-

01 H

MA

O

s01t

0719

600-

01 H

MA

O

s10t

0209

700-

01 H

MA

O

s03t

0571

700-

01 M

atE

Os0

8t02

0750

0-01

Zip

O

s04t

0571

600-

01 M

atE

Os0

3t02

2950

0-00

Mat

E O

s11t

0129

000-

00 M

atE

Os0

3t08

3920

0-01

Mat

E O

s12t

0581

600-

01 N

ram

p O

s03t

0388

100-

02 H

MA

O

s02t

0775

100-

01 C

atio

n_effl

ux

Os0

1t03

0980

0-01

HM

A

Os0

3t06

0660

0-00

Nra

mp

Os0

8t04

2220

0-00

Cat

ion_

efflux

O

s11t

0129

200-

01 M

atE

Os1

1t01

2610

0-01

Mat

E O

s03t

0751

600-

02 H

MA

O

s03t

0751

600-

01 H

MA

O

s03t

0858

800-

01 M

atE

Os0

3t02

1670

0-01

Mat

E O

s01t

0733

001-

00 N

ram

p O

s08t

0480

000-

01 M

atE

Os0

5t05

5400

0-02

Mat

E O

s05t

0554

000-

01 M

atE

Os0

5t03

6860

0-01

HM

A

Os0

7t02

9890

0-01

HM

AO

s03t

0570

800-

01 M

atE

Os0

7t02

5720

0-01

Nra

mp

Os1

0t01

9500

0-01

Mat

E O

s07t

0232

900-

00 H

MA

O

s08t

0562

800-

01 M

atE

Os0

9t05

4830

0-01

Mat

E O

s03t

0819

400-

01 H

MA

O

s01t

0927

300-

01 H

MA

O

s12t

0106

600-

01 M

atE

Os0

7t06

7140

0-01

HM

A

Os1

2t06

1570

0-01

Mat

E

Os0

5t04

6190

0-00

Cat

ion_

efflux

O

s08t

0562

800-

02 M

atE

Os0

7t05

1660

0-01

Mat

E O

s05t

0198

400-

01 Z

ip

Os0

5t04

7270

0-01

Zip

O

s10t

0206

800-

01 M

atE

Os1

0t02

0680

0-02

Mat

E

Os0

1t08

3780

0-01

Cat

ion_

efflux

O

s01t

0837

800-

02 C

atio

n_effl

ux

Os0

1t05

0450

0-02

Mat

E O

s07t

0502

200-

01 M

atE

Os0

3t05

7290

0-01

Mat

E O

s08t

0403

300-

00 H

MA

O

s01t

0504

500-

01 M

atE

Os0

3t01

1140

0-01

HM

A

Os0

4t05

5600

0-01

HM

A

Os0

8t03

8450

0-01

PD

R_as

soc

Os0

8t02

0540

0-01

HM

A

Os0

2t01

7260

0-00

HM

A

Os0

3t02

0850

0-01

Nra

mp

Os0

3t05

7190

0-01

Mat

E O

s07t

0232

800-

00 Z

ip

Os0

4t05

3390

0-01

HM

A

Os0

6t04

9440

0-01

Mat

E O

s01t

0972

200-

00 Z

ip

Os1

0t01

9090

0-01

Mat

E O

s06t

0495

100-

00 M

atE

Os1

2t04

2100

0-01

HM

A

Os0

2t01

9660

0-01

HM

A

Os1

0t05

3230

0-01

HM

A

Os0

4t03

7340

0-01

Mat

E O

s03t

0126

700-

01 H

MA

O

s01t

0130

000-

02 C

atio

n_effl

ux

Os0

1t01

3000

0-01

Cat

ion_

efflux

O

s03t

0178

100-

00 H

MA

O

s01t

0595

201-

00 H

MA

O

s01t

0503

400-

05 N

ram

p O

s01t

0503

400-

04 N

ram

p O

s03t

0667

300-

00 Z

ip

Os0

1t05

0340

0-03

Nra

mp

Os1

2t01

2600

0-01

Mat

E O

s06t

0554

800-

01 P

DR_

asso

c O

s04t

0590

100-

00 H

MA

O

s05t

0128

400-

01 C

atio

n_effl

ux

Os0

1t06

0900

0-00

PD

R_as

soc

Os0

1t06

0920

0-00

PD

R_as

soc

Os0

1t06

8490

0-01

Mat

E O

s04t

0581

800-

01 H

MA

O

s03t

0700

800-

02 N

ram

pO

s02t

0122

200-

00 M

atE

Os0

2t06

7640

0-00

Mat

E O

s03t

0700

800-

01 N

ram

p O

s06t

0707

100-

01 M

atE

Os0

8t04

6740

0-01

Zip

O

s08t

0467

400-

02 Z

ip

Os0

8t04

6740

0-03

Zip

O

s01t

0507

700-

01 H

MA

O

s05t

0316

100-

01 Z

ip

Os0

5t03

1610

0-02

Zip

O

s03t

0607

400-

01 N

ram

p O

s01t

0516

900-

00 P

DR_

asso

c O

s01t

0249

700-

00 H

MA

Os0

1t07

5800

0-00

HM

AO

s01t

0766

000-

00 M

atE

Os0

2t08

1900

0-00

HM

A

Os0

2t08

2160

0-00

Mat

E O

s03t

0156

600-

01 H

MA

O

s03t

0383

900-

01 H

MA

O

s05t

0164

800-

01 Z

ip

Os0

5t01

6480

0-02

Zip

O

s06t

0558

300-

00 M

atE

Os0

9t03

3230

0-00

PD

R_as

soc

Os0

9t03

3300

0-00

PD

R _a

ssoc

O

s12t

0552

600-

00 M

atE

Os0

9t05

2430

0-00

Mat

E O

s07t

0623

200-

02 H

MA

O

s07t

0623

200-

03 H

MA

O

s01t

0724

500-

01 P

DR_

asso

c O

s07t

0623

200-

01 H

MA

O

s05t

0534

500-

01 H

MA

O

s08t

0545

900-

00 M

atE

Os0

7t02

5840

0-01

Nra

mp

Os0

7t02

5840

0-02

Nra

mp

Os1

0t03

4450

0-00

Mat

E O

s03t

0372

600-

00 H

MA

O

s01t

0342

750-

01 P

DR_

asso

c O

s04t

0613

000-

01 Z

ip

Os1

2t01

2580

0-00

Mat

E O

s10t

0537

400-

00 H

MA

O

s10t

0506

100-

01 H

MA

O

s03t

0626

700-

01 M

atE

Os0

6t06

7600

0-01

Nra

mp

Os0

6t07

0070

0-01

HM

A

Os0

4t06

6110

0-00

HM

A

Os0

1t09

3320

0-00

HM

A

Os0

1t08

2600

0-00

HM

A

Os0

2t05

3010

0-02

HM

A

Os0

2t05

3010

0-01

HM

A

Os0

3t06

6750

0-01

Zip

O

s03t

0411

800-

01 Z

ip

Os0

1t09

7630

0-01

HM

A

Os0

3t01

8810

0-01

Mat

E O

s04t

0244

800-

01 H

MA

O

s06t

0665

800-

01 H

MA

O

s10t

0345

100-

01 M

atE

Os0

6t05

4230

0-01

HM

A

02120583

M1120583

M50120583

MRo

otSh

oot

4

2

0

minus2

minus4

Log 2 fold change at 1d heatmap of transporters

02120583

M1120583

M50120583

M

Os0

3t03

8810

0-01

HM

A

Os0

3t03

4680

0-00

Cat

ion_

efflux

Figure 4 Expression profiling of metal ion transporter genes in roots and shoots under Cd exposure at 1 d demonstrates Cd concentration-dependent differences Heatmap analysis of metal ion transporters containing Pfam domains [PF01554 (MatE) PF08370 (PDR assoc)PF01545 (Cation efflux) PF02535 (Zip) PF00403 (HMA) and PF01566 (Nramp)] The relative expression values under 02 1 and 50120583MCd (data from [4]) are presented The color scale shows log2-transformed transcript levels for each gene

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Youko Oono and Takashi Matsumoto conceived and desig-ned the experiments Takashi Matsumoto performed sam-pling Hiroyuki Kanamori Harumi Sasaki and Satomi Moriperformed the experiments Youko Oono Takayuki Yazawaand Hiroyuki Kanamori analyzed the data and contributedanalysis tools Youko Oono wrote the paper Hirokazu Handaand Takashi Matsumoto contributed valuable insights intothe discussion and revision of the paper Youko Oono andTakayuki Yazawa contributed equally to this work

Acknowledgments

The authors thank Ms F Aota Ms K Ohtsu and Ms KYamada for technical assistance This work was supported

by a grant from the Ministry of Agriculture Forestry andFisheries of Japan (Genomics for Agricultural InnovationRTR-0001)

References

[1] CODEX ldquoReport of the 38th session of the CODEXCommitteeon Food Additives and Contaminantsrdquo ALINORM 062912Codex Alimentarius Commission 2006

[2] B Halliwell and J M C Gutteridge ldquoOxygen-toxicity oxygenradicals transition-metals and diseaserdquo Biochemical Journalvol 219 no 1 pp 1ndash14 1984

[3] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[4] Y Oono T Yazawa Y Kawahara et al ldquoGenome-wide tran-scriptome analysis reveals that cadmium stress signaling con-trols the expression of genes in drought stress signal pathwaysin ricerdquo PLoS ONE vol 9 no 5 Article ID e96946 2014

[5] S Yoshida D A Forno J H Cock and K A Gomez Labora-tory Manual for Physiological Studies of Rice International RiceResearch Institute Manila Philippines 3rd edition 1976

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

[6] S Sauve W A Norvell M McBride and W HendershotldquoSpeciation and complexation of cadmium in extracted soilsolutionsrdquo Environmental Science amp Technology vol 34 no 2pp 291ndash296 2000

[7] Y Kawahara Y Oono H Wakimoto et al ldquoTENOR databasefor comprehensive mRNA-Seq experiments in ricerdquo Plant andCell Physiology vol 57 no 1 article e7 2016

[8] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1 pp10ndash12 2011

[9] Y Oono Y Kawahara H Kanamori et al ldquomRNA-seq revealsa comprehensive transcriptome profile of rice under phosphatestressrdquo Rice vol 4 no 2 pp 50ndash65 2011

[10] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[11] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[12] M Zhang X Liu L Yuan et al ldquoTranscriptional profiling incadmium-treated rice seedling roots using suppressive subtrac-tive hybridizationrdquo Plant Physiology and Biochemistry vol 50no 1 pp 79ndash86 2012

[13] K Lee D W Bae S H Kim et al ldquoComparative proteomicanalysis of the short-term responses of rice roots and leaves tocadmiumrdquo The Journal of Plant Physiology vol 167 no 3 pp161ndash168 2010

[14] K Shah R G Kumar S Verma and R S Dubey ldquoEffect ofcadmium on lipid peroxidation superoxide anion generationand activities of antioxidant enzymes in growing rice seedlingsrdquoPlant Science vol 161 no 6 pp 1135ndash1144 2001

[15] L Perfus-Barbeoch N Leonhardt A Vavasseur and CForestier ldquoHeavy metal toxicity cadmium permeates throughcalcium channels and disturbs the plant water statusrdquo PlantJournal vol 32 no 4 pp 539ndash548 2002

[16] RMittler S VanderauweraN Suzuki et al ldquoROS signaling thenew waverdquo Trends in Plant Science vol 16 no 6 pp 300ndash3092011

[17] C Frova ldquoThe plant glutathione transferase gene familygenomic structure functions expression and evolutionrdquo Physi-ologia Plantarum vol 119 no 4 pp 469ndash479 2003

[18] C Cosio and C Dunand ldquoSpecific functions of individual classIII peroxidase genesrdquo Journal of Experimental Botany vol 60no 2 pp 391ndash408 2009

[19] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[20] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[21] R Cailliatte A Schikora J-F Briat S Mari and C CurieldquoHigh-affinity manganese uptake by the metal transporterNRAMP1 is essential for Arabidopsis growth in low manganeseconditionsrdquo Plant Cell vol 22 no 3 pp 904ndash917 2010

[22] A Sasaki N Yamaji K Yokosho and J F Ma ldquoNramp5 isa major transporter responsible for manganese and cadmiumuptake in ricerdquo Plant Cell vol 24 no 5 pp 2155ndash2167 2012

[23] N Satoh-Nagasawa MMori N Nakazawa et al ldquoMutations inrice (Oryza sativa) heavymetalATPase 2 (OsHMA2) restrict thetranslocation of zinc and cadmiumrdquo Plant and Cell Physiologyvol 53 no 1 pp 213ndash224 2012

[24] Y O Korshunova D Eide W G Clark M L Guerinot andH B Pakrasi ldquoThe IRT1 protein from Arabidopsis thaliana is ametal transporter with a broad substrate rangerdquo PlantMolecularBiology vol 40 no 1 pp 37ndash44 1999

[25] N E Grossoehme S AkileshM L Guerinot andD EWilcoxldquoMetal-binding thermodynamics of the histidine-rich sequencefrom themetal-transport protein IRT1 of Arabidopsis thalianardquoInorganic Chemistry vol 45 no 21 pp 8500ndash8508 2006

[26] S Lee andGAn ldquoOver-expression ofOsIRT1 leads to increasediron and zinc accumulations in ricerdquo Plant Cell and Environ-ment vol 32 no 4 pp 408ndash416 2009

[27] LWangWXie Y Chen et al ldquoA dynamic gene expression atlascovering the entire life cycle of ricerdquo Plant Journal vol 61 no 5pp 752ndash766 2010

[28] G M He X P Zhu A A Elling et al ldquoGlobal epigeneticand transcriptional trends among two rice subspecies and theirreciprocal hybridsrdquo Plant Cell vol 22 no 1 pp 17ndash33 2010

[29] T T Lu G J Lu D L Fan et al ldquoFunction annotation of therice transcriptome at single-nucleotide resolution by RNA-seqrdquoGenome Research vol 20 no 9 pp 1238ndash1249 2010

[30] P Pedas C K Ytting A T Fuglsang T P Jahn J K Schjoerringand S Husted ldquoManganese efficiency in Barley identificationand characterization of themetal ion transporterHvIRT1rdquoPlantPhysiology vol 148 no 1 pp 455ndash466 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology