Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP, OsNRAMP1, in arsenic transport and tolerance

Original Article

Expression in Arabidopsis and cellular localization revealinvolvement of rice NRAMP, OsNRAMP1, in arsenictransport and tolerance

Manish Tiwari1, Deepika Sharma1, Sanjay Dwivedi1, Munna Singh2, Rudra Deo Tripathi1 & Prabodh Kumar Trivedi1

1CSIR-National Botanical Research Institute (CSIR-NBRI), Rana Pratap Marg, Lucknow 226001, India and 2Department ofBotany, University of Lucknow, Lucknow 226007, India

ABSTRACT

Irrigation of paddy fields to arsenic (As) containing ground-water leads to As accumulation in rice grains and causesserious health risk to the people worldwide. To reduce Asintake via consumption of contaminated rice grain, identifica-tion of the mechanisms for As accumulation and detoxifica-tion in rice is a prerequisite. Herein, we report involvement ofa member of rice NRAMP (Natural Resistance-AssociatedMacrophage Protein) transporter, OsNRAMP1, in As, inaddition to cadmium (Cd), accumulation through expressionin yeast and Arabidopsis. Expression of OsNRAMP1 in yeastmutant (fet3fet4) rescued iron (Fe) uptake and exhibitedenhanced accumulation of As and Cd. Expression of OsN-RAMP1 in Arabidopsis provided tolerance with enhancedAs and Cd accumulation in root and shoot. Cellular localiza-tion revealed that OsNRAMP1 resides on plasma membraneof endodermis and pericycle cells and may assist in xylemloading for root to shoot mobilization. This is the first reportdemonstrating role of NRAMP in xylem mediated loadingand enhanced accumulation of As and Cd in plants. Wepropose that genetic modification of OsNRAMP1 in ricemight be helpful in developing rice with low As and Cdcontent in grain and minimize the risk of food chain contami-nation to these toxic metals.

Key-words: Cadmium; fet3fet4 yeast mutant; heavy metal;iron uptake; xylem loading.

INTRODUCTION

Arsenic (As) is a highly toxic environmental pollutant whichcauses chronic and epidaemic effects on humans across globe,particularly in Southeast Asian countries, through water andcrop contamination.The exposure ofAs has been described asthe largest mass poisoning of the population in history and aneglected cancer risk (Stone 2008; Zhao, McGrath & Meharg2010a). In coastal belt of Bangladesh, India and China, As inground water exceeds the safe limit of 10 mg L-1 as describedby World Health Organization (WHO 2001) and over 100million people are exposed to As-contaminated drinking

water (Mukherjee et al. 2008; Brammer & Ravenscroft 2009;Rosen & Liu 2009). Use of As contaminated groundwater forthe irrigation of paddy alone inserts more than 1000 tonnes ofAs to the soil each year in Bangladesh alone (Ali et al. 2003).This leads to accumulation of high levels of As in paddy fieldswhich ranges from 4 to 8 mg kg-1 and can reach up to 83 mgkg-1 in summer season (Abedin et al. 2002). As present in thepaddy field is readily absorbed by rice plants and translocatedto the grain. This results in approximately 10-fold higher Ascontent in rice grain than other cereal grains grown in sameconditions (Williams et al. 2007; Khan et al. 2010; Tuli et al.2010), constituting a menace for populations that rely mostlyon rice for diet (Zhao et al. 2010a). Consequently, rice culti-vars with low As in grain are highly desirable to minimizenutritional As intake through the consumption of contami-nated rice grains.

The mechanisms for As accumulation, movement anddetoxification in plants are poorly known and need in-depthinvestigation. Over the last few years, studies related to themolecular mechanisms of As uptake, metabolism and translo-cation in plants have gained momentum (Tripathi et al. 2007;Ma et al. 2008; Zhao et al. 2010b; Kumar et al. 2011). It hasbeen demonstrated that arsenate [As(V)] move into plants viaphosphate transporters, while As(III) enters through Lsi1,(Nodulin-like intrinsic protein), a major influx transporter forthe silicic acid (Ma et al. 2008; Zhao et al. 2009; Tripathi et al.2012). Recently, a plasma membrane intrinsic protein (PIP)has been shown to be involved in As tolerance and transport(Mosa et al. 2012). It has also been demonstrated that tono-plast oriented efflux transporter (PvACR3) mediates As(III)sequestration into the vacuoles in hyper accumulator fern,Pteris vittata, (Indriolo et al. 2010).TwoABCC-type transport-ers from Arabidopsis have been shown as the major vacuolarAs(III)-phytochelatin complex transporters suggesting theirrole in As(III) detoxification (Song et al. 2010).

Intriguingly, it is now evocative with few reports thatmechanism of Fe uptake correlates with the As accumulationand affects As transport in plants (Dwivedi et al. 2010; Zhaoet al. 2010a; Rahman et al. 2011). It has been demonstratedthat presence of Fe plaques adjacent to the root enhances theAs uptake in root of rice (Meharg & Rahman 2003; Liu et al.2004). Similarly, rice seedlings accumulate more As from soilwhen additional Fe was supplied (Rahman et al. 2011) due to

Correspondence: P. K. Trivedi. Fax: +91 522 220 5836, +91 522 2202205839; e-mail: [email protected], [email protected]

Plant, Cell and Environment (2014) 37, 140–152 doi: 10.1111/pce.12138

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increased physicochemical adsorption of As on Fe-plaquenear rice root surface (Robinson et al. 2006). The m-X-rayfluorescence (mXRF) imaging of rice tissues has shown thatabundance of As correlates with the presence of Fe at theroot surface (Smith et al. 2009). All these studies raised pos-sibility that uptake of As in rice may follow the similar trans-port route like Fe.

NRAMP was identified as Fe transporter in macrophagesof rat (Vidal et al. 1993). Since then, several similar as well asother divalent cation transporters have been characterized.The identified NRAMPs from various organisms primarilyrepresent Fe transport behaviour in yeast (Eide et al. 1996;Pinner et al. 1997; Curie et al. 2000; Thomine et al. 2000).NRAMPs are in general, membrane spanning protein, owingnearly 12 highly hydrophobic transmembrane domains arenow recognized as a ubiquitous family of metal transporterswith several homologues in fungi, animals, plants and bacte-ria (Cellier et al. 1995). Some Arabidopsis NRAMPs (AtN-RAMP1, AtNRAMP3 and AtNRAMP4) are high affinity Fetransporter and rescue low Fe sensitive phenotype of yeastmutant fet3fet4 (Curie et al. 2000; Thomine et al. 2000, 2003).Similar to Fe uptake, Mn transport ability of AtNRAMP1and AtNRAMP3 and AtNRAMP4 has been demonstrated(Cailliatte et al. 2010; Lanquar et al. 2010). In addition,several studies reported that NRAMPs also retain heavymetal transport (Ni and Cd) ability (Thomine et al. 2000,2003; Mizuno et al. 2005; Cailliatte et al. 2009; Oomen et al.2009). It has been demonstrated that Nrat1, NRAMP-likeprotein, mediate and significantly contributes in Al3+ trans-port and uptake in rice (Xia et al. 2010).

Although, several studies have been carried out to elucidatefunction of Arabidopsis NRAMPs in Fe and other metaluptake, limited efforts have been made for characterization ofrice NRAMP (Belouchi, Kwan & Gros 1997; Curie et al. 2000;Sasaki et al. 2009; Takahashi et al. 2011; Ishimaru et al. 2012).Our previous study (Chakrabarty et al. 2009) suggested thatOsNRAMP1 expression is up-regulated during As exposure inrice while its role is hitherto unknown in As transport. Herein,we report involvement of OsNRAMP1 inAs(III),in addition toCd and Fe, transport and accumulation through heterologousexpression in Arabidopsis and yeast (fet3fet4) mutant.Our datasuggest that localization of OsNRAMP1 within pericyle regionmay offer the quick mobilization of toxic metals in aerial tissuesthrough assisting xylem mediated transport and reducing theextent of toxicity exerted by these metals.

MATERIALS AND METHODS

Plants materials, growth condition and heavymetal treatments

Rice (Oryza sativa) seeds were dehusked and sterilized with0.1% mercuric chloride and were grown on half strength MS(Murashige & Skoog 1962) plates containing 1.5% sucroseunder 22 °C temperature and 16 h light/8 h dark cycle up to10 d. For As treatment, seeds of different cultivars were ger-minated and allowed to grow in hydroponic condition for10 d followed by addition of As(III) (25) mM in the nutrient

solution and plants were further grown for 3 d followed byharvesting. For the tissue-specific expression analyses, riceplants were grown in hydroponic culture for four weeksunder control environments and root, stem and leaf bladeswere harvested. Mature panicle tissues were collected fromthe rice plants grown in the field.

Arabidopsis thaliana (Col-0) plants were grown in soilriteunder controlled environments (16 h light/8 h dark cycle,22 °C, 150 mM m-1s-1 light intensity and 50% relative humid-ity), watered with nutrients solution and reverse osmosiswater alternatively in every week till plant completes its lifecycle. Arabidopsis were transformed according to floral dipprotocol (Clough & Bent 1998) by using Agrobacteriumtumefaciens strain GV3101 and transformants were selectedon kanamycin (50 mg mL-1) containing half strength MSmedia. Tolerance assessment of transgenic lines was carriedout on ABIS media with Fe (50 mM) devised by Thomineet al. (2003). For As(III) treatment to wild type (WT) and alltransgenic lines, As(III) (NaAsO2; Sigma, St. Louis, MO,USA) or CdCl2 were used in different concentrations.

RNA isolation and gene expression analysis

Total RNA from different tissues was extracted using theSpectrum Plant Total RNA Kit (Sigma), followed by treat-ment with RNAase-free DNase (Fermantas, Life Sciences,Ontario, Canada). Two microgram of total RNA was synthe-sized into single stranded cDNA using RevertAid H minusfirst strand cDNA synthesis Kit (Fermantas) as per manufac-turer’s instructions. Semi-quantitative real-time polymerasechain reaction (RT-PCR) was carried out with one fifth of thiscDNA taken as template in 20 mL reaction volume using genespecific primers.The list of oligonucleotide primers (EurofinsMWG Operon,Bangalore,India) used in the study is providedin Supporting Information Table S1. The PCR reaction wasperformed as following conditions: an initial denaturation at94 °C for 2 min, 30 cycles at 94 °C for 15 s, 55 °C for 15 s, and72 °C for 30 s, followed by a final 5 min extension at 72 °C.Theprimers for ubiquitin and actin gene were used as endogenouscontrol for rice and Arabidopsis respectively to ensure thatequal amounts of cDNA were used in all the reactions.The PCR products were analysed on agarose gels by EtBrstaining. For RT-PCR, synthesized cDNA was diluted 1:10(v/v) in diethylpyrocarbonate-treated water and subjectedto quantitative RT-PCR (qRT-PCR) analysis using SYBRGreen Supermix (ABI, Grand Island, NY, USA) using ABI7500 instrument (ABI Biosystems). Microarray analyses fordifferent tissues was carried out using publicly available CELfiles in the GEO database (GSE5630; GSE5631; GSE5633;GSE19259) as described earlier (Kumar et al. 2013a).

Cloning, constructs preparation andtransformation

Full-length cDNA encoding OsNRAMP1 was amplified fromcDNA library prepared from root of rice through PCR usinggene specific primers NRAMPFI and NRAMPRI (Support-ing Information Table S1) and cloned using InsTAclone PCR

OsNRAMP1 in arsenic transport and tolerance 141

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 140–152

Cloning Kit (Fermentas).Nucleotide sequence of cloned frag-ment was confirmed through sequencing of both the strands.Full-length cDNA was cloned in plant expression vector(pBI121) between Xba1 and Sac1 restriction sites after ampli-fication using oligonucleotide primers (NRAMP1XbaI andNRAMP1SacI) having XbaI and SacI restriction sites. Thisconstruct carries CaMV35S promoter for driving expressionof OsNRAMP1 and was used for transformation of Arabidop-sis (Col-0). For transformation in yeast, construct (pDR195-OsNRAMP1) was prepared in yeast expression vectorpDR195 at SmaI and EcoRI restriction sites. The constructused for cellular localization was prepared in plant expressionvector pCAMBIA1303 carrying CaMV35S promoter. TheOsNRAMP1 cDNA was amplified without stop codon andcloned in pCAMBIA1303 at NcoI and SpeI restriction sites,respectively. All the constructs were sequenced before use toconfirm error free amplification and cloning. List of primersand their uses for preparation of specific constructs is given inSupporting Information Table S1.

Yeast experiments

Plasmids pDR195-OsNRAMP1 and pDR195 were trans-formed in fet3fet4 (Eide et al. 1996) yeast strain used in thisstudy. Empty vector pDR195 transformed yeast cells wereconsidered as control in each experiment. Yeast cells weregrown in yeast extract-peptone-dextrose media before trans-formation. Transformation of yeast cells was performedusing Yeast Transformation Kit (Sigma). After transforma-tion, transformants were selected on SD (synthetic defined)-URA containing 2% glucose and agar. For Fe uptake assay,control and OsNRAMP1 expressing yeast cells were grownup to OD600 of 1 followed by serial dilution and spotting onSD-URA agar plates supplemented with FeCl3 and BPS atvarious serial dilutions. For growth inhibition assessment ofAs(III) and Cd, cells were grown in 5 mL of primary culturesupplemented with Fe (50 mM) followed by spotting onmetal containing SD plates as described by Oomen et al.(2009) and incubated at 30 °C for 2 d before photographtaken. The relative growth inhibition of fet3fet4 transformedwith pDR195-OsNRAMP1 and pDR195, primary culture(150 mL) was inoculated in SD-URA medium (5 mL) withand without supplementation of heavy metals. Relativegrowth was determined through measuring OD600 at differ-ent time intervals.

Root length measurements

WT and OsNRAMP1 expressing Arabidopsis transgenic lineswere used for tolerance/sensitivity measurements of theplants towards Fe,As(III) and Cd stress.Plants were verticallygrown on Petri plates having ABIS containing media(Thomine et al. 2003) for 10 d after stratification (4 °C for48 h in dark). Root length was considered as tolerance/sensitivity parameter for WT and transgenic lines. Differentconcentrations of Fe,As(III) and Cd, as indicated in the figurelegends, were taken in the study.The variables were expressedas mean � standard deviation (SD) of data collected for

15–20 independent plants for each line. Statistical differencesbetween control and all genotypes were calculated by studentt-test (P < 0.05).

Metal measurement in Arabidopsis and yeast

For metal measurement in WT and OsNRAMP1 expressingArabidopsis lines were grown on ABIS media supplementedwith As(III) and Cd for 2 weeks. Roots and shoots wereseparated and washed with Milli-Q (MQ) water morethan three times. To remove surface and apoplastic heavymetals, roots were washed and desorbed for 10 min with2 mM CaSO4 and 10 mM ethylenediaminetetraacetic acid(Cailliatte et al. 2010). For metal measurement in yeast,control and OsNRAMP1 expressing yeast cells were over-night grown in SD-URA liquid media containing 200 mMFeCl3. After maintaining the yeast OD600 of 1, secondary cul-tures were grown in 50 mL volume. At OD600 of 1.8, Cd andAs(III) were added and secondary culture was further grownfor 3 h. Cells were centrifuged and washed with MQ water atleast five times to remove extracellular metals. All sampleswere oven dried for 4 d at 60 °C and biomass was determinedbefore digesting in HNO3 (for mineralization). Mineralizedsamples were used for the determination of As and Cdcontent as well as other metals through ICP-MS (Agilent7500; Santa Clara, CA, USA). The variables were expressedas mean � SD of data collected of three biological replicateof WT and transgenic Arabidopsis lines as well as three inde-pendent replicate cultures of control and yeast expressingOsNRAMP1. Level of significance was calculated by studentt-test (P < 0.05).

Localization of OsNRAMP1

Roots of 1-week-old OsNRAMP1:GFP expressing stabletransgenic plants under control of CaMV35S promoter wereexamined to study the cell specific localization of OsN-RAMP1. GFP fluorescence was examined using confocalmicroscope (LSM 510, Zeiss, Jena, Germany).

Statistical analysis

Each experiment was carried out under a completely rand-omized design with independent experiments with at leastthree replications. The data were analysed by Student’spaired t-test, and mean values under each treatment werecompared at P � 0.05–0.001.

RESULTS

OsNRAMP1 expression is induced duringAs stress

Global gene expression analyses of rice during As stressshowed up-regulation of one locus, LOC_Os07g15460,having homology with NRAMP transporters (Chakrabartyet al. 2009). Among the six rice putative NRAMPs presentin the rice genome, LOC_Os07g15460 was designated as

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OsNRAMP1 due to clustering with AtNRAMP1 and AtN-RAMP6 from Arabidopsis and other homologues frommonocotyledonous and dicotyledonous plants (Migeon et al.2010; Takahashi et al. 2011). In order to investigate the spatio-temporal expression of OsNRAMP1 in rice, publically avail-able microarray data was analysed. Our analysis indicated adifferential expression pattern of OsNRAMP1 in varioustissues with maximum transcript abundance in root andpanicle tissues (Fig. 1a). To corroborate microarray expres-sion data, semiquantitative RT-PCR analysis was carried outusing total RNA from root and shoot of ten day old riceseedling; root, stem and leaf blade of 4-week-old rice plantand panicles. RT-PCR analysis was in agreement to the insilico observation of OsNRAMP1 expression data (Fig. 1b).Transcript of OsNRAMP1 was detected in all the tissueswhereas a preferential expression was observed in root andpanicle tissue.

To further confirm As-induced expression of OsNRAMP1reported by Chakrabarty et al. (2009), the expression of OsN-RAMP1 was analysed in As(III) tolerant (IC-340072) andsensitive (IC-115730) rice cultivars (Dave et al. 2013) underAs(III) stress. Our RT-PCR result suggest that the expression

of OsNRAMP1 get induced after As(III) exposure in boththe rice cultivars (Fig. 1c).

Expression of OsNRAMP1 in DEY1453 (fet3fet4)

Members of NRAMP protein family including AtNRAMP1,AtNRAMP3 and AtNRAMP4 in Arabidopsis as well as ofother higher plants have shown the Fe transport ability byrescuing the phenotype of Saccharomyces cerevisiae mutantDEY1453 (fet3fet4), defective in Fe uptake.To examine capa-bility of OsNRAMP1 to rescue fet3fet4 phenotype as well asAs and Cd uptake potential, yeast expression vector pDR195containing OsNRAMP1 was transformed into fet3fet4, whichgrows poorly in medium containing low Fe concentration dueto disrupted high affinity Fe transporter (Eide et al. 1996).Alongside empty vector pDR195 transformed yeast cells wasconsidered as control in each experiment.The fet3fet4 mutanttransformed with OsNRAMP1 was able to grow on low Fe(30 mM) concentration at different dilutions whereas growthof control was arrested (Fig. 2a). At the same time, bothcontrol as well as OsNRAMP1 expressing yeast cells grewsimilarly at high Fe (200 mM) concentration. These observa-tions suggest that OsNRAMP1 was able to rescue inability offet3fet4 to grow on low Fe which shows its relevance for Fetransport as reported earlier (Curie et al. 2000).

In addition to Fe, NRAMPs have been shown to beinvolved in the transport of toxic metals Cd (Cailliatte et al.2009; Oomen et al. 2009) or Ni (Mizuno et al. 2005) but theirinvolvement in As transport is still unexplored.Very recently,Cd transport ability of OsNRAMP1 has been demonstratedusing Cd sensitive yeast mutant (ycf1) (Takahashi et al.2011). To study involvement of OsNRAMP1 in As(III) andCd transport control and OsNRAMP1 complementedfet3fet4 cells were grown in SD-URA medium for overnight.Overnight grown cells were used for spotting on SD-URA-agar plates supplemented with As(III) (100 mM) and Cd(30 mM) at indicated dilutions. Supplementation of Cd inmedium caused considerable growth inhibition in yeast cellshaving functional OsNRAMP1 (Fig. 2b) whereas a marginalgrowth inhibition occurred in case of As(III) which was muchlower as compared to Cd. The growth inhibition due to pres-ence of functional OsNRAMP1 in fet3fet4 towards Cd andAs(III) suggest that OsNRAMP1 may facilitate import ofthese metals inside the yeast.

To strengthen our hypothesis that OsNRAMP1 maymediate the Cd and As(III) uptake, metal content was meas-ured in the yeast cells expressing OsNRAMP1 grown in thepresence of Cd and As(III). Significantly enhanced accumu-lation of Cd (5-fold) was observed in yeast cells expressingOsNRAMP1 in comparison to control. In case of As(III),enhancement in accumulation was much lower (Fig. 2c). Nosignificant change in content of other metals (Fe, Zn and Mn)was observed in yeast cells expressing OsNRAMP1. We alsoanalysed relative growth inhibition in liquid media in thepresence of Cd and As(III) in yeast cells. The relative growthof fet3fet4 gradually decreased in case of Cd (Fig. 2d) butno significant change was observed with As(III) supple-mentation (Data not shown). Our observations suggest that

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Figure 1. Expression of OsNRAMP1 in different tissues andunder arsenic stress. (a) Expression data of OsNRAMP1 in variousdevelopmental stages of rice, mature leaves (ML), ovary, stages ofpanicle maturation (P1–P6), root (R1), seed development stages(S1–S5), shoot apical meristem (SAM), stigma and young leaves(YL) of Affymetrix Rice Genome Array as cluster display. Thecolour scale (representing log signal values) is shown at thebottom. (b) Validation of in silico data via RT-PCR in differenttissues. (c) OsNRAMP1 expression profile in root of IC-340072and IC-115730 rice cultivars under As(III) exposure. Seeds of bothcultivars were germinated and allowed to grow in hydroponic for10 d followed by 3 d exposure to 25 mM As(III). Rice ubiquitin isused as internal control for equal amount of cDNA in theexperiment.



OsNRAMP1 facilitate enhanced Cd uptake in yeast withgrowth inhibition. However, As(III) supplementation couldnot significantly affect the growth of fet3fet4 which might bedue to marginal enhancement in As content.

Expression of OsNRAMP1 in Arabidopsis

To determine the role of OsNRAMP1 in As(III) and Cdtransport, stable homozygous transgenic lines expressingOsNRAMP1 under control of CaMV35S promoter inA. thaliana (Col-0) background were developed. Transgeneexpression was confirmed through RT-PCR analysis andthree OsNRAMP1 expressing lines were selected on thebasis of similar expression for further analyses (SupportingInformation Fig. S1a). In general, growth and developmentof transgenic lines and WT were comparable in pots or onhalf-strength MS plates (Supporting Information Fig. S1a,b).There was no phenotypic change observed between WT andtransgenic lines grown on ABIS media (Thomine et al. 2003)

containing Fe (50 mM) for 10 d (Supporting InformationFig. S1b). Therefore, ABIS media supplemented with Fe(50 mM) was considered optimal for growth of the plants andused as basal concentration in all further experiments. Inorder to study the effect of Fe on transgenic lines, plants weregrown in excess Fe environment (750 mM) and no phenotypicchange appeared in transgenic lines compared to WT (Sup-porting Information Fig. S1c) contrary to earlier report of itsorthologues AtNRAMP1 overexpression in Arabidopsis thatconferred tolerance to high Fe content (Curie et al. 2000).Thus, expression of OsNRAMP1 in Arabidopsis does notaffect growth of Arabidopsis at high Fe regime regardless ofprominent Fe rescuing ability in fet3fet4 yeast mutant.

Transgenic lines display tolerance to As(III)and Cd

To determine the response of OsNRAMP1 expressing Ara-bidopsis lines to As(III) and Cd, WT and transgenic lines

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Figure 2. OsNRAMP1 transport abilities in yeast. OsNRAMP1 was expressed in the DEY1453 (fet3fet4) yeast mutants (deficient in Feuptake) and subsequently studied for their capability to rescue the mutant phenotype. An empty vector transformant (pDR195) was used ascontrol. (a) Control and transformed DEY1453 yeast cells growth on synthetic dextrose-URA (pH 5.5) supplemented with low FeCl3

(30 mM) and high Fe (200 mM FeCl3) chelated with 80 mM BPS. (b) Growth of yeast cells with and without CdCl2 (30 mM) and As(III)(100 mM) on SD-URA agar plates (pH 5.5). (c) Elemental analysis in cells grown in the presence of Cd 30 mM and As(III) (100 mM) in liquidSD-URA media. (d) Relative growth inhibition of OsNRAMP1 expressing yeast cells grown in the presence of Cd (10 mM). Eachexperiment was carried out at least three times. Bars show the mean of triplicate cultures and error bars represent the �SD (*** and *represent P < 0.001 and P < 0.01). For Fe uptake assay, yeast pre-cultures having OD600-1.0 were serially diluted to 10-1, 10-2 and 10-3 and10 mL were spotted and incubated at 30 °C for 3 d.



were germinated on ABIS media containing As(III) and Cd(Fig. 3a). WT as well as transgenic lines showed root growthinhibition in the presence of the As(III) in concentrationdependent manner. However, tolerance was observed intransgenic lines in comparison to WT (Fig. 3) which was moresignificant at 10 mM As(III) concentration.This tolerance wasalso visible in aerial tissue on the plates (Fig. 3a). To studylong-term effect on biomass of these plants, overall weight ofseedlings grown on heavy metal for 20 d was measured(Fig. 4). More biomass was observed in the transgenic lines incomparison to WT plants grown on As(III) and Cd whichsuggests that OsNRAMP1 provide tolerance to As(III)(Figs 3 & 4). This ability of OsNRAMP1 differ from Arabi-dopsis NRAMPs that preceded the sensitivity to Cd(Takahashi et al. 2011). Together, these observations indicatethat expression of OsNRAMP1 in Arabidopsis conferred tol-erance for As(III) and Cd.

OsNRAMP1 expression in Arabidopsis leads toenhanced As and Cd accumulation

Expression of OsNRAMP1 conferred tolerance to As(III)and Cd in Arabidopsis. To study whether tolerance is accom-panied with accumulation of these heavy metals, measure-ment of As and Cd were performed in root and shoot tissues.WT and transgenic lines expressing OsNRAMP1 were grownfor two week on ABIS media in the presence of As(III)(5 mM) and Cd (10 mM), respectively. All the OsNRAMP1expressing transgenic lines accumulated more As(III) and Cdin root and shoot tissues (Fig. 5). In case of As(III), root andshoot of transgenic lines accumulated nearly twofold Ascompared to WT. Similarly, enhanced Cd content (approxi-mately 40%) was observed in root of transgenic lines. Shoottissue of transgenic lines rather showed enhanced Cd accu-mulation in comparison to WT but this enhancement was less

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Figure 3. Expression of OsNRAMP1 in Arabidopsis confers tolerance for As(III) and Cd. (a) Phenotypic changes of WT and transgeniclines grown on different metals. Seeds of Arabidopsis thaliana (WT; accession Col-0) and three transgenic lines (L1, L2 and L3) weregerminated on ABIS medium supplemented basal amount of Fe-EDTA (50 mM) and 10 mM As(III) and 25 mM Cd grown vertically for 10 d.(b) Root length of the plants after growth of 10 d on ABIS media supplemented with As(III) (5 and 10 mM) and Cd (10 and 25 mM). Barsrepresent the mean of root length of 15–20 plants of each genotype and error bars represent �SD. *, ** and *** significantly different atP < 0.05, P < 0.05 and 0.001 respectively. Experiments are performed more than three times and result is the representative of one set.



relative to accumulation in root tissue. In addition to As andCd, enhanced accumulation of Fe and Mn was also observedin shoot tissue of transgenic lines in comparison to WT.However, the Fe and Mn content in root tissue of transgeniclines were not affected in comparison to WT (Fig. 6). Anincreased As and Cd content in both tissues, root and shoot,as well as Fe and Mn in shoot tissue in OsNRAMP1 express-ing transgenic lines indicate that OsNRAMP1 might haverole in root to shoot mobilization of these metals.

OsNRAMP1 specifically expressed inendodermis and pericycle cells in Arabidopsis

Interestingly,heterologous expression of OsNRAMP1 in Ara-bidopsis in our study contributed to As and Cd accumulationand tolerance in spite of plasma membrane localizationas reported by Takahashi et al. (2011). This concluded thatOsNRAMP1 facilitate the entry of these metals inside thecells, however, raised questions about tolerance of transgeniclines to heavy metals. To answer this, stable transgenic linesof Arabidopsis expressing OsNRAMP1:green fluorescentprotein (GFP) fusion protein under control of CaMV35Spromoter were developed and cell-specific localization of thefusion protein was studied. Surprisingly, green fluorescencewas visualized near active zone (quiescent centre) of root intransgenic plants (Fig. 7) even under the control of constitu-tive promoter. The detailed analyses of GFP fluorescence invarious regions of root including elongation and maturationzone suggested that OsNRAMP1 precisely expressed in innercells of root, mainly in pericycle region of maturation zone asgreen fluorescence was confined to this region (Fig. 7). Therestricted cell-specific expression of OsNRAMP1 in the peri-cycle cells seems to facilitate the movement of heavy metals inthe vascular region and may assist in distribution from root toaerial region (shoot) of the plants. This could provide reasonfor tolerance of OsNRAMP1 expressing Arabidopsis lines

associated with enhanced metal accumulation in root andshoot in proportional manner.

Enhanced expression of metal transporters intransgenic lines

To elucidate the rationale behind tolerance of transgeniclines to As(III) and Cd, we examined expression of ABCC-type transporters (AtABCC1, AtABCC2) and AtHMA4which are known to help in transport and mobilization ofCd and As(III) in vacuoles. An enhanced expression ofAtABCC1 and AtABCC2 transporters was observed in trans-genic lines and might be the plausible reason of higher accu-mulation of heavy metals (Fig. 8). As anticipated, enhancedtranscript accumulation of AtHMA4 was also observedwhich is known transporter of pericycle region involved inxylem loading of metals such as Cd and Zn.

DISCUSSION

NRAMPs are prominently recognized for transportation ofnutrients such as Fe, Zn and Mn in plants, have ubiquitousoccurrence in bacteria, fungi, animals and plants (Colangelo

Figure 4. Biomass of Arabidopsis seedlings grown under As(III)and Cd stress. Seeds of transgenic lines and WT were germinatedon ABIS media containing As(III) (10 mM) and Cd(25 mM)respectively for 20 d. Fresh biomass of 15–20 seedlings of eachtransgenic line was measured. Bars represent the mean of rootlength of 15–20 plants of WT and transgenic lines and error barsrepresent �SD. ** and *** significantly different at P < 0.05 and0.001, respectively from WT.

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Figure 5. Expression of OsNRAMP1 in Arabidopsis leads toenhanced accumulation of As(III) and Cd. Seeds of Arabidopsisthaliana (WT; accession Col-0) and three transgenic lines (L1, L2and L3) were germinated on ABIS medium supplemented withFe-EDTA (50 mM) and 5 mM As(III) and 10 mM Cd grown for twoweeks. As and Cd accumulation in root and shoot of the plantswere measured as described detail in Experimental Procedures.Values are the average of three independent experiments errorbars represent �SD (** P, 0.01; *** P, 0.001).



& Guerinot 2006; Nevo & Nelson 2006). In addition to essen-tial divalent cations (Fe, Zn, Mn), NRAMPs have oftenshown capacity to mobilize toxic metals like Cd, Ni (Mizunoet al. 2005; Cailliatte et al. 2009; Oomen et al. 2009) andAl (Xia et al. 2010). In this study, our focus was to elucidatethe role of OsNRAMP1 in As as well as Cd transport andaccumulation using yeast and Arabidopsis systems. Earlierstudies reported that expression of OsNRAMP1 was inducedduring As stress in rice in addition to defence and stress-responsive genes, transporters, heat-shock proteins, metal-lothioneins, sulphate-metabolizing proteins (Norton et al.2008; Chakrabarty et al. 2009; Kumar et al. 2011, 2013a,b;Gautam et al. 2012). In this study, similar results wereobserved through RT-PCR analysis carried out in two ricecultivars with contrasting features (Dave et al. 2013), uponAs(III) exposure (Fig. 1c) supporting possible involvementin As(III) transport. Furthermore, a few reports haverevealed that As uptake in rice root is related to Fe availabil-ity in the soil and its accumulation was correlated with Fe inrice tissues (Dwivedi et al. 2010; Zhao et al. 2010a; Rahmanet al. 2011).Therefore, it is reminiscent that transporters of Femight be involved directly or indirectly in As uptake in rice.We have demonstrated this through expression of OsN-RAMP1 in Arabidopsis in this study.

Plant NRAMPs are foremost known for Fe uptake activityas demonstrated using yeast mutants. Similarly, in this study,

we report that expression of OsNRAMP1 isolated fromindica variety of rice (IR-64) into fet3fet4, rescued the growthat low Fe in the medium. Concomitant to Fe transport,expression of OsNRAMP1 in yeast mutant enhanced theuptake of Cd and As(III) when supplemented in SD-URAagar plates (Fig. 2b,c). Our results regarding Cd toxicity infet3fet4 are in agreement to earlier study with Cd sensitivestrain (ycf1) (Takahashi et al. 2011). However, toxicity con-ferred by As(III) in yeast was much lower in comparison toCd which might be due to less enhancement in As or indirectconsequence of OsNRAMP1 expression.

In our study, Arabidopsis transgenic lines expressing OsN-RAMP1 did not display any morphological changes whengrown on half strength MS medium or in soil under normalcondition (Supporting Information Fig. S1) as observed instudies related to plant NRAMPs (Thomine et al. 2003;Oomen et al. 2009). By contrast, the overexpression ofOsNRAMP1 in rice ensued in reduced growth perhaps dueto affecting the distribution of essential metals (Takahashiet al. 2011). Present study revealed the Cd transport activityof OsNRAMP1 and suggested that OsNRAMP1 participatein cellular Cd transport within plants without exerting muchtoxic effects possibly due to retaining efficient detoxificationsystem. Parallel to rice, increase of Cd content in aerial tissueindicates the critical role of OsNRAMP1 in root to shoottranslocation as predicted earlier (Takahashi et al. 2011) and

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Figure 6. Modulation in Fe and Mn level in OsNRAMP1 expressing transgenic lines under As(III) and Cd stress. Fe and Mn content weremeasured in root and shoot of two week old plants grown in medium with or without supplementation of As(III) (5 mM) and Cd treated(10 mM) using ICP-MS. Values are the average of three independent experiments error bars represent �SD (* P; 0.05; ** P, 0.01; *** P,0.001).



verified in this study. However, transgenic lines displayedsignificant level of tolerance as measured in terms of rootlength and total biomass corresponding to WT in the pres-ence of As(III) as well as Cd (Fig. 4). This study, for the firsttime, shows role of NRAMPs, especially OsNRAMP1, exhib-iting tolerance to As(III) and Cd with enhanced accumula-tion in roots and shoots. In the transgenic lines, significanthigher accumulation of As was recorded in comparison to

WT (Fig. 5). In general, Arabidopsis NRAMPs includingAtNRAMP1, AtNRAMP3, AtNRAMP4 and AtNRAMP6were demonstrated to increase Cd sensitivity while expressingin yeast (Thomine et al. 2000;Cailliatte et al. 2009).Apart fromArabidopsis NRAMPs,TcNRAMPs have also been shown Cduptake activity (Oomen et al. 2009; Wei et al. 2009). The overexpression of AtNRAMP1 and AtNRAMP6 in Arabidopsiswere shown to confer Cd sensitivity (Curie et al. 2000;

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Figure 7. GFP fluorescence in root apex of OsNRAMP1:GFP stable transformed Arabidopsis plant. GFP fluorescence in dividing (a)–(c)and in maturation (differentiation) (d–f) zones. (g–i) are the enlarged images of individual cells. Arrowhead marked the position of cylinderconsist by endodermis and pericycle cells (d). Xv and Pc are xylem vessels and endodermis or pericycle cells and arrowhead indicate theirpositions in the cross sections. Excitation wavelength for GFP was set at 488 nm and emitted light were collected through 505–530 nm bandpass filter.



Cailliatte et al. 2009). AtNRAMP1 are later shown to lie atplasma membrane of root hairs and might be involved inenhanced influx of Cd into the cells, causing Cd toxicity(Cailliatte et al. 2010) whereas AtNRAMP6 due to its vesicu-lar localization could release the Cd in cytosol and therebyexerts toxic effect of Cd (Cailliatte et al. 2009). Notwithstand-ing, OsNRAMP1 provided tolerance for Cd as well as As(III)in Arabidopsis and this could only be explained on the basis ofdetailed tissue-specific localization analysis of OsNRAMP1.

The cell specific localization revealed that GFP fluores-cence was predominantly confined within pericycle region(Fig. 7). The in-depth examination GFP fluorescence in cellsof maturation zone of root suggests that OsNRAMP1 pre-cisely expressed in inner cells of root, mainly in pericycle

region consisting of endodermis and pericycle. The cell-specific expression of OsNRAMP1 under constitutive pro-moter, CaMV35S promoter, can only be explained due topost-transcriptional regulation which might be a regulatorymechanism for this transporter. Based on our localizationand expression analyses, we suggest that these metals enter inpericycle cells through OsNRAMP1. We hypothesize thatfrom pericycle cells, Cd may be exported outside from peri-cycle cells into xylem parenchyma cells by HMA4 trans-porter (Hussain et al. 2004). At the same time, As(III) couldbe mobilized into xylem via unknown transporter. The iden-tification of such a transporter will be important in studyingroot to shoot movement of such toxic metal. The remainingcytosolic As(III) and Cd may bind to cellular thiols, mainlywith phytochelatins and then sequestered in vacuoles viaAtABCC1 and AtABCC2 transporters (Fig. 9). This pro-posed premise was supported with enhanced expression ofAtHMA4 and AtABCCs transporters in transgenic lines(Fig. 8). The efficient loading of these metals inside the con-ductive tissues and thereafter rapid mobilization in aerialtissues and vacuoles may provide tolerance for heavy metalsin transgenic lines. This might be reason behind tolerancewith these metals rather than sensitivity which was observedin case of AtNRAMPs (Curie et al. 2000; Thomine et al. 2003;Oomen et al. 2009). Recently, It has been demonstrated thatrice low-affinity cation transporter (OsLCT1) functions atthe nodes in Cd transport into grains and suggested that theregulation of OsLCT1 may enable the generation of ‘low-Cd rice’ without negative effects on agronomical traits(Uraguchi et al. 2011). Taken together, we propose that OsN-RAMP1 is targeted to plasma membrane of endodermis andpericycle cells and specific orientation assisting in xylemloading and distribution of heavy metals from root to shoot.

Identification of mechanisms for As transport in rice ishighly required to develop strategy for generating genotypesof rice containing lesser As in grains. An As(III) efflux trans-porter (PvACR3) localized to the tonoplast in As hyperaccu-mulators has been shown to mediateAs(III) transport into thevacuoles and to play an important role in As detoxification(Indriolo et al. 2010), but none of its homologues were foundin higher plants. Two ABCC-type transporters have beenidentified in Arabidopsis as the major vacuolar transportersfor As(III)-phytochelatin complexes (Song et al. 2010) whichare required for As detoxification. Interestingly, thesetransporters showed enhance expression in transgenic linesexpressing OsNRAMP1 developed in this study and mightcontribute to considerable accumulation of Cd andAs in root.This is the first report for identification of a member ofNRAMPs family transporter,OsNRAMP1, involved in xylemmediated transfer of Cd andAs.The observations of this studyare of a central importance for development of rice genotypeswith less accumulation of As in their grains through knockingdown of OsNRAMP1 to meet the perspective of As safe rice.

ACKNOWLEDGMENTS

This work was supported by a research grant from theCouncil of Scientific and Industrial Research, New Delhi,

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Figure 8. Enhanced expression of metal transporters intransgenic lines expressing OsNRAMP1. Real time-PCR analysesof AtABCC1, AtABCC2 and AtHMA4 was carried out in the roottissue of two week old transgenic lines and WT plants. Expressionof AtABCC1, and AtABCC2 was studied in two week old plantsgrown in medium supplemented with As(III) (5 mM) whereasexpression of AtHMA4 was analyzed in plants grown in mediumsupplemented with Cd (10 mM). Values are the average of threeindependent experiments error bars represent �SD (* P, 0.05;** P, 0.01; *** P, 0.001).



as Network Project (BSC-0107). Authors thank ProfessorOomen Ronald, Biochimie & Physiologie Moléculaire desPlantes, UMR5004, CNRS/INRA/SupAgro/UM II, F-34060Montpellier Cedex 1, France for providing DEY1453(fet3,fet4) strain of yeast. M.T. and D.S. thankfully acknowl-edge the Indian Council of Medical Research, India, andCouncil of Scientific and Industrial Research, India for JuniorResearch Fellowships to them respectively.

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PXP V

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Received 20 March 2012; received in revised form 2 May 2013;accepted for publication 13 May 2013



SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:

Figure S1. Growth of OsNRAMP1 expressing Arabidopsislines in response to Fe. (a) Growth of Col-0 and OsNRAMP1expressing Arabidopsis lines (L1, L2 and L3) in soil after 3weeks. Lower panel represents expression of OsNRAMP1 indifferent transgenic lines. (b) Growth of WT and different

transgenic lines under different Fe concentrations. Seeds ofArabidopsis thaliana (WT; accession Col-0) and three trans-genic lines were germinated on ABIS medium supplementedwith 50 and 750 mM Fe-EDTA and grown vertically for 10 d.(c) Root length of the WT and transgenic lines grown ondifferent Fe concentrations after 10 d. Values are the averageof 15–20 plants root of each genotype and error bars repre-sent �SD (P < 0.05).Table S1. List of oligonucleotides and their use in the study.



Documents

Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP, OsNRAMP1, in arsenic transport and tolerance