Arsenic tolerance in Arabidopsis is mediated by twoABCC-type phytochelatin transportersWon-Yong Songa,b,1, Jiyoung Parka,1, David G. Mendoza-Cózatlc,1, Marianne Suter-Grotemeyerd, Donghwan Shima,Stefan Hörtensteinerb, Markus Geislerb, Barbara Wederb, Philip A. Reae, Doris Rentschd, Julian I. Schroederc,2,3,Youngsook Leea,2,3, and Enrico Martinoiaa,b,2,3

aPohang University of Science and Technology–University of Zurich Cooperative Laboratory, Department of Integrative Bioscience and Biotechnology, WorldClass University Program, Pohang University of Science and Technology, Pohang 790-784, Korea; bInstitute of Plant Biology, University of Zurich, 8008 Zurich,Switzerland; cDivision of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA 92093-0116; dInstituteof Plant Sciences, University of Bern, 3013 Bern, Switzerland; and ePlant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA19104-6018

Edited by Maarten Koornneef, The Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved October 19, 2010 (received for reviewSeptember 20, 2010)

Arsenic is an extremely toxic metalloid causing serious health pro-blems. In Southeast Asia, aquifers providing drinking and agricul-tural water for tens of millions of people are contaminated witharsenic. To reduce nutritional arsenic intake through the consump-tion of contaminated plants, identification of the mechanisms forarsenic accumulation and detoxification in plants is a prerequisite.Phytochelatins (PCs) are glutathione-derived peptides that chelateheavy metals and metalloids such as arsenic, thereby functioning asthefirst step in their detoxification. Plant vacuoles act asfinal detox-ification stores for heavy metals and arsenic. The essential PC–metal(loid) transporters that sequester toxicmetal(loid)s in plant vacuoleshave long been sought but remain unidentified in plants. Here weshow that in the absence of two ABCC-type transporters, AtABCC1and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenicand arsenic-based herbicides. Heterologous expression of theseABCC transporters in phytochelatin-producing Saccharomyces cere-visiae enhanced arsenic tolerance and accumulation. Furthermore,membrane vesicles isolated from these yeasts exhibited a pro-nounced arsenite [As(III)]–PC2 transport activity. Vacuoles isolatedfrom atabcc1 atabcc2 double knockout plants exhibited a very lowresidual As(III)–PC2 transport activity, and interestingly, less PC wasproduced in mutant plants when exposed to arsenic. Overexpres-sion of AtPCS1 and AtABCC1 resulted in plants exhibiting increasedarsenic tolerance. Our findings demonstrate that AtABCC1 andAtABCC2 are the long-sought and major vacuolar PC transporters.Modulation of vacuolar PC transporters in other plants may allowengineering of plants suited either for phytoremediation or reducedaccumulation of arsenic in edible organs.

ABC transporter | vacuolar sequestration | multidrug resistance-associatedprotein

Arsenic has been widely used in medicine, industry, and ag-riculture. In medicine, it was used to treat diseases such as

syphilis, trypanosomiasis, or amoebic dysentery. In agriculture,arsenic-based herbicides, such as disodium methanearsonate(DSMA), continue to be applied for weed and pest control.However, arsenic is a highly toxic environmental pollutant thatcauses global health problems. In Southeast Asia as well as inregions with extensive mining, such as China, Thailand, and theUnited States, arsenic concentrations in water can be far abovethe World Health Organization limit of 10 μg/L (133 nM), con-centrations that are known to cause health problems. People areexposed to arsenic poisoning both by drinking contaminatedwater and by ingesting crops cultivated in soils irrigated withpolluted water. It has been shown that arsenic in rice paddy fieldsis readily taken up by rice plants and translocated to the grains,constituting a danger for populations that rely mostly on rice fortheir diet (1, 2). In Bangladesh alone an estimated 25 millionpeople are exposed to water contaminated with arsenic exceeding50 μg/L, the Bangladesh government standard, and more than 2

million people are estimated to face a risk of death from cancercaused by arsenic (3–5).To reduce nutritional arsenic intake, identification of the

mechanisms implicated in arsenic accumulation and detoxificationof plants is a prerequisite. Considerable progress has been madeduring the last years in the identification of the molecular mecha-nisms of arsenic (As) uptake, metabolism, and translocation (forreviews see refs. 6–8). After entering into roots through high-affinity phosphate transporters, arsenate [As(V)] is readily reducedto arsenite [As(III)]. Alternatively, under reducing conditions, As(III) is taken up bymembrane proteins belonging to the aquaporinfamily (9, 10). In rice, it has been shown that As(III) is exportedsubsequently to the xylem by the silicon efflux transporter Lsi2,resulting in root-to-shoot transport of arsenic (11). Thefinal step ofarsenic detoxification in the cell is the sequestration into vacuoles,which depends mainly on glutathione (GSH) and phytochelatins(PCs). PCs are GSH-derived metal-binding peptides whose syn-thesis is induced by arsenic as well as other toxic metals (12, 13).PCs chelate As(III) and heavy metals with their thiol groups, andthe metal(loid)–PC complexes are sequestered into vacuoles, pro-tecting cellular components from the reactive metal(loid)s.PC-mediated detoxification is unique to plants and a few other

PC-producing organisms but different from that of budding yeastor humans, in which PCs or PC synthase (PCS) do not exist. Thetransporters responsible for active transport of PC-conjugatedAs(III) as well as for transport of PC-conjugated heavy metalsinto plant vacuoles (14–17) are yet to be identified but have beenproposed to be ABC transporters, as is the case with glutathione(GS)-conjugated As(III) (As(GS)3) in other organisms (18, 19).For example, in Saccharomyces cerevisiae it has been shown thatarsenic is detoxified by YCF1, an ABC protein transporting As(GS)3 into vacuoles (18). In humans, it has been shown thatHsABCC1 and HsABCC2 are involved in arsenic detoxificationby transporting As(III) conjugated to GSH (19). Another ABCtransporter, HMT1, has been proposed to act as a vacuolar PCtransporter in Schizosaccharomyces pombe, although an hmt1 de-

Author contributions: W.-Y.S., J.P., D.G.M.-C., J.I.S., Y.L., and E.M. designed research;W.-Y.S., J.P., D.G.M.-C., M.S.-G., D.S., S.H., M.G., B.W., P.A.R., and D.R. performed research;and J.P., J.I.S., Y.L., and E.M. wrote the paper.

Conflict of interest statement: Y.L., E.M., J.I.S., W.-Y.S., J.P., and D.G.M.-C. have filed apatent on the reduction of arsenic in crops and the use of ABCCs for phytoremediationbased on the discovery reported in the manuscript.

This article is a PNAS Direct Submission.

See Commentary on page 20853.1W.-Y.S., J.P., and D.G.M.-C. contributed equally to this work.2J.I.S., Y.L., and E.M. contributed equally to this work.3Towhom correspondencemay be addressed. E-mail: jischroeder@ucsd.edu, ylee@postech.ac.kr, or enrico.martinoia@botinst.unizh.ch.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1013964107/-/DCSupplemental.

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letion mutant continued to exhibit PC accumulation in vacuoles,and HMT1 did not confer arsenic resistance (20, 21). In plants, noortholog for HMT1 has been identified.Here we report the identification of two ABC transporters re-

quired for arsenic detoxification. These transporters are plant PCtransporters that have been sought since the discovery of PCs (12,22). Thus this finding provides a key to understanding the detoxifi-cation of many xenobiotic molecules, heavy metals, and metalloids,including arsenic, that are conjugated with PCs for detoxification.

Resultsatabcc1 atabcc2 Double Knockout Mutant Is Hypersensitive to Arsenicand Arsenic-Based Herbicide. To identify candidate transporterslikely involved in arsenic detoxification in plants, we focused onscreening the ABCC subfamily of ABC transporters, also knownas the multidrug resistance-associated proteins (MRPs). Thissubfamily includes ABC transporters implicated in heavy metalresistance, such as ScYCF1, the yeast vacuolar As(GS)3 trans-porter (18), and the two human arsenic-detoxifying ABC trans-porters (19). Members of this family have also been shown totransport GSH conjugates and to confer cadmium resistance inplants and humans (23–26). In addition, for many ABCC proteinsin Arabidopsis, vacuolar localization has been demonstrated orproposed on the basis of data obtained using either Westernblotting of membrane fractions, GFP fusion proteins, or vacu-olar proteomics approaches (27–30). We have therefore isolatedhomozygous knockout lines for all 15ArabidopsisABCC genes andhave grown these mutants in the presence of arsenic and arsenic-containing herbicides. Two different forms of arsenic were usedbecause they have different entry pathways to the cell, as well asdifferential metabolism. Whereas As(V) is taken up by the high-affinity phosphate transporter (31, 32), DSMA is much more hy-drophobic and is rapidly absorbed by plant roots. Furthermore, As(V) is reduced to As(III) in the cell, whereas in DSMA arsenic isalready present in the As(III) form. Although no As(V)-sensitiveatabcc knockout mutant was found, the growth of two deletionmutants, atabcc1/atmrp1 and atabcc2/atmrp2, was impaired in thepresence of the arsenic herbicide DSMA, compared with that ofthe wild type (Fig. 1 and Fig. S1). To determine whether the rel-atively moderate arsenic sensitivity could be due to overlappingfunctions of the two ABC transporters, we generated a doubleknockout atabcc1 atabcc2 mutant. AtABCC1 and AtABCC2,which belong to clade I of theABCC subfamily, share a high aminoacid sequence similarity. Furthermore, both have been localized tothe vacuolar membrane in former studies (27, 28). Whereas undercontrol conditions no difference in growth was observed betweenthe wild type and the double knockout mutant, growth of theatabcc1 atabcc2 double knockout line was dramatically impairedon plates containingDSMAaswell as low level ofAs(V) comparedwith the wild type (Fig. 1 A and B). Quantification of the freshweight and root length of plants grown in As(V)-containing mediaconfirmed that a significant difference could only be observed forthe double knockout, which exhibited a strongly reduced freshweight even at low As(V) concentrations (Fig. 1 C and D). In thepresence of 30 μM As(V), root growth was reduced by more than60%. The sensitivity of the atabcc1 atabcc2 knockout line wascomparable to that observed for cad1-3, an Arabidopsis mutantimpaired in PC synthesis (Fig. S2) (33). To verify whetherAtABCC1 and AtABCC2 confer arsenic tolerance under naturalconditions, we grew atabcc1 and atabcc2 single and doubleknockout lines, and the corresponding wild type on soil for 3 wkand then irrigated the plants with 133.5 μMAs(V) for an additional8 d. The atabcc1 atabcc2 double mutants were extremely sensitiveto As(V) and did not survive this treatment, whereas the wild-typeand single knockout plants were not visibly affected (Fig. 1E).

AtABCC1 and AtABCC2 Enhance Arsenic Tolerance and Accumulationin the Presence of TaPCS1 in Budding Yeasts. To test whether

AtABCC1 and AtABCC2 confer arsenic resistance by compart-mentalization of a GS–As complex into the vacuole, we expressedthe transporters in SM4, a budding yeast mutant strain lackingYCF1 and the three other vacuolar ABCC-type ABC transporters,YHL035c, YLL015w, and YLL048c. AtABCC1- or AtABCC2-expressing SM4 yeast lines showed only a marginal increase inarsenic resistance, compared with the empty vector control (Fig.2A). These results indicate that in contrast to Arabidopsis,AtABCC1 and AtABCC2 cannot mediate arsenic tolerance inbudding yeast, which does not synthesize PCs. These experimentsalso suggest that AtABCC1 and AtABCC2 are not efficient GS–As complex transporters and thus cannot complement ycf1.In contrast to S. cerevisiae and humans, which depend on GSH

for arsenic detoxification, in plants detoxification of arsenic hasbeen shown to mainly depend on PCs (8, 16, 33, 34). We there-fore tested whether AtABCC1 and AtABCC2 confer arsenic re-sistance by transporting PC–As complexes. As a first approach, wegenerated a yeast mutant expressing the wheat PCS TaPCS1 (35,36) in the SM4 background and named it SM7. This yeast strainwas then used to express either AtABCC1 or AtABCC2. Whengrown on arsenic-free control medium, no large difference couldbe observed between the SM7 cells transformed with the emptyvector or expressing either of the ABC transporters. However,when the yeast cells were exposed to 100 μM As(III), the linesexpressing AtABCC1 or AtABCC2 grew dramatically better than

Fig. 1. Hypersensitivity of atabcc1 atabcc2 double knockout mutants to ar-senic [As(V)] and DSMA, an arsenic-based herbicide. (A and B) Wild-type,AtABCC1 knockout (abcc1-3), AtABCC2 knockout (abcc2-2), and doubleknockout (abcc1 abcc2) seedlings grown in 100 mg/L of DSMA (A) and 50 μMof As(V) (B) containing half-strength MS media for 16 d. Note that singleknockout mutants of AtABCC1 or AtABCC2 are also sensitive to the arsenicherbicide (A) but not to As(V) (B). (Scale bar, 1 cm.) (C and D) Fresh weight (C)and root length (D) of each genotype of plants grown on 0–50 μM As(V)-containing 1/2 MS media. Mean ± SEM (for fresh weights, n = 16, from fourindependent experiments; for root lengths, n = 332–336, from three in-dependent experiments). *P < 0.04, **P < 0.01 (Student’s t test). (E) Arsenic-sensitive phenotype of atabcc1 atabcc2 double knockout plants grown in soil.Three-week-old plants were watered with 133.5 μM As(V). Four representa-tive plants out of 12; each was photographed 8 d after arsenic treatment.

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controls (Fig. 2A). In this condition, yeast cells expressing theABCtransporters accumulated more arsenic than the correspondingcontrol (Fig. 2B). Furthermore, in the presence of arsenic,AtABCC1- and AtABCC2-expressing SM7 strains contained muchhigher PC levels compared with SM7 strains transformed with theempty vector (Fig. S3), indicating that efficient PC synthesis is onlyachieved when PCs are transported into the vacuole.

Together, these results indicate that AtABCC1 and AtABCC2can mediate vacuolar PC–As sequestration, allowing the PC-producing yeast strains to cope with arsenic. These heterologousreconstruction assays also suggest that chelation of arsenic by PCalone cannot detoxify arsenic, but transporters of PC–As com-plex are required, because expression of PCS in the SM4 back-ground (SM7) did not increase arsenic tolerance (Fig. 2A).

AtABCC1 and AtABCC2 Mediate PC Transport. To directly determinewhether AtABCC1 and AtABCC2 mediate PC–As transport, weperformed transport experiments using microsomal vesicles iso-lated from SM7 yeast transformed with the empty vector or ex-pressing AtABCC1 and AtABCC2. As shown in Fig. 3A, vesiclesisolated from yeast cells expressing AtABCC1 or AtABCC2 effi-ciently took up As(III)–PC2 in a time-dependent manner,whereas in vesicles isolated from the empty vector control theuptake was negligible. As expected for an ABC-type transporter,As(III)–PC2 uptake into yeast vesicles was not inhibited by dis-sipating the proton gradient with NH4

+, whereas in the presenceof vanadate the transport activity was completely inhibited (Fig.3B). Comparison of the uptake activity of apoPC2 and As(III)–PC2 revealed that the As(III)–PC2 complex is transported moreefficiently than apoPC2 by both AtABCC1 and AtABCC2 (Fig.3B). The ratio of the transport activity between apoPC2 and As(III)–PC2 varied between the different batches of PC2 produced,probably owing to variable stability of the PC2–As complex.The discrimination between apoPC2 and As(III)–PC2 was morepronounced for AtABCC2 than for AtABCC1. As shown for As(III)–PC2, apoPC2 uptake was also inhibited by vanadate but notby NH4

+ (Fig. S4A). We further tested whether AtABCC2 cantransport arsenic in a non-chelated form using microsomalvesicles of the SM7 cells expressing the transporter. Arseniccontent measured using inductively coupled plasma mass spec-trometry (ICP-MS) showed that without PCs no arsenic wastransported via AtABCC2 (Fig. 3C). Furthermore, by comparisonof concentrations of PC2 and arsenic transported into these vesi-cles, we could estimate that AtABCC2 mediates the transport oftwo molecules of PC2 per molecule of arsenic (Fig. 3C).Surprisingly, the concentration-dependent As(III)–PC2 uptake

did not follow classic saturation kinetics, but exhibited an ap-parent sigmoid curve for both AtABCC1 and AtABCC2 (Fig.3D). At PC2 concentrations below 10 μM, As(III)–PC2 uptakeactivity was negligible but was strongly stimulated by increasing

Fig. 2. AtABCC1 and AtABCC2 enhance arsenic tolerance and accumulationin PCS-expressing budding yeasts. (A) Expression of AtABCC1 (ABCC1) orAtABCC2 (ABCC2) in PC-producing yeast (SM7) enhanced tolerance to 100 μMof As(III). EV, empty vector control. In SM4 strain without PCS expression, nogrowth difference between genotypes was apparent. Cells were grown inhalf-strength SD-Ura (synthetic dextrose lacking uracil) media supplementedwith 100 μM As(III) for 2 d. O.D.600, optical density at 600 nm of the yeastsuspension that was initially spotted on the plates. (B) Arsenic content of SM7yeast cells expressing AtABCC1 or AtABCC2 was higher than that of cellstransformed with the empty vector. Cells were grown in half-strength SD-Uramedia supplemented with 100 μM As(III) for 12 h. Mean ± SEM (n = 21, fromseven independent experiments). *P < 0.03, **P < 0.005 (Student’s t test).

Fig. 3. AtABCC1 and AtABCC2 mediate PC transport inmicrosomes isolated from yeast transformed with AtABCC1and AtABCC2 transporters. (A) Time-dependent uptake ofPC2–As in yeast microsomes isolated from S. cerevisiaetransformed with the empty vector (EV), AtABCC1, andAtABCC2. PC concentration was 25 μM. (B) Comparison oftransport activities for apoPC2 and PC2–As, and inhibition ofPC2–As transport by NH4

+ and vanadate. PC concentrationwas 25 μM. (C) Relationship between arsenic (Left) and PC(Right) transport activity. PC2 (500 μM) with 1 mM As(III) or1 mM As(III) alone was used, and the concentrations oftransported PC2 and arsenic were measured. (D) Concen-tration dependence of PC2–As (solid line) and apoPC2(dotted line) transport activity of vesicles isolated fromAtABCC1 (filled squares) and AtABCC2 (filled triangles)expressing SM7 S. cerevisiae. Inset: Enlarged version at lowconcentrations of the substrate. Mean ± SEM (from threeindependent experiments with four replicates each).

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PC concentrations. The PC concentration for half-maximal up-take activity was estimated to be 350 μM. This high concentra-tion suggests a rather low affinity of AtABCC1 and AtABCC2for PCs. Previous reports demonstrated that PCs can accumulateto concentrations exceeding 1,000 μM under metal(loid) stress(37), in good agreement with the concentrations that exhibitedoptimal uptake in these experiments (Fig. 3D). Interestingly, theconcentration dependence for apoPC2 was linear and did notreveal a saturation curve up to 1,000 μM apoPC2 (Fig. 3D).

AtABCC1 and AtABCC2 Are the Major As(III)–PC2 Transporters in PlantVacuoles. To determine whether AtABCC1 and AtABCC2 indeedencode the long-sought functional PC transporters in planta and todetermine their contribution to vacuolar PC uptake, we comparedthe transport activities for As(III)–PC2 and apoPC2 of intactvacuoles isolated from the atabcc1 atabcc2 double knockoutand wild-type plants. As shown in Fig. 4A, vacuolar uptake activ-ities of both As(III)–PC2 and apoPC2 were dramatically reducedby 85% and 95%, respectively, in the mutant plant, indicatingthat these two ABC transporters are by far the most important PCand PC-conjugate transporters in Arabidopsis. AtABCC11 andAtABCC12, very close homologs of AtABCC1 and AtABCC2,could be responsible for the very low residual PC uptake activity.Comparison of the transport rates of apoPC2 and As(III)–PC2 invacuoles isolated from wild-type Arabidopsis showed that the dif-ferences in transport rates for these two forms of PC were lesspronounced in Arabidopsis vacuoles than in yeast vesicles. Thismight be due to the more than twofold higher expression level ofAtABCC1 compared with AtABCC2 in fully expanded Arabidopsisleaves, the tissue used for vacuole isolation (Genenvestigator orhttp://www.bar.utoronto.ca/). In yeast experiments, the selectivity ofAtABCC1 for As(III)–PC2 was much lower than that of AtABCC2.An alternative explanation could be that the different lipid envi-ronment in plant and yeast vesicles also affects the selectivity. Asobserved for yeasts, vacuolar PC transport in Arabidopsis was notinhibited by NH4

+ (residual transport activity 98% ± 4%) butstrongly inhibitedby vanadate (residual transport activity 25%±7%)(Fig. S4B), which are typical features of ABC transporters.

PC contents, integrated over 2 d of continuous arsenic expo-sure, were reduced in atabcc1 atabcc2 double knockout plants,when total plant PC contents, reflecting the vacuolar and cyto-plasmic pools, were measured (Fig. 4B). The difference betweenwild-type plants and the mutants become more pronounced athigh arsenic concentrations, which increased production of PCs.This observation is in line with the sigmoid kinetics of the vacuolartransport observed in vesicles isolated from the yeasts expressingthe two AtABCC transporters and again supports the necessity oftransporting PCs into the vacuole for continuous PC synthesis.Surprisingly, double mutant plants took up slightly more arsenic,but the transfer of arsenic to the shoot was reduced in these plants(Fig. S5). The same result was obtained in plants grown on platesas well as under hydroponic conditions.

Overexpression of AtABCC1 Increases Arsenic Tolerance Only WhenCoexpressed with PCS. To determine whether arsenic tolerance canbe increased by expressing one of the PC transporters, we trans-formed wild-type plants with a construct in which AtABCC1 isdriven by the 35S promoter. We could identify only plants in whichthe transcript levels were maximally increased by 20–40%. How-ever, these lines did not confer increased arsenic tolerance (Fig. S6),which may be explained by the circumstances that the over-expression was not strong enough or the PC transport activity wasnot the limiting factor in conferring arsenic tolerance.Therefore, wegenerated AtPCS1 single and AtPCS1 AtABCC1 co-overexpressinglines. No differences in arsenic tolerance could bedetected betweenthe wild type and seven AtPCS1 single overexpressing lines, despitetranscript levels of AtPCS1 exceeding that of the wild type by860–3,260% in those lines (Fig. S7). In contrast, the two plants co-transformed with AtPCS1 and AtABCC1 (MP-1 and MP-2)exhibited a consistent increase in arsenic tolerance (Fig. 5). Besidesthe less-chlorotic phenotype, shoot fresh weight was also increasedin the plants overexpressing both genes.When exposed to 70 μMAs(V), the shoot weight of these two lines corresponds to 161% and147% of those of the wild-type and AtPCS1-overexpressing plants(Fig. 5C). Even after normalization with the growth in control me-dium, to take into account the slightly better growth of the doubletransformant in normal medium, the twoAtPCS1AtABCC1 doubletransformants exhibited an increased arsenic tolerance (124% and114% of the wild type, respectively). These results show that coex-pression of an ABC-type PC transporter and PCS can result inplants with enhanced arsenic tolerance.

DiscussionCooperation of PC Transport and Synthesis in Arsenic Detoxification.Previous studies have demonstrated that PCs are required for ar-senic tolerance in higher plants (33, 36). PC-conjugated substrateshave been suggested to be sequestered into vacuoles. However,until now no plant transporter for PCs has been identified. Ourresults demonstrate that AtABCC1 and AtABCC2 are both vac-uolar PC transporters required for arsenic resistance inA. thaliana.The similar phenotypes of the atabcc1 atabcc2 double knockoutand the cad1 mutant, which lacks PC synthesis, suggest that thesequestration of PC–As complexes into vacuoles is as critical forthe detoxification of arsenic as the synthesis of PCs. A similarmechanism may work for other metal(loid)s, or alternatively, theymay form bis–GS complex and be sequestered for detoxification.The main transporters for the GS-metal(loid) complexes in plantsremain to be identified.The AtABCC1 and AtABCC2 transporters and PCS may

function in a concerted way in the arsenic detoxification pathway.The PCS enzyme, although present even when plants are not ex-posed to heavy metals or metalloids, remains inactive under nor-mal conditions and is rapidly activated when plants take up arsenicor other toxic metal(loid)s (12, 38). Similarly, expression ofAtABCC1 and AtABCC2 is not induced by arsenic in Arabidopsis,nor does the absence of one of the ABCCs result in increased

Fig. 4. Vacuoles isolated from the atabcc1 atabcc2 double knockout exhibitonly a very low residual PC transport activity, and atabcc1 atabcc2 doubleknockouts show decreased content of PCs at the seedling stage. (A) Vacuoleswere isolated from wild-type and the atabcc1 atabcc2 double knockout plantsand tested for transport activities for PC2–As and apoPC2. As a negative con-trol, ATP was replaced by ADP. PC concentration was 250 μM. Mean ± SEM(from two independent experiments with four replicates each). (B) Twelve-day-old seedlings were exposed to different concentrations of potassium ar-senate for 96 h, and the integrated accumulation of PCs was determined byHPLC-MS. Mean of two replicates (0 μM of arsenic) or three replicates ± SEM(50 and 100 μM of arsenic).

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transcription of the other (Fig. S8B) (39). AtABCC1 andAtABCC2 are not synthesized de novo but constitutively present ina plant cell to rapidly respond to toxic metal(loid)s and xenobioticstresses in a way similar to PCS1. Furthermore, the synthesis ofPCs seems to be positively correlated with the presence of the PCtransporters (Fig. 4B and Fig. S3). In both yeasts and plants, PCsare increased when cells also express PC transporters. This ob-servation suggests that PC synthesis undergoes a feedback regu-lation and that removal of PCs from the cytosol leads to anincrease of its synthesis. A puzzling result was the observation inthe double knockout that arsenic concentration was higher inroots and that the root-to-shoot transfer was diminished (Fig. S5),which is in contrast to the observations made in plants lacking GSHor PCs (34). This cannot be due to an alternative PC transporter,because the atabcc1 atabcc2 double mutant is very susceptible toarsenic exposure. Even at lower concentrations the atabcc1 atabcc2knockout plants still produce PCs (Fig. 4B); however, they accu-mulate these peptides in the cytosol. As(V) exhibits a complex me-tabolism in plants, and apparently accumulation of PC–As com-plexes in the cytosol induces mechanisms that reduce the transferof arsenic to the shoot, either by inducing genes that may be re-sponsible for the translocation of phosphate from the root to theshoot or by inducing a toxicity response that has the same effect.Consistent with this possibility, atabcc1 atabcc2 plants grown hy-droponically in the presence of 5 μM As(V) were reduced inphosphate contents by 16% (P = 0.05) in shoots, whereas in rootstheir phosphate contentwas similar to that of thewild type (Fig. S5).Under our experimental conditions, overexpressing AtPCS1

alone did not result in an increased arsenic tolerance (Fig. S7).Inconsistent results have been reported using a similar approach.Li et al. (40) showed a pronounced increase when AtPCS1 wasexpressed in Arabidopsis, whereas Guo et al. (41) could only ob-serve an increased tolerance when both AsPCS1 and ScGSH1were coexpressed, indicating that thus-far-unknown parametersmay play a role when PCS1s are overexpressed in Arabidopsis. Wealso could not observe any increase in tolerance when AtABCC1was overexpressed alone. Only transgenic plants overexpressingboth AtPCS1 and AtABCC1 exhibited an increased arsenic tol-erance. Therefore, it has to be assumed that efficient increase inarsenic tolerance requires both PCS and the PC transporter.Modification of ABCC transporter and PCS expression in plants

may therefore enable future engineering of reduction in the ac-cumulation of arsenic in edible organs of plants. A similar casewas reported recently for reduction of cadmium content in ricegrain by overexpression of OsHMA3, a P-type ATPase (42).It will also be interesting to learn whether these two differenttransport systems can give a synergistic effect.

Biochemical Characteristics of AtABCC1 and AtABCC2. Both AtABCC1andAtABCC2 transported apoPC2 as well as As(III)–PC2 (Fig. 3B),althoughAs(III)–PC2was the substratemore efficiently transported.The transport of apoPC2 by the ABC transporters may supply thevacuolar apoPC2 required for stabilization of toxic metal(loid)stransported into the vacuole by other transporters that carry un-bound forms ofmetals andmetalloids. Alternatively, or additionally,apoPC2 might also be used to produce high-molecular-weight thiol–As complexes, which possess high binding capacity for arsenic.Kinetic studies of the As(III)–PC2 uptakemediated by AtABCC1

and AtABCC2 revealed that the transport activity was low at lowconcentrations of As(III)–PC2 and increased steadily as the sub-strate concentration was raised (Fig. 3D). The minimal transportactivity at low PC concentrations may allow detoxification of toxicmetals more efficiently. PCs are synthesized in response to thepresence of arsenic or heavy metals by activation of PCS (12, 38).It is likely that at the beginning of this reaction, PCs are accumu-lated, which subsequently complex arsenic or other heavy metals.If apoPC was efficiently transported into the vacuole even at verylow concentrations, fewer PCswould be available in the cytosol forimmediate chelation of arsenic when plants are exposed to arse-nic. However, after As(III)–PC2 is accumulated in the cytosol, ithas presumably to be transported rapidly into the vacuole to avoidinhibition of PC synthesis by a feedback regulation mechanism(Fig. 4B and Fig. S3). Biochemically, the sigmoidal concentrationdependence indicates allosteric regulation by multiple substratebinding sites or interaction with other proteins.In addition to their central role as PC transporters, AtABCC1

and AtABCC2 have been reported to be involved in a broad rangeof detoxification processes. Both proteins transport GS conjugates(23, 26), which are formed by the attack of theGS thiolate anion toan electrophilic and potentially toxic substrate. Furthermore,AtABCC2 has been shown to transport chlorophyll catabolites,which are potentially toxic, because they can absorb lightand produce harmful reactive oxygen species (43). This versatile

Fig. 5. Co-overexpression of PC synthase (AtPCS1) andAtABCC1 results in increased arsenic tolerance. (A) Wild-typeplants, plants of a representative AtPCS1-overexpressingline (PCS1), and plants overexpressing AtPCS1 and AtABCC1(MP-1 and MP-2) grown either on 110 μM As(V)-containingor control half-strength MS plates. (B) Transcript levelsof AtABCC1 and AtPCS1 in the plants used in A, deter-mined by qPCR. (C) Shoot fresh weight of wild-type andtransgenic plants grown under control conditions or in thepresence of 70 μM As(V). Mean ± SEM (from two inde-pendent experiments with four replicates each). *P < 0.02 byStudent’s t test.

Song et al. PNAS | December 7, 2010 | vol. 107 | no. 49 | 21191

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and important role in plant detoxification requires that AtABCC1and AtABCC2 are expressed in all tissues in a constitutive way.

Materials and MethodsPlant Materials, Growth Conditions, and Arsenical Treatments. Arabidopsisseeds of wild-type and transgenic plants (ecotype Columbia-0) were grownon half-strength Murashige-Skoog (MS) agar plates with 1.5% sucrose in theabsence or presence of the indicated concentrations of As(V) (Na2HAsO4) orDSMA, an arsenic herbicide (Supleco), in a controlled environment witha 16-h light/8-h dark cycle at 22 °C/18 °C for the indicated times. All ABCC/MRP knockout mutants were obtained from the Salk Genomic AnalysisLaboratory, except abcc2-2, abcc11-1, and abcc12-2, which were provided byDr. Markus Klein. (Philip Morris International, Switzerland).

Isolation of Intact Vacuoles from Arabidopsis Protoplasts and Transport Assays.Vacuoles were prepared from Arabidopsis mesophyll protoplasts as de-scribed previously (44, 45). Transport studies with Arabidopsis mesophyllvacuoles were performed as described for barley (44, 45) but using siliconeoil poly(dimethylsiloxane-co-methylphenylsiloxane) 550 (Aldrich) insteadof AR200. The assay for apoPC2 contained 200 μM PC2 and 200 μM DTT; thatfor As(III)–PC2 contained 200 μM PC2, 400 μM As(III), and 200 μM DTT. 35S-Labeled PC2 [50 nCi (1,840 Bq)] and 3H2O [50 nCi (1,840 Bq)] were used astracer in every assay. The volume of vacuoles was determined using 3H2O.

Transport rates were calculated by subtracting the radioactivity taken upafter 20 min from that after 2.5 min incubation.

For other methods, see SI Materials and Methods.

Note Added in Proof. A report describing the independent identification ofthe related Abc2 as a vacuolar phytochelatin uptake transporter in S. pombeis in press (46).

ACKNOWLEDGMENTS. We thank the Salk Genomic Analysis Laboratory forproviding the Arabidopsis mutants; Dr. Markus Klein for the abcc mutantseeds; Dr. Christopher Cobbett (University of Melbourne, Victoria, Australia)for cad1-3 mutant seeds; Dr. Karl Kuchler (University of Vienna, Vienna,Austria) for YMM31 and YMM34 yeast strains; Dr. David Somers for hiscritical reading of the manuscript; and the Korea Forest Research Institutefor kindly allowing the use of their ICP-MS spectrometer. This work wassupported by the Global Research Laboratory program of the Ministry ofEducation, Science and Technology of Korea Grant K20607000006 (to Y.L.and E.M.), by the European Union project PHIME (Public health aspects oflong-term, low-level mixed element exposure in susceptible populationstrata) Contract FOOD-CT-2006-0016253 and the Swiss National Foundation(E.M. and M.G.), by National Institute of Environmental Health SciencesGrant P42 ES010337 (to J.I.S.), and by the Division of Chemical, Geo andBiosciences at the Office of Energy Biosciences of the Department of EnergyGrant DE-FG02-03ER15449 (to J.I.S.). D.G.M.-C. is recipient of a Pew LatinAmerican Fellowship.

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21192 | www.pnas.org/cgi/doi/10.1073/pnas.1013964107 Song et al.

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