Supporting InformationSong et al. 10.1073/pnas.1013964107SI Materials and MethodsPlant Materials, Growth Conditions, and Arsenical Treatments. AllABCC/MRP knockout mutants were obtained from the SalkGenomic Analysis Laboratory, except abcc2-2, abcc11-1, andabcc12-2, which were provided by Dr. Markus Klein (1). Acces-sion numbers and Salk number for all 15 abccmutants are listed inTable S1. A new mutant allele for AtABCC1, Salk_017431, wasused in this study and named abcc1-3 after two previously studiedalleles, abcc1-1 (1) and abcc1-2 (2). The homozygous knockoutof abcc1-3 was isolated by RT-PCR using AtABCC1-s andAtABCC1-as primers (Fig. S8A). Sequences for primers are de-scribed in Table S2. The abcc1 abcc2 double knockout homozy-gote lines were selected by genomic PCR using AtABCC1,2Fand AtABCC1,2R primers containing common sequences ofAtABCC1 and AtABCC2, and by RT-PCR using AtABCC1-s andAtABCC1-as primers for AtABCC1 and AtABCC2RT-F andAtABCC2RT-R primers for AtABCC2 (Fig. S8A). The cad1-3 (3)knockout mutant of phytochelatin synthase 1 (AtPCS1) was usedto analyze the relationship between AtABCC1 and AtABCC2,and AtPCS1.Arabidopsis seeds of wild-type and transgenic plants (ecotype

Columbia-0) were grown on half-strength Murashige-Skoog(MS) agar plates with 1.5% sucrose in the absence or presenceof the indicated concentrations of arsenate [As(V), Na2HAsO4]or disodium methanearsonate (DSMA), an arsenic herbicide(Supleco), in a controlled environment with a 16 h light/8 h darkcycle at 22 °C/18 °C for the indicated times. For phenotype anal-ysis, for each condition and line, seven plants were collected,weighed, and root length was determined. For soil experiments,Arabidopsis seeds were germinated on half-strength MS agarplates, and 10 d later seedlings with similar growth were trans-ferred to the soil. Plants were watered with 133.5 μM As(V) so-lution at 10 d after transfer.

Generation of Mutant Yeast Strain and Heterologous Expressionin Yeast. To generated SM4 (ycf1::His3, yhl035c::HIS3-MX6,yll015w::Kan-MX6, yll048c::TRP1-MX6) mutant, PCR productsof yhl035c::HIS3-MX6, yll015w::Kan-MX6 and yll048c::TRP1-MX6 were inserted into the Cd-sensitive mutant DTY167 (4) byhomologous recombination. Yeast strains used in this study aredescribed in Table S2. To amplify the DNA fragment for homol-ogous recombination, primers and genomic DNA from single nullmutants were used: His3MX6-YHL035F andHis3MX6-YHL035Rprimers for yhl035c::HIS3-MX6 and genomic DNA from YMM31strain, YLL015F and YLL015R primers for yll015w::Kan-MX6and DNA from Y11503 strain, and YLL048CF and YLL048CRprimers for yll048c::TRP1-MX6 and DNA from YMM34 strain.To express the TaPCS1 under the CUP1-1 promoter, the SM7

(ycf1:: His3, yhl035c::HIS3-MX6, yll015w::Kan-MX6, yll048c::TRP1-MX6, TaPCS1::cup1-1) mutant was generated by insertingTaPCS1 into CUP1-1 in the SM4 strain. TaPCS1::cup1-1F andTaPCS1::cup1-1R primers and TaPCS1 cDNA template wereused to amplify TaPCS1::cup1-1. To express AtABCC1 in yeast,AtABCC1/AtMRP1 cDNA was subcloned from pBsc-MRP1 (5)into SacII/SalI sites of pGEM-Teasy (Promega). An oligonu-cleotide NotI linker was inserted into the BstXI site of pGEM-Teasy, allowing NotI/NotI subcloning of the entire cDNA intothe NotI site of pNEV, resulting in pNEV-AtABCC1. Yeast cellswere transformed with pNEV empty vector, pNEV-AtABCC1 andpYES3-AtABCC2 (6) by the lithium acetate method (7), andselected on synthetic dextrose agar plates lacking uracil (SD-Ura). For the arsenic tolerance test, yeast strains expressing the

indicated constructs were grown in half-strength minimal mediaminus uracil (1/2 SD-Ura) in the presence or absence of 100 μMarsenite [As(III)] at 30 °C for 2 d.

Generation of AtPCS1 and AtABCC1 Overexpression Arabidopsis. TogenerateAtPCS1andAtABCC1 co-overexpressionplants,AtABCC1andAtPCS1 plant binary vectors were developed. For cloning of thetwo genes, modified-pCAMBIA (MPC) vectors were generated bychanging the location of 35S promoter to the front of the multi-cloning sites, using the BstXI and EcoRI sites in the pCAMBIA1300and pCAMBIA3300 vectors. The AtABCC1 construct, MPC3300-AtABCC1, was generated by inserting the AtABCC1 cDNA intothe SalI and SmaI cloning sites of the modified-pCAMBIA3300(MPC3300). The AtPCS1 binary vector, MPC1300-AtPCS1, wasconstructed by inserting the AtPCS1 cDNA into EcoRI and XbaIsites of the modified-pCAMBIA1300 (MPC1300). The constructswere introduced into theAgrobacterium tumefaciensGV3301 strain,whichwas then used to transformArabidopsis thaliana by the dippingmethod (8). The transgenic plants were selected on 1/2 MS agarmedium containing hygromycin and Basta (DL-phosphinothricin).Two independent lines of plants with the highest AtABCC1 tran-script levels were selected among 30 independent double trans-formants and used for experiments shown in Fig. 5 (main text).

Measurement of Arsenic and Phosphorus Content. To measure ar-senic contents in yeast cells, SM7 yeast cells transformed with theempty vector (pNEV-Ura), pNEV-AtABCC1 or pYES3-AtABCC2were grown in 1/2 SD-Ura supplemented with 100 μM As(III)for 12 h. The same number of cells from each strain was used forthe measurement of arsenic contents. Cells were rinsed with 10mM K+-phosphate buffer (pH 7) twice and then with ice-coldwater once. To measure ion contents in wild-type and atabcc1atabcc2 double knockout plants, seedlings grown in half-strengthMS agar plates for 2 wk were treated with 20 μM As(V) solutionfor 3, 5, or 10 d. Hydroponically grown plants as describedpreviously (9) were treated with 5 μM As(V) for 5 d. Shoots androots were collected separately and rinsed with 2 mM K+-phosphate buffer (pH 5.7) twice and then with ice-cold wateronce. Samples were then digested with 11 N HNO3 at 100 °C for6 h to 1 d. After samples were completely digested they werediluted with distilled water, and ion contents were analyzed usingan ICP-MS spectrometer (ELAN DRC-e; Perkin-Elmer).

35S-Phytochelatin-2 Synthesis. 35S-PC2 was synthesized using re-combinant AtPCS1-6xHis protein and 35S-GSH. Briefly, theAtPCS1 coding region was cloned without stop codon usingpENTR-D/TOPO from Arabidopsis cDNA (Col-0) and re-combined into pDEST42 according to the manufacturer’s rec-ommendations (Gateway cloning; Invitrogen). AtPCS1-6xHis wasexpressed in BL21 cells and purified as described in ref. 10, exceptthat purified protein was eluted in 0.25 M imidazole. After pu-rification, the buffer containing AtPCS1-6xHis was exchanged for0.1 M Tris (pH 8.0) using PD-10 columns (GE Healthcare).Phytochelatin (PC) synthase reaction (100 μL) was performed in0.1 M Tris (pH 8.0) containing 5 μg AtPCS1-6xHis, 0.1 mMCdCl2, 3 mM glutathione (GSH), and 30 μL of 35S-GSH (60 μCi,2.22 MBq; Perkin-Elmer) at 37 °C for 60 min. PCs were separatedby HPLC as previously described (11), without any derivatization,and PC2 elution was monitored at 220 nm. Purity was confirmedby mass spectrometry (11). 35S-PC2 was finally resuspended in0.1 M Tris (pH 8.0), 0.5 mMDTT, and 0.5 mM of nonradioactivePC2 (AnaSpec).

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Isolation of Membrane Vesicles from Yeast and Transport Assays.SM7 yeast cells transformed with the empty vector (pNEV-Ura),pNEV-AtABCC1, or pYES3-AtABCC2 were grown on SD-Uraliquid medium and harvested by centrifugation. All cell lines wereincubated in YPD medium for 30 min, collected by centrifuga-tion, and digested with lyticase (1,000 U/g fresh weight cells;Sigma), and then microsomal vesicles were isolated as describedpreviously (12). For PC2 transport experiments, 25 nCi (920 Bq)of 35S-PC2 was used as tracer in every assay. To generate com-plexes of PC2 and As(III), As(III), PC2, and DTT were mixed ina 2:1:1 ratio and incubated at room temperature for 40 min.Transport experiments were carried out using transport buffer[4 mM ATP, 5 mM MgCl2, 10 mM creatine phosphate, 16 units/mL creatine kinase, 1 mg/mL BSA, 100 mM KCl, and 25 mMTris-Mes (pH 7.4)] containing 25 μM PC2 for time-dependenttransport assay, with concentrations indicated in Fig. 3D (maintext) for concentration-dependent assay, and 0.5 mM PC2 with1 mM arsenic in assays to determine the ratio of PC2 and arsenic.Before starting the transport experiments 10 μL of thawed vesi-cles (100 μg of protein) were resuspended in 90 μL of transportbuffer and kept on ice for 5 min before incubating at 25 °C. At thetimes indicated in the figures, the reactions were diluted to 1 mLwith ice-cold washing buffer [100 mM KCl and 25 mM Tris-Mes(pH 7.4)], filtered immediately under vacuum through a 0.45-μm–

diameter pore size nitrocellulose filters (Millipore), and washedwith 2 mL of washing buffer. The inhibitory effects of vanadateand NH4Cl were tested by including 1 mM and 5 mM of thesecompounds, respectively, to the transport assay.

Isolation of Intact Vacuoles from Arabidopsis Protoplasts andTransport Assays. Vacuoles were prepared from Arabidopsis me-sophyll protoplasts as described previously (13, 14). Transportstudies with Arabidopsis mesophyll vacuoles were performed as

described for barley (13, 14), but using silicone oil poly(dime-thylsiloxane-co-methylphenylsiloxane) 550 (Aldrich) instead ofAR200. The assay for apoPC2 contained 200 μM PC2 and 200 μMDTT; that for As(III)–PC2 contained 200 μM PC2, 400 μM As(III), and 200 μM DTT. 35S-Labeled PC2 [50 nCi (1,840 Bq)] and3H2O [50 nCi (1,840 Bq)] were used as tracer in every assay. Thevolume of vacuoles was determined using 3H2O. Transport rateswere calculated by subtracting the radioactivity taken up after20 min from that after 2.5 min incubation.

Phytochelatin Measurements in Plants and Saccharomyces cerevisiae.Arabidopsis wild-type and atabcc1 atabcc2 double knockoutplants were stratified for 2 d at 4 °C and germinated in 1/4 MSmedia as described in ref. 15. Plants were grown for 12 d beforebeing transferred to 1/4 MS media containing 50 and 100 μMK2HAsO4. PCs were extracted and quantified as described in ref.15. Saccharomyces cerevisiae strains SM4 and SM7 containingeither empty plasmid, AtABCC1, or AtABBC2 were grown inSD-Ura containing 100 μMCuSO4 to an OD600nm 1.0. Cells wereexposed to 100 μM As2O3 for 20 h, harvested by centrifugation,and broken with glass beads as described in ref. 16. Cell extractswere used for PC analyses as described in ref. 11.

Quantitative Real-Time RT-PCR. Total RNAwas extracted from wild-type or transgenic Arabidopsis seedlings. For arsenic inductionanalysis, wild-typeplantswere treatedwithhalf-strengthMSsolutionsupplemented with or without 180 μM As(V) for 24 h. To quantifythe transcript levels of AtABCC1, AtABCC2, and AtPCS1, real-timeRT-PCR was performed with the SYBR kit (Takara). AtABCC1-sand AtABCC1-as primers were used to amplifyAtABCC1, whereasAtABCC2RT-F, AtABCC2RT-R, and AtPCS1_F2, AtPCS1_R2primers for AtABCC2 and AtPCS1, respectively. Amplified sam-ples were normalized against Tubulin8 levels.

1. Frelet-Barrand A, et al. (2008) Comparative mutant analysis of Arabidopsis ABCC-typeABC transporters: AtMRP2 contributes to detoxification, vacuolar organic aniontransport and chlorophyll degradation. Plant Cell Physiol 49:557–569.

2. Raichaudhuri A, et al. (2009) Plant vacuolar ATP-binding cassette transporters thattranslocate folates and antifolates in vitro and contribute to antifolate tolerance invivo. J Biol Chem 284:8449–8460.

3. Howden R, Goldsbrough PB, Andersen CR, Cobbett CS (1995) Cadmium-sensitive, cad1mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107:1059–1066.

4. Szczypka MS, Wemmie JA, Moye-Rowley WS, Thiele DJ (1994) A yeast metal resistanceprotein similar to human cystic fibrosis transmembrane conductance regulator (CFTR)and multidrug resistance-associated protein. J Biol Chem 269:22853–22857.

5. Geisler M, et al. (2004) Arabidopsis immunophilin-like TWD1 functionally interactswith vacuolar ABC transporters. Mol Biol Cell 15:3393–3405.

6. Lu YP, et al. (1998) AtMRP2, an Arabidopsis ATP binding cassette transporter able totransport glutathione S-conjugates and chlorophyll catabolites: Functional compar-isons with Atmrp1. Plant Cell 10:267–282.

7. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cellstreated with alkali cations. J Bacteriol 153:163–168.

8. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743.

9. Chen A, Komives EA, Schroeder JI (2006) An improved grafting technique for matureArabidopsis plants demonstrates long-distance shoot-to-root transport of phyto-chelatins in Arabidopsis. Plant Physiol 141:108–120.

10. Saavedra E, Encalada R, Pineda E, Jasso-Chavez R, Moreno-Sanchez R (2005) Glycolysisin Entamoeba histolytica. Biochemical characterization of recombinant glycolyticenzymes and flux control analysis. FEBS J 272:1767–1783.

11. Mendoza-Cozatl DG, et al. (2008) Identification of high levels of phytochelatins,glutathione and cadmium in the phloem sap ofBrassica napus. A role for thiol-peptidesin the long-distance transport of cadmium and the effect of cadmium on iron trans-location. Plant J 54:249–259.

12. Tommasini R, et al. (1996) The human multidrug resistance-associated protein func-tionally complements the yeast cadmium resistance factor 1. Proc Natl Acad Sci USA 93:6743–6748.

13. Song WY, et al. (2003) Engineering tolerance and accumulation of lead and cadmiumin transgenic plants. Nat Biotechnol 21:914–919.

14. FrangneN, et al. (2002) Flavone glucoside uptake into barleymesophyll andArabidopsiscell culture vacuoles. Energization occurs by H(+)-antiport and ATP-binding cassette-type mechanisms. Plant Physiol 128:726–733.

15. Sung DY, Kim TH, Komives EA, Mendoza-Cozatl DG, Schroeder JI (2009) ARS5 isa component of the 26S proteasome complex, and negatively regulates thiolbiosynthesis and arsenic tolerance in Arabidopsis. Plant J 59:802–813.

16. Vande Weghe JG, Ow DW (2001) Accumulation of metal-binding peptides in fissionyeast requires hmt2+. Mol Microbiol 42:29–36.

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wt abcc1 abcc2 abcc3 abcc4 wt abcc5 abcc6 abcc7 abcc8 wt abcc9

DSMA 125 mg/L

DSMA 150 mg/L

½ MS

abcc10 abcc11 abcc12 wt wt abcc13 abcc14 abcc15

DSMA 125 mg/L

DSMA 150 mg/L

½ MS

Fig. S1. Arsenic herbicide sensitivity test for knockout mutants of all 15 members of the Arabidopsis ABCC/MRP subfamily. Arrows indicate the atabcc1 andatabcc2 mutants, whose growth was reduced compared with wild type. Mutant and wild-type plants were grown in half-strength MS (1/2 MS) media in theabsence or presence of 125 or 150 mg/L DSMA for 2 wk. (Scale bar, 1 cm; applies to all panels.)

WT 3abcc1 abcc2 cad1-

As 30 µM

As 10 µM

½ MS

Fig. S2. atabcc1 atabcc2 double knockout mutant is as sensitive to arsenic as the phytochelatin synthesis-deficient cad1-3 mutant. Wild-type, atabcc1 atabcc2double knockout (abcc1 abcc2), and AtPCS1 mutant (cad1-3) plants were grown in 10 or 30 μM of As(V) containing half-strength MS (1/2 MS) media for 13 d.(Scale bar, 1 cm.)

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Fig. S3. Phytochelatin levels in AtABCC1- and AtABCC2-expressing SM7 yeast strains. Yeast strains expressing phytochelatin synthesis (SM7) either alone(empty vector, EV) or together with AtABCC1 and AtABCC2, respectively, were grown in SD-Ura supplemented with 0.1 mM CuSO4 to an OD 600 nm 1.0 andthen exposed to 100 μM arsenic for 20 h. Phytochelatin content was measured in cell extracts by HPLC. Mean ± SEM (from three independent experiments).

Fig. S4. ApoPC2 and As(III)–PC2 transport in AtABCC2-expressing yeast vesicle (A) and wild-type Arabidopsis vacuoles (B) is inhibited by vanadate but not byNH4

+. (A) Inhibition of AtABCC2-mediated apoPC2 transport by vanadate treatment. Experimental procedures were the same as described for Fig. 3B (maintext), except that the apoPC2 concentration was 250 μM. (B) Inhibition of As(III)–PC2 transport in Arabidopsis vacuoles. Experimental procedures were the sameas described for Fig. 4 (main text). Mean ± SEM (from two independent experiments with four replicates each).

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A BShoot Root

DCcinopordyHlatoT

E P contents

Fig. S5. Arsenic accumulation was decreased in shoots and increased in roots of atabcc1 atabcc2 knockout mutants. (A–C) Arsenic contents of shoots (A) androots (B) were measured in wild-type (WT) and atabcc1 atabcc2 double knockout (abcc1 abcc2) seedlings. Arabidopsis plants were grown in half-strength MSagar plates for 2 wk and treated with 20 μM As(V) for 3, 5, or 10 d. Total arsenic contents per each plant are shown in C. (D and E) Arsenic (D) and phosphorus(E) contents of wild-type and the double mutant plants, which were grown hydroponically and treated with 5 μM As(V) for 5 d. Mean ± SEM (n = 5–10, fromthree independent experiments for A–C; n = 3–5 for D and E).

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A WT PCS2 PCS3WT PCS2 PCS3

WT PCS6 PCS7

PCS4 PCS5 PCS4 PCS5

WT PCS6 PCS7

As 70 µM ½ MS

B

Fig. S7. Phenotype of AtPCS1-overexpressing plants in As(V)-containing media. (A) Six lines of AtPCS1-overexpressing plants and the wild type were grown for3 wk in the presence or absence of 70 μM of As(V). (Scale bar = 1 cm.) (B) Transcript level of AtPCS1 was detected in wild-type and AtPCS1-overexpressing plantsby quantitative RT-PCR analyses.

WT WT

A

As 70 µM

M-1 M-2

½ MS

M-1 M-2

B

Fig. S6. Overexpression of AtABCC1 alone does not increase tolerance to arsenic. (A) Two AtABCC1-overexpressing lines (M-1 and M-2) show similar arsenicresistance to wild-type plants (WT). Plants were grown in half-strength MS media with or without As 70 μM for 14 d. (B) Transcript level of AtABCC1 wasdetected in AtABCC1-overexpressing lines by quantitative RT-PCR analyses. Total RNA was isolated from shoots and roots of 2-wk-old seedlings. Data werenormalized using Tubulin8. Mean ± SD. (Scale bar, 1 cm.)

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A

ABCC1

WTabcc1-3

abcc2-2

abcc1abcc2

B

ABCC2

ACT

Fig. S8. Transcript levels of AtABCC1 and AtABCC2 in single and double knockout plants of AtABCC1 and AtABCC2. (A) AtABCC1 or AtABCC2 transcripts werenot detectable in atabcc1-3 or atabcc2-2 knockout plants, respectively. Neither of the transcripts was detectable in atabcc1 atabcc2 double knockout plants.AtABCC1-s and AtABCC1-as primers were used to amplify AtABCC1 transcripts, and AtABCC2RT-F and AtABCC2RT-R primers were used for AtABCC2 transcriptanalysis. Actin was used as a loading control. (B) Expression of AtABCC1 and AtABCC2 is not induced by arsenate exposure or by the absence of AtABCC1 orAtABCC2. Transcript levels of AtABCC1 or AtABCC2 were determined by quantitative RT-PCR analyses using total RNA isolated from the wild-type plantstreated with or without 180 μM of As(V) for 24 h, and from atabcc1 or atabcc2 knockout plants. Data were normalized using transcript level of Tubulin8 andpresented as fold changes to the wild-type value. Mean ± SEM (from two independent experiments with two replicates each).

Table S1. Arabidopsis ABCC/MRP knockout mutants used in thisstudy

Name Accession no. Salk no. Reference

abcc1-3 At1g30400 Salk_017431 This studyabcc2-2 At2g34660 Salk_127425 (1)abcc3 At3g13080 WiscDsLox481-484C11 This studyabcc4 At2g47800 Salk_093541 This studyabcc5 At1g04120 Salk_002438 This studyabcc6 At3g13090 Salk_110544 This studyabcc7 At3g13100 Salk_068478 This studyabcc8 At3g21250 Salk_110195 This studyabcc9 At3g60160 Salk_087700 This studyabcc10 At3g59140 Salk_148625 This studyabcc11-1 At1g30420 Salk_137031 (1)abcc12-2 At1g30410 Salk_057394 (1)abcc13 At2g07680 Salk_044759 This studyabcc14 At3g62700 Salk_091563 This studyabcc15 At3g60970 Salk_123843 This study

1. Frelet-Barrand A, et al. (2008) Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport andchlorophyll degradation. Plant Cell Physiol 49:557–569.

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Table S2. Yeast strains and primers used in this study

Strain or primer Genotype or sequence Reference

DTY167 MATa ura3 leu2 his3 trp3 lys2 suc2 ycf::hisG (1)YMM31 MATa, ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 ycf1:: HIS3-MX6 From Dr. Karl KuchlerY11503 Matα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YLL015w::kanMX4 EUROSCARFYMM34 MATα ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 yll048c:: TRP1 From Dr. Karl KuchlerSM4 ycf1:: His3, yhl035c::HIS3-MX6, yll015w::Kan-MX6, yll048c:: TRP1-MX6 This studySM7 ycf1:: His3, yhl035c::HIS3-MX6, yll015w::Kan-MX6, yll048c:: TRP1-MX6, TaPCS1:: cup1-1 This studyAtABCC1-s CCGCAGAAATCCTCTTGGTCTTGATG This studyAtABCC1-as GTGAATCATCACCGTTAGCTTCTCTGG This studyAtABCC1,2F ATCCGTGAACTCCGTTGGTATGTC This studyAtABCC1,2R ACCACATGGTATGAAGTGATTGGC This studyAtABCC2RT-F AGCGTGCCAAAGATGACTCACACCAC This studyAtABCC2RT-R TACTTATCACGAAGAACACAACAGGG This studyHis3MX6-YHL035F GGGAACGGATCCCCTTATTATCCGAAATAATGGTTCATTTTGGGAAATCTGTTTAGCTTGC This studyHis3MX6-YHL035R GCTCTAGGCCCCCACTGTCACGACACATACTATAAAATATACCGCGTTTCTGCGCACTTAAC This studyYLL015F ACCAAAACCAAGAAAGAAAGTCGTTCC This studyYLL015R TGACTGAGAATACTGTTGTCATGTC This studyYLL048CF AAAACTGCTCCTGATCTCAAGCCAC This studyYLL048CR AATGTTCTCTCGAGCTTCCAAGGCC This studyTaPCS1:: cup1-1F ATAGATATTAAGAAAAACAAACTGTACAATCAATCAATCAATCATCACATAAAatggaggtggcgtcgctg This studyTaPCS1:: cup1-1R TTTAAAAATTAAAAACAGCAAATAGTTAGATGAATATATTAAAGACTATTCGTTTCAagtgagcagattgtcgcag This studyAtPCS1_F2 GGAAGCCATGGACAGTATTG (2)AtPCS1_R2 TTCTCCTCTGCGCTGAGATT (2)Tubulin8-F CTCACAGTCCCGGAGCTGACAC This studyTubulin8-R GCTTCAGTGAACTCCATCTCGT This study

1. Szczypka MS, Wemmie JA, Moye-Rowley WS, Thiele DJ (1994) A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) andmultidrug resistance-associated protein. J Biol Chem 269:22853–22857.

2. Blum R, et al. (2007) Function of phytochelatin synthase in catabolism of glutathione-conjugates. Plant J 49:740–749.

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