Arsenic Tolerance in Arabidopsis Is Mediated by Two ABCC-Type Phytochelatin Transporters

Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters

Won-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, World Class 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; dInstitute of Plant Sciences, University of Bern, 3013 Bern, Switzerland; and ePlant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018

Edited by Maarten Koornneef, The Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved October 19, 2010 (received for review September 20, 2010) Arsenic is an extremely toxic metalloid causing serious health pro- million people are estimated to face a risk of death from cancer blems. In Southeast Asia, aquifers providing drinking and agricul- caused by arsenic (3–5). tural water for tens of millions of people are contaminated with To reduce nutritional arsenic intake, identification of the arsenic. To reduce nutritional arsenic intake through the consump- mechanisms implicated in arsenic accumulation and detoxification tion of contaminated plants, identification of the mechanisms for of plants is a prerequisite. Considerable progress has been made arsenic accumulation and detoxification in plants is a prerequisite. during the last years in the identification of the molecular mecha- Phytochelatins (PCs) are glutathione-derived peptides that chelate nisms of arsenic (As) uptake, metabolism, and translocation (for heavy metals and metalloids such as arsenic, thereby functioning as reviews see refs. 6–8). After entering into roots through high- the first step in their detoxification. Plant vacuoles act as final detox- affinity phosphate transporters, arsenate [As(V)] is readily reduced ification stores for heavy metals and arsenic. The essential PC–metal to arsenite [As(III)]. Alternatively, under reducing conditions, As (loid) transporters that sequester toxic metal(loid)s in plant vacuoles (III) is taken up by membrane proteins belonging to the aquaporin have long been sought but remain unidentified in plants. Here we family (9, 10). In rice, it has been shown that As(III) is exported PLANT BIOLOGY show that in the absence of two ABCC-type transporters, AtABCC1 subsequently to the xylem by the silicon efflux transporter Lsi2, and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic resulting in root-to-shoot transport of arsenic (11). The final step of and arsenic-based herbicides. Heterologous expression of these arsenic detoxification in the cell is the sequestration into vacuoles, ABCC transporters in phytochelatin-producing Saccharomyces cere- which depends mainly on glutathione (GSH) and phytochelatins visiae enhanced arsenic tolerance and accumulation. Furthermore, (PCs). PCs are GSH-derived metal-binding peptides whose syn- membrane vesicles isolated from these yeasts exhibited a pro- thesis is induced by arsenic as well as other toxic metals (12, 13). nounced arsenite [As(III)]–PC2 transport activity. Vacuoles isolated PCs chelate As(III) and heavy metals with their thiol groups, and from atabcc1 atabcc2 double knockout plants exhibited a very low the metal(loid)–PC complexes are sequestered into vacuoles, pro- residual As(III)–PC2 transport activity, and interestingly, less PC was tecting cellular components from the reactive metal(loid)s. produced in mutant plants when exposed to arsenic. Overexpres- PC-mediated detoxification is unique to plants and a few other sion of AtPCS1 and AtABCC1 resulted in plants exhibiting increased PC-producing organisms but different from that of budding yeast arsenic tolerance. Our findings demonstrate that AtABCC1 and or humans, in which PCs or PC synthase (PCS) do not exist. The AtABCC2 are the long-sought and major vacuolar PC transporters. transporters responsible for active transport of PC-conjugated Modulation of vacuolar PC transporters in other plants may allow As(III) as well as for transport of PC-conjugated heavy metals engineering of plants suited either for phytoremediation or reduced into plant vacuoles (14–17) are yet to be identified but have been accumulation of arsenic in edible organs. proposed to be ABC transporters, as is the case with glutathione (GS)-conjugated As(III) (As(GS)3) in other organisms (18, 19). ABC transporter | vacuolar sequestration | multidrug resistance-associated For example, in Saccharomyces cerevisiae it has been shown that protein arsenic is detoxified by YCF1, an ABC protein transporting As (GS)3 into vacuoles (18). In humans, it has been shown that fi rsenic has been widely used in medicine, industry, and ag- HsABCC1 and HsABCC2 are involved in arsenic detoxi cation Ariculture. In medicine, it was used to treat diseases such as by transporting As(III) conjugated to GSH (19). Another ABC syphilis, trypanosomiasis, or amoebic dysentery. In agriculture, transporter, HMT1, has been proposed to act as a vacuolar PC Schizosaccharomyces pombe hmt1 arsenic-based herbicides, such as disodium methanearsonate transporter in , although an de- (DSMA), continue to be applied for weed and pest control. letion mutant continued to exhibit PC accumulation in vacuoles, However, arsenic is a highly toxic environmental pollutant that causes global health problems. In Southeast Asia as well as in regions with extensive mining, such as China, Thailand, and the 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; United States, arsenic concentrations in water can be far above and J.P., J.I.S., Y.L., and E.M. wrote the paper. the World Health Organization limit of 10 μg/L (133 nM), con- Conflict of interest statement: Y.L., E.M., J.I.S., W.-Y.S., J.P., and D.G.M.-C. have filed a centrations that are known to cause health problems. People are patent on the reduction of arsenic in crops and the use of ABCCs for phytoremediation exposed to arsenic poisoning both by drinking contaminated based on the discovery reported in the manuscript. water and by ingesting crops cultivated in soils irrigated with This article is a PNAS Direct Submission. polluted water. It has been shown that arsenic in rice paddy fields 1W.-Y.S., J.P., and D.G.M.-C. contributed equally to this work. is readily taken up by rice plants and translocated to the grains, 2J.I.S., Y.L., and E.M. contributed equally to this work. constituting a danger for populations that rely mostly on rice for 3To whom correspondence may be addressed. E-mail: [email protected], ylee@postech. their diet (1, 2). In Bangladesh alone an estimated 25 million ac.kr, or [email protected]. people are exposed to water contaminated with arsenic exceeding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 50 μg/L, the Bangladesh government standard, and more than 2 1073/pnas.1013964107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1013964107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 and HMT1 did not confer arsenic resistance (20, 21). In plants, no ortholog for HMT1 has been identified. Here we report the identification of two ABC transporters required for arsenic detoxification. These transporters are plant PC transporters that have been sought since the discovery of PCs (12, 22). Thus this finding provides a key to understanding the detoxification of many xenobiotic molecules, heavy metals, and metalloids, including arsenic, that are conjugated with PCs for detoxification. Results atabcc1 atabcc2 Double Knockout Mutant Is Hypersensitive to Arsenic and Arsenic-Based Herbicide. To identify candidate transporters likely involved in arsenic detoxification in plants, we focused on screening the ABCC subfamily of ABC transporters, also known as the multidrug resistance-associated proteins (MRPs). This subfamily includes ABC transporters implicated in heavy metal resistance, such as ScYCF1, the yeast vacuolar As(GS)3 transporter (18), and the two human arsenic-detoxifying ABC transporters (19). Members of this family have also been shown to transport GSH conjugates and to confer cadmium resistance in plants and humans (23–26). In addition, for many ABCC proteins in Arabidopsis, vacuolar localization has been demonstrated or proposed on the basis of data obtained using either Western blotting of membrane fractions, GFP fusion proteins, or vacuolar proteomics approaches (27–30). We have therefore isolated homozygous knockout lines for all 15 Arabidopsis ABCC genes and have grown these mutants in the presence of arsenic and arsenic- containing herbicides. Two different forms of arsenic were used because they have different entry pathways to the cell, as well as Fig. 1. Hypersensitivity of atabcc1 atabcc2 double knockout mutants to ar- differential metabolism. Whereas As(V) is taken up by the high- senic [As(V)] and DSMA, an arsenic-based herbicide. (A and B) Wild-type, affinity phosphate transporter (31, 32), DSMA is much more hy- AtABCC1 knockout (abcc1-3), AtABCC2 knockout (abcc2-2), and double drophobic and is rapidly absorbed by plant roots. Furthermore, As knockout (abcc1 abcc2) seedlings grown in 100 mg/L of DSMA (A) and 50 μM (V) is reduced to As(III) in the cell, whereas in DSMA arsenic is of As(V) (B) containing half-strength MS media for 16 d. Note that single already present in the As(III) form. Although no As(V)-sensitive knockout mutants of AtABCC1 or AtABCC2 are also sensitive to the arsenic atabcc knockout mutant was found, the growth of two deletion herbicide (A) but not to As(V) (B). (Scale bar, 1 cm.) (C and D) Fresh weight (C) atabcc1/atmrp1 atabcc2/atmrp2 and root length (D) of each genotype of plants grown on 0–50 μM As(V)- mutants, and , was impaired in the containing 1/2 MS media. Mean ± SEM (for fresh weights, n = 16, from four presence of the arsenic herbicide DSMA, compared with that of independent experiments; for root lengths, n = 332–336, from three in- the wild type (Fig. 1 and Fig. S1). To determine whether the rel- dependent experiments). *P < 0.04, **P < 0.01 (Student’s t test). (E) Arsenic- atively moderate arsenic sensitivity could be due to overlapping sensitive phenotype of atabcc1 atabcc2 double knockout plants grown in soil. functions of the two ABC transporters, we generated a double Three-week-old plants were watered with 133.5 μM As(V). Four representa- knockout atabcc1 atabcc2 mutant. AtABCC1 and AtABCC2, tive plants out of 12; each was photographed 8 d after arsenic treatment. which belong to clade I of the ABCC subfamily, share a high amino acid sequence similarity. Furthermore, both have been localized to – the vacuolar membrane in former studies (27, 28). Whereas under mentalization of a GS As complex into the vacuole, we expressed control conditions no difference in growth was observed between the transporters in SM4, a budding yeast mutant strain lacking YCF1 the wild type and the double knockout mutant, growth of the and the three other vacuolar ABCC-type ABC transporters, YHL035c YLL015w YLL048c AtABCC1 AtABCC2 atabcc1 atabcc2 double knockout line was dramatically impaired , , and . -or - on plates containing DSMA as well as low level of As(V) compared expressing SM4 yeast lines showed only a marginal increase in with the wild type (Fig. 1 A and B). Quantification of the fresh arsenic resistance, compared with the empty vector control (Fig. A Arabidopsis weight and root length of plants grown in As(V)-containing media 2 ). These results indicate that in contrast to , confirmed that a significant difference could only be observed for AtABCC1 and AtABCC2 cannot mediate arsenic tolerance in the double knockout, which exhibited a strongly reduced fresh budding yeast, which does not synthesize PCs. These experiments weight even at low As(V) concentrations (Fig. 1 C and D). In the also suggest that AtABCC1 and AtABCC2 are not efficient GS– presence of 30 μM As(V), root growth was reduced by more than As complex transporters and thus cannot complement ycf1. 60%. The sensitivity of the atabcc1 atabcc2 knockout line was In contrast to S. cerevisiae and humans, which depend on GSH comparable to that observed for cad1-3,anArabidopsis mutant for arsenic detoxification, in plants detoxification of arsenic has impaired in PC synthesis (Fig. S2) (33). To verify whether been shown to mainly depend on PCs (8, 16, 33, 34). We there- AtABCC1 and AtABCC2 confer arsenic tolerance under natural fore tested whether AtABCC1 and AtABCC2 confer arsenic conditions, we grew atabcc1 and atabcc2 single and double resistance by transporting PC–As complexes. As a first approach, knockout lines, and the corresponding wild type on soil for 3 wk we generated a yeast mutant expressing the wheat PCS TaPCS1 and then irrigated the plants with 133.5 μM As(V) for an additional (35, 36) in the SM4 background and named it SM7. This yeast 8 d. The atabcc1 atabcc2 double mutants were extremely sensitive strain was then used to express either AtABCC1 or AtABCC2. to As(V) and did not survive this treatment, whereas the wild-type When grown on arsenic-free control medium, no large difference and single knockout plants were not visibly affected (Fig. 1E). could be observed between the SM7 cells transformed with the empty vector or expressing either of the ABC transporters. AtABCC1 and AtABCC2 Enhance Arsenic Tolerance and Accumulation However, when the yeast cells were exposed to 100 μM As(III), in the Presence of TaPCS1 in Budding Yeasts. To test whether the lines expressing AtABCC1 or AtABCC2 grew dramatically AtABCC1 and AtABCC2 confer arsenic resistance by compart- better than controls (Fig. 2A). In this condition, yeast cells

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1013964107 Song et al. Downloaded by guest on September 29, 2021 Together, these results indicate that AtABCC1 and AtABCC2 can mediate vacuolar PC–As sequestration, allowing the PC- producing yeast strains to cope with arsenic. These heterologous reconstruction assays also suggest that chelation of arsenic by PC alone cannot detoxify arsenic, but transporters of PC–As complex are required, because expression of PCS in the SM4 background (SM7) did not increase arsenic tolerance (Fig. 2A).

AtABCC1 and AtABCC2 Mediate PC Transport. To directly determine whether AtABCC1 and AtABCC2 mediate PC–As transport, we performed transport experiments using microsomal vesicles isolated from SM7 yeast transformed with the empty vector or expressing AtABCC1 and AtABCC2. As shown in Fig. 3A, vesicles isolated 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 the uptake 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 presence of 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 more efﬁciently than apoPC2 by both AtABCC1 and AtABCC2 (Fig. 3B). The ratio of the transport activity between apoPC2 and As Fig. 2. AtABCC1 and AtABCC2 enhance arsenic tolerance and accumulation – in PCS-expressing budding yeasts. (A) Expression of AtABCC1 (ABCC1) or (III) PC2 varied between the different batches of PC2 produced, AtABCC2 (ABCC2) in PC-producing yeast (SM7) enhanced tolerance to 100 μM probably owing to variable stability of the PC2–As complex. of As(III). EV, empty vector control. In SM4 strain without PCS expression, no The discrimination between apoPC2 and As(III)–PC2 was more growth difference between genotypes was apparent. Cells were grown in

pronounced for AtABCC2 than for AtABCC1. As shown for As PLANT BIOLOGY half-strength SD-Ura (synthetic dextrose lacking uracil) media supplemented (III)–PC2, apoPC2 uptake was also inhibited by vanadate but not with 100 μM As(III) for 2 d. O.D. , optical density at 600 nm of the yeast + 600 by NH4 (Fig. S4A). We further tested whether AtABCC2 can suspension that was initially spotted on the plates. (B) Arsenic content of SM7 transport arsenic in a non-chelated form using microsomal yeast cells expressing AtABCC1 or AtABCC2 was higher than that of cells vesicles of the SM7 cells expressing the transporter. Arsenic transformed with the empty vector. Cells were grown in half-strength SD-Ura media supplemented with 100 μM As(III) for 12 h. Mean ± SEM (n = 21, from content measured using inductively coupled plasma mass spec- seven independent experiments). *P < 0.03, **P < 0.005 (Student’s t test). trometry (ICP-MS) showed that without PCs no arsenic was transported via AtABCC2 (Fig. 3C). Furthermore, by comparison of concentrations of PC2 and arsenic transported into these vesi- expressing the ABC transporters accumulated more arsenic than cles, we could estimate that AtABCC2 mediates the transport of the corresponding control (Fig. 2B). Furthermore, in the presence two molecules of PC2 per molecule of arsenic (Fig. 3C). of arsenic, AtABCC1- and AtABCC2-expressing SM7 strains con- Surprisingly, the concentration-dependent As(III)–PC2 uptake tained much higher PC levels compared with SM7 strains trans- did not follow classic saturation kinetics, but exhibited an ap- formed with the empty vector (Fig. S3), indicating that efﬁcient parent sigmoid curve for both AtABCC1 and AtABCC2 (Fig. PC synthesis is only achieved when PCs are transported into 3D). At PC2 concentrations below 10 μM, As(III)–PC2 uptake the vacuole. activity was negligible but was strongly stimulated by increasing

Fig. 3. AtABCC1 and AtABCC2 mediate PC transport in microsomes isolated from yeast transformed with AtABCC1 and AtABCC2 transporters. (A) Time-dependent uptake of

PC2–As in yeast microsomes isolated from S. cerevisiae transformed with the empty vector (EV), AtABCC1,and AtABCC2. PC concentration was 25 μM. (B) Comparison of

transport activities for apoPC2 and PC2–As, and inhibition of + PC2–As transport by NH4 and vanadate. PC concentration was 25 μM. (C) Relationship between arsenic (Left) and PC

(Right) transport activity. PC2 (500 μM) with 1 mM As(III) or 1 mM As(III) alone was used, and the concentrations of

transported PC2 and arsenic were measured. (D) Concen- tration dependence of PC2–As (solid line) and apoPC2 (dotted line) transport activity of vesicles isolated from AtABCC1 (ﬁlled squares) and AtABCC2 (ﬁlled triangles) expressing SM7 S. cerevisiae. Inset: Enlarged version at low concentrations of the substrate. Mean ± SEM (from three independent experiments with four replicates each).

Song et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 PC concentrations. The PC concentration for half-maximal up- PC contents, integrated over 2 d of continuous arsenic expo- take activity was estimated to be 350 μM. This high concentra- sure, were reduced in atabcc1 atabcc2 double knockout plants, tion suggests a rather low affinity of AtABCC1 and AtABCC2 when total plant PC contents, reflecting the vacuolar and cyto- for PCs. Previous reports demonstrated that PCs can accumulate plasmic pools, were measured (Fig. 4B). The difference between to concentrations exceeding 1,000 μM under metal(loid) stress wild-type plants and the mutants become more pronounced at (37), in good agreement with the concentrations that exhibited high arsenic concentrations, which increased production of PCs. optimal uptake in these experiments (Fig. 3D). Interestingly, the This observation is in line with the sigmoid kinetics of the vacuolar concentration dependence for apoPC2 was linear and did not transport observed in vesicles isolated from the yeasts expressing reveal a saturation curve up to 1,000 μM apoPC2 (Fig. 3D). the two AtABCC transporters and again supports the necessity of transporting PCs into the vacuole for continuous PC synthesis. – AtABCC1 and AtABCC2 Are the Major As(III) PC2 Transporters in Plant Surprisingly, double mutant plants took up slightly more arsenic, Vacuoles. To determine whether AtABCC1 and AtABCC2 indeed but the transfer of arsenic to the shoot was reduced in these plants encode the long-sought functional PC transporters in planta and to (Fig. S5). The same result was obtained in plants grown on plates determine their contribution to vacuolar PC uptake, we compared as well as under hydroponic conditions. the transport activities for As(III)–PC2 and apoPC2 of intact vacuoles isolated from the atabcc1 atabcc2 double knockout Overexpression of AtABCC1 Increases Arsenic Tolerance Only When and wild-type plants. As shown in Fig. 4A, vacuolar uptake activ- Coexpressed with PCS. To determine whether arsenic tolerance can ities of both As(III)–PC2 and apoPC2 were dramatically reduced be increased by expressing one of the PC transporters, we trans- by 85% and 95%, respectively, in the mutant plant, indicating formed wild-type plants with a construct in which AtABCC1 is that these two ABC transporters are by far the most important PC driven by the 35S promoter. We could identify only plants in which and PC-conjugate transporters in Arabidopsis. AtABCC11 and the transcript levels were maximally increased by 20–40%. How- AtABCC12, very close homologs of AtABCC1 and AtABCC2, ever, these lines did not confer increased arsenic tolerance (Fig. S6), could be responsible for the very low residual PC uptake activity. which may be explained by the circumstances that the over- Comparison of the transport rates of apoPC2 and As(III)–PC2 in expression was not strong enough or the PC transport activity was vacuoles isolated from wild-type Arabidopsis showed that the dif- not the limiting factor in conferring arsenic tolerance. Therefore, we ferences in transport rates for these two forms of PC were less generated AtPCS1 single and AtPCS1 AtABCC1 co-overexpressing pronounced in Arabidopsis vacuoles than in yeast vesicles. This lines. No differences in arsenic tolerance could be detected between might be due to the more than twofold higher expression level of the wild type and seven AtPCS1 single overexpressing lines, despite AtABCC1 compared with AtABCC2 in fully expanded Arabidopsis transcript levels of AtPCS1 exceeding that of the wild type by leaves, the tissue used for vacuole isolation (Genenvestigator or 860–3,260% in those lines (Fig. S7). In contrast, the two plants co- http://www.bar.utoronto.ca/). In yeast experiments, the selectivity of transformed with AtPCS1 and AtABCC1 (MP-1 and MP-2) – AtABCC1 for As(III) PC2 was much lower than that of AtABCC2. exhibited a consistent increase in arsenic tolerance (Fig. 5). Besides An alternative explanation could be that the different lipid envi- the less-chlorotic phenotype, shoot fresh weight was also increased ronment in plant and yeast vesicles also affects the selectivity. As in the plants overexpressing both genes. When exposed to 70 μMAs observed for yeasts, vacuolar PC transport in Arabidopsis was not (V), the shoot weight of these two lines corresponds to 161% and + ± inhibited by NH4 (residual transport activity 98% 4%) but 147% of those of the wild-type and AtPCS1-overexpressing plants strongly inhibited by vanadate (residual transport activity 25% ± 7%) (Fig. 5C). Even after normalization with the growth in control me- (Fig. S4B), which are typical features of ABC transporters. dium, to take into account the slightly better growth of the double transformant in normal medium, the two AtPCS1 AtABCC1 double transformants exhibited an increased arsenic tolerance (124% and 114% of the wild type, respectively). These results show that coex- pression of an ABC-type PC transporter and PCS can result in plants with enhanced arsenic tolerance. Discussion Cooperation of PC Transport and Synthesis in Arsenic Detoxification. Previous studies have demonstrated that PCs are required for arsenic tolerance in higher plants (33, 36). PC-conjugated substrates have been suggested to be sequestered into vacuoles. However, until now no plant transporter for PCs has been identified. Our results demonstrate that AtABCC1 and AtABCC2 are both vacuolar PC transporters required for arsenic resistance in A. thaliana. The similar phenotypes of the atabcc1 atabcc2 double knockout and the cad1 mutant, which lacks PC synthesis, suggest that the sequestration of PC–As complexes into vacuoles is as critical for the detoxification of arsenic as the synthesis of PCs. A similar mechanism may work for other metal(loid)s, or alternatively, they may form bis–GS complex and be sequestered for detoxification. Fig. 4. Vacuoles isolated from the atabcc1 atabcc2 double knockout exhibit The main transporters for the GS-metal(loid) complexes in plants only a very low residual PC transport activity, and atabcc1 atabcc2 double remain to be identified. knockouts show decreased content of PCs at the seedling stage. (A) Vacuoles The AtABCC1 and AtABCC2 transporters and PCS may were isolated from wild-type and the atabcc1 atabcc2 double knockout plants function in a concerted way in the arsenic detoxification pathway. – and tested for transport activities for PC2 As and apoPC2. As a negative con- The PCS enzyme, although present even when plants are not ex- trol, ATP was replaced by ADP. PC concentration was 250 μM. Mean ± SEM (from two independent experiments with four replicates each). (B)Twelve- posed to heavy metals or metalloids, remains inactive under nor- day-old seedlings were exposed to different concentrations of potassium ar- mal conditions and is rapidly activated when plants take up arsenic senate for 96 h, and the integrated accumulation of PCs was determined by or other toxic metal(loid)s (12, 38). Similarly, expression of HPLC-MS. Mean of two replicates (0 μM of arsenic) or three replicates ± SEM AtABCC1 and AtABCC2 is not induced by arsenic in Arabidopsis, (50 and 100 μMofarsenic). nor does the absence of one of the ABCCs result in increased

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1013964107 Song et al. Downloaded by guest on September 29, 2021 Fig. 5. Co-overexpression of PC synthase (AtPCS1)and AtABCC1 results in increased arsenic tolerance. (A) Wild-type plants, plants of a representative AtPCS1-overexpressing line (PCS1), and plants overexpressing AtPCS1 and AtABCC1 (MP-1 and MP-2) grown either on 110 μM As(V)-containing or control half-strength MS plates. (B) Transcript levels of AtABCC1 and AtPCS1 in the plants used in A, determined by qPCR. (C) Shoot fresh weight of wild-type and transgenic plants grown under control conditions or in the presence of 70 μM As(V). Mean ± SEM (from two independent experiments with four replicates each). *P < 0.02 by Student’s t test.

transcription of the other (Fig. S8B) (39). AtABCC1 and may therefore enable future engineering of reduction in the ac- AtABCC2 are not synthesized de novo but constitutively present in cumulation of arsenic in edible organs of plants. A similar case PLANT BIOLOGY a plant cell to rapidly respond to toxic metal(loid)s and xenobiotic was reported recently for reduction of cadmium content in rice stresses in a way similar to PCS1. Furthermore, the synthesis of grain by overexpression of OsHMA3, a P-type ATPase (42). PCs seems to be positively correlated with the presence of the PC It will also be interesting to learn whether these two different transporters (Fig. 4B and Fig. S3). In both yeasts and plants, PCs transport systems can give a synergistic effect. are increased when cells also express PC transporters. This observation suggests that PC synthesis undergoes a feedback regu- Biochemical Characteristics of AtABCC1 and AtABCC2. Both AtABCC1 – B lation and that removal of PCs from the cytosol leads to an and AtABCC2 transported apoPC2 as well as As(III) PC2 (Fig. 3 ), – fi increase of its synthesis. A puzzling result was the observation in although As(III) PC2 was the substrate more ef ciently transported. the double knockout that arsenic concentration was higher in The transport of apoPC2 by the ABC transporters may supply the roots and that the root-to-shoot transfer was diminished (Fig. S5), vacuolar apoPC2 required for stabilization of toxic metal(loid)s which is in contrast to the observations made in plants lacking GSH transported into the vacuole by other transporters that carry un- bound forms of metals and metalloids. Alternatively, or additionally, or PCs (34). This cannot be due to an alternative PC transporter, – because the atabcc1 atabcc2 double mutant is very susceptible to apoPC2 might also be used to produce high-molecular-weight thiol atabcc1 atabcc2 As complexes, which possess high binding capacity for arsenic. arsenic exposure. Even at lower concentrations the – knockout plants still produce PCs (Fig. 4B); however, they accu- Kinetic studies of the As(III) PC2 uptake mediated by AtABCC1 and AtABCC2 revealed that the transport activity was low at low mulate these peptides in the cytosol. As(V) exhibits a complex me- concentrations of As(III)–PC and increased steadily as the sub- tabolism in plants, and apparently accumulation of PC–As com- 2 strate concentration was raised (Fig. 3D). The minimal transport plexes in the cytosol induces mechanisms that reduce the transfer activity at low PC concentrations may allow detoxification of toxic of arsenic to the shoot, either by inducing genes that may be re- metals more efficiently. PCs are synthesized in response to the sponsible for the translocation of phosphate from the root to the presence of arsenic or heavy metals by activation of PCS (12, 38). shoot or by inducing a toxicity response that has the same effect. atabcc1 atabcc2 It is likely that at the beginning of this reaction, PCs are accumu- Consistent with this possibility, plants grown hy- lated, which subsequently complex arsenic or other heavy metals. droponically in the presence of 5 μM As(V) were reduced in fi P If apoPC was ef ciently transported into the vacuole even at very phosphate contents by 16% ( = 0.05) in shoots, whereas in roots low concentrations, fewer PCs would be available in the cytosol for their phosphate content was similar to that of the wild type (Fig. S5). immediate chelation of arsenic when plants are exposed to arse- Under our experimental conditions, overexpressing AtPCS1 nic. However, after As(III)–PC2 is accumulated in the cytosol, it alone did not result in an increased arsenic tolerance (Fig. S7). has presumably to be transported rapidly into the vacuole to avoid Inconsistent results have been reported using a similar approach. inhibition of PC synthesis by a feedback regulation mechanism Li et al. (40) showed a pronounced increase when AtPCS1 was (Fig. 4B and Fig. S3). Biochemically, the sigmoidal concentration expressed in Arabidopsis, whereas Guo et al. (41) could only ob- dependence indicates allosteric regulation by multiple substrate serve an increased tolerance when both AsPCS1 and ScGSH1 binding sites or interaction with other proteins. were coexpressed, indicating that thus-far-unknown parameters In addition to their central role as PC transporters, AtABCC1 may play a role when PCS1s are overexpressed in Arabidopsis.We and AtABCC2 have been reported to be involved in a broad range also could not observe any increase in tolerance when AtABCC1 of detoxification processes. Both proteins transport GS conjugates was overexpressed alone. Only transgenic plants overexpressing (23, 26), which are formed by the attack of the GS thiolate anion to both AtPCS1 and AtABCC1 exhibited an increased arsenic tol- an electrophilic and potentially toxic substrate. Furthermore, erance. Therefore, it has to be assumed that efficient increase in AtABCC2 has been shown to transport chlorophyll catabolites, arsenic tolerance requires both PCS and the PC transporter. which are potentially toxic, because they can absorb light Modification of ABCC transporter and PCS expression in plants and produce harmful reactive oxygen species (43). This versatile

Song et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 and important role in plant detoxiﬁcation requires that AtABCC1 Transport rates were calculated by subtracting the radioactivity taken up and AtABCC2 are expressed in all tissues in a constitutive way. after 20 min from that after 2.5 min incubation. For other methods, see SI Materials and Methods. Materials and Methods ﬁ Plant Materials, Growth Conditions, and Arsenical Treatments. Arabidopsis Note Added in Proof. A report describing the independent identi cation of seeds of wild-type and transgenic plants (ecotype Columbia-0) were grown the related Abc2 as a vacuolar phytochelatin uptake transporter in S. pombe on half-strength Murashige-Skoog (MS) agar plates with 1.5% sucrose in the is in press (46).

absence or presence of the indicated concentrations of As(V) (Na2HAsO4)or DSMA, an arsenic herbicide (Supleco), in a controlled environment with ACKNOWLEDGMENTS. We thank the Salk Genomic Analysis Laboratory for a 16-h light/8-h dark cycle at 22 °C/18 °C for the indicated times. All ABCC/ providing the Arabidopsis mutants; Dr. Markus Klein for the abcc mutant MRP knockout mutants were obtained from the Salk Genomic Analysis seeds; Dr. Christopher Cobbett (University of Melbourne, Victoria, Australia) for cad1-3 mutant seeds; Dr. Karl Kuchler (University of Vienna, Vienna, Laboratory, except abcc2-2, abcc11-1, and abcc12-2, which were provided by Austria) for YMM31 and YMM34 yeast strains; Dr. David Somers for his Dr. Markus Klein. (Philip Morris International, Switzerland). critical reading of the manuscript; and the Korea Forest Research Institute for kindly allowing the use of their ICP-MS spectrometer. This work was Isolation of Intact Vacuoles from Arabidopsis Protoplasts and Transport Assays. supported by the Global Research Laboratory program of the Ministry of Vacuoles were prepared from Arabidopsis mesophyll protoplasts as de- Education, Science and Technology of Korea Grant K20607000006 (to Y.L. scribed previously (44, 45). Transport studies with Arabidopsis mesophyll and E.M.), by the European Union project PHIME (Public health aspects of vacuoles were performed as described for barley (44, 45) but using silicone long-term, low-level mixed element exposure in susceptible population strata) Contract FOOD-CT-2006-0016253 and the Swiss National Foundation oil poly(dimethylsiloxane-co-methylphenylsiloxane) 550 (Aldrich) instead μ μ (E.M. and M.G.), by National Institute of Environmental Health Sciences of AR200. The assay for apoPC2 contained 200 MPC2 and 200 M DTT; that Grant P42 ES010337 (to J.I.S.), and by the Division of Chemical, Geo and – μ μ μ 35 for As(III) PC2 contained 200 MPC2, 400 M As(III), and 200 M DTT. S- Biosciences at the Ofﬁce of Energy Biosciences of the Department of Energy 3 Labeled PC2 [50 nCi (1,840 Bq)] and H2O [50 nCi (1,840 Bq)] were used as Grant DE-FG02-03ER15449 (to J.I.S.). D.G.M.-C. is recipient of a Pew Latin 3 tracer in every assay. The volume of vacuoles was determined using H2O. American Fellowship.

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