ABCC1)

FEBS Letters 580 (2006) 6891–6897

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1) Landry Gayeta,1, Nathalie Picaulta,1,2, Anne-Claire Cazale´b, Audrey Beylyb, Philippe Lucasa, He´le`ne Jacqueta, Henri-Pierre Susoa,3, Alain Vavasseura, Gilles Peltierb, Cyrille Forestiera,* a

CEA Cadarache, DSV-DEVM – LEMS, UMR 6191 CNRS-CEA-Universite´ Aix-Marseille II, 13108 St Paul lez Durance, France CEA Cadarache, DSV-DEVM – LEP, UMR 6191 CNRS-CEA-Universite´ Aix-Marseille II, 13108 St Paul lez Durance, France

b

Received 2 November 2006; revised 17 November 2006; accepted 17 November 2006 Available online 29 November 2006 Edited by Ulf-Ingo Flu¨gge

Abstract ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of ‘‘safe-food’’ strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: ABC transporter; MRP1/ABCC1; Antimony; Glibenclamide; MK571; Arabidopsis

*

Corresponding author. Fax: +33 4 4225 2364. E-mail address: [email protected] (C. Forestier). URL: http://www-dsv.cea.fr/thema/lems/lems.htm. 1

L. Gayet and N. Picault contributed equally to this work. Present address: Laboratoire Ge´noˆme et De´veloppement des Plantes, UMR5096, CNRS-Universite´ de Perpignan-IRD, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France. 3 Present address: Elopak R&D, Food Science and Technology, 3431 Spikkestad, Norway. 2

1. Introduction Heavy metals in soil pose a serious risk to public safety and groundwater supplies. Indeed, cadmium, lead, arsenic and antimony are often found at many contaminated sites. These contaminants are frequently incorporated into trophic chains via water, microorganisms and plants. These heavy metals are usually detoxified after chelation to glutathion by membrane-located transport proteins such as glutathione-conjugate transporters (GS-X pumps), a concept originally described by Ishikawa [1]. GS-X pumps, also known as multidrug resistance-associated proteins (MRPs), constitute a sub-family of the ATP-binding cassette superfamily of proteins (ABC proteins) implicated in the transport of various compounds, such as vincristine, daunorubicin, leukotriene-C4, metolachlor-GS, 17b-estradiol-17-(b-D -glucuronide) and antimony [2]. ABC proteins are ubiquitous and form the largest gene family of transporters known, with more than 5000 distinct genes ([3]; http://www.pasteur.fr/recherche/unites/pmtg/abc/database.iphtml). It is now well established that several yeast (HMT1, YCF1), Arabidopsis (ATM3, ALS3, PDR12) and mammalian (MRP1, MRP2) ABC transporters are responsible for the detoxification of heavy metals, such as Cd, Ni, As, Sb, Hg, Pb, Gd, Al and Pt [4–16]. In Schyzosaccharomyces pombe, HMT1, the heavy metal transporter 1, is localized at the vacuolar membrane and detoxifies Cd-phytochelatins complexes into the vacuole [11,12]. From a functional point of view, YCF1, the yeast cadmium factor 1 from Saccharomyces cerevisiae, is the homolog of HMT1. YCF1 is a GS-X pump that confers to this organism resistance to Cd, As, Sb, Hg and Pb [4,6,7,17–20]. YCF1 is located at the vacuolar membrane and transports glutathione-S-chelates, such as Cd(GS)2, Sb(GS)3 or As(GS)3, from the cytosol to the vacuole [4,17–20]. YCF1 and MRP1 are closely related, not only in terms of sequence (43% identity, 63% similarity), but also in function [21]. MRP1 can restore Cd resistance in Dycf1 yeast strains [22]. However, to our knowledge, a direct demonstration that MRP1 is a Cd(GS)2 transporter has not been obtained yet. Besides Cd(II), MRP1 is involved in the cellular detoxification of different heavy metals in humans, such as Hg(II), As(III), As(V), Sb(III) and Sb(V) [5,9,10,23]. For instance, Sb(III), forming tri-glutathione chelates, is very efficiently detoxified by MRP1. Thanks to this property, an antimony-selected cell line overexpressing MRP1 has been generated that is 35-fold more resistant to Sb(III) than the parental counterpart [9].

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Theoretically, the engineering of ABC transporters into plants could serve two opposite objectives. First, overexpression of effluxing plasma membrane MRP pumps could reduce the quantity of xenobiotics in the plant, thus lowering their entry into trophic chains (’’safe-food’’ strategies). Conversely, overexpression of vacuolar ABC transporters could induce accumulation of xenobiotics into the vacuole, thus participating in the development of phytoremediation strategies. This second strategy seems promising since overexpression of YCF1 in Arabidopsis thaliana confers to this model plant enhanced tolerance and accumulation properties for Pb and Cd [7]. In both strategies, the soil composition (pH, percentage of humic fractions, content of metals, etc.) is a major parameter determining the availability of the metals for the plant; thus, the success of the approach. Moreover, soils are usually multi-contaminated (for instance, the association of Sb and Pb), and it is thus relevant to evaluate the contribution of different ABC transporters that present different substrate specificities. Since MRP1 is a very efficient transporter implicated in the excretion of numerous substances toxic for humans, its introduction in plant cells could offer new potentialities limiting the penetration of xenobiotics, notably heavy metals, in human foods. To test this hypothesis, we investigated in this study the consequence of the introduction of the human MRP1 in the context of plants naturally expressing various AtMRPs (more than 14 genes in the Arabidopsis genome; cf. [24,25]). Since MRP1 is an effective transporter of antimony in mammalian cells [5,9,10], our study has been focused on this substrate.

2. Materials and methods 2.1. Plant material and growth conditions Arabidopsis thaliana ecotype Wassilewskija plants were grown in sand as previously described [26]. During heavy metal and anticancer drug treatments, transgenic (T3 generation) and wild-type plants were maintained in a growth chamber with a short-day photoperiod of 8-h light and 16-h darkness at 21 C/18 C, respectively, at a light intensity of 100 lmol m 2 s 1. Sterile seeds were plated onto half-strength Murashige and Skoog medium, pH 5.6, and 0.7 g l 1 agar. After 1 day at 4C, seeds were germinated in a vertical orientation for 5 days in a short-day growth chamber. The plantlets were then transferred to half-strength Murashige and Skoog medium in absence or in presence of various xenobiotics (As(III), As(IV), Sb(III), Cd(II), doxorubicin, vinblastine) and were grown for an additional 9 day period, following which root lengths were measured. Typically, the assays involved 10 seedlings per genotype and per growth condition. 2.2. Plant expression constructs, transformation and selection In order to localize HsMRP1, the C-terminal part of MRP1 was epitope-tagged with GFP. The plasmids pEGFP-N2 (from BD Biosciences) and pFast-Bac1-MRP1-His were used to generate the HsMRP1EGFP-N2 fusion by the splicing by overlap extension technique [27]. The oligonucleotides and conditions used for the fusion are given in supplementary data. The HsMRP1-EGFP-N2 fusion sequence was subcloned into the pCR–XL-TOPO vector (Invitrogen) and sequenced. After digestion with EcoRV, the CaMV35S cassette was subcloned into the EcoRV/StuI sites of the pGreen0179 binary vector, giving the pVF90 clone [28]. The enhanced green fluorescent protein (EGFP) was cloned into the EcoRI site of the pVF90 described above. The HsMRP1-EGFP-N2 sequence was extracted by XbaI/SacI digestion and cloned at the same sites in the pVF90 vector. Following introduction of the constructs or of the empty vector with the pSOUP [29] in Agrobacterium tumefaciens AGL1 by electroporation, the Arabidopsis plants were transformed by the floral dip method [30]. The transformed plants were selected on solid medium supplemented with hygromycin B (30 mg l 1).

L. Gayet et al. / FEBS Letters 580 (2006) 6891–6897 2.3. Northern and Western-blot analysis Total RNA was isolated from leaves of A. thaliana using TRIzol (Life Technologies). Northern blot analyses were carried out on T1 transformant plant using an EGFP probe labelled with DIG-dUTP as described [31]. RNA loading was checked with ethidium bromide. Total protein or microsomes extracted from shoots or mesophyll cell protoplasts (MCPs) were denaturated and subjected to SDS–PAGE electrophoresis. After transfer onto nylon membrane, proteins were detected with an anti-GFP antibody (Monoclonal antibody JL-8, BD Biosciences). 2.4. Localization of MRP1-EGFP in plants and transport experiments MCPs from A. thaliana were isolated from 5-week-old plant leaves by enzymatic digestion (1-h at 30 C) in 8 mM CaCl2, 0.5 M Mannitol, 5 mM MES, 1% Cellulase R10, 0.25% macerozyme, 1 mM KH2PO4, 0.5 mM ascorbate, 0.1% PVP40, pH 5.6 KOH. Obtention of MCPs was checked under the microscope and cells were washed and resuspended in the MC buffer (0.5 M sorbitol, 1 mM CaCl2, 10 mM MES, pH 5.6 KOH). Root cell protoplasts (RCPs) were obtained by using the same protocol, except that Cellulase R10 was increased to 1.5%, macerozyme was substituted by 0.03% pectolyase Y-23 (enzymatic digestion during 3-h at 27 C, in darkness). Expression of the EGFP was monitored using an epifluorescence microscope (Nikon, Optiphot 2) fitted with a CCD camera (AxioCam, Zeiss, Gottingen, Germany) to capture pictures. EGFP-N2 has an excitation peak at 488 nm with an emission peak at 507 nm. For heavy metal transport experiments or cell integrity tests, MCPs diluted in the MC buffer were exposed for 1-h to appropriate Sb(III)Cl3 concentrations. To determine the metal content, cells were gently washed in MC buffer, pelleted, dried for 48-h at 50 C and mineralized by 200 ll of 70% nitric acid before analysis by ICP-OES (Vista MPX, Varian). To evaluate cell integrity, the number of intact MCPs was determined before and after treatment by counting under the microscope with a Malassez cell. Similar results were obtained in control experiments when FDA was used as a marker of cell viability. In all cases, MCPs exhibiting a shrinking or partial disruption of the chloroplast envelopes were not taken into account. Transport of 0.5 lM calcein-AM (Molecular probes) by MCPs was determined by a 1-h incubation at 30 C in MC buffer. MCPs were centrifuged and quantification of the fluorescent dye released in the supernatant was performed as previously described [9]. Detection of the calcein was performed with a microplate fluorescence reader (FLX-800, Bio-Tek Instruments Inc.), using a 485 ± 20 nm excitation filter and a 528 ± 20 nm emission filter. In order to measure the Sb(III) efflux (Fig. 5C), cells were first loaded with 200 lM Sb(III)Cl3 in the MC buffer for 1-h (at 4 C in order to limit the efflux), washed and then incubated in a Sb(III)-free medium at 30 C. The metal content was determined by ICP-OES as described above. Effluxes were blocked either with MK571 (kindly provided by Dr. Ford-Hutchinson, Merck-Frosst Inc., Canada), a specific inhibitor of MRP1 known to specifically reverse MRP1-mediated anticancer drug resistance [32], or by glibenclamide (from Sigma).

3. Results and discussion 3.1. Generation of MRP1-EGFP over-expressing plants A. thaliana ecotype Wassilewskija plants were engineered to overexpress the exogenous human HsMRP1 under the control of the CaMV 35S strong constitutive promoter in a modified pGREEN vector. EGFP was fused at the C-terminal part of HsMRP1 to localize its expression in the transgenic plants. As a result, 26 independent transgenic Arabidopsis lines designated 35S-MRP1-EGFP#1 to 35S-MRP1-EGFP#26 were produced. As a control, 9 independent lines, 35S-EGFP#1 to 35S-EGFP#9, over-expressing EGFP alone under the control of a CaMV 35S promoter were also generated. T2 lines were obtained from individual T1 plants by self-fertilization. Transgenic lines that displayed 3:1 segregation for hygromycin resistance in the T2 generation and that were 100% hygromycin resistant in the T3 generation were selected for further analy-

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sis. T1 plant lines were first screened by Northern blot analysis to select plants over-expressing Hs MRP1 (Fig. 1). Among them, two lines over-expressing Hs MRP1::EGFP (35SMRP1-EGFP#4 and 35S-MRP1-EGFP#22) and one line over-expressing EGFP (35S-EGFP#6) were selected for their capacity to overexpress the corresponding mRNA. Transgenic plants T1, T2 and T3 did not show any noticeable difference compared to wild-type (WT) plants grown under normal conditions (data not shown). Different control plants, including transformation controls and wild-type plants, were used as references. In the following, transgenic lines 35S-EGFP#6, 35SMRP1-EGFP#4 and 35S-MRP1-EGFP#22 were renamed as GFP6.9, MRP4.18 and MRP22.14, respectively.

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3.2. MRP1-EGFP is addressed to the plasma membrane but not to the vacuolar membrane of transgenic plants In order to investigate the expression of MRP1-EGFP in transgenic plants, Western-blot analyses were carried out.

Total protein from either root cell protoplasts (RCPs, Fig. 2A) or mesophyll cell protoplasts (MCPs, Fig. 2B) were extracted from all transgenic lines. EGFP alone was detected at an apparent molecular mass of 31-kDa (expected at 27kDa) in the GFP6.9 transgenic line (Fig. 2A, GFP6.9 lane). As illustrated in Fig. 2, in all cell-types tested, anti-GFP antibodies revealed a polypeptide with an apparent molecular mass close to 200-kDa in both MRP1-EGFP transgenic lines (theoretical molecular mass 198 kDa). No obvious cleavage of the GFP was observed whatever the MRP1-EGFP-expressing tissue tested. These results indicate that MRP1-EGFP is fully translated by the plant cellular machinery in the root, as well as the shoot, compartment. In a second step, the sub-cellular localization of MRP1EGFP in transgenic plants was investigated in MCPs by fluorescence microscopy. Fig. 3A and B shows a clear plasma membrane localization of the transporter in MCPs (white arrows) as well as in epidermal cells (black arrows). In order

35S-MRP1-EGFP 11 14 15 21 22 23 26 HsMRP1::EGFP mRNA

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Fig. 1. RNA gel blot analysis of wild-type (WT), transgenic 35S, 35S-EGFP and 35S-MRP1-EGFP T1 lines plants. The EGFP cDNA was used to probe an RNA gel blot containing RNA (5 lg) prepared from the leaves of the Arabidopsis lines. Ethidium bromide-stained rRNA is shown as a loading control.

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Fig. 2. The MRP1-EGFP fusion protein is fully translated in A. thaliana. Immunodetection of either root cell protoplasts (RCPs, A) or mesophyll cell protoplasts (MCPs, B) extracted from wild type (WT), GFP6.9, MRP4.18 and MRP22.14 transgenic lines using an anti-EGFP antibody.

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to investigate a possible localization of the protein at the vacuolar membrane, the MC buffer was gently diluted in order to induce a slow turgescence of the protoplasts. Vacuoles were obtained from bursting MCPs. As illustrated in Fig. 3C and D, no GFP fluorescence was observed at the vacuolar membrane, either in isolated vacuoles (black arrows) or in vacuoles still in release from the MCPs (white arrows). Similar results were obtained with both MRP4.18 and MRP22.14 transgenic lines. Detection by Western-blots of MRP1-EGFP in roots (Fig. 2A) was confirmed by the analysis of the EGFP epifluorescence in the primary root apex (Fig. 3E–H). For these experiments, a first capture of the fluorescent signal from the GFP6.9 line was obtained (Fig. 3F) and the exposure time of the CDD camera used for this image was then kept constant for all subsequent acquisitions. As a result, no auto-fluorescence was observed in wild-type plants (Fig. 3E), whereas a clear GFP fluorescent emission was present in MRP1-EGFP lines (Fig. 3G–H). Altogether, these results demonstrate that the human MRP1 is expressed throughout A. thaliana transgenic plants and localized at the plasma membrane. 3.3. Plant tolerance to heavy metals and anticancer drugs In humans, MRP1 confers an ATP-dependent resistance to several natural products (e.g., anthracyclines or Vinca alka-

loids), folic acid analogues, arsenic and antimonial oxyanions, but is also a primary active transporter of glutathione-, glucuronate- and sulphate-conjugated organic anions [2]. In order to evaluate the functional activity of MRP1-EGFP in Arabidopsis, MRP4.18 and MRP22.14 transgenic plantlets were grown in the presence of various heavy metals (1–10 lM As(III), 1–300 lM As(V), 1–40 lM Sb(III) or 1–100 lM Cd(II)). In all cases, as illustrated for 20 lM Sb(III) in Fig. 4, MRP4.18 and MRP22.14 transgenic plants were indistinguishable from wild-type plants, plants overexpressing the empty vector or plants overexpressing GFP alone (GFP6.9). Similar results were obtained when different culture mediums (halfstrength Murashige and Skoog or Hoagland medium, with/ without 1% sugar) and different growth temperatures (22 or 30 C) were used. Moreover, no difference was observed between Sb(III), Cd(II) or As(III) accumulation in plants, as determined by ICP-OES analysis (data not shown). It is worth noting that the effects of cytotoxic drugs, such as doxorubicin or vinblastine (1–100 lM), were also investigated. However, a degradation of these compounds was observed as a decoloration of the plant medium within a few hours in the phytotron. This photosensitivity of the molecules has also been recently observed [33] and prevents further investigation of anticancer drugs under these plant culture conditions.

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Fig. 3. The MRP1-EGFP fusion protein is expressed at the plasma membrane in A. thaliana transgenic lines. MRP1-EGFP localization was determined by fluorescence microscopy in MCPs (A–D). MCPs extracted from the MRP22.14 transgenic line were observed in bright light (A). In the corresponding cells, chloroplasts were revealed by red fluorescence emission, whereas MRP1-EGFP was detected by the green fluorescence at the plasma membrane (B), either in MCPs (white arrows) or in epidermal cells (black arrows). As observed in bright light (C), when a dilution of the MC buffer was applied, a swelling of the cells was initiated leading to the progressive bursting of the protoplasts (white arrows), leaving intact vacuoles (black arrows). When these vacuoles were observed by fluorescence microscopy, no GFP was detected (D). (E–H). Images of the primary root apex collected from the different plant genotypes obtained by fluorescence microscopy (upper panel) or in bright light (lower panel) after standardization of the fluorescence signal with GFP6.9 plants. Arrows in the bright field images highlight the diffraction of the light surrounding the root tissue at the interface between the primary root and the nutrient solid medium.

L. Gayet et al. / FEBS Letters 580 (2006) 6891–6897

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Fig. 4. In intact plants, MRP1-EGFP over-expression does not affect root growth on Sb(III)-contaminated medium. Seeds from the MRP4.18 line were sown on half-strength MS media, grown for 5 days and then transferred to media with or without 20 lM Sb(III) for 9 additional days (lower and upper panels, respectively). Similar results were observed with the MRP22.14 transgenic line (data not shown).

3.4. MRP1-EGFP participates in the cellular transport of calcein and antimony salts Our results suggest either that MRP1-EGFP is not functional in plants or that the cellular efflux of heavy metals by MRP1-EGFP is insufficient to confer an increased tolerance to antimony salts to the transgenic plants. To answer this question, MCPs were isolated and the transport activity supported by MRP1-EGFP was directly measured. In a first step, we took advantage of the fact that MRP1 activity can be probed using a calcein assay [9,34]. As shown in Fig. 5A, the cellular efflux of the fluorescent dye was strongly increased in MRP1-EGFP-overexpressing cells in comparison to that observed in wild type or EGFP-expressing cells. In a second step, we investigated the ability of MRP1-EGFP to transport Sb(III). Indeed, in mammals, MRP1-overexpressing GLC4/ Sb30 cells have been demonstrated to be 35-fold more resistant to antimonial salts [9]. For this purpose, the sensitivity of MCPs to the cytotoxic effects of Sb(III) was determined by counting intact cells. Under control conditions, after 1-h, almost 100% of the cells were intact, whatever the genotype. When MCPs were exposed to 200 lM Sb(III) for 1-h, less than 50% of the wild type cells survived, whereas more than 70% of MCPs isolated from the two transgenic lines overexpressing MRP1 survived (Fig. 5B). A concomitant application of Sb(III) with either glibenclamide, an inhibitor of MRP1 [35] and a useful tool to trap anionic fluorescent dyes in the cytosol of plant cells [36], or MK571, a potent inhibitor of MRP1 known to specifically reverse MRP1-mediated anticancer drug resistance [32], markedly diminished the resistance of MRP1overexpressing cells. In contrast, these compounds did not affect the sensitivity to antimony of wild type cells, indicating that endogenous AtMRPs were either not involved in the large Sb(III) efflux observed or, alternatively, that these plant proteins are insensitive to MK571 and glibenclamide. In a third step, the kinetics of Sb(III) efflux were measured (Fig. 5C). After 10-min, 75% of the metalloid was effluxed by control cells, a value reaching 95% in MRP1-EGFP-overexpressing

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cells. Compared to MRP1 expressed in GLC4/Sb30 cells [9], these plant protoplasts exhibit a rapid efflux of antimony. Finally, the direct contribution of MRP1-EGFP in the cellular transport of Sb(III) was investigated by the measurement of the cellular accumulation of antimony in the different transgenic lines. Antimony content in MCPs, which reached a mean of 8.5 lg per mg of protein within 1-h in wild type and GFP transgenic lines, was reduced by 45% to reach a mean of 4.6 lg per mg of protein in MRP1-EGFP-overexpressing lines (Fig. 5D). The co-application of Sb(III) with either glibenclamide or MK571 markedly enhanced the antimony retention in MCPs from both MRP1-EGFP transgenic lines. In humans, it has already been demonstrated that a C-terminal GFP fusion of the human MRP1 keeps its intracellular localization and is still functional [37]. In the present study, the fusion protein was correctly localized and active in detoxifying calcein or antimony salts. The fact that transgenic plants did not exhibit a resistance phenotype to MRP1 substrates was unexpected but could be explained by the following reasons. First, in plant cells, the vacuole is the main organelle for the sequestration of toxic compounds once they have penetrated the cytoplasm. As we have seen, MRP1 is not localized to the vacuolar membrane in A. thaliana transgenic plants. In contrast, when YCF1 fused to GFP (either at the N- or C-terminal part) was overexpressed in Arabidopsis, both vacuolar and plasma membrane localizations were observed. This was accompanied by an increased Cd influx into the vacuole measured during uptake experiments, and explains why transgenic plants were more tolerant to Cd [7]. Such a critical importance of vacuolar sequestration of toxic compounds in plant cells may explain why the unique presence of MRP1 at the plasma membrane is insufficient to confer Sb(III) tolerance in planta. Interestingly, as published in a very recent work [33], MRP1 expression in tobacco plants under a 35S promoter equipped with a tandem enhancer sequence El2 resulted in a vacuolar membrane localization of the protein. These plants were shown to be more tolerant to Cd, although they did not accumulate more metal. This underlines the uncertainties inherent to heterologous expression concerning protein localization depending on the host or the vector used. It is possible that in our study using Arabidopsis and expression of MRP1EGFP under the control of a single 35S promoter, the level of active protein is insufficient to confer a whole plant phenotype. This is supported by the observation that the activity of YCF1 in transgenic plants was strongly improved when four copies of the CaMV 35S enhancer were inserted into the plant transformation vector [7]. Moreover, in our study, a high Sb(III) efflux activity carried out by endogenous transporters was already observed in control plants (Fig. 5C), diminishing the benefit of MRP1 expression. A final reason that could explain the absence of a resistance phenotype might be due to Sb(III) speciation. Antimony–(GS)3 complexes effluxed by MRP1 from the cytosol could be dissociated in the more acidic apoplastic medium to release Sb(III). In such case, Sb(III) could re-enter into the cell for a new cycle without any benefit for the plant. In conclusion, these results demonstrate that a human ABC transporter can be functionally introduced into A. thaliana and is addressed to the plasma membrane, which corresponds to its localization in human cells. The transporter is functional in transferring calcein and Sb(III) and is still sensitive to its classical modulators, MK571 and glibenclamide. It is likely that

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Fig. 5. The MRP1-EGFP fusion protein drives the efflux of calcein and Sb(III) in MCPs of A. thaliana transgenic lines. (A) MRP1-EGFP participates in the cellular efflux of calcein from A. thaliana transgenic MCPs. MCPs were incubated with 0.5 lM calcein-AM for 1-h at 30 C. The calcein efflux from MCPs isolated from wild type plants (WT), GFP6.9 plants, MRP4.18 plants and MRP22.14 plants was quantified by spectrofluorimetry. (B) Cells overexpressing MRP1-EGFP exhibited a better tolerance to antimony salts compared to wild type cells. MCPs isolated from the different transgenic lines were incubated with 200 lM Sb(III)Cl3 for 1-h at 30 C in the presence or absence of 10 lM glibenclamide or 50 lM MK571. Under each condition, the number of cells was calculated as the ratio of the number of intact MCPs in the presence vs. the absence of treatment. One hundred percent corresponds to 1 · 105 MCPs per ml. (C) Sb(III) efflux was enhanced in MRP1-EGFP-overexpressing cells. Cells were first loaded with 200 lM Sb(III)Cl3 for 1-h (at 4 C), washed and then incubated in a Sb(III)-free medium. Intracellular antimony content was measured at different times and expressed as a percentage of the initial intracellular Sb(III) content. (D) Antimony accumulation was reduced in MCPs isolated from MRP4.18 and MRP22.14 plants. Experimental conditions were similar to those described in (B). The Sb content measured by ICP-OES was expressed on a protein basis. (A–D) Each bar is the mean ± S.E. of three independent experiments.

the level of expression of the protein is insufficient to confer to transgenic plants a strong resistance phenotype to xenobiotics, but potentially, this goal could be achieved by using stronger promoters. On the other hand, stable A. thaliana cell cultures over-expressing human MRP1, where endogenous plants MRPs either have or have not been knocked out, could offer a novel tool to screen for new modulators of this protein in a context devoid of human ABC proteins. Acknowledgments: The authors thank Dr. T. Furukawa (Kagoshima University, Japan) for the pFast-Bac1-MRP1-His plasmid, Dr. P. Richaud, P. Carrier and P. Auroy (CEA Cadarache, France) for help in ICP analysis, M.A. Roncato (CEA Cadarache, France) for technical assistance. This work was supported by the Commissariat a` l’Energie Atomique, by the European Commission Marie Curie Research Training Network and by the ‘‘Toxicologie Nucle´aire Environnementale’’ program.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2006. 11.051.

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