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First published online May 27, 2005; 10.1104/pp.104.058107 Plant Physiology 138:827-836 (2005) © 2005 American Society of Plant Biologists AtPDR12 Contributes to Lead Resistance in Arabidopsis1National Research Laboratory of Phytoremediation, Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790–784, Korea (M.L., K.L., J.L., Y.L.); and Korea Forest Research Institute, Suwon, 441–350, Korea (E.W.N., Y.L.)
Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.
Heavy metals contaminate soils as a result of industrial processes and agricultural practices (Ross, 1994  ) and are a dangerous stress to plants that grow in the contaminated soils. Plants grown in contaminated agricultural fields contain the heavy metals and thus endanger animals who feed on them. Among the heavy metals, lead (Pb) is particularly important, since it is toxic to many organisms, widespread in the human environment, and causes multiple serious health problems in growing children and adults (as stated in a U.S. Environmental Protection Agency fact sheet at http://www.epa.gov/iaq/lead.html).
Many transporters have been identified that detoxify heavy metals in diverse organisms. Since cells have to remove heavy metals from the cytoplasm to avoid toxicity, some transporters extrude them across the plasma membrane and others sequester them into inactive organelles. Transporters that have been shown to extrude heavy metals across the plasma membrane are P-type pumps, including ZntA from Escherichia coli, a Pb(II)/Cd(II)/Zn(II)-transporting ATPase that mediates resistance to toxic concentrations of Pb(II), Cd(II), and Zn(II) by pumping these metals out of cells (Rensing et al., 1997
ABC transporters have been reported to transport many cytotoxic compounds, such as drugs, in addition to heavy metals (Rogers et al., 2001 In this paper, we present evidence that a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters in Arabidopsis has a role in Pb(II) detoxification. We show that AtPDR12 expression is strongly induced by Pb(II) treatment and that AtPDR12-knockout plants are more sensitive to Pb(II) than wild-type plants. In addition, AtPDR12-knockout plants contain more Pb than wild-type plants and AtPDR12-overexpressing plants are more resistant to Pb(II) than wild-type plants. These results, as well as localization of AtPDR12 to the plasma membrane, suggest that AtPDR12 confers Pb(II) resistance by pumping Pb(II) and/or Pb(II)-containing toxic compounds out of the cell from the cytoplasm.
Expression of AtPDR12 Is Elevated by Pb(II) Treatment As a first step in the identification of ABC transporters involved in Pb(II) transport, we searched databases (http://www.ncbi.nlm.nih.gov/ and http://www.ddbj.nig.ac.jp/E-mail/clustalw-e.html) to select Arabidopsis ABC transporters with high amino acid sequence homology to ABC transporters of other organisms that are reported to transport toxic substances. We then performed reverse transcription (RT)-PCR experiments to test whether the transcript levels of these candidate ABC transporters are up-regulated when Arabidopsis is treated with Pb(II). The expression of AtMRP4, AtPDR1, AtPDR4, AtPDR6, and AtPDR9 did not change much upon Pb(II) treatment (data not shown), whereas that of AtPDR12 increased both in shoots and in roots (Fig. 1). Without Pb(II) treatment, the AtPDR12 transcript was expressed in shoots but not detected in roots.
AtPDR12-Knockout Plants Are Less Resistant to Pb(II) Than Wild-Type Plants To test whether AtPDR12 contributes to Pb(II) resistance in plants, we obtained seeds of 2 AtPDR12-knockout alleles from the Salk Institute Genomic Analysis Laboratory. These knockout plants have T-DNA inserted into 2 different sites of the 21st exon of the AtPDR12 gene (Fig. 2A; http://signal.salk.edu/cgi-bin/tdnaexpress, SALK stock nos. SALK_ 013945 and SALK_005635). Homozygous knockout plants (atpdr12-1 and atpdr12-2) were selected by PCR using 1 T-DNA-specific primer and 2 AtPDR12-specific primers with genomic DNA as a template. The PCR using 2 AtPDR12-specific primers did not produce any product (right lanes of Fig. 2B, subsections a and c), but the PCR using a T-DNA-specific primer and 1 AtPDR12-specific primer produced 730- and 760-bp-size bands from genomic DNA of atpdr12-1 and 2, respectively, as expected (right lanes of Fig. 2B, subsections b and d). These PCR products were sequenced using T-DNA-specific primers, and insertion sites were confirmed. To confirm that the homozygous lines do not have the AtPDR12 transcript, RT-PCR was performed using total RNA extracted from the plants. PCR using 2 AtPDR12-specific primers did not produce any product from the homozygous knockout plants, as it did from wild-type plants (Fig. 2C).
We obtained the next generation of seeds from the selected AtPDR12-homozygous knockout plants and tested their resistance to Pb(II) and Cd(II). In control medium, the growth of AtPDR12-knockout plants (atpdr12-1 and atpdr12-2) was similar to that of wild-type plants, and they reproduced normally (Fig. 3, A and B, top). In Pb(II)-containing medium, the growth of roots and shoots was reduced more in AtPDR12-knockout plants than in wild-type plants (Fig. 3, A and B, bottom). Although addition of Pb(NO3)2 may have caused some precipitation of P and S, the plant symptoms did not resemble those of P and S deficiency, which cause dark-green coloration and chlorosis of young leaves, respectively. Rather, the Pb(II)-treated plants displayed the typical symptom of Pb(II)-treated plants, reduced root growth. Growth of AtPDR12-knockout plants was not different from that of wild-type plants in 30, 40, or 50 µM Cd(II)-containing media (Fig. 3C; data not shown) or in media containing Zn(II), Cu(II), Al(III), or the antifungal drug sclareol (data not shown). These results demonstrate that AtPDR12 specifically confers Pb(II) resistance, at least under our experimental conditions.
For quantitative analyses of the phenotypes, we tested various concentrations of Pb(NO3)2 and chose two concentrations that showed the most striking differences between the knockout and wild-type plants. Figure 4 shows the fresh weight and root length of AtPDR12-knockout and wild-type plants grown on Pb(II)-containing one-half Murashige and Skoog (MS) agar plates without (control) and with 0.7 mM or 0.75 mM Pb(NO3)2 for 4 weeks. In the control medium without additional Pb(II), atpdr12-1 plants had 1.2-fold higher fresh weight and 1.1-fold higher root length than wild-type plants. In contrast, atpdr12-2 plants did not display any differences in fresh weight and root length compared to wild-type plants (Fig. 4, A and B). However, in Pb(NO3)2-containing media, both fresh weight and root length were more severely reduced in AtPDR12-knockout plants than in wild-type plants; the fresh weights of atpdr12-1 and atpdr12-2 were reduced to 52% and 87% of wild type, and root lengths were decreased to 67% and 79% of wild type in medium containing 0.75 mM Pb(NO3)2 (Fig. 4, A and B).
AtPDR12-Knockout Plants Contain More Pb We measured the Pb contents of 2-week-old wild-type and AtPDR12-knockout plants whose roots were soaked in 1 mM Pb(NO3)2 for 2 d. Shoots of atpdr12-1 plants contained 1.4-fold more Pb than those of wild-type plants (P < 0.001; Fig. 5). Although the Pb content of atpdr12-2 plants seemed slightly higher than that of wild-type plants in Pb(NO3)2-containing media, the difference was not statistically significant (data not shown). Additionally, the phenotypes of atpdr12-2 plants were less pronounced than those of atpdr12-1 plants with respect to fresh weight and root length. We have no valid explanation for this difference, except perhaps for the observation that the T-DNA insertion site of atpdr12-2 is closer to the stop codon than that of atpdr12-1. We then examined the possibility that Pb treatment of the knockout plants causes changes in the levels of other metal ions, using an inductively coupled plasma spectrometer. While potassium, copper, magnesium, iron, zinc, and manganese levels remained the same, the Pb level was confirmed to be higher in AtPDR12-knockout plants (atpdr12-1) than in the wild type (data not shown).
AtPDR12-Overexpressing Arabidopsis Plants Are More Resistant to Pb(II) If AtPDR12 contributes to Pb(II) resistance, AtPDR12-overexpressing plants may be more resistant to Pb(II) than wild-type plants. The AtPDR12 gene was inserted into the pBI121 vector with a 35S promoter (pBI121:AtPDR12) and the construct was introduced into wild-type Arabidopsis plants. We selected 3 T2 lines that had only 1 copy of pBI121:AtPDR12, and T3 homozygous lines were chosen from the next generation and tested for their resistance to Pb(II). In one-half MS control medium without Pb(NO3)2, growth of AtPDR12-overexpressing and wild-type plants was similar (Fig. 6A). However, in medium containing 0.75 mM Pb(NO3)2, the shoots and roots of AtPDR12-overexpressing plants grew slightly better than those of wild-type plants (Fig. 6B). Quantitative analyses showed that the fresh weights and root lengths of AtPDR12-overexpressing and wild-type plants were similar in control medium except for one AtPDR12-overexpressing line, which had a slightly reduced fresh weight (Fig. 7, A and C). In Pb(NO3)2-containing medium, fresh weight and root length were consistently higher in the 3 AtPDR12-overexpressing plants than in the wild-type plant; the fresh weight of AtPDR12-overexpressing lines were 1.14-, 1.37-, and 1.28-fold higher than that of the wild type (Fig. 7B), and root lengths of 2 AtPDR12-overexpressing lines were 1.23- and 1.29-fold longer than that of the wild-type plant (Fig. 7D). To investigate the AtPDR12 transcript level in AtPDR12-overexpressing plants, total RNA was extracted from Pb(II)-treated AtPDR12-overexpressing and wild-type plants. The 3 AtPDR12-overexpressing plants had a higher AtPDR12 transcript level compared to wild-type plants (Fig. 7E).
To test whether AtPDR12-overexpressing plants had reduced Pb contents compared to wild-type plants, AtPDR12-overexpressing and wild-type plants were grown on 0.7 mM Pb(NO3)2-containing medium for 2 weeks and then their shoots were prepared. Pb contents of 3 independent lines of AtPDR12-overexpressing plants (P12-1, P12-2, and P12-2) had Pb contents that were 73%, 68%, and 72% of that of wild-type plants (P < 0.05 for all 3 lines; Fig. 8).
Pb(II) Resistance Conferred by AtPDR12 Is Independent of Glutathione Because Pb(II) can form complexes with thiol compounds, we investigated whether the Pb(II) detoxification mechanism of AtPDR12 is related to glutathione. To test this possibility, we compared the growth of wild-type and AtPDR12-transgenic plants in medium containing buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. In control or 1 mM BSO-containing media, the growth rates of AtPDR12-overexpressing (P12-1), -knockout (atpdr12-1), and wild-type plants were similar (Fig. 9, A and B). In 0.7 mM Pb(NO3)2-containing medium, AtPDR12-overexpressing and -knockout plants showed slightly enhanced and slightly reduced growth, respectively, compared to the wild-type plants (Fig. 9C), as found in previous experiments. In medium containing both 1 mM BSO and 0.7 mM Pb(NO3)2, this growth difference was even more striking than in medium containing Pb(NO3)2 alone (Fig. 9D). This amplified phenotypes under low-glutathione condition indicates that AtPDR12 does not use glutathione for Pb(II) detoxification.
AtPDR12 Is Localized at the Plasma Membrane and Expressed in Many Organs of Arabidopsis
To investigate the localization of AtPDR12, we isolated protoplasts from Arabidopsis whole seedlings and introduced a green fluorescent protein (GFP):AtPDR12 construct (GFP gene fused to the 5' end of AtPDR12 gene) into them using the polyethylene glycol transformation method (Jin et al., 2001
To investigate the tissue-specific expression pattern of AtPDR12 in wild-type plants, we extracted total RNA from each tissue and performed RT-PCR. AtPDR12 transcripts were detected in rosette leaves, stems, cauline leaves, and flowers, but not in roots (Fig. 10B).
AtPDR12 has been studied before (Van den Brule and Smart, 2002 ; Campbell et al., 2003), but information about it has been limited to its induction by defense-related molecules and the susceptibility of its knockout plants to sclareol. In this study, we cloned the AtPDR12 gene and studied its function in Pb(II) resistance. Results we obtained are highly suggestive of a role for AtPDR12 in Pb(II) resistance. First, we showed that the expression level of AtPDR12 was strongly induced by Pb(II) treatment (Fig. 1). Second, we showed that the shoots and roots of AtPDR12-knockout plants grow less on Pb(II)-containing medium than those of wild-type plants (Figs. 3 and 4). Third, we showed that AtPDR12-overexpressing plants, when grown on Pb(II)-containing medium, had improved growth of shoots and roots compared to wild-type plants (Figs. 6 and 7). We propose that the improved resistance of the AtPDR12-overexpressing plants is due to the exclusion of Pb(II), because GFP:AtPDR12 was localized to the plasma membrane (Fig. 10), and the AtPDR12-knockout and -overexpressing plants had increased or reduced levels of Pb, respectively (Figs. 5 and 8). It remains unknown whether AtPDR12 transports Pb(II) and/or Pb-containing compounds directly or indirectly. It may also transport other substrates generated by Pb(II) toxicity, or it may even function as a regulator of an unknown Pb(II) transporter. This problem will be difficult to resolve until a flux assay for Pb(II) becomes available. To test whether AtPDR12 responds specifically to Pb(II), AtPDR12-knockout plants were tested for growth on media containing various metal ions such as Zn(II), Cd(II), Cu(II), and Al(III). However, their growth rates were not different from those of wild-type plants (data not shown). AtPDR12 is unique in the sense that it confers resistance to Pb(II) but not to Cd(II), whereas the heavy metal transporters of other organisms, including ZntA and Ycf1, confer resistance to both Pb(II) and Cd(II). Thus, AtPDR12 appears to be the first ABC transporter gene that contributes specifically to Pb(II) resistance.
Not much is known about the Pb(II) resistance mechanisms of plants. We previously showed that oxalate secretion is important for Pb resistance in certain varieties of rice (Yang et al., 2000
AtPDR12 may function in plant defense responses, since previous reports showed that the AtPDR12 transcript was highly elevated under sclareol treatment (Van den Brule and Smart., 2002
AtPDR12 may have functions unrelated to Pb detoxification as well, because AtPDR12 is expressed in shoots and flowers under normal conditions (Fig. 9). It is commonly found in many organisms that one protein has multiple functions that are not related to each other (Lee et al., 2002 In summary, our results strongly support the possibility that AtPDR12 contributes to the resistance of Arabidopsis to Pb(II) by pumping or regulating transport of Pb(II) or Pb(II)-related toxic compounds to the exterior of the cell.
Plant Material and Growth Conditions
Seeds of Arabidopsis (Arabidopsis thaliana) wild-type ecotype Colombia-0, AtPDR12-overexpressing, and AtPDR12-knockout plants were surface sterilized, placed in the dark at 4°C for 2 d, and then sown on agar plates of one-half MS with 1.5% Suc (Murashige and Skoog, 1962
The full AtPDR12 sequence was isolated from a cDNA by PCR. The cDNA was synthesized using total RNA extracted from Pb(II)-treated roots of Arabidopsis wild-type plants. The primers used were AtPDR12-TFF (5'-CCCGGGGGGGATCCATGGAGGGAACTAGTTT TCACCAAGCGAGTA-3') and AtPDR12-TFR (5'-GGATCCGCGGCCGCCTATCGTTTTTGGAAATTGAAACTCTTGATTC-3'). The primers contained two restriction enzyme sites for easy cloning. The PCR product was ligated into the pGEM-T easy vector (Promega, Madison, WI) using T4 DNA ligase. The fidelity of the AtPDR12 sequence (GenBank accession no. NM_101421) was confirmed by automated DNA sequencing (ABI 3100, Perkin-Elmer Applied Biosystems, Foster City, CA).
To test expression levels of ABC proteins under Pb(II) stress, total RNA was extracted from Pb(II)-treated and water-treated roots of Arabidopsis. RT-PCR was performed using an RT-PCR kit (Invitrogen, Carlsbad, CA) with each ABC protein-specific primer set. The primer sequences employed are listed in Table I. As a control for RT-PCR, Tub1 and Tub2 primers were used.
Selection of AtPDR12-Knockout Arabidopsis Two alleles of AtPDR12-knockout Arabidopsis seeds, SALK_013945 and SALK_005635, were obtained from the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/cgi-bin/tdnaexpress). The seeds were sown and grown on soil for 4 weeks, after which genomic DNA was extracted from the plants. Homozygous AtPDR12-knockout plants were selected by PCR using 3 primers. In the case of the SALK_013945 line, a T-DNA-specific primer (pROKLBb1, 5'-GCGTGGACCGCTTGCTGCAACT-3') and 2 AtPDR12-specific primers (AtPDR12-TDNA-F, 5'-ATCAACACGGTCCCAGGAATGC-3' and AtPDR12-TDNA-R, 5'-CCATACGTTCTCGTGCAAGCGA-3') were used. In the case of the SALK_005635 line, a T-DNA-specific primer and 2 gene-specific primers (AtP12K/O-005635-LP, 5'-CTAATCCAGTGTTGTTGATCATCCT-3' and AtP12K/O-005635-RP, 5'-GAGCTAATCAAGGAGCTAAGCCAGC-3') were used.
To make AtPDR12-overexpressing plants, AtPDR12 was fused to pBI121 using the BamHI site of the pBI121 vector (Jefferson et al., 1987
Arabidopsis plants were grown on one-half MS-agar medium supplemented with 1.5% (w/v) Suc without (control) or with 0.7 mM or 0.75 mM Pb(NO3)2 for 4 weeks or 2 weeks. Plants were collected and weighed and their root length was measured. The shoots of plants were digested with 11 N HNO3 at 200°C overnight. Digested samples were diluted with 0.5 N HNO3 and analyzed using an atomic absorption spectrometer (SpectrAA-800, Varian, Palo Alto, CA) or inductively coupled plasma spectrometer (IRIS/AP, Thermo Jarrell Ash, Franklin, MA).
To investigate AtPDR12 localization, we made 2 different AtPDR12-GFP fusion constructs, an N-terminal and a C-terminal GFP construct, which we denoted GFP:AtPDR12 and AtPDR12:GFP, respectively. The former construct was made by inserting AtPDR12 into the 326GFP-3G vector (CLONTECH Laboratories, Palo Alto, CA) using the SmaI and SalI sites, and the latter construct was made by inserting AtPDR12 into the 326GFP vector (CLONTECH) using the BamHI site. AtAHA2:RFP was used as a plasma membrane marker (Kim et al., 2001
To investigate the tissue-specific RNA level of AtPDR12, RT-PCR was performed using total RNA extracted from each organ. Rosette leaves and roots were harvested from wild-type plants grown on one-half MS plate for 2 weeks. Flowers, stems, and cauline leaves were harvested from wild-type plants grown in soil for 5 weeks. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number NM_101421.
We thank the Salk Institute Genomic Analysis Laboratory for the AtPDR12-knockout seeds and Mrs. Jung and Han for technical assistance. Received December 13, 2004; returned for revision January 25, 2005; accepted February 2, 2005.
1 This work was supported by Pohang Steel Company (grants to Y.L.) and by the National Research Laboratory program of the Ministry of Science and Technology of Korea. 
2 Present address: Dartmouth College, Biological Science Department, 6044 Gilman, Hanover, NH, 03755. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058107. * Corresponding author; e-mail ylee{at}postech.ac.kr; fax 82–54–279–2199.
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ycf1 mutant (Tommasini et al., 1998
-Tubulin was used as a loading control.
-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc Natl Acad Sci USA 86: 6838–6842
