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Identification and characterization of MtMTP1, a Zn transporter of CDF family, in the Medicago truncatula
Plant Physiology and Biochemistry 47 (2009) 1089–1094
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Research article
Identification and characterization of MtMTP1, a Zn transporter of CDF family, in the Medicago truncatula Mingliang Chen, Xiaoye Shen, Daofeng Li, Lei Ma, Jiangli Dong, Tao Wang* State Key Laboratory for Agro-Biotechnology, College of Biological Science, China Agricultural University, 100193 Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2008 Accepted 21 August 2009 Available online 4 September 2009
Zn is an essential micronutrient in plants, and the mechanisms of Zn homeostasis are under intensive study. In this report, we have identified MtMTP1, a Zn transporter of the CDF family in the legume model plant Medicago truncatula. The ORF of the MtMTP1 cDNA encodes a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. Like other metal tolerance proteins (MTPs) in plants, heterologous expression of MtMTP1 can complement the Zn-susceptible zrc1 cot1 yeast double mutant. The expression pattern was studied by quantitative fluorescent PCR. The expression of MtMTP1 was detected in all vegetative organs with the highest level of expression observed in leaves. With Zn supplementation its expression in roots was reduced while its expression in stems was increased in the first 2 days. No obvious changes were detected in leaves. Inoculation with Rhizobium meliloti downregulated its expression in roots. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Medicago truncatula MTP gene Zinc Gene expression Rhizobium inoculation
1. Introduction Zn is an essential micronutrient in plants due to its important role in various physiological and metabolic processes. It is involved in the activity of more than 400 enzymes as a structural component [1], and regulates many transcriptional and signaling reactions through numerous Zn-finger proteins [2]. Zn deficiency is a widespread problem, particularly in high-pH soils with low Zn activity, and can lead to various severe symptoms in plants. However, excessive accumulation of Zn in plants causes toxicity by inhibition of root growth, decreased photosynthesis and other effects, probably via competition with Fe and Mg [3]. Thus, plants have developed a precise homeostatic system to control the uptake, transport, storage, and use of Zn. Membrane transporters play an important role in Zn homeostasis. Several kinds of zinc transport proteins have been identified, including the ZIP (ZRT, IRT-like protein) [4,5], MTP (metal tolerance protein) [6,7], HMA (heavy metal ATPase) [8,9], and ZIF (zincinduced facilitator) families [10]. The MTPs belong to the cation diffusion facilitator (CDF) family of proteins that are widespread in bacteria, fungi, plants, and animals. These proteins function in transporting various metal ions in the cytoplasm, including Zn, Mn, Fe, Ni, Cd and Co, to the outside of the cells or into the vacuole [11]. The first MTP gene identified in plants was ZAT1 in Arabidopsis thaliana, which was then renamed MTP1 [6]. The expression of * Corresponding author. Tel./fax: þ86 10 62733969. E-mail address: [email protected] (T. Wang). 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.08.006
AtMTP1 is detected in all organs, and is not affected by Zn supplementation [6,13]. The localization of the AtMTP1:GFP fusion protein in Arabidopsis protoplasts and cells showed that AtMTP1 is located at the vacuolar membrane [12,13]. Heterologous expression of the MTP1 protein can complement the Zn-sensitive mutant of Stylosanthes cerevisiae or Ralstonia metallidurans, and mediates the influx of Zn in Xenopus oocytes [12–14]. Plants transformed with AtMTP1 exhibit enhanced Zn resistance with increasing Zn concentration in roots, and knock-out plants show Zn hypersensitivity with reduced Zn content in stems and leaves [6,12]. These results suggest that AtMTP1 participates in Zn homeostasis by transporting Zn into the vacuole. Although there are twelve predicted CDF family members in A. thaliana [15], only two members have been identified; the other gene is AtMTP3, which can also complement the yeast zrc1 cot1 double mutant. However, AtMTP3 is primarily expressed in roots, and is strongly induced in epidermal and cortex cells of the root hair zone by high but non-toxic concentrations of Zn or Co, or Fe deficiency. Knock-out AtMTP3 mutants also show increased sensitivity, and the Zn content in above-ground organs is increased. These results demonstrate that the function of AtMTP3 differs from that of AtMTP1; it may act to mediate the exclusion of Zn from shoots upon Zn supplementation [16]. An AtMTP1 homolog, AhMTP1, has been identified in the closely related species Arabidopsis halleri, which is also a Zn hyperaccumulator plant. The expression of AhMTP1 is substantially higher than AtMTP1 in roots and leaves. Perhaps this is the reason for Zn tolerance in A. halleri [17]. Another well-studied MTP gene in plants is TgMTP1 from the metal hyperaccumulator Thlaspi goesingense. Interestingly, there
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are two variant transcripts. The difference between the two variants is a His-rich putative metal-binding domain that is spliced out in one but not the other. This can alter the metal specificities of the MTP transporters. In shoots the constitutive expression of TgMTP1 is higher than homologous genes in the non-accumulators A. thaliana, Thlaspi arvense, and Brassica juncea [18,19]. In the closely related metal hyperaccumulator species, Thlaspi caerulescens, TcMTP1 is mainly expressed in leaves and expression in highly Zn-tolerant accessions is much higher than in less Zn-tolerant accessions [20]. The MTP gene in hybrid poplar has been identified and named PtdMTP1 [7]. Expression of PtdMTP1 is detected in all organs, and is not regulated by Zn supplementation. Its overexpression can increase Zn tolerance in yeast and plants, and is considered to contribute to Zn sequestration in the vacuole. Three MTPs in Nicotiana glauca and Nicotiana tabacum have also been identified. Heterologous expression of each of these three genes complements Zn-susceptible yeast mutants [21]. Furthermore, the MTP gene ShMTP1 in Stylosanthes hamata, a tropical legume, has been identified. Its expression in yeast, however, enhances resistance to Mn, but not to Zn [22]. Legumes are second only to the Graminae in their importance to humans. Some species, including Medicago sativa (alfalfa), are considered a potential source of biomaterial for the removal and recovery of heavy metal ions. Alfalfa has been found to tolerate heavy metals and grow well in contaminated soils [23–25]. Further studies have shown that alfalfa has the ability to bind various heavy metals in contaminated waters, including Zn, and its shoot biomass can bind reasonably large amounts of heavy metals from aqueous solutions [26–28]. Previous study in hyperaccumulator T. goesingense and A. halleri showed the metal transporters played an important role in metal accumulation [17,20]. To date, only a few Zn transporters have been identified in legumes. Seven ZIP transporters in Medicago truncatula have been identified [29]. In soybeans, GmZIP1 is a symbiosis-specific zinc transporter [30]. Several ZIP Zn transporters have also been identified in Lotus japonicus (Cvitanich unpublished). Here we report the identification of a homologous MTP gene, MtMTP1, in M. truncatula. It can complement the zinc hypersensitivity yeast zrc1 cot1 double mutant. Expression of MtMTP1 is observed in all vegetative organs, especially the leaves. Expression is down-regulated in roots when supplemented with Zn or are inoculated with Rhizobium meliloti. 2. Results 2.1. MtMTP1 is a member of the zinc transporter CDF family A tBLASTx search of M. truncatula BAC sequences was carried out using AtMTP1 AT2G46800 as a model. An ORF that translated a protein sequence showed strong homology in the M. truncatula BAC clone mth1-63a17. Hence, the ORF cDNA was named MtMTP1. The MtMTP1 clone was 1221 bp in length and was predicted to encode a 45-kDa protein of 407 amino acids. Comparison with the BAC sequence demonstrated that the gene did not contain introns. The predicted protein sequence contained the conservative domains of MTPs, including a hydrophilic C-terminal domain, six transmembrane domains (TMs), a signature N-terminal sequence, and a His-rich region between TM4 and TM5. The MTP1 of M. sativa was nearly identical to MtMTP1 except for a few amino acids. The identity between the two proteins was as high as 94% (Fig. 1). The GenBank accession number of MtMTP1 has been assigned as FJ389717, and that of MsMTP1 as FJ389718. A phylogenetic tree was constructed using MtMTP1 and MsMTP1 plus the sequences of another 13 known or predicted plant MTPs (Fig. 2). All the plant
MTPs were highly conserved except in the His-rich region that was predicted to bind zinc ions. The predicted protein LjMTP1 from another leguminous model plant, L. japonicus, shared the highest sequence identity (73%) with MtMTP1. The identified protein PtdMTP1 had a shared identity of 69% with MtMTP1. The identity between MtMTP1 and the rest of other identified proteins was highly conservative, ranging between 55% and 65%. These results suggest that MtMTP1 can be considered a member of the zinc transporter CDF family. 2.2. MtMTP1 can restore Zn resistance in the yeast zrc1 cot1 double mutant The yeast mutant CM137 (Dzrc1::HIS3 Dcot1::KanR) was used in complementation experiments to test whether MtMTP1 functions in the transport of Zn. This yeast mutant is sensitive to high concentrations of Zn and Co due to a defect in vacuolar Zn and Co sequestration. The wild-type CM100 grew well on SC medium supplemented with 500 mM Zn, but the mutants CM137 and CM137 transformed with an empty pYES2 expression vector had no growth on the same medium. However, CM137 transformed with the pYES2 expression vector containing the MtMTP1 cDNA could grow on SC medium supplemented with 500 mM Zn (Fig. 3A). To examine Zn tolerance in more detail, liquid culture assays containing different concentrations of Zn were performed. The growth of CM137 rapidly declined with Zn supplementation, and was entirely inhibited with 100 mM Zn. The growth of CM137 transformed with pYES2-MtMTP1 slowly declined with increasing Zn concentrations until Zn concentration reached approximately 1000 mM (Fig. 3B). The same type of Co-tolerance liquid assays were carried out. Although some MTPs can enhance Co tolerance in the zrc1 cot1 yeast double mutant [8,17], MtMTP1 can’t enhance the tolerance. These results suggest that MtMTP1 can restore Zn resistance, but not Co tolerance in the yeast zrc1 cot1 double mutant. 2.3. The expression of MtMTP1 is regulated by Zn supplementation Quantitative fluorescent PCR was carried out to investigate the expression of MtMTP1 in different organs of M. truncatula plants fertilized with Zn (Fig. 4). Expression of MtMTP1 was observed in all vegetative organs. The level of expression was the highest in leaves – nearly twofold higher than was observed in roots, while the expression level in stems was slightly lower than that in roots. The expression of MtMTP1 was affected by Zn supplementation. Expression in roots declined with Zn supplementation, especially after 7 days. Twelve hours after Zn supplementation the expression in roots declined to almost 2/3 of the level of the expression without Zn supplementation and maintained a similar level of expression. After 7 days its expression further declined to 1/3 of expression level prior to Zn supplement. The 12-h Zn exposure caused the expression in stems to increase to as much as 1.5-fold of that without Zn supplementation, and in the next 2 days the expression in stems was kept at a similar level. After 7 days, however, it had declined back to a value close to the level observed prior to Zn supplementation. High levels of expression were maintained in leaves, with no obvious changes after Zn supplementation. This suggests that expression of MtMTP1 is regulated by Zn supplementation. 2.4. MtMTP1 is down-regulated in roots by Rhizobium inoculation To determine whether the expression of MtMTP1 was regulated by Rhizobium inoculation, quantitative fluorescent PCR was carried out to detect expression in roots, beginning 2 days after inoculation
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Fig. 1. Multiple alignment of the predicted amino acid sequence of MtMTP1 with other identified Zn-transport MTPs. The multiple alignment was performed with software DNAMAN5.2. Fully conserved residues are boxed in black while semi-conserved residues are boxed in gray. The six predicted TMs are shown as lines under the sequences. The GenBank accession numbers of aligned MTPs are: AtMTP1 (GenBank accession no. AAD11757), AtMTP3 (GenBank accession no. AM231755), PtdMTP1 (GenBank accession no. AAR23528), TgMTP1 (GenBank accession no. AAS67024) LjMTP1 (GenBank accession no. GQ1220914), MtMTP1 (GenBank accession no. FJ389717) and MsMTP1 (GenBank accession no. FJ389718).
with R. meliloti (Fig. 5). The expression on day 1 declined to 1/3 of the level of expression before inoculation, and the expression on day 2 remained at the level observed for day 1. MtMTP1 expression exhibited no obvious changes during the testing period for the control that had no Rhizobium inoculation. This suggests that MtMTP1 is down-regulated in roots by Rhizobium inoculation.
3. Discussion Here, we identified the CDF protein MtMTP1 in M. truncatula from the M. truncatula BAC clone mth1-63a17. The gene had no intron, and the ORF of the cDNA encoded a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. The
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Fig. 2. Phylogenetic tree of selected Zn-transport MTPs. Phylogenetic analysis was performed with ClustalW (www2.ebi.ac.uk/clustalw/). Species designations and corresponding GenBank accession numbers of MTPs are: AtMTP1 (GenBank accession no. AAD11757), AtMTP3 (GenBank accession no. AM231755), PtdMTP1 (GenBank accession no. AAR23528), TgMTP1 (GenBank accession no. AAS67024), Arabidopsis halleri AhMTP1 (GenBank accession no. CAD89013), Nicotiana glauca NgMTP1 (GenBank accession no. BAD89561), Nicotiana tabacum NtMTP1A (GenBank accession no. BAD89562), Cucumis sativus CsMTP1 (GenBank accession no. ABS12731), Brassica juncea BjMTP1 (GenBank accession no. AAO83659), Eucalyptus grandis EgMTP1 (GenBank accession no. AAL25646), Vitis vinifera VvMTP1 (GenBank accession no. CAN75453), Oryza sativa OsMTP1 (GenBank accession no. NM_001061074), and LjMTP1 (GenBank accession no. GQ1220914).
MTP1 from the closely related species M. sativa was nearly identical to MtMTP1, with the exception of a few amino acids. Sequence comparisons revealed that the MTPs in plants are highly evolutionarily conserved. The conserved domains in CDF families are readily found in the MTPs of plants [11]. Only the His-rich region exists in many variations. Blaudez et al. reported that a deletion of several amino acids in TM2 and TM6 causes a loss of the ability to transport Zn [7]. Our alignment studies showed that these same amino acids are highly conserved in all Zn-transport MTPs in plants.
Fig. 3. Zn resistance of the yeast zrc1 cot1 double mutant expressing MtMTP1. A, The yeast zrc1 cot1 double mutant CM137 was transformed with pYES2-MtMTP1. CM137 is a negative control, and CM100 (wild-type) is a positive control. Serially diluted yeast cultures were spotted on SC (2% galactose-ura) plates with 500 mM ZnCl2 (b) or no ZnCl2 (a). Plates were incubated for 3 days at 30 C. B, Zinc tolerance of yeast cells in liquid culture. CM137 transformed with MtMTP1 (triangles) or empty vector (squares), and CM100 (diamonds) were incubated in liquid SC (2% galactose-ura) medium supplemented with indicated concentration of ZnCl2. The density of culture was measured after culturing for 24 h at 30 C with shaking at 150 rpm. The OD600 of the initial density of cell cultures was 0.01.
Fig. 4. Expression of MtMTP1 in vegetative organs with Zn supplement. The expression in vegetative organs of four weeks old plants, without Zn supplement (black), with 30 mM ZnSO4 supplement for 12 h (gray), for 24 h (spot), for 48 h (vertical line) and for 7 days (white) were assessed by quantitative fluorescence PCR. DDCT Values were calculated as follows: DDCT ¼ [CT (MtMTP1) CT (TC106785)] [CT (MtMTP1CKroots) CT (TC106785CK roots)]. Relative transcript levels were calculated as follows: RTL ¼ 2DDCT. PCRs of each sample were performed in triplicate.
Like other MTPs identified in plant, MtMTP1 can also complement the yeast zrc1 cot1 double mutant to restore Zn resistance. However, the Zn resistance of the yeast mutant transformed with MtMTP1 is weaker than that of the mutant transformed with AtMTP1. The yeast mutant transformed with MtMTP1 can tolerate only 500 mM Zn. In comparison, the same mutant transformed with other MTP genes can tolerate 5000 mM Zn [7,16]. This might be due to the longer His-rich region in MtMTP1. The His-rich region is essential for transporting Zn, and its structure may change the MTPs’ ability to transport Zn. The yeast mutant transformed with TgMTP1 lacking the His-rich region does not survive when exposed to 500 mM Zn. Expression of the His-rich region alone does not produce Zn resistance, however [16]. A similar result was observed for MtMTP1 (data not shown). Deletion of the His-rich loop (32 amino acid residues from 185 to 216) from AtMTP1 results in enhancement of the ability to transport Zn [33]. Perhaps the function of the His-rich region is to bind Zn in the cytoplast for transport by the TMs. An extremely long His-rich region would result in keeping adequate concentrations of the cytoplasmic Zn. Quantitative fluorescent PCR analysis demonstrated that MtMTP1 is constitutively expressed in all vegetative organs. The same results have been reported for other MTP genes [7,13,16,17].
Fig. 5. Expression of MtMTP1 in roots at the beginning of inoculation. The seven-dayold seedlings grown in nitrogen-free HY medium were inoculated with Rhizobium meliloti at a density of 106. The expression in roots at the first 2 days were assessed by fluorescence quantitative PCR. Relative transcript levels were calculated as described in Fig. 4.
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The expression level of MtMTP1 in different organs varied with its expression level almost twofold higher in leaves than in roots and stems. The TcMTP1 gene from the well-known metal hyperaccumulator T. caerulescens also showed the highest expression in leaves [20]. One of the characteristics of metal hyperaccumulators is a high rate of root-to-shoot transport that results in the accumulation of high concentrations of heavy metals in shoots [34–36]. The closely related species M. sativa is considered a good organism for phytoremediation. Previous studies have shown that alfalfa shoot biomass can bind a significant amount of heavy metals, including Zn, from aqueous solutions [28]. The high expression of MTP may contribute to Zn accumulation in leaves. M. truncatula may have metal tolerance similar to that of M. sativa. Furthermore, the expression of MtMTP1 was regulated by Zn supplementation. While supplementation with Zn resulted in no obvious changes in expression in leaves, expression in roots declined to 1/3 of the expression level without Zn supplementation by the end of the experiment. This phenomenon has also been observed in T. caerulescens accessions with low Zn tolerance [20]. This suggests that there is a gap in Zn tolerance between M. truncatula and T. caerulescens. Expression in stems was increased from 12 h to 48 h after Zn supplementation, but declined back to the levels observed without Zn supplement after 7 days. This may be due to an initial reaction to Zn elevation. The expression results suggest that M. truncatula may be a Zn-tolerant plant similar to M. sativa; and that MtMTP1 takes part in Zn tolerance by accumulating Zn in leaves. The metal tolerance of M. truncatula should be further studied. The expression of MtMTP1 in roots inoculated with Rhizobium declined to 1/3 of that before inoculation after 2 days. Grewal found that adequate Zn nutrition may improve alfalfa nodulation on low Zn soils [37]. The soybean Zn-transport protein GmZIP1, detected only in nodules and not in vegetative organs, has been found to enhance the uptake of Zn in symbiosomes [30]. In barley, high affinity phosphate transporters in roots were up-regulated by Zn deficiency [38]. Increasing phosphorus content promoted nodule formation at low nitrate concentrations [39,40]. These results suggest that there is a relationship between Zn and nodulation. Zn may play an important role in nodulation through regulation of phosphorus content. 4. Materials and methods 4.1. Plant material and growth conditions M. truncatula Gaertn ‘Jemalong’ A17 (obtained from Dasharath Lohar) and M. sativa cv. Baoding were used for the experiments. Plants were grown in growth chambers at 20–25 C, 50–60% relative humidity, and a 16-h day with illumination at a light intensity of 200–400 mmol m2 s1. Seeds were chemically scarified with anhydrous sulfuric acid, sterilized in 3% active chlorine solution, washed with sterilized water and germinated on filter paper for 4 days. For hydroponic cultures, once M. truncatula seedlings had a root length of about 3 cm, the plantlets were transferred onto a raft floating on HY nutrient solution [31] mixed with an aquarium pump. All nutrient solutions were buffered by the addition of 1 mM MES, pH 5.5. The following environmental growth conditions were used: light/dark cycle 8 h/16 h, 22 C, 85% relative humidity, light intensity 250 mmol m2 s1. Nutrient solutions were renewed once a week. At the fourth week, plants were transferred into fresh nutrient solutions containing 30 mM Zn for 12 h, 24 h, 48 h or 7 days. At harvest, roots, stems and leaves were washed in deionized water and immediately frozen in liquid nitrogen. For rapid inoculation, M. truncatula seedlings were grown directly in paper pouches overlaid with nitrogen-free HY medium.
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At the seventh day, the seedlings were transferred to fresh nitrogen-free HY medium with 106 R. meliloti USDA1002T (obtained from Wenxin Chen) for 24 or 48 h. The environmental growth conditions were the same as for the hydroponic cultures. For more detailed information on inoculation, refer to the chapter ‘‘Rhizobial inoculation and nodulation of M. truncatula’’ in the Medicago Handbook. Available via DIALOG. http://www.noble. org/MedicagoHandbook/pdf/Rhizobial_Inoculation_Nodulation.pdf. At harvest, roots were washed in deionized water and immediately frozen in liquid nitrogen. 4.2. Cloning of MTP1 genes and computational analyses The AtMTP1-like sequence MtMTP1 was found using tBLASTx and M. truncatula bacterial artificial chromosome (BAC) sequences from the NSF Medicago Genome Program (www.medicago.org) using AtMTP1 as a model. M. truncatula RNA was isolated using an RNeasy Plant Mini Kit (Qiagen), treated with RNase-free DnaseI (Takara), and reverse transcribed to cDNA using M-MLV Reverse Transcriptase (Promega). Primers were designed (50 primer: 50 -CCGGAATTCATGGAAGCACAAAGCTCACCA-30 and 30 primer: 50 -CCATCGATTTAGCCCAATACCAAAGCCAT-30 ) to clone the open reading frame. The primers were also used to amplify the homologous gene from M. sativa cDNA. All PCRs used High Fidelity polymerase Probest (Takara). PCRs were carried out for 5 min at 95 C, followed by 35 cycles of 45 s at 95 C, 45 s at 55 C, and 90 s at 72 C, with a final extension period of 10 min at 72 C. The purified PCR fragments were ligated into the pMD18 vector (Takara) for sequencing. A homologous gene search was performed using the NCBI Blast Server (www.ncbi.nlm.nih.gov); the multiple sequence alignment was performed using DNAMAN5.2 software; and the phylogenetic tree of Zn-transport MTPs was constructed using Clustal W (www2. ebi.ac.uk/clustalw/). 4.3. Yeast complementation studies The plasmid pYES2 (Invitrogen) was used for yeast transformation. The open reading frame of MtMTP1 was excised with EcoRI and ClaI from pMD18 and subcloned into the vector. The final constructs were sequenced for verification. The yeast strain CM100 (wt) and the zinc-sensitive strain CM137 (Dzrc1::HIS3 Dcot1::KanR) [32] (obtained from David J. Eide) were used in this experiment. The constructed vectors were transformed into yeast strains as per the Invitrogen yeast handbook, and transformants were selected on SC-ura/2% glucose medium. For metal tolerance assays, yeast clones containing vectors were grown to an OD600 of 1.5 in liquid SC-ura/2% glucose medium. One milliliter of each culture was washed twice with sterilized water and inoculated into 50 ml fresh liquid SC-ura/2% galactose medium and shaken at 150 rpm for 24 h at 30 C. For the Zn-tolerance plate assay, cultures were adjusted to an OD600 of 1.0, and diluted to three concentrations: 1:10, 1:100, or 1:1000. Five microliters of each dilution were spotted onto SC-ura/2% galactose medium/2% agar plates with 500 mM ZnCl2 and grown for 3 days at 30 C. For Zn- and Co-tolerance assays in liquid medium, cultures were adjusted to an OD600 of 0.01 with SC-ura/2% galactose medium. Each 10-ml subculture was treated with appropriate Zn or Co concentrations, and cultured at 30 C with shaking at 150 rpm. The cultures treated with Zn were grown for 24 h while Co cultures were grown for 48 h. 4.4. Quantitative fluorescent PCR experiments RNA from different plant tissues from various treatments was isolated as described above. RNA was isolated from five plants of
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each treatment and the sample was pooled. Three micrograms of total RNA from each sample were reverse transcribed to cDNA as described above in a total volume of 20 ml. One microliter of the RT reaction was used for PCR. The MtMTP1-specific primers 50 -TGTGCACCATCATGACCATG-30 and 50 -CTTCCAGGATGTTTCTCAGCA-30 specifically amplified a 269-bp fragment. Primers specific for the constitutively expressed control gene M. truncatula b-actin (TC106785), 50 -AGCTACGAATTGCCTGATGG-30 and 50 -CTCATTCTATCAGCAATGCCTG-30 , specifically amplified a 227-bp fragment. The real-time PCRs were performed with EvaGreen (Biotium) in a volume of 25 ml. The PCR operation was performed on an SLAN real-time quantitative fluorescent PCR detection system (Huongshi). Relative transcript levels were calculated as follows: RTL ¼ 2DDCT. PCRs of each sample were performed in triplicate. Acknowledgements This work was supported by the Hi-Tech Research and Development (863) Program of China (2006AA10Z105, 2006AA100109). We thank Dr. Dasharath Lohar for providing seeds of M. truncatula Gaertn ‘Jemalong’ A17, Dr. David Eide for the CM100 and CM137 yeast strains, and Professor Wenxin Chen for Rhizobium meliloti strains.
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Chaney, Soil pH rhizobia, and vesicular–arbuscular mycorrhizae inoculation effects on growth and heavy metal uptake of alfalfa (Medicago sativa). Biol. Fertil. Soils 8 (1989) 61–68. L.J. Cajuste, R.G. Carrillo, E. Cota, R.J. Laird, The distribution of metals from waste water in the Mexican valley of mezquital. Water Air Soil Pollut. 57 (1991) 763–781. V.C. Baligar, T.A. Campbell, R.J. Wright, Differential Responses of alfalfa clones to aluminum-toxic acid soil. Plant Nutr. 16 (1993) 219–223. J.L. Gardea-Torresdey, K.J. Tiemann, J.H. Gonzalez, J.A. Henning, M.S. Towsend, Ability of silica-immobilized Medicago sativa (Alfalfa) to remove copper ions from solution. J. Hazard. Mater. 48 (1996) 181–190. J.L. Gardea-Torresdey, K.J. Tiemann, J.H. Gonzalez, J.A. Henning, M.S. Townsend, Uptake of copper ions from solution by different populations of Medicago Sativa (Alfalfa). Solv. Extract. Ion-Exch. 14 (1996) 119–140. J.L. Gardea-Torresdey, J.H. Gonzalez, K.J. Tiemann, O. Rodriguez, G. 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Research article
Identification and characterization of MtMTP1, a Zn transporter of CDF family, in the Medicago truncatula Mingliang Chen, Xiaoye Shen, Daofeng Li, Lei Ma, Jiangli Dong, Tao Wang* State Key Laboratory for Agro-Biotechnology, College of Biological Science, China Agricultural University, 100193 Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2008 Accepted 21 August 2009 Available online 4 September 2009
Zn is an essential micronutrient in plants, and the mechanisms of Zn homeostasis are under intensive study. In this report, we have identified MtMTP1, a Zn transporter of the CDF family in the legume model plant Medicago truncatula. The ORF of the MtMTP1 cDNA encodes a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. Like other metal tolerance proteins (MTPs) in plants, heterologous expression of MtMTP1 can complement the Zn-susceptible zrc1 cot1 yeast double mutant. The expression pattern was studied by quantitative fluorescent PCR. The expression of MtMTP1 was detected in all vegetative organs with the highest level of expression observed in leaves. With Zn supplementation its expression in roots was reduced while its expression in stems was increased in the first 2 days. No obvious changes were detected in leaves. Inoculation with Rhizobium meliloti downregulated its expression in roots. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Medicago truncatula MTP gene Zinc Gene expression Rhizobium inoculation
1. Introduction Zn is an essential micronutrient in plants due to its important role in various physiological and metabolic processes. It is involved in the activity of more than 400 enzymes as a structural component [1], and regulates many transcriptional and signaling reactions through numerous Zn-finger proteins [2]. Zn deficiency is a widespread problem, particularly in high-pH soils with low Zn activity, and can lead to various severe symptoms in plants. However, excessive accumulation of Zn in plants causes toxicity by inhibition of root growth, decreased photosynthesis and other effects, probably via competition with Fe and Mg [3]. Thus, plants have developed a precise homeostatic system to control the uptake, transport, storage, and use of Zn. Membrane transporters play an important role in Zn homeostasis. Several kinds of zinc transport proteins have been identified, including the ZIP (ZRT, IRT-like protein) [4,5], MTP (metal tolerance protein) [6,7], HMA (heavy metal ATPase) [8,9], and ZIF (zincinduced facilitator) families [10]. The MTPs belong to the cation diffusion facilitator (CDF) family of proteins that are widespread in bacteria, fungi, plants, and animals. These proteins function in transporting various metal ions in the cytoplasm, including Zn, Mn, Fe, Ni, Cd and Co, to the outside of the cells or into the vacuole [11]. The first MTP gene identified in plants was ZAT1 in Arabidopsis thaliana, which was then renamed MTP1 [6]. The expression of * Corresponding author. Tel./fax: þ86 10 62733969. E-mail address: [email protected] (T. Wang). 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.08.006
AtMTP1 is detected in all organs, and is not affected by Zn supplementation [6,13]. The localization of the AtMTP1:GFP fusion protein in Arabidopsis protoplasts and cells showed that AtMTP1 is located at the vacuolar membrane [12,13]. Heterologous expression of the MTP1 protein can complement the Zn-sensitive mutant of Stylosanthes cerevisiae or Ralstonia metallidurans, and mediates the influx of Zn in Xenopus oocytes [12–14]. Plants transformed with AtMTP1 exhibit enhanced Zn resistance with increasing Zn concentration in roots, and knock-out plants show Zn hypersensitivity with reduced Zn content in stems and leaves [6,12]. These results suggest that AtMTP1 participates in Zn homeostasis by transporting Zn into the vacuole. Although there are twelve predicted CDF family members in A. thaliana [15], only two members have been identified; the other gene is AtMTP3, which can also complement the yeast zrc1 cot1 double mutant. However, AtMTP3 is primarily expressed in roots, and is strongly induced in epidermal and cortex cells of the root hair zone by high but non-toxic concentrations of Zn or Co, or Fe deficiency. Knock-out AtMTP3 mutants also show increased sensitivity, and the Zn content in above-ground organs is increased. These results demonstrate that the function of AtMTP3 differs from that of AtMTP1; it may act to mediate the exclusion of Zn from shoots upon Zn supplementation [16]. An AtMTP1 homolog, AhMTP1, has been identified in the closely related species Arabidopsis halleri, which is also a Zn hyperaccumulator plant. The expression of AhMTP1 is substantially higher than AtMTP1 in roots and leaves. Perhaps this is the reason for Zn tolerance in A. halleri [17]. Another well-studied MTP gene in plants is TgMTP1 from the metal hyperaccumulator Thlaspi goesingense. Interestingly, there
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are two variant transcripts. The difference between the two variants is a His-rich putative metal-binding domain that is spliced out in one but not the other. This can alter the metal specificities of the MTP transporters. In shoots the constitutive expression of TgMTP1 is higher than homologous genes in the non-accumulators A. thaliana, Thlaspi arvense, and Brassica juncea [18,19]. In the closely related metal hyperaccumulator species, Thlaspi caerulescens, TcMTP1 is mainly expressed in leaves and expression in highly Zn-tolerant accessions is much higher than in less Zn-tolerant accessions [20]. The MTP gene in hybrid poplar has been identified and named PtdMTP1 [7]. Expression of PtdMTP1 is detected in all organs, and is not regulated by Zn supplementation. Its overexpression can increase Zn tolerance in yeast and plants, and is considered to contribute to Zn sequestration in the vacuole. Three MTPs in Nicotiana glauca and Nicotiana tabacum have also been identified. Heterologous expression of each of these three genes complements Zn-susceptible yeast mutants [21]. Furthermore, the MTP gene ShMTP1 in Stylosanthes hamata, a tropical legume, has been identified. Its expression in yeast, however, enhances resistance to Mn, but not to Zn [22]. Legumes are second only to the Graminae in their importance to humans. Some species, including Medicago sativa (alfalfa), are considered a potential source of biomaterial for the removal and recovery of heavy metal ions. Alfalfa has been found to tolerate heavy metals and grow well in contaminated soils [23–25]. Further studies have shown that alfalfa has the ability to bind various heavy metals in contaminated waters, including Zn, and its shoot biomass can bind reasonably large amounts of heavy metals from aqueous solutions [26–28]. Previous study in hyperaccumulator T. goesingense and A. halleri showed the metal transporters played an important role in metal accumulation [17,20]. To date, only a few Zn transporters have been identified in legumes. Seven ZIP transporters in Medicago truncatula have been identified [29]. In soybeans, GmZIP1 is a symbiosis-specific zinc transporter [30]. Several ZIP Zn transporters have also been identified in Lotus japonicus (Cvitanich unpublished). Here we report the identification of a homologous MTP gene, MtMTP1, in M. truncatula. It can complement the zinc hypersensitivity yeast zrc1 cot1 double mutant. Expression of MtMTP1 is observed in all vegetative organs, especially the leaves. Expression is down-regulated in roots when supplemented with Zn or are inoculated with Rhizobium meliloti. 2. Results 2.1. MtMTP1 is a member of the zinc transporter CDF family A tBLASTx search of M. truncatula BAC sequences was carried out using AtMTP1 AT2G46800 as a model. An ORF that translated a protein sequence showed strong homology in the M. truncatula BAC clone mth1-63a17. Hence, the ORF cDNA was named MtMTP1. The MtMTP1 clone was 1221 bp in length and was predicted to encode a 45-kDa protein of 407 amino acids. Comparison with the BAC sequence demonstrated that the gene did not contain introns. The predicted protein sequence contained the conservative domains of MTPs, including a hydrophilic C-terminal domain, six transmembrane domains (TMs), a signature N-terminal sequence, and a His-rich region between TM4 and TM5. The MTP1 of M. sativa was nearly identical to MtMTP1 except for a few amino acids. The identity between the two proteins was as high as 94% (Fig. 1). The GenBank accession number of MtMTP1 has been assigned as FJ389717, and that of MsMTP1 as FJ389718. A phylogenetic tree was constructed using MtMTP1 and MsMTP1 plus the sequences of another 13 known or predicted plant MTPs (Fig. 2). All the plant
MTPs were highly conserved except in the His-rich region that was predicted to bind zinc ions. The predicted protein LjMTP1 from another leguminous model plant, L. japonicus, shared the highest sequence identity (73%) with MtMTP1. The identified protein PtdMTP1 had a shared identity of 69% with MtMTP1. The identity between MtMTP1 and the rest of other identified proteins was highly conservative, ranging between 55% and 65%. These results suggest that MtMTP1 can be considered a member of the zinc transporter CDF family. 2.2. MtMTP1 can restore Zn resistance in the yeast zrc1 cot1 double mutant The yeast mutant CM137 (Dzrc1::HIS3 Dcot1::KanR) was used in complementation experiments to test whether MtMTP1 functions in the transport of Zn. This yeast mutant is sensitive to high concentrations of Zn and Co due to a defect in vacuolar Zn and Co sequestration. The wild-type CM100 grew well on SC medium supplemented with 500 mM Zn, but the mutants CM137 and CM137 transformed with an empty pYES2 expression vector had no growth on the same medium. However, CM137 transformed with the pYES2 expression vector containing the MtMTP1 cDNA could grow on SC medium supplemented with 500 mM Zn (Fig. 3A). To examine Zn tolerance in more detail, liquid culture assays containing different concentrations of Zn were performed. The growth of CM137 rapidly declined with Zn supplementation, and was entirely inhibited with 100 mM Zn. The growth of CM137 transformed with pYES2-MtMTP1 slowly declined with increasing Zn concentrations until Zn concentration reached approximately 1000 mM (Fig. 3B). The same type of Co-tolerance liquid assays were carried out. Although some MTPs can enhance Co tolerance in the zrc1 cot1 yeast double mutant [8,17], MtMTP1 can’t enhance the tolerance. These results suggest that MtMTP1 can restore Zn resistance, but not Co tolerance in the yeast zrc1 cot1 double mutant. 2.3. The expression of MtMTP1 is regulated by Zn supplementation Quantitative fluorescent PCR was carried out to investigate the expression of MtMTP1 in different organs of M. truncatula plants fertilized with Zn (Fig. 4). Expression of MtMTP1 was observed in all vegetative organs. The level of expression was the highest in leaves – nearly twofold higher than was observed in roots, while the expression level in stems was slightly lower than that in roots. The expression of MtMTP1 was affected by Zn supplementation. Expression in roots declined with Zn supplementation, especially after 7 days. Twelve hours after Zn supplementation the expression in roots declined to almost 2/3 of the level of the expression without Zn supplementation and maintained a similar level of expression. After 7 days its expression further declined to 1/3 of expression level prior to Zn supplement. The 12-h Zn exposure caused the expression in stems to increase to as much as 1.5-fold of that without Zn supplementation, and in the next 2 days the expression in stems was kept at a similar level. After 7 days, however, it had declined back to a value close to the level observed prior to Zn supplementation. High levels of expression were maintained in leaves, with no obvious changes after Zn supplementation. This suggests that expression of MtMTP1 is regulated by Zn supplementation. 2.4. MtMTP1 is down-regulated in roots by Rhizobium inoculation To determine whether the expression of MtMTP1 was regulated by Rhizobium inoculation, quantitative fluorescent PCR was carried out to detect expression in roots, beginning 2 days after inoculation
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Fig. 1. Multiple alignment of the predicted amino acid sequence of MtMTP1 with other identified Zn-transport MTPs. The multiple alignment was performed with software DNAMAN5.2. Fully conserved residues are boxed in black while semi-conserved residues are boxed in gray. The six predicted TMs are shown as lines under the sequences. The GenBank accession numbers of aligned MTPs are: AtMTP1 (GenBank accession no. AAD11757), AtMTP3 (GenBank accession no. AM231755), PtdMTP1 (GenBank accession no. AAR23528), TgMTP1 (GenBank accession no. AAS67024) LjMTP1 (GenBank accession no. GQ1220914), MtMTP1 (GenBank accession no. FJ389717) and MsMTP1 (GenBank accession no. FJ389718).
with R. meliloti (Fig. 5). The expression on day 1 declined to 1/3 of the level of expression before inoculation, and the expression on day 2 remained at the level observed for day 1. MtMTP1 expression exhibited no obvious changes during the testing period for the control that had no Rhizobium inoculation. This suggests that MtMTP1 is down-regulated in roots by Rhizobium inoculation.
3. Discussion Here, we identified the CDF protein MtMTP1 in M. truncatula from the M. truncatula BAC clone mth1-63a17. The gene had no intron, and the ORF of the cDNA encoded a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. The
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Fig. 2. Phylogenetic tree of selected Zn-transport MTPs. Phylogenetic analysis was performed with ClustalW (www2.ebi.ac.uk/clustalw/). Species designations and corresponding GenBank accession numbers of MTPs are: AtMTP1 (GenBank accession no. AAD11757), AtMTP3 (GenBank accession no. AM231755), PtdMTP1 (GenBank accession no. AAR23528), TgMTP1 (GenBank accession no. AAS67024), Arabidopsis halleri AhMTP1 (GenBank accession no. CAD89013), Nicotiana glauca NgMTP1 (GenBank accession no. BAD89561), Nicotiana tabacum NtMTP1A (GenBank accession no. BAD89562), Cucumis sativus CsMTP1 (GenBank accession no. ABS12731), Brassica juncea BjMTP1 (GenBank accession no. AAO83659), Eucalyptus grandis EgMTP1 (GenBank accession no. AAL25646), Vitis vinifera VvMTP1 (GenBank accession no. CAN75453), Oryza sativa OsMTP1 (GenBank accession no. NM_001061074), and LjMTP1 (GenBank accession no. GQ1220914).
MTP1 from the closely related species M. sativa was nearly identical to MtMTP1, with the exception of a few amino acids. Sequence comparisons revealed that the MTPs in plants are highly evolutionarily conserved. The conserved domains in CDF families are readily found in the MTPs of plants [11]. Only the His-rich region exists in many variations. Blaudez et al. reported that a deletion of several amino acids in TM2 and TM6 causes a loss of the ability to transport Zn [7]. Our alignment studies showed that these same amino acids are highly conserved in all Zn-transport MTPs in plants.
Fig. 3. Zn resistance of the yeast zrc1 cot1 double mutant expressing MtMTP1. A, The yeast zrc1 cot1 double mutant CM137 was transformed with pYES2-MtMTP1. CM137 is a negative control, and CM100 (wild-type) is a positive control. Serially diluted yeast cultures were spotted on SC (2% galactose-ura) plates with 500 mM ZnCl2 (b) or no ZnCl2 (a). Plates were incubated for 3 days at 30 C. B, Zinc tolerance of yeast cells in liquid culture. CM137 transformed with MtMTP1 (triangles) or empty vector (squares), and CM100 (diamonds) were incubated in liquid SC (2% galactose-ura) medium supplemented with indicated concentration of ZnCl2. The density of culture was measured after culturing for 24 h at 30 C with shaking at 150 rpm. The OD600 of the initial density of cell cultures was 0.01.
Fig. 4. Expression of MtMTP1 in vegetative organs with Zn supplement. The expression in vegetative organs of four weeks old plants, without Zn supplement (black), with 30 mM ZnSO4 supplement for 12 h (gray), for 24 h (spot), for 48 h (vertical line) and for 7 days (white) were assessed by quantitative fluorescence PCR. DDCT Values were calculated as follows: DDCT ¼ [CT (MtMTP1) CT (TC106785)] [CT (MtMTP1CKroots) CT (TC106785CK roots)]. Relative transcript levels were calculated as follows: RTL ¼ 2DDCT. PCRs of each sample were performed in triplicate.
Like other MTPs identified in plant, MtMTP1 can also complement the yeast zrc1 cot1 double mutant to restore Zn resistance. However, the Zn resistance of the yeast mutant transformed with MtMTP1 is weaker than that of the mutant transformed with AtMTP1. The yeast mutant transformed with MtMTP1 can tolerate only 500 mM Zn. In comparison, the same mutant transformed with other MTP genes can tolerate 5000 mM Zn [7,16]. This might be due to the longer His-rich region in MtMTP1. The His-rich region is essential for transporting Zn, and its structure may change the MTPs’ ability to transport Zn. The yeast mutant transformed with TgMTP1 lacking the His-rich region does not survive when exposed to 500 mM Zn. Expression of the His-rich region alone does not produce Zn resistance, however [16]. A similar result was observed for MtMTP1 (data not shown). Deletion of the His-rich loop (32 amino acid residues from 185 to 216) from AtMTP1 results in enhancement of the ability to transport Zn [33]. Perhaps the function of the His-rich region is to bind Zn in the cytoplast for transport by the TMs. An extremely long His-rich region would result in keeping adequate concentrations of the cytoplasmic Zn. Quantitative fluorescent PCR analysis demonstrated that MtMTP1 is constitutively expressed in all vegetative organs. The same results have been reported for other MTP genes [7,13,16,17].
Fig. 5. Expression of MtMTP1 in roots at the beginning of inoculation. The seven-dayold seedlings grown in nitrogen-free HY medium were inoculated with Rhizobium meliloti at a density of 106. The expression in roots at the first 2 days were assessed by fluorescence quantitative PCR. Relative transcript levels were calculated as described in Fig. 4.
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The expression level of MtMTP1 in different organs varied with its expression level almost twofold higher in leaves than in roots and stems. The TcMTP1 gene from the well-known metal hyperaccumulator T. caerulescens also showed the highest expression in leaves [20]. One of the characteristics of metal hyperaccumulators is a high rate of root-to-shoot transport that results in the accumulation of high concentrations of heavy metals in shoots [34–36]. The closely related species M. sativa is considered a good organism for phytoremediation. Previous studies have shown that alfalfa shoot biomass can bind a significant amount of heavy metals, including Zn, from aqueous solutions [28]. The high expression of MTP may contribute to Zn accumulation in leaves. M. truncatula may have metal tolerance similar to that of M. sativa. Furthermore, the expression of MtMTP1 was regulated by Zn supplementation. While supplementation with Zn resulted in no obvious changes in expression in leaves, expression in roots declined to 1/3 of the expression level without Zn supplementation by the end of the experiment. This phenomenon has also been observed in T. caerulescens accessions with low Zn tolerance [20]. This suggests that there is a gap in Zn tolerance between M. truncatula and T. caerulescens. Expression in stems was increased from 12 h to 48 h after Zn supplementation, but declined back to the levels observed without Zn supplement after 7 days. This may be due to an initial reaction to Zn elevation. The expression results suggest that M. truncatula may be a Zn-tolerant plant similar to M. sativa; and that MtMTP1 takes part in Zn tolerance by accumulating Zn in leaves. The metal tolerance of M. truncatula should be further studied. The expression of MtMTP1 in roots inoculated with Rhizobium declined to 1/3 of that before inoculation after 2 days. Grewal found that adequate Zn nutrition may improve alfalfa nodulation on low Zn soils [37]. The soybean Zn-transport protein GmZIP1, detected only in nodules and not in vegetative organs, has been found to enhance the uptake of Zn in symbiosomes [30]. In barley, high affinity phosphate transporters in roots were up-regulated by Zn deficiency [38]. Increasing phosphorus content promoted nodule formation at low nitrate concentrations [39,40]. These results suggest that there is a relationship between Zn and nodulation. Zn may play an important role in nodulation through regulation of phosphorus content. 4. Materials and methods 4.1. Plant material and growth conditions M. truncatula Gaertn ‘Jemalong’ A17 (obtained from Dasharath Lohar) and M. sativa cv. Baoding were used for the experiments. Plants were grown in growth chambers at 20–25 C, 50–60% relative humidity, and a 16-h day with illumination at a light intensity of 200–400 mmol m2 s1. Seeds were chemically scarified with anhydrous sulfuric acid, sterilized in 3% active chlorine solution, washed with sterilized water and germinated on filter paper for 4 days. For hydroponic cultures, once M. truncatula seedlings had a root length of about 3 cm, the plantlets were transferred onto a raft floating on HY nutrient solution [31] mixed with an aquarium pump. All nutrient solutions were buffered by the addition of 1 mM MES, pH 5.5. The following environmental growth conditions were used: light/dark cycle 8 h/16 h, 22 C, 85% relative humidity, light intensity 250 mmol m2 s1. Nutrient solutions were renewed once a week. At the fourth week, plants were transferred into fresh nutrient solutions containing 30 mM Zn for 12 h, 24 h, 48 h or 7 days. At harvest, roots, stems and leaves were washed in deionized water and immediately frozen in liquid nitrogen. For rapid inoculation, M. truncatula seedlings were grown directly in paper pouches overlaid with nitrogen-free HY medium.
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At the seventh day, the seedlings were transferred to fresh nitrogen-free HY medium with 106 R. meliloti USDA1002T (obtained from Wenxin Chen) for 24 or 48 h. The environmental growth conditions were the same as for the hydroponic cultures. For more detailed information on inoculation, refer to the chapter ‘‘Rhizobial inoculation and nodulation of M. truncatula’’ in the Medicago Handbook. Available via DIALOG. http://www.noble. org/MedicagoHandbook/pdf/Rhizobial_Inoculation_Nodulation.pdf. At harvest, roots were washed in deionized water and immediately frozen in liquid nitrogen. 4.2. Cloning of MTP1 genes and computational analyses The AtMTP1-like sequence MtMTP1 was found using tBLASTx and M. truncatula bacterial artificial chromosome (BAC) sequences from the NSF Medicago Genome Program (www.medicago.org) using AtMTP1 as a model. M. truncatula RNA was isolated using an RNeasy Plant Mini Kit (Qiagen), treated with RNase-free DnaseI (Takara), and reverse transcribed to cDNA using M-MLV Reverse Transcriptase (Promega). Primers were designed (50 primer: 50 -CCGGAATTCATGGAAGCACAAAGCTCACCA-30 and 30 primer: 50 -CCATCGATTTAGCCCAATACCAAAGCCAT-30 ) to clone the open reading frame. The primers were also used to amplify the homologous gene from M. sativa cDNA. All PCRs used High Fidelity polymerase Probest (Takara). PCRs were carried out for 5 min at 95 C, followed by 35 cycles of 45 s at 95 C, 45 s at 55 C, and 90 s at 72 C, with a final extension period of 10 min at 72 C. The purified PCR fragments were ligated into the pMD18 vector (Takara) for sequencing. A homologous gene search was performed using the NCBI Blast Server (www.ncbi.nlm.nih.gov); the multiple sequence alignment was performed using DNAMAN5.2 software; and the phylogenetic tree of Zn-transport MTPs was constructed using Clustal W (www2. ebi.ac.uk/clustalw/). 4.3. Yeast complementation studies The plasmid pYES2 (Invitrogen) was used for yeast transformation. The open reading frame of MtMTP1 was excised with EcoRI and ClaI from pMD18 and subcloned into the vector. The final constructs were sequenced for verification. The yeast strain CM100 (wt) and the zinc-sensitive strain CM137 (Dzrc1::HIS3 Dcot1::KanR) [32] (obtained from David J. Eide) were used in this experiment. The constructed vectors were transformed into yeast strains as per the Invitrogen yeast handbook, and transformants were selected on SC-ura/2% glucose medium. For metal tolerance assays, yeast clones containing vectors were grown to an OD600 of 1.5 in liquid SC-ura/2% glucose medium. One milliliter of each culture was washed twice with sterilized water and inoculated into 50 ml fresh liquid SC-ura/2% galactose medium and shaken at 150 rpm for 24 h at 30 C. For the Zn-tolerance plate assay, cultures were adjusted to an OD600 of 1.0, and diluted to three concentrations: 1:10, 1:100, or 1:1000. Five microliters of each dilution were spotted onto SC-ura/2% galactose medium/2% agar plates with 500 mM ZnCl2 and grown for 3 days at 30 C. For Zn- and Co-tolerance assays in liquid medium, cultures were adjusted to an OD600 of 0.01 with SC-ura/2% galactose medium. Each 10-ml subculture was treated with appropriate Zn or Co concentrations, and cultured at 30 C with shaking at 150 rpm. The cultures treated with Zn were grown for 24 h while Co cultures were grown for 48 h. 4.4. Quantitative fluorescent PCR experiments RNA from different plant tissues from various treatments was isolated as described above. RNA was isolated from five plants of
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each treatment and the sample was pooled. Three micrograms of total RNA from each sample were reverse transcribed to cDNA as described above in a total volume of 20 ml. One microliter of the RT reaction was used for PCR. The MtMTP1-specific primers 50 -TGTGCACCATCATGACCATG-30 and 50 -CTTCCAGGATGTTTCTCAGCA-30 specifically amplified a 269-bp fragment. Primers specific for the constitutively expressed control gene M. truncatula b-actin (TC106785), 50 -AGCTACGAATTGCCTGATGG-30 and 50 -CTCATTCTATCAGCAATGCCTG-30 , specifically amplified a 227-bp fragment. The real-time PCRs were performed with EvaGreen (Biotium) in a volume of 25 ml. The PCR operation was performed on an SLAN real-time quantitative fluorescent PCR detection system (Huongshi). Relative transcript levels were calculated as follows: RTL ¼ 2DDCT. PCRs of each sample were performed in triplicate. Acknowledgements This work was supported by the Hi-Tech Research and Development (863) Program of China (2006AA10Z105, 2006AA100109). We thank Dr. Dasharath Lohar for providing seeds of M. truncatula Gaertn ‘Jemalong’ A17, Dr. David Eide for the CM100 and CM137 yeast strains, and Professor Wenxin Chen for Rhizobium meliloti strains.
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