JXB Advance Access originally published online on September 6, 2006
Journal of Experimental Botany 2006 57(14):3575-3582; doi:10.1093/jxb/erl102
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RESEARCH PAPER |
Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings
1Northeast Forestry, University Key Laboratory of Forest Plant Ecology, Ministry of Education, Hexing Road 26, Harbin 150040, PR China
2HIP, INF 360, D-69120-Heidelberg, Germany
* To whom correspondence should be addressed. E-mail: zan{at}hip.uni-hd.de
Received 21 September 2005; Accepted 29 June 2006
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Abstract |
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The protective function of a plant type-2 metallothionein was analysed after expression in Escherichia coli and in Arabidopsis thaliana seedlings. BjMT2 from Brassica juncea was expressed in E. coli as a TrxA::BjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. Escherichia coli cells expressing the TrxA::BjMT2 fusion were more tolerant to Cu2+ and Cd2+ exposure than control strains. Likewise, when BjMT2 cDNA was expressed in A. thaliana under the regulation of the 35S promoter, seedlings exhibited an increased tolerance against Cu2+ and Cd2+ based on shoot growth and chlorophyll content. Analysis of transiently transformed cells of A. thaliana and tobacco leaves by confocal laser scanning microscopy (CLSM) revealed exclusive cytosolic localization of a BjMT2::EGFP (enhanced green fluorescent protein) fusion protein in control and heavy metal-exposed plant cells. Remarkably, ectopic expression of BjMT2 reduced root growth in the absence of heavy metal exposure, whereas in the presence of 50 or 100 µM Cu2+ root growth in control and transgenic lines was identical. The results indicate that in A. thaliana, root and shoot development are differentially affected by ectopic expression of BjMT2.
Key words: Arabidopsis thaliana, BjMT2::EGFP, cadmium (Cd), compartmentation, copper (Cu), Escherichia coli, expression, metallothionein 2, root growth, tobacco
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Introduction |
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Copper and zinc are essential micronutrients for most organisms, but are cytotoxic at higher concentrations. Higher plants extract these ions with their root system from the soil and, after allocation via the xylem to different target tissues, incorporate these ions as cofactors into different enzymes or regulatory proteins (Cobbett and Goldsbrough, 2002; Hall, 2002). During senescence, copper and zinc ions become remobilized and redistributed within the plant via the phloem system. During this entire process of uptake, transport, and reversible incorporation into proteins, the cytosolic levels of these heavy metal ions have to be kept below the toxicity threshold (Cobbett and Goldsbrough, 2002).
In eukaryotic organisms, several specialized peptides are involved in heavy metal ion homeostasis and detoxification. To achieve these functions, higher plants make use of two types of peptides: the protein family of gene-encoded metallothioneins (MTs; Zhou and Goldsbrough, 1994; Murphy et al., 1997) and the phytochelatins (PCs; Cobbett, 2000), which are enzymatically formed from glutathione. While MTs are thought to be primarily involved in cellular copper (and perhaps zinc) homeostasis, PCs are particularly effective in detoxifying cadmium.
For higher plants, the presence of multiple MT isoforms has been demonstrated (Zhou and Goldsbrough, 1994; Schäfer et al., 1998; Guo et al., 2003). For the genome of Arabidopsis thaliana, seven putative MT genes have been annotated (plus one pseudogene), including the previously characterized genes AtMT1, AtMT2a/2b, and AtMT3 (Guo et al., 2003). Expression studies with promoter–ß-glucuronidase (GUS) plants have revealed that the different MT isoforms exhibit overlapping expression patterns, pointing to partial functional redundancy (Garcia-Hernandez et al., 1998; Guo et al., 2003). Plant MT1 and MT2 genes have been shown to complement MT-deficient yeast (cup1D), demonstrating their function in copper detoxification (Zhou and Goldsbrough, 1994); expression of plant MT1 and MT2 in the yeast mutant also increased cadmium tolerance. Likewise, expression of plant MTs in Escherichia coli led to increased tolerance towards copper and cadmium (Kille et al., 1991; Evans et al., 1992). Based on these findings, plant MTs are thought to play a major role in cellular copper homeostasis during plant development.
Although it is widely accepted that MTs play an important role for copper (and possibly zinc) homeostasis, only a few studies have further explored their in vivo function in plants. Progress was hampered by the difficulty in monitoring MT protein expression, these cysteine-rich proteins being extremely labile (Murphy et al., 1997). In 1992, Evans et al. for the first time overexpressed an MT-like gene from pea, PsMTA, in E. coli and A. thaliana. For both organisms, a significantly increased accumulation of copper was reported (Evans et al., 1992). When an MT2 gene from A. thaliana was expressed in a Synechocystis mutant deficient in its Zn2+-metallothionein gene smtA, a partial complementation was achieved, pointing to a possible role for MT2 in plant zinc homeostasis (Robinson et al., 1996). More recently, Lee et al. (2004) transiently expressed MT2a and MT3 of A. thaliana in Vicia faba guard cells and observed an enhanced resistance to cadmium.
In the present study, the role of MT2 for copper tolerance in plants has been readdressed. In particular, the effect of MT2 overexpression on seedling growth and development in the absence or presence of toxic copper levels was analysed, an important aspect not yet addressed in previous studies. For this purpose, an MT2 protein from Brassica juncea, BjMT2, was overexpressed in A. thaliana under the control of the 35S promoter. Following proof of function of BjMT2 in E. coli, shoot and root development of transgenic A. thaliana seedlings was analysed. The constitutive overexpression of BjMT2 differentially affected shoot and root development. Transient transformation of plant cells with a BjMT2::EGFP (enhanced green fluorescent protein) fusion protein indicated that heavy metal exposure did not affect the subcellular distribution of BjMT2 protein, the protein remaining evenly distributed throughout the cytoplasm.
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Materials and methods |
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Cloning of TrxA::BjMT2 for expression in E. coli
The coding region of the BjMT2 cDNA (Y10853 [GenBank] ; Schäfer et al., 1998) was amplified by polymerase chain reaction (PCR) with oligonucleotide primers, containing a KpnI and a NcoI site, respectively: sense primer 5'-GACCATGGCTTGCTGTGGAGGAAACTGTGG-3'; antisense primer 5'-GGGGTACCGTGGTTATCTATTTGCAGG-3' (restriction sites underlined). The resulting PCR fragment was digested with KpnI and NcoI and gel purified before ligation into the same sites of the pETM20 vector. In this vector, the introduced BjMT2-encoding sequence is fused at its N-terminus with TrxA (including a 6x His tag) and a cleavage site for tobacco etch virus (TEV) protease. The resulting TrxA::BjMT2-encoding plasmid was transformed into E. coli cells (strain Rosetta-gamiTM).
Expression and purification of TrxA::BjMT2 fusion protein
Escherichia coli cells transformed with the TrxA::BjMT2-encoding plasmid were inoculated into 5 ml of LB (Luria–Bertani) medium supplemented with ampicillin and chloramphenicol, and shaken at 180 rpm and 37 °C. These overnight cultures were then transferred into 45 ml of LB medium with antibiotics and shaken at 180 rpm, at 37 °C, until the OD600nm reached 0.6–0.8. Thereafter, cells were cooled down to 22 °C, and expression of the TrxA::BjMT2 fusion protein was induced by addition of 0.5 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) (at this temperature, a maximum yield of soluble BjMT2 protein was obtained as compared with BjMT2 protein in inclusion bodies). Escherichia coli cells were shaken at 180 rpm, at 22 °C, for 18–22 h. Cells were collected by centrifugation at 12 000 rpm for 5 min. About 0.25 g of bacterial pellet was then resuspended in 5 ml of TRIS–HCl buffer (50 mM, pH 8), and cells were disrupted by sonication. The resulting suspension was centrifuged again at 12 000 rpm for 15 min. The supernatant was mixed with 1 ml of Ni-agarose (Quiagen, Hilden, Germany), gently shaken at 4 °C for 1 h, and then transferred into a column, followed by washing with buffer containing increasing concentrations of imidazole. The TrxA::BjMT2 fusion protein was eluted with buffer containing 250 mM imidazole.
BjMT2 protein was obtained from the TrxA::BjMT2 fusion protein by cleavage with TEV protease. Prior to cleavage, the fusion protein was dialysed overnight against 50 mM Na-Pi buffer, 200 mM NaCl, pH 8.0, to remove imidazole. After dialysis, the fusion protein was mixed with TEV protease at an approximate ratio of 10:1, and incubated at 30 °C for 3–4 h. Thereafter, the mixture was incubated again with Ni-agarose and shaken gently at 4 °C for 1 h. Since TEV protease and the cleaved-off TrxA domain both contain 6x His tags, the pure BjMT2 protein was recovered in the flow-through fraction.
Labelling of TrxA::BjMT2 and BjMT2 with the thiol reagent monobromobimane (MBB)
For both recombinant proteins, TrxA::BjMT2 and BjMT2, 20 µl (
5 µg of protein) were incubated with 5 µl of 500 mM CHES buffer (pH 8.4), 5 µl of 10 mM dithiothreitol (DTT), 10 µl of 30 mM MBB, and 10 µl of H2O. After mixing and incubation at room temperature for 15 min, 20 µl of each mixture was subjected to SDS–PAGE, using non-reducing sample buffer. After PAGE, the gel was examined under UV light.
Bacterial growth of TrxA::BjMT2-expressing E. coli cells in the presence of copper or cadmium
To address the effect of BjMT2 expression in E. coli, bacterial growth of non-transformed E. coli cells (strain Rosetta-gamiTM) was compared with cells of the same strain bearing either the empty pETM20 vector or the TrxA::BjMT2-encoding plasmid (pETM20 vector with RAGE protein replaced by BjMT2). Each strain was separately inoculated (OD600nm=0.1) into 50 ml of LB medium with appropriate antibiotics, 0.5 mM IPTG, and either 5 mM Cu(NO3)2 or 1.5 mM Cd(NO3)2. Cultures were shaken at 200 rpm and 37 °C. The OD600nm was measured at 2 h intervals.
Stable transformation of A. thaliana (ecotype Columbia) with a CaMV35S-BjMT2 cassette
Arabidopsis thaliana, ecotype Columbia, was transformed via floral dip (Clough and Bent, 1998) with a cauliflower mosaic virus (CaMV)35S-BjMT2 cassette cloned into the pBinAR vector (Hoefgen and Willmitzer, 1992) and mobilized into Agrobacterium tumefaciens (C58C1). Recombinant A. tumefaciens were selected on medium containing kanamycin, rifampicin, and carbenicillin. Arabidopsis thaliana T0 seeds were germinated on 0.5x MS (Murashige–Skoog) medium-containing agar plates under kanamycin selection. Leaves from kanamycin-resistant seedlings were collected and the presence of the transgene was confirmed by PCR with pBinAR-specific primers. Independent confirmation of BjMT2 expression was obtained by northern blot analysis, using total RNA isolated from leaf tissue (0.5 g fresh weight from leaves of 20-d-old plants) according to Logemann et al. (1987). From each sample, 20 µg of total RNA was separated and, after blotting, detected with a biotin-labelled PCR probe against full-length BjMT2.
Transient transformation of different plant tissues with a BjMT2::EGFP fusion
Arabidopsis thaliana leaf epidermal cells and onion peel cells were transformed by gene gun procedure as described earlier (Lehr et al., 1999). Tobacco leaf transformation was performed by Agrobacterium leaf infiltration (Wroblewski et al., 2005).
CLSM analysis of transient BjMT2::EGFP expression in plant cells
Transiently transformed cells were analysed by confocal laser scaning microscopy (CLSM) using LSM410 (Zeiss, Jena, Germany) with the following settings: for EGFP excitation 488 nm and emission 515 nm longpass; for chlorophyll autofluorescence excitation 488 nm and emission 645–700 nm.
Growth analysis of transformed Arabidopsis seedlings after heavy metal exposure
Seeds from transgenic (T2) and wild-type plants were germinated on 0.5x MS medium-containing agar plates (control), or plates supplemented with 50 µM Cu(NO3)2 or 50 µM Cd(NO3)2. The plates were incubated at 22 °C with 8 h light/16 h dark. After cultivation for 15 d, seedling phenotypes were recorded visually and by determination of fresh and dry weight, respectively.
Chlorophyll determination
After 20 d of growth on 0.5x MS agar plates, shoots of transgenic (T2) and wild-type seedlings were carefully removed and extracted in dimethylsulphoxide (DMSO) for 60 min at 65 °C. After centrifugation, chlorophyll content was determined spectrophotometrically.
Determination of root growth
Seeds of transgenic (T2) and wild-type plants were germinated on horizontally placed 0.5x MS agar plates for 4 d. Thereafter, plates were turned into the vertical position and plants were further cultivated at 22 °C for 20 d with 8 h light/16 h dark, before total root length was determined for 20 seedlings per treatment.
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Results |
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Efficient expression of functional BjMT2 as a TrxA fusion protein in E. coli
It has previously been shown that in the allotetraploid species B. juncea, MT2 proteins form a small gene family (Schäfer et al., 1998). The BjMT2 gene chosen for the present study (Y10853 [GenBank] ) shows high homology to the corresponding MT2a and MT2b genes from A. thaliana. For independent proof of function, BjMT2 was expressed as a fusion protein with thioredoxin in E. coli. Soluble TrxA::BjMT2 fusion protein could be extracted from bacterial lysate and affinity purified on an Ni-agarose matrix (Fig. 1). After splitting the fusion protein with TEV protease, pure BjMT2 protein could be recovered from the affinity chromatography flow-through fraction, the thioredoxin domain and TEV protease both carrying a His tag. After incubation with the thiol reagent MBB and subsequent separation by SDS–PAGE, the fusion protein and the purified BjMT2 protein exhibited a strong fluorescence (in contrast to proteins from total E. coli lysate), indicative of multiple MBB binding to cysteine residues.
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To confirm BjMT2 function in vivo, bacterial growth under heavy metal exposure was compared for non-transformed E. coli cells, cells bearing the empty vector only (i.e. expressing thioredoxin), and TrxA::BjMT2-expressing cells. While in the presence of 5 mM Cu(NO3)2 the expression of thioredoxin alone slightly improved bacterial growth, the expression of the TrxA::BjMT2 fusion protein provided substantial tolerance towards copper exposure (Fig. 2). Similarly, when grown in the presence of 1.5 mM Cd(NO3)2, TrxA::BjMT2-expressing bacteria showed a significantly improved growth as compared with the other two bacterial strains. In the absence of heavy metals, all bacterial strains exhibited identical growth kinetics. Together, these data confirm that functional BjMT2 protein was expressed in E. coli.
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Expression of BjMT2 in A. thaliana seedlings under control of the CaMV 35S promoter increases tolerance towards Cu and Cd
Arabidopsis thaliana was transformed with a 35S-BjMT2 construct, using the floral dip procedure. A large number of independent primary transformants (T0) were obtained. After prescreening via kanamycin resistance and PCR with primers directed against the untranslated regions, positive transformants were tentatively identified and confirmed by northern blot for transgene expression (Fig. 3). For further analysis, plants showing strong expression of BjMT2 mRNA (individuals 4–7) were selected, and T2 seeds were used for analysis of copper and cadmium tolerance. T2 seeds were germinated on agar plates (0.5x MS medium) in the absence or presence of 50 µM Cu(NO3)2 and 50 µM Cd(NO3)2, respectively (Fig. 4). On control medium, shoot development of wild-type and BjMT2-expressing seedlings was indistinguishable. In contrast, BjMT2-expressing seedlings were more tolerant towards copper and cadmium exposure as based on seedling growth (Table 1). In wild-type seedlings, 50 µM Cu(NO3)2 caused a minor growth inhibition, whereas in BjMT2-expressing seedlings Cu treatment even promoted growth. In the presence of 50 µM Cd(NO3)2, growth of wild-type seedlings was significantly reduced, whereas BjMT2-expressing seedlings were apparently unaffected by Cd treatment.
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Note that under both stress treatments, germination of wild-type seeds was significantly affected, which was not the case for BjMT2-expressing seedlings. In the presence of 50 µM Cu(NO3)2 and 50 µM Cd(NO3)2, wild-type seed germination was reduced by 17% and 37%, respectively. For the determination of mean seedling weight (Table 1), ungerminated seeds were not considered.
Interestingly, even in the absence of copper stress, BjMT2-expressing seedlings showed a slightly higher chlorophyll content as compared with wild-type seedlings. In the presence of 100 µM Cu(NO3)2, the chlorophyll content of transformant seedlings was increased by >50% as compared with wild-type seedlings. The results confirmed that in A. thaliana seedlings, the expression of BjMT2 under the control of the constitutive 35S promoter protected shoot development against copper and cadmium exposure.
BjMT2 remains exclusively confined to the cytosolic compartment in the absence and presence of Cu exposure
Plant MT genes do not contain signal peptides or sequence motifs indicative of subcellular compartmentation to particular organelles. Therefore, it is generally assumed that MT proteins are confined to the cytosolic compartment. However, it could be speculated that upon binding of MT to metal ions, the MT–metal ion complexes could be sequestered within the cytosol. To address the question of BjMT2 compartmentation, a BjMT2::EGFP fusion protein was expressed in different cell types (Fig. 6). Transient transformation of epidermal leaf cells was performed with the particle gun (Fig. 6a, b; A. thaliana, onion). Alternatively, tobacco leaves were infiltrated with A. tumefaciens bearing the BjMT2::EGFP cassette in the pBinAR vector (Fig. 6c, d). Efficient transformation was achieved in all cases. Consistently, EGFP fluorescence was exclusively confined to the cytosolic compartment.
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To assess the possibility of heavy metal-induced sequestration of BjMT2, the leaf infiltration technique was used for tobacco leaves (see above). For this experiment, A. tumefaciens were taken up either in water or, alternatively, in 50 µM Cu(NO3)2 or 50 µM Cd(NO3)2 solutions and injected into leaves. Inspection of >20 cells of each treatment over a period of 3–6 d did not reveal any change in EGFP localization under the exposure to copper or cadmium (data not shown). It is concluded that in intact leaves, the binding of copper or cadmium to BjMT2 does not affect its subcellular distribution.
In the absence of heavy metal exposure, overexpression of BjMT2 inhibits root elongation in seedlings of A. thaliana
Systematic inspection of wild-type and transformed A. thaliana seedlings expressing BjMT2 had indicated that root growth appeared to be affected on control plates. In a detailed analysis, this effect was quantified. While in the presence of 50 or 100 µM Cu(NO3)2, root growth of wild-type and transformant seedlings did not differ, transformant seedlings exhibited a significantly reduced root elongation growth on control media (Fig. 7). The observed inhibitory effect of ectopic BjMT2 expression on control medium was highly reproducible. In four independent experiments, the inhibition of root elongation on control medium was 35±6, 63±7, 53±9, and 41±6%, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Expression of functional plant MT2 as a thioredoxin fusion protein in E. coli
MTs from higher plants have been previously expressed in E. coli as GST fusion proteins (Evans et al., 1992; Mir et al., 2004). In particular, the expression of an MT2-type metallothionein from Quercus suber was shown to form complexes not only with copper but also with zinc (Mir et al., 2004). However, in previous studies, the effect of recombinant plant MT expression on the growth kinetics of transformed E. coli cells was not reported. The present observations with E. coli cells expressing a TrxA::BjMT2 fusion protein confirm that due to the heavy metal-complexing activity of BjMT2, cells were significantly more tolerant to copper and cadmium exposure. Our results indicate that BjMT2 protein can be expressed in E. coli as a functional TrxA fusion protein with high yield.
Expression of BjMT2 in Arabidopsis differentially affects shoot and root development
Regarding the overlapping expression profiles of plant MT genes (Garcia-Hernandez et al., 1998), possible effects of ectopic expression of a single MT2 protein in stable transformants were difficult to predict. Recently, the transient expression of the Arabidopsis MTs AtMT2a and AtMT3 was shown to enhance the resistance of V. faba guard cells to cadmium (Lee et al., 2004). The present study demonstrates for the first time that ectopic expression of a plant MT2 protein under the regulation of the strong and constitutive 35S promoter conferred a higher copper and cadmium tolerance in plants. The expression of BjMT2 produced a strong phenotype in A. thaliana seedling development. The causal relationship between increased MT2 expression and the protective effects on seedling shoot growth and chlorophyll content is certainly complex and likely to be multifactorial. It may include protection from heavy metal-induced oxidative stress, but, in the case of copper, it may also involve more subtle effects on heavy metal homeostasis.
An unexpected observation was the inhibitory effect of BjMT2 expression on seedling root growth in the absence of copper exposure. One possible explanation may be that the overexpression of MT2 protein interfered with copper homeostasis. In agreement with this assumption, root elongation growth was similar in wild-type and BjMT2-expressing seedlings when grown on medium supplemented with copper. Another possibility may be interference of ectopic BjMT2 expression with zinc homeostasis. It has been previously shown that the expression of AtMT2 in a Zn2+-MT-deficient Synechococcus strain partly complemented Zn2+ hypersensitivity (Robinson et al., 1996). Thus, BjMT2 may reduce the free Zn2+ level, thereby interfering with growth processes depending on Zn2+-binding proteins. In this case, the addition of copper would displace Zn2+ from BjMT2 and restore zinc homeostasis. Finally, the MT2-mediated reduction of root elongation could also be related to interference of excess MT2 with the cellular redox balance and/or signalling processes via reactive oxygen species (Mir et al., 2004; Thomas et al., 2005). This is an attractive hypothesis to explain the differential effects on roots as opposed to shoots (i.e. organ-specific differences in redox signalling and defence against reactive oxygen species). Experiments are under way to elucidate the mechanism of root growth inhibition by BjMT2.
Exposure to toxic concentrations of copper or cadmium does not affect cytosolic MT2 compartmentation
The protein sequences of plant MT2 proteins do not contain any sequence signatures indicative of organellar sequestration. Therefore, it is generally assumed that plant MTs are cytosolic proteins. However, assuming a ‘shuttle’ function for MT proteins, one could speculate that depending on the protein status, i.e. free MT2 versus MT2 complexed with copper (or zinc), the protein could be differentially distributed within the cytoplasm, i.e. free or associated with organellar membranes. This intriguing question was only recently addressed in a study with transient expression of AtMT2a and AtMT3 in Cd-exposed V. faba guard cells (Lee et al., 2004; see above). The authors did not find any indication for a change of MT localization when cells were exposed to Cd. In the present study, a detailed analysis of transient BjMT2::EGFP expression in tobacco leaf cells exposed to copper stress [50/100 µM Cu(NO3)2] did also not reveal any change in BjMT2::EGFP localization. In particular, there was no apparent binding of BjMT2::EGFP to any particular organelle, indicating that the heavy metal binding status of BjMT2 protein does not affect its localization within the cytoplasm.
It is concluded that the ectopic expression of BjMT2 in A. thaliana not only increases copper and cadmium tolerance in the seedling stage but also interferes strongly with root development. The heavy metal binding status of BjMT2 does not affect its intracellular compartmentation.
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Acknowledgements |
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We gratefully acknowledge critical reading of this manuscript by Steffen Greiner and Sebastian Wolf. This work was supported by grants to ZY and TR (DFG-FOR383).
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Abbreviations |
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CaMV, cauliflower mosaic virus; CLSM, confocal laser scanning microscopy; IPTG, isopropyl-ß-D-thiogalactopyranoside; MBB, monobromobimane; MT, metallothionein; PC, phyochelatin; TEV, tobacco etch virus; TrxA, thioredoxin A.
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References |
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