Plant, Cell and Environment (2001) 24, 217–226
Elevated expression of metal transporter genes in three
accessions of the metal hyperaccumulator Thlaspi
caerulescens
A. G. L. ASSUNÇÃO,1 P. DA COSTA MARTINS,2* S. DE FOLTER,2 R. VOOIJS,1 H. SCHAT1 & M. G. M. AARTS2
1
Department of Ecology and Ecotoxicology of Plants, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV
Amsterdam, The Netherlands and 2BU Plant Development and Reproduction, Plant Research International, Postbus 16,
6700 AA Wageningen, The Netherlands
ABSTRACT
Heavy metal hyperaccumulation in plants is an intriguing
and poorly understood phenomenon. Transmembrane
metal transporters are assumed to play a key role in this
process. We describe the cloning and isolation of three zinc
transporter cDNAs from the Zn hyperaccumulator Thlaspi
caerulescens. The ZTP1 gene is highly similar to the
Arabidopsis ZAT gene. Of the other two, one is most probably an allele of the recently cloned ZNT1 gene from
T. caerulescens (Pence et al; Proceedings of the National
Academy of Science USA 97, 4956–4960, 2000). The second,
called ZNT2, is a close homologue of ZNT1. All three
zinc transporter genes show increased expression in
T. caerulescens compared with the non-hyperaccumulator
congener T. arvense, suggesting an important role in heavy
metal hyperaccumulation. ZNT1 and ZNT2 are predominantly expressed in roots and ZTP1 is mainly expressed in
leaves but also in roots. In T. arvense, ZNT1 and ZNT2 are
exclusively expressed under conditions of Zn deficiency.
Their expression in T. caerulescens is barely Zn-responsive,
suggesting that Zn hyperaccumulation might rely on a
decreased Zn-induced transcriptional downregulation of
these genes. ZTP1 expression was higher in plants from
calamine soil than in plants from serpentine or normal soil.
The calamine plants were also the most Zn tolerant,
suggesting that high ZTP1 expression might contribute to
Zn tolerance.
Key-words: Thlaspi caerulescens; heavy metals; hyperaccumulation; tolerance; transporters; zinc/nickel.
INTRODUCTION
A limited number of plant species, called hyperaccumulators, accumulate certain heavy metals to extremely high,
Correspondence: Dr M.G.M. Aarts. Fax: + 31 317418094;
e-mail: [email protected]
*Present address: Academisch Ziekenhuis Utrecht, Department of
Haematology, Heidelberglaan 100, 3508 GA Utrecht.
© 2001 Blackwell Science Ltd
normally severely toxic concentrations in their shoots
(Ernst 1968; Baker & Brooks 1989; Reeves 1992). Most of
these species are more or less restricted to strongly metalenriched soil types but some of them are also commonly
found on non-metalliferous soil (Ingrouille & Smirnoff
1986; Baker & Proctor 1990; Schat, Llugany & Bernhard
1999). There is considerable debate concerning the ultimate
evolutionary explanation of the hyperaccumulation trait.
The herbivore defence hypothesis, which states that metal
hyperaccumulation is a way to reduce damage by herbivory
and parasitism (Boyd & Martens 1992) is presently
favoured by most authors and supported by circumstantial
experimental evidence (Boyd & Martens 1994; Martens &
Boyd 1994; Boyd, Shaw & Martens 1994; Pollard & Baker
1997; Davis & Boyd, 2000; Ghaderian, Lyon & Baker 2000).
In comparison with normal plants, hyperaccumulators
are characterized by strongly enhanced rates of uptake, tolerance and root-to-shoot transport of the metals in question (Lasat, Baker & Kochian 1996; Schat et al. 1999). The
underlying mechanisms and their precise inter-relationships are largely unknown yet. In F2 crosses between the
Zn hyperaccumulator Arabidopsis halleri and the nonhyperaccumulator Arabidopsis petraea, the Zn hyperaccumulation and tolerance traits segregated independently
(Macnair et al. 1999). Additionally, hyperaccumulation, tolerance and root-to-shoot transport of Zn varied independently between Thlaspi caerulescens accessions from
different soil types (Schat et al. 1999). These results suggest
that hyperaccumulation might be a complex trait, with
uptake, internal transport and tolerance being at least
partly under independent genetic control. Transmembrane
metal transporters may be decisively involved in uptake,
xylem loading and unloading (Lasat, Baker & Kochian
1998) and vacuolar sequestration of heavy metals, particularly in the leaf epidermal cells (Vázquez et al. 1994;
Küpper, Zhao & McGrath 1999), trichomes (Krämer et al.
1997), or stomatal guard cells (Heath, Southworth &
Dallura 1997). The molecular basis of Zn uptake and transport in plants is, as yet, largely unexplored.
Grotz et al. (1998) have isolated and functionally characterized three Zn transporter genes from Arabidopsis, called
ZIP1, ZIP2 and ZIP3 (ZIP: ZRT, IRT-like protein), by
217
218 A. G. L. Assunção et al.
functional complementation of a yeast mutant defective in
Zn uptake. They also identified a related genomic DNA
sequence predicted to encode the ZIP4 protein. ZIP genes
belong to a growing family of putative metal transporter
genes with members in the fungal, plant and animal
kingdom (Grotz et al. 1998; Eng et al. 1998). The proteins
encoded by ZIP genes have a high degree of similarity with
the yeast ZRT1 and ZRT2 proteins that are involved in the
high and low-affinity Zn uptake system (Zhao & Eide
1996a,b), and with the Arabidopsis IRT1 transporter that
mediates Fe uptake (Eide et al. 1996). ZIP4 is more closely
related to Arabidopsis IRT1 than to ZIP1, ZIP2 or ZIP3
(Eng et al. 1998). ZIP gene expression is Zn-regulated.
ZIP1 and ZIP3 are induced in roots and ZIP4 in both roots
and shoots of Zn-limited plants. The ZIP1 and ZIP3 proteins, which are presumably plasma membrane located, are
suggested to play a role in the uptake of Zn from the rhizosphere, whereas ZIP4, which contains a potential chloroplast targeting sequence, was suggested to mediate
transport of Zn into plastids (Grotz et al. 1998).
Recently, using the complementation strategy applied by
Grotz et al. (1998), a Zn transporter cDNA was isolated
from T. caerulescens by Pence et al. (2000). This transporter
gene named ZNT1, is also a member of the ZIP gene family
and is highly homologous to Arabidopsis ZIP4 (Grotz et al.
1998). ZNT1 is highly expressed in roots and shoots of
T. caerulescens, both under conditions of Zn deficiency and
at normal nutritional Zn supply. In the related non-hyperaccumulator species, Thlaspi arvense, it is expressed under
Zn deficient conditions, but shows strong downregulation
at normal Zn supply. The high expression of Zn transporters in T. caerulescens, irrespective of Zn availability, has
been suggested to be the major reason for the enhanced Zn
uptake of this species. In general, alterations of the patterns
of Zn-responsive transcriptional regulation of Zn transporters might play a pivotal role in Zn hyperaccumulation
(Lasat et al. 2000; Pence et al. 2000).
The ZAT gene encodes another Zn transporter known in
plants. The ZAT cDNA was isolated from Arabidopsis (Van
der Zaal et al. 1999) and is homologous to the mammalian
Zn transporter genes ZnT2 (Palmiter, Cole & Findley 1996)
and ZnT3 (Wenzel et al. 1997), which are involved in Zn
vesicular sequestration, and ZnT4 (Huang & Gitschier
1997), which is involved in Zn transport into milk. Transgenic Arabidopsis which overexpressed the ZAT gene
exhibited enhanced Zn resistance and an increased Zn
content in roots under high Zn exposure suggesting that the
ZAT protein is involved in the plant-internal compartmentation of this metal (Van der Zaal et al. 1999).
The present study aims to identify additional Zn transporters in T. caerulescens and to characterize the variation
in metal preference patterns with respect to uptake, rootto-shoot transport and tolerance among T. caerulescens
accessions from contrasting soil types (calamine, serpentine
and non-metalliferous soil) in relation to the expression of
Zn transporter genes.We identified three cDNAs putatively
encoding Zn transporter proteins and tried to establish
their role in T. caerulescens Zn hyperaccumulation.
MATERIALS AND METHODS
Plant material and plant culture
Seeds were collected from three T. caerulescens accessions.
Accession La Calamine (LC) originated from a calamine
ore waste, enriched in Zn, Cd and Pb, at La Calamine,
Belgium. Accession Monte Prinzera (MP) originated from
Ni-enriched serpentine soil at Monte Prinzera, Italy and
accession Lellingen (LE) originated from a nonmetalliferous soil, at Lellingen, Luxembourg. The T. arvense
non-hyperaccumulator reference accession originated from
a roadside in Amsterdam, the Netherlands. Arabidopsis
thaliana, ecotype Columbia, was obtained from the
Nottingham Arabidopsis Stock Centre, UK.
To grow plants, seeds of T. caerulescens and T. arvense
accessions were sown on moist peat. Three-week-old
seedlings were transferred to 600 mL polyethylene pots
(one plant per pot), filled with modified half-strength
Hoagland’s nutrient solution (Schat et al. 1996), supplemented with ZnSO4 and/or NiSO4 at the desired concentrations. The solutions were replaced twice a week.
Germination and plant culture were performed in a climate
chamber (20/15 °C day/night; 250 mmol m-2 s-1 at plant
level, 14 h d-1; 75% relative humidity).
Zn and Ni uptake, root-to-shoot transport and
tolerance assays
For the measurement of Zn and Ni uptake, plants of each
of the T. caerulescens and T. arvense accessions were
exposed to nutrient solution supplemented with 1, 10 and
100 mm of ZnSO4 or 0, 1, 10 and 100 mm of NiSO4, supplied
alone or together in factorial combinations. Five plants
were used per treatment. After 3 weeks of growth, the
plants were harvested, after desorbing the roots systems
with ice-cold 5 mm PbNO3 (1 h). Roots and shoots were
dried at 80 °C, wet-ashed in a 4 : 1 mixture of HNO3 (65%)
and HCl (37%), in Teflon bombs at 140 °C for 7 h and
analysed for Zn and Ni, using flame atomic absorption spectrometry (Perkin Elmer 1100B; Perkin Elmes, Norwalk, CT,
USA). Total uptake was calculated on a total plant dry
weight basis. Shoot-to-root metal concentration ratios were
used as an estimate of root-to-shoot transport.
To measure tolerance, plants of each of the T.
caerulescens and T. arvense accessions were exposed to
nutrient solution supplemented with a series of Zn or Ni
concentrations. The tolerance was inferred from the presence or absence of chlorosis after 3 weeks of growth under
metal exposure and represented by the first concentration
of an increasing series at which chlorosis was observed.
Library construction and screening
A cDNA library was prepared from mRNA extracted from
roots of T. caerulescens, accession LC, grown hydroponically in a solution containing 10 mm ZnSO4. The cDNA
library with a primary titre of 1·9 ¥ 107 pfu mL-1 was constructed in a phagemid vector (pAD-GAL4–2·1) of the
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
Zinc transporters of Thlaspi caerulescens 219
HybriZap-2·1 two-hybrid cDNA cloning system (Stratagene, La Jolla, CA, USA). About 9.5 ¥ 104 plaques of the
amplified library were screened. An Arabidopsis ZAT1
(Van der Zaal et al. 1999) partial cDNA clone (EST
143A21, GenBank accession no. 46380; Newman et al. 1994)
was used as probe, together with cloned DNA fragments
obtained by polymerase chain reaction (PCR) using degenerate primers ZTPfor (5¢-TTY GCI GCI GGI GTI ATH
CTN GCN AC-3¢) and ZTPrev (5¢-GCI AGI ARR TCI
ACN AGN GCC ATR TA-3¢). Degenerate primers were
based on conserved DNA sequences in the Arabidopsis
ZIP genes (Grotz et al. 1998) and six additional Arabidopsis homologues. Plaques were lifted and blotted onto a
nylon membrane (Amersham Pharmacia Biotech, Uppsala,
Sweden), according to the recommended procedures and
hybridized with the 32P-labelled DNA probes (randomprimed DNA labelling system, Amersham Pharmacia
Biotech, Uppsala, Sweden). Pre-hybridization and
hybridization were performed in hybridization solution
[10% dextran sulphate, 1 m NaCl, 1% sodium dodecyl
sulphate (SDS)] supplemented with denatured salmon
sperm DNA (100 mg mL-1). After an overnight incubation
at 65 °C the membranes were rinsed twice in 2 ¥ SSC
(300 mm NaCl, 30 mm Na Citrate, pH 7.0) for 2 min at room
temperature and twice in 2 ¥ SSC, 1% SDS for 20 min at
65 °C. Positive plaques were purified and the phagemid
vector was extracted by in vivo excision according to the
instruction provided by the manufacturer.
DNA and predicted amino acid
sequence analysis
DNA sequences of both strands of each cDNA were determined by automated sequencing. DNA homology searches
and sequence analyses were performed using the Basic
Local Alignment Search Tool (BLAST) (Altschul et al.
1997). Multiple sequence alignments were performed by
using the CLUSTAL program (DNASTAR, Madison, WI,
USA). Potential protein targeting signals were predicted by
PSORT (Klein, Kanehisa & DeLisi 1985), potential transmembrane sequences were predicted using TMHMM
(Sonnhammer, von Heijne & Krogh 1998).
RNA isolation and RNA blot analysis
Total RNA was extracted using the RNeasy Extraction Kit
(Qiagen GmbH, Hilden, Germany) from leaves and roots
of T. caerulescens, accessions LC and MP and Arabidopsis
ecotype Col (all grown on normal potting soil) and from
leaves and roots of hydroponically grown T. arvense and T.
caerulescens accessions LC, MP and LE, exposed to 0, 2 and
10 mm Zn. Ten micrograms of total RNA was separated by
gel-electrophoresis using a 1% agarose gel and capillary
blotted onto Hybond N+ nylon membrane (Amersham),
according to standard procedures.
Genomic DNA fragments representing ZIP2, ZIP3 and
ZIP4 (obtained by PCR) and the insert of EST clone
143A21 (Acc. no. T46380) representing ZAT1, as well as
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
DNA fragments representing the full cDNAs (ZTP1, ZNT1
and ZNT2) were used as probes for RNA blot hybridization. A PCR fragment of 0·6 kb representing the T. arvense
ZTP1 homologue (ZTP1-arvense) was also used as probe
for RNA blot hybridization. The forward primer used in
this PCR reaction was 5¢-GGC AGA CTT ACG GGT TCT
TCA GG-3¢ and the reverse primer was 5¢-GTG AGA
ACG GAA AAG CCA ATA GC-3¢. Membranes were prehybridized, hybridized and washed as described above
except that additional washes were performed at high stringency (in 0·1 ¥ SSC, 1% SDS for 20 min at 65 °C). Membranes were stripped using a solution of 0·1% SDS, 2 mm
Tris-HCl (pH 8·0), 1 mm EDTA for 10 min at 65 °C. The
membranes were checked for removal of the probe before
re-probing. The hybridization signals were scanned using a
Fuji Phosphor Imager (BAS 2000; Fuji Photo Film Co.,
Tokyo, Japan) and, if necessary, quantified using the TINA
programme provided by the manufacturer.
DNA isolation and DNA blot analysis
DNA was extracted from leaves of T. arvense,
T. caerulescens, accessions LC, MP and LE and Arabidopsis as described by Aarts, Koncz & Pereira (2000). One
microgram of Thlaspi and 0·5 mg of Arabidopsis genomic
DNA, separately digested with HindIII, was separated by
gel-electrophoresis using a 1% agarose-TAE (Tris-Acetate
40 mm, EDTA 1 mm) gel and vacuum blotted onto Hybond
N+ nylon membrane (Amersham) according to standard
procedures. DNA blot hybridization was performed as the
RNA blot hybridization described before.
RESULTS
Physiological characterization of T.
caerulescens accessions
The Zn and Ni uptake, shoot-to-root concentration ratio
and tolerance characteristics were determined for hydroponically grown T. caerulescens and non-hyperaccumulator
T. arvense plants (Tables 1 and 2). In comparison with
T. arvense, the T. caerulescens accessions La Calamine (LC),
Monte Prinzera (MP) and Lellingen (LE) were all characterized by a strongly enhanced Zn uptake, Zn shoot-to-root
concentration ratio and Zn tolerance. However, there were
pronounced differences between these T. caerulescens
Table 1. Tolerance to Zn and Ni of Thlaspi arvense and
T. caerulescens plants. Tolerance was inferred from the presence
or absence of chlorosis after 3 weeks of growth under metal
exposure. The figures represent the first concentration of an
increasing series at which chlorosis was observed
T. arvense
T. caerulescens LC
T. caerulescens MP
T. caerulescens LE
Zn (mm)
Ni (mm)
< 25
1000
100
50
< 25
100
250
100
Table 2. Uptake (a), shoot concentration (b), and shoot-to-root concentration ratio (c) of Zn and Ni in the non-hyperaccumulator
Thlaspi arvense (Ta) and accessions (acc.) LC, MP and LE of the hyperaccumulator T. caerulescens (Tc), after 3 weeks of growth in a
nutrient solution with factorial combinations of Zn and Ni concentrations. Uptake is expressed on a whole plant dry weight basis (mmol
metal g-1 total plant dry weight), shoot concentration is given as mmol metal g-1 shoot dry weight. nt = not tested. Standard errors varied
between 3 and 20% of the means
Zn supply (mm)
Species (acc.)
Ni supply (mm)
1
10
100
1
10
100
nt
nt
nt
nt
nt
0·2
1·7
17·8
nt
1·3
10·0
34·2
nt
0·7
2·9
nt
nt
nt
nt
nt
nt
0·2
1·9
14·2
nt
0·6
3·6
20·5
nt
nt
nt
nt
(a) Uptake of Zn and Ni
Zn uptake
Ta
Tc (LC)
Tc (MP)
Tc (LE)
0
1
10
100
0
1
10
100
0
1
10
100
0
1
10
100
1·0
nt
nt
nt
4·6
5·5
5·0
7·4
13·2
15·5
12·2
11·6
6·9
4·4
5·8
nt
Ni uptake
3·8
nt
nt
nt
8·1
9·4
10·0
9·9
53·9
61·8
55·1
42·1
42·1
30·6
28·4
nt
nt
nt
nt
nt
35·7
37·5
21·3
30·5
118·7
125·1
134·3
108·9
nt
nt
nt
nt
nt
0·2
1·8
nt
nt
0·2
1·5
21·8
nt
8·4
48·3
104·7
nt
1·4
17·3
nt
(b) Shoot concentration of Zn and Ni
Zn shoot concentration
Ta
Tc (LC)
Tc (MP)
Tc (LE)
0
1
10
100
0
1
10
100
0
1
10
100
0
1
10
100
0·8
nt
nt
nt
5·6
6·5
5·8
9·0
14·1
15·2
9·5
7·9
8·5
5·4
7·2
nt
1·1
nt
nt
nt
4·4
11·0
13·1
11·5
51·5
54·3
48·8
33·7
52·5
38·0
34·8
nt
Ni shoot concentration
nt
nt
nt
nt
42·2
44·8
25·8
34·8
98·6
106·6
116·6
90·9
nt
nt
nt
nt
nt
0·1
1·1
nt
nt
0·2
1·5
25·5
nt
9·9
49·0
106·8
nt
1·8
20·8
nt
nt
nt
nt
nt
nt
0·2
1·9
20·1
nt
1·4
10·3
35·2
nt
0·8
3·1
nt
nt
nt
nt
nt
nt
0·2
2·0
15·8
nt
0·6
4·1
23·8
nt
nt
nt
nt
(c) Shoot-to-root concentration ratio of Zn and Ni
Zn shoot-to-root ratio
Ta
Tc (LC)
Tc (MP)
Tc (LE)
0
1
10
100
0
1
10
100
0
1
10
100
0
1
10
100
0·5
nt
nt
nt
7·1
4·8
4·1
5·7
1·5
0·9
0·4
0·3
25·0
13·8
16·3
nt
0·4
nt
nt
nt
4·1
4·2
5·9
4·5
0·8
0·6
0·6
0·5
62·5
55·1
13·7
nt
Ni shoot-to-root ratio
nt
nt
nt
nt
4·5
6·1
7·7
6·3
0·5
0·5
0·6
0·5
nt
nt
nt
nt
nt
0·1
0·2
nt
nt
0·9
0·9
3·6
nt
4·2
1·1
1·1
nt
9·7
6·1
nt
nt
nt
nt
nt
nt
0·8
1·6
2·4
nt
1·7
1·2
1·2
nt
2·2
1·4
nt
nt
nt
nt
nt
nt
1·0
1·9
2·1
nt
1·8
2·7
2·4
nt
nt
nt
nt
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
Zinc transporters of Thlaspi caerulescens 221
accessions. As far as tolerance is concerned, the LC plants
maintained normal growth and leaf pigmentation at
1000 mm Zn in the nutrient solution. The MP and LE plants
already showed chlorosis at 100 and 50 mm Zn, respectively
(Table 1). The analysis of Zn uptake showed that LC plants
accumulated significantly less Zn (P < 0·01) than MP and
LE plants, both at low and high supply levels (Table 2a.).
Root-to-shoot transport of Zn, as estimated from the ratio
between shoot and root Zn concentration, was significantly
higher (P < 0·01) in all T. caerulescens accessions than in
T. arvense and, among accessions, higher in LE than in LC
and MP plants (Table 2c).
The Ni tolerance was higher in T. caerulescens than in
T. arvense, particularly in the MP plants originating from
serpentine soils (Table 1). The Ni accumulation varied
strongly between the accessions (P < 0·01). The MP plants
and, to a lower degree also the LE plants, hyperaccumulated Ni, although only at low external Zn concentrations
(1 mm). Higher Zn concentrations strongly inhibited Ni
uptake whereas Ni had no effect on Zn uptake (Table 2a).
The LC plants did not show any Ni hyperaccumulation at
all, irrespective of the concentration of Zn in the nutrient
solution (Table 2a). Remarkably, there was no inhibitory
effect of Zn on Ni uptake in these plants. Transport of Ni
was consistently higher in T. caerulescens than in T. arvense
with the highest shoot-to-root concentration ratios for LE
and the lowest for LC. The MP and LE plants exhibited a
pronounced inhibitory effect of Ni on Zn transport and vice
versa. This was not apparent in LC plants (Table 2c).
Identification of Zn transporter genes in
T. caerulescens
To assess whether any of the known Arabidopsis Zn transporter genes might be differentially expressed in
T. caerulescens, Northern blot experiments were performed
using genomic DNA fragments representing the ZIP2,
ZIP3 and ZIP4 genes and a partial cDNA clone representing ZAT1 as probes. The blots contained RNA from leaves
and roots of T. caerulescens accessions LC and MP and Arabidopsis ecotype Col, all grown on normal potting soil.
Figure 1. Northern blot analysis of Arabidopsis ZAT gene
expression in leaves and roots of T. caerulescens, accessions LC
and MP (TcLC and TcMP) and Arabidopsis thaliana ecotype
Columbia (AtCol), grown in normal potting soil. The second row
represents the hybridization of the blot with 16S rRNA used as a
loading control. The blot was washed under low-stringency
conditions.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
ZAT1 was clearly overexpressed in T. caerulescens leaves
compared to roots, or to Arabidopsis leaves and roots
(Fig. 1). Transcription of ZIP2 and ZIP3 was not detected
in any of the samples, but weak ZIP4 transcription was
found for all samples (data not shown). The ZIP2 and ZIP3
probes hybridized strongly to a T. caerulescens DNA-blot
(data not shown), proving that the inability to detect ZIP2
and ZIP3 transcripts was not due to lack of homology.
In order to obtain Thlaspi-specific probes for ZIP homologues, a degenerate PCR approach was chosen. From database searches it became apparent that in Arabidopsis at least
10 ZIP-like gene sequences are present,including the known
ZIP and IRT genes. Degenerate primers were designed on
two conserved DNA sequences found within this gene
family (see ‘Materials and Methods’). In total, PCR fragments for five different ZIP-like genes were isolated from T.
caerulescens accession LC, which were homologous to Arabidopsis genes ZIP1, ZIP3 and ZIP4 (data not shown).
Isolation of T. caerulescens Zn
transporter cDNAs
The Arabidopsis partial ZAT1 cDNA insert and DNA
fragments L13 and L3, homologous to, respectively, ZIP1
and ZIP4, were used as probes to screen a T. caerulescens
cDNA library. The library was made with root RNA from
LC plants grown hydroponically on medium containing
10 mm Zn. With probe L13 no positive clones were found.
With each of the other two probes about 40 positive clones
were obtained. After sequencing the 5¢ ends of the longest
inserts, three different full-length cDNA sequences were
identified, one from a ZAT1-specific clone and two from L3specific clones. The complete DNA sequence for each fulllength clone was determined and compared to sequences in
the GenBank database. The cDNA fragment obtained with
the Arabidopsis ZAT1 partial cDNA probe, is 1340 base
pairs (bp) long and encodes a predicted protein of 393
amino acids. The cDNA has 90% DNA identity and 75%
amino acid identity with Arabidopsis ZAT1 cDNA (Van der
Zaal et al. 1999). We called this gene ZTP1 for Zn TransPorter 1 (Fig. 2).The ZTP1 protein is predicted to be an integral membrane protein with six potential transmembrane
domains (Fig. 2), just like the Arabidopsis ZAT protein of
which it is most likely the T. caerulescens orthologue. The
subcellular location of ZTP1 is unclear. Although previous
studies with the Arabidopsis ZAT gene suggested targeting
of the protein to the vacuole (Van der Zaal et al. 1999), an
obvious vacuolar targeting signal was not detected.
The cDNA clones obtained with the L3 probe are 1375
and 1520 bp long and encode predicted proteins of 409 and
423 amino acids, respectively. They share 90 and 83% DNA
identity and 76 and 65% amino acid identity with the predicted Arabidopsis ZIP4 DNA and protein sequences
(Grotz et al. 1998). By database search we found the first
clone to be nearly identical (99% DNA identity) to the
ZNT1 Zn transporter gene recently cloned from
T. caerulescens accession Prayon (ZNT1-PR; Pence et al.
2000) and we believe it to be the LC allele of ZNT1 (ZNT1-
222 A. G. L. Assunção et al.
Figure 2. Amino acid sequence alignment of the T. caerulescens
ZTP1 zinc transporter with the A. thaliana ZAT zinc transporter
(GenBank accession no. AF072858). The sequences were aligned
using the CLUSTAL method (DNAstar). The putative transmembrane domains, predicted by TMHMM (Sonnhammer et al. 1998),
are overlined and numbered. Identical residues are shaded.
LC). The second clone, which is a close homologue of
ZNT1-LC with 80% DNA identity, was labelled ZNT2
(Fig. 3). One recently deposited EST sequence representing the ZIP4 gene was found in the GenBank database
(Acc. No. AV441840), which starts at nearly the same position as the ZNT1-LC cDNA clone and downstream of the
5¢ end of the ZNT2 cDNA. After comparison of this EST
sequence to the genomic DNA sequence of ZIP4 in Arabidopsis, we detected an in-frame stop codon only 3 bases
upstream of the EST 5¢ end. As no putative intron–exon
boundary sequences were found between the stop codon
and the EST sequence start, we concluded that the predicted start codon of ZIP4 corresponds to the first ATG
codon we identified in both the ZNT1-LC and the ZNT2
cDNA clones, which is thus most likely the protein translation start codon. With this new ZIP4 cDNA sequence, the
predicted ZIP4 protein sequence is extended at the Nterminus by 34 amino acids compared with earlier publications (Grotz et al. 1998; Pence et al. 2000). This start codon
is not present in the ZNT1-PR cDNA sequence reported
by Pence et al. (2000) and consequently the predicted amino
acid sequence of ZNT1-LC is 30 amino acids longer at its
N-terminus compared to ZNT1-PR. The ZNT1-LC and
ZNT2 proteins are predicted to have a N-terminal signal
sequence (Fig. 3), as well as the eight potential transmembrane domains previously reported for ZNT1-PR by Pence
et al. (2000). ZNT1-LC and ZNT2 are likely to be targeted
to the plasma membrane.
T. caerulescens accessions and the non-hyperaccumulator
T. arvense. Compared to T. arvense, ZTP1 is higher
expressed in both roots and leaves of T. caerulescens accessions LC, MP and LE, grown hydroponically at 0, 2 and 10 mm
Zn (Fig. 4).The expression in T. caerulescens LC was slightly
higher than in the other two T. caerulescens accessions. In
MP and LE the difference in expression between roots and
leaves was more pronounced, with enhanced mRNA levels
in leaves. To confirm that the overexpression of ZTP1 in LC
was not due to preferential hybridization of the LC probe to
the LC RNA, an additional hybridization was performed
using a 0·6 kb PCR-fragment of the ZTP1 homologue from
T. arvense as a probe (ZTP1-arvense). The T. arvense probe
preferentially hybridized to T. arvense RNA compared to T.
caerulescens RNA. However, as with the ZTP1 probe, the
ZTP1-arvense probe hybridized stronger to T. caerulescens
LC RNA than to T. arvense RNA. After image analysis, we
estimated that the ZTP1 mRNA hybridization signal was
about five times higher in LC than in T. arvense.
The ZNT1 expression levels in root and leaf of all accessions are very similar to the ZNT2 expression levels. In
comparison with T. arvense, both genes are highly expressed
in all three T. caerulescens accessions, at all tested Zn concentrations (Fig. 5). The expression is higher in roots than
in leaves, which is most pronounced for ZNT1. The
Figure 3. Amino acid sequence alignment of ZNT1 (LC) and
Expression of Zn transporter genes in different
T. caerulescens accessions and in T. arvense
To determine the expression of the identified Zn transporter
genes, low stringency hybridizations were performed using
the ZTP1, ZNT1-LC and ZNT2 cDNA inserts as probes
on blots containing RNA from the three different
ZNT2 with ZNT1 (Prayon) (GenBank accession no. AF133267)
and ZIP4, predicted from genomic and partial cDNA sequences
(GenBank accessions no. ATU95973 and AV441840). The
sequences were aligned using the CLUSTAL method included in
the DNAstar programme (DNAstar). The putative
transmembrane domains, predicted by TMHMM (Sonnhammer
et al. 1998), are numbered and overlined. Identical residues are
shaded.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
Zinc transporters of Thlaspi caerulescens 223
Figure 4. Northern blot analysis of ZTP1 (A)
and ZTP1-arvense (B) expression in leaves and
roots of T. arvense (Ta) and T. caerulescens,
accessions LC, MP and LE (TcLC, TcMP and
TcLE), grown at 0, 2 and 10 mm Zn.
Approximately equal loading of the RNAs in
blots A and B is shown in C and D,
respectively, after hybridization with a 16S
rRNA-specific probe. The blots were washed
under low-stringency conditions.
T. arvense ZNT1 transcript was only detected in roots and
leaves of plants grown at 0 mm Zn. Also ZNT2 is expressed
only at 0 mm Zn, although it is barely detectable after
hybridization (Fig. 5). Under these conditions, and especially in roots, the expression is much lower in T. arvense
than in T. caerulescens. To establish whether ZNT1 is downregulated by high zinc concentrations, we hybridized a
RNA-blot, containing root and leaf RNA of LC plants
sampled in a 48 h time period after transfer from 0 mm Zn
to 1 mm Zn, with a ZNT1-LC-specific probe (Fig. 6). There
was no apparent reduction in hybridization signal in this
48 h period, either in roots or in shoots. To determine the
level of cross hybridization of the T. caerulescens LC probes
to MP, LE and T. arvense DNA and RNA, low and high
stringency DNA blot hybridizations were performed.
Under low stringency conditions, the ZTP1 probe detected
the corresponding ZTP1 genomic fragment as well as one
cross-hybridizing ZTP1-homologous sequence, present in
both Thlaspi species and in Arabidopsis (Fig. 7). Upon high
stringency washing only the ZTP1-containing fragments
were observed. The Southern analysis with ZNT1 and
ZNT2 probes showed the respective homologues in
T. arvense, Arabidopsis and all T. caerulescens accessions.
Under reduced stringency conditions each of the two
probes cross-hybridized to their respective DNA fragments, but not to any additional DNA fragments (Fig. 7),
suggesting that these two genes are the only closely related
ZIP4 homologues present in T. caerulescens and T. arvense.
DISCUSSION
The physiological analysis of three Zn hyperaccumulator
T. caerulescens accessions and a non-hyperaccumulator
T. arvense accession confirms that the Zn hyperaccumulator species is characterized by a much higher Zn uptake,
shoot-to-root concentration ratio and tolerance than the
related non-hyperaccumulator species. Additionally, we
have observed a high and independent inter-accession variability for these physiological properties in T. caerulescens.
The metallicolous T. caerulescens accessions LC and MP
are specifically adapted to their native soils, since the
observed metal tolerance characteristics correspond well
with the soil metal composition at the sites of seed collection. The LC and MP plants exhibited elevated tolerance
to, respectively, Zn and Ni. The high level of Zn tolerance
in LC plants is associated with decreased uptake and transport of this metal, compared to the non-metallicolous
accession LE. Reduced Zn accumulation and transport in
T. caerulescens and Arabidopsis halleri accessions from
calamine soil, as compared to accessions from non-metalliferous soil, has been reported previously (Meerts & Van
Isacker 1997; Bert et al. 2000; Escarré et al. 2000), indicating that Zn tolerance and accumulation in these species are
independent traits. Schat et al. (1999) however, reported a
very low Zn and Cd shoot-to-root concentration ratio in a
non-metallicolous accession originating from another site
near Lellingen, about 4 km distant from the site of origin
of the accession LE used in the present study. More extensive comparisons of non-metallicolous and metallicolous
accessions have shown that the low transport in the former
accession from Lellingen is probably highly exceptional
(data not shown).
In contrast to the observations for Zn uptake by the Zntolerant LC accession, the highly Ni-tolerant MP plants
showed an increased rather than decreased uptake of Ni
and Zn, compared to the non-metallicolous accession LE.
Figure 5. Northern blot analysis of ZNT1 (A)
and ZNT2 (B) expression in leaves and roots of
T. arvense (Ta) and T. caerulescens accessions
LC, MP and LE (TcLC, TcMP and TcLE),
grown at 0, 2 and 10 mm Zn. Approximately
equal loading of the RNAs in blots A and B is
shown in C and D, respectively, after
hybridization with a 16S rRNA-specific probe.
The blots were washed under low-stringency
conditions.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
224 A. G. L. Assunção et al.
Figure 6. Northern blot analysis of ZNT1
expression in roots (R) and leaves (L) of
T. caerulescens accessions LC grown at
1 mm Zn for a 48 h period. The blot was
washed under low-stringency conditions.
This indicates once more that metal tolerance and metal
uptake are independent traits.
The observed metal uptake, transport and tolerance
characteristics suggest an important role for transmembrane metal transporters in the metal hyperaccumulation
mechanism. Thorough genetic and physiological analysis of
the inter-accession variability in respect to the expression
of T. caerulescens Zn transporter genes could give more
insight into the function of this genes and the mechanism
of metal hyperaccumulation. In this work it was shown that
T. caerulescens contains at least three different expressed
genes with strong homology to Zn transporters. ZTP1 is the
closest homologue of the Arabidopsis ZAT Zn transporter
gene and most likely the Thlaspi orthologue. Based on
Southern analysis there appears to be one other ZAT/ZTP1
homologous DNA sequence present in both Thlaspi and
Arabidopsis. A corresponding cDNA has not been found
and expression has thus not been tested. Overexpressing
the ZAT gene in Arabidopsis caused an increased Zn
content in roots as well as enhanced Zn tolerance. This suggests that the ZAT protein is involved in the internal compartmentation of this metal (Van der Zaal et al. 1999).
Based on the Northern analysis, we conclude that ZTP1 is
clearly overexpressed in T. caerulescens compared to
T. arvense, with the highest expression in LC. LC is also the
accession with the highest tolerance to Zn. Together with
its predominant expression in the leaves this emphasizes
the proposed role for ZTP1/ZAT-like transporters in Zn
compartmentation and also suggests an important contribution of ZTP1 expression to Zn tolerance.
ZNT1 and ZNT2 resemble the Arabidopsis ZIP4 gene,
suggested to encode a Zn transporter (Grotz et al. 1998).
Experimental evidence supporting this view was recently
provided by Pence et al. (2000), also mentioned by Lasat
et al. (2000), who described the cloning of a ZNT1 partial
cDNA from T. caerulescens accession Prayon, by functional
complementation of a Zn uptake deficient yeast mutant.We
have been unable to show any functional complementation
using the same zinc uptake-deficient yeast mutant zhy3
(Zhao & Eide 1996b) transformed with overexpression constructs containing either the ZTP1, ZNT1 or ZNT2 cDNA
sequences. It may be that the presence of the plant Nterminal signal sequence interfered with proper intracellular localization of the heterologous protein.The presence of
an N-terminal signal sequence may be also the reason that
the ZNT2 cDNA was not identified in the functional complementation experiment that yielded the ZNT1 cDNA
(Pence et al. 2000; Lasat et al. 2000), although the ZNT2
mRNA is not considerably less abundant than the ZNT1
mRNA. On the other hand, the presence of a signal
sequence did not disturb the functional complementation of
the yeast mutant by the Arabidopsis ZIP1, ZIP2 and ZIP3
genes (Grotz et al. 1998), which belong to the same ZIP-like
gene family, although not as closely related to ZNT1 and
ZNT2 as ZIP4. As for ZNT2 in T. caerulescens, neither a
full-length nor a partial ZIP4 cDNA clone was picked up
from the Arabidopsis seedling cDNA-expression library
that yielded the ZIP1, ZIP2 and ZIP3 cDNAs (Grotz et al.
1998), although its transcript should not be less abundant.
Initially the ZIP4 protein was predicted to contain a
potential chloroplast targeting sequence. Based on the
recently deposited ZIP4 partial cDNA sequence (acc. no.
AV441840) the predicted protein contains an N-terminal
signal sequence and is most likely targeted to the plasma
membrane (PSORT, Klein et al. 1985).This is more in accordance with expression in both roots and shoots.
Figure 7. Southern blot analysis of ZTP1
(A), ZNT1 (B) and ZNT2 (C).Genomic
DNA from T. arvense (Ta), T. caerulescens
accessions LC (TcLC), MP (TcMP), LE
(TcLE) and A. thaliana ecotype Columbia
(At) was digested with HindIII. The figure
represents low-stringency (A and C) and
high-stringency (B) washings. Under low
stringency washing, the ZTP1 (A) and
ZNT2 (C) probes cross-hybridize to only
one other homologous gene copy (*). For
the ZNT2 probe, this is the ZNT1 gene, as
the weaker hybridization signals in (C) are
the very strong hybridizing signals in (B).
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
Zinc transporters of Thlaspi caerulescens 225
The ZNT1 and ZNT2 genes are clearly part of the ZIPlike gene family. First of all they encode proteins with eight
predicted transmembrane domains, as found in the Arabidopsis ZIP4 protein and other related ZIP proteins. The
predicted proteins contain a histidine-rich region between
transmembrane domains III and IV (Fig. 3), which is proposed to be the heavy metal binding sequence (Eng et al.
1998). Finally, the proteins contain the conserved ZIP signature sequence in transmembrane domain IV. This
sequence is fully conserved among all members of the ZIP
family (Grotz et al. 1998; Eng et al. 1998) and suggested to
play a role in substrate transport over a membrane.
In comparison with T. arvense, ZNT1 and ZNT2 are
highly expressed in at least four, respectively, three,
T. caerulescens accessions. The high expression concerns
predominantly root tissue, which is a strong indication of a
role for these genes in enhanced Zn uptake from the soil.
This is in line with the strongly enhanced Zn uptake in
T. caerulescens compared with T. arvense (Table 2). The differences in ZNT1/ZNT2 expression among T. caerulescens
accessions are less pronounced and also much less associated with differences in Zn uptake between these accessions. It is clear that expression of ZNT1 and ZNT2 does
not account for all of the observed inter-accession differences in Zn uptake, transport to the shoots, or in Zn tolerance. Of course it would be very unlikely that zinc uptake
is solely controlled by the expression of two zinc transporter genes. Additional zinc transporters, like the nonmetal-specific Nramp transporters (Thomine et al. 2000) or
other ZIP-like proteins are also likely to be involved in zinc
uptake. Alternatively, zinc transporter regulation may act
on the post-transcriptional level as was reported for the
yeast ZRT1 zinc transporter (Gitan et al. 1998; Gitan &
Eide, 2000).
The Ni hyperaccumulation in MP and, to a lower degree,
in LE and the complete absence of this phenomenon in
LC, suggests the presence of at least two different
uptake systems involved in Zn/Ni hyperaccumulation in
T. caerulescens: a Zn-specific high-affinity uptake system and
a low-affinity Zn/Ni uptake system, which prefers Zn over
Ni. The latter seems to be suppressed in the LC accession,
but overexpressed in the MP accession. The relatively small
difference in ZNT1 and ZNT2 expression between the different accessions suggests that other genes are responsible
for the enhanced Ni accumulation in MP and LE.
All three Zn transporters are highly expressed in
T. caerulescens at all Zn concentrations tested. The highest
concentration of 10 mm approaches the Zn concentration
which is available as water-soluble Zn in heavily Zncontaminated soils (Ernst & Nelissen, 2000). Pence et al.
(2000) observed that ZNT1 expression is downregulated
only after prolonged exposure to 50 mm Zn, whereas in T.
arvense it is downregulated at 1 mm. In addition, we
observed that the transcriptional downregulation of ZNT1
was not yet obvious after 48 h exposure to 1 mm Zn. The
apparent decrease in Zn-imposed downregulation of zinc
transporter genes in T. caerulescens has been conserved
among at least four accessions and may well be the first evo© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226
lutionary step that led to the ability to accumulate and tolerate high Zn levels in this hyperaccumulator species.
ACKNOWLEDGMENTS
We thank Martijn Fiers for making the cDNA library,
Richard Immink and Marco Busscher for their support in
performing the automatic sequence reactions, Dr David
Eide for providing the zinc uptake-deficient yeast strain, Dr
Arle Kruckeberg for providing plasmids and technical
support and Paul Koevoets and Professor Dr Wilfried Ernst
for critical reading of the manuscript.
Part of this work was supported by the Portuguese Foundation for Science and Technology, programme PRAXIS
XXI (grant no. BD/16152/98).
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Received 14 June 2000; received in revised form 9 October 2000;
accepted for publication 9 October 2000
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226