|
Plant Physiol. (1999) 119: 1047-1056
Overexpression of a Novel Arabidopsis Gene Related to Putative
Zinc-Transporter Genes from Animals Can Lead to Enhanced Zinc
Resistance and Accumulation
Bert J. van der Zaal*,
Leon W. Neuteboom,
Johan E. Pinas,
Agnes N. Chardonnens,
Henk Schat,
Jos A.C. Verkleij, and
Paul J.J. Hooykaas
Institute of Molecular Plant Sciences, Leiden University, Clusius
Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
(B.J.v.d.Z., L.W.N., J.E.P., P.J.J.H.); and Department of Ecology and
Ecotoxicology, Faculty of Biology, Vrije Universiteit Amsterdam, De
Boelelaan 1087, 1081 HV Amsterdam, The Netherlands (A.N.C., H.S.,
J.A.C.V.)
 |
ABSTRACT |
We describe the isolation of an
Arabidopsis gene that is closely related to the animal
ZnT genes (Zn
transporter). The protein
encoded by the ZAT (Zn transporter of
Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning
domains. To obtain evidence for the postulated function of the
Arabidopsis gene, transgenic plants with the ZAT coding sequence under
control of the cauliflower mosaic virus 35S promoter were analyzed.
Plants obtained with ZAT in the sense orientation exhibited enhanced Zn
resistance and strongly increased Zn content in the roots under high Zn
exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better
understanding of the mechanism of Zn homeostasis and resistance in
plants.
| |
INTRODUCTION |
Several heavy metals are essential during plant growth and
development, but their excess can easily lead to toxic effects. Contamination of soils with heavy metals, either by natural causes or
due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, heavy-metal-tolerant plants. The precise mechanisms of uptake, transport, and accumulation of heavy metals in plants are poorly understood, but several genes likely to be involved in these processes have been described. Recently,
a family of ZIP genes that are expressed in roots upon Zn
deficiency was isolated from Arabidopsis (Grotz et al., 1998 ). The
proteins encoded by the ZIP genes have eight predicted TM regions and a high degree of similarity to the ZRT genes
from yeast that are involved in Zn uptake. Expression of the
ZIP genes in yeast conferred Zn-uptake activities to these
cells, demonstrating that they are probably functional homologs of the
yeast ZRT genes (Grotz et al., 1998). The only other
metal-transporting protein recently identified in plants belongs to the
large family of cation-transporting P-type ATPases (Tabata et al.,
1997), but these proteins are structurally very different from the
metal-transporting proteins mentioned above.
Recent data have provided more insight into the mechanisms of
heavy-metal tolerance. Metallophytes often exhibit tolerance to several
different heavy metals, but all of these metals need not be present at
toxic levels in their habitat (Schat and ten Bookum, 1992a; Schat and
Vooijs, 1997, and refs. therein; Schat and Verkleij, 1998). Although
such a feature is suggestive of a general mechanism of heavy-metal
tolerance, recent genetic evidence has shown that a number of different
mechanisms must exist, each with its own metal specificity (Schat and
Vooijs, 1997). In Arabidopsis, a plant species with a typical level of
tolerance to heavy metals, it has been demonstrated that the
Cd-sensitive mutants cad1 and cad2 are defective
in the synthesis of the metal-binding compound phytochelatin (Howden et
al., 1995). cad1 plants were only slightly more
sensitive to Cu and Zn, indicating that phytochelatin-mediated detoxification is not sufficient for Cu and Zn detoxification (Howden
et al., 1995b). Metallothioneins appear to be of major importance for
constitutive Cu tolerance in Arabidopsis (Zhou and Goldsbrough, 1994).
Aside from complexation of heavy metals by heavy-metal-binding
proteins, there is evidence that transport-mediated sequestration can
contribute to heavy-metal tolerance. In the Zn-tolerant plant Silene vulgaris it was shown that Zn transport across the
tonoplast was about 2.5 times higher than in Zn-sensitive plants of the same species (Verkleij et al., 1998). The ZRC1 gene from the
yeast Saccharomyces cerevisiae encodes a protein with six
putative TM regions; when overexpressed, this gene confers elevated
resistance to Zn and Cd (Kamizono et al., 1989). A structurally very
similar gene, COT1, was later found to be involved in Co
accumulation in yeast (Conklin et al., 1992).
Recently, several genes homologous to ZRC1 and
COT1 have been described in mammalian cells. The first gene
discovered, ZnT-1 (Zn transporter
1), was cloned by virtue of its ability to complement a
mutated, Zn-sensitive cell line (Palmiter and Finley, 1995). Subsequently, ZnT-2 (Palmiter et al., 1996),
ZnT-3 (Wenzel et al., 1997), and ZnT-4 (Huang and
Gitschier, 1997) have been described. The ZnT-1 protein most likely
transports Zn out of the cells (Palmiter and Finley, 1995), whereas
ZnT-2 confers Zn resistance by facilitating vesicular sequestration
(Palmiter et al., 1996a). Other proteins related to yeast ZRC1/COT1 and
mammalian ZnT have been found in several bacteria; for example, the
CzcD protein from Alcaligenes eutrophus might be involved in
Zn efflux (Nies, 1992).
A family of proteins with six TM regions thus seems to be involved in
the transport of heavy metals, mostly Zn, thereby conferring enhanced
resistance toward these metals. To our knowledge, no plant homologs of
this rather widespread gene family have yet been described. In this
paper we describe an Arabidopsis cDNA clone encoding a protein closely
related to the ZnT family of mammalian Zn transporters, demonstrating
that plants do contain these types of genes. Experiments were performed
to analyze the functional properties of the gene. We demonstrate that
overexpression of the complete protein-coding domain results in
enhanced Zn resistance and increased accumulation of Zn in the root.
The relevance of these findings is discussed.
The accession number for the nucleotide sequence of the ZAT cDNA
reported in this paper is AF072858.
| |
MATERIALS AND METHODS |
cDNA Isolation and Sequence Analysis
The cDNA sequence encoding ZAT (Zn transporter of
Arabidopsis
thaliana) was isolated fortuitously.
A cDNA library prepared with a cDNA-synthesis kit (ZAP, Stratagene)
from RNA isolated from auxin-treated root cultures of Arabidopsis
ecotype C24 was differentially screened for cDNA clones corresponding to auxin-induced mRNAs (Neuteboom et al., 1999). While characterizing the clones obtained, we noticed that in one of these clones two cDNA
sequences were present. Apparently due to a defective XhoI site, the cDNA sequence encoding ZAT preceded the cDNA sequence of an
auxin-inducible mRNA. When the total cDNA insert of the original clone
was used as a probe for northern-blot analysis, two signals were found.
The signal corresponding to the mRNA of the lowest molecular mass was
auxin inducible, whereas the other relatively weak signal corresponding
to ZAT mRNA was not changed after auxin treatment. Probing northern
blots with subcloned fragments confirmed that two cDNA inserts were
present in the original clone, each giving rise to only one of the
signals observed with the original clone (data not shown).
Sequence reactions on both strands were performed according to
the dideoxy chain-termination method (Sanger et al., 1977) using the T7
sequencing mix (Pharmacia). DNA homology searches and sequence analysis
were performed using the Basic Local Alignment Search Tool (BLAST,
National Center for Biotechnology Information, Bethesda, MD), the
sequence-analysis software package from the Genetics Computer Group
(Devereux et al., 1984), the CBS SignalP version 1.1 World Wide Web
Prediction Server (Nielsen et al., 1997), and miscellaneous links
that can be found at the CMS Molecular Biology Resource home page
(http://www.unl.edu/stc-95/ResTools/cmshp.html). Multiple-sequence alignments and the dendrogram were
made with the SeqLab program using the PileUp function with the
end-weight option (Genetics Computer Group).
Plasmid Constructs
A 1460-bp fragment was isolated from the original cDNA clone by
digestion with BamHI (site in the SK+
polylinker) and BglII (site just downstream of the TAA
termination codon). This ZAT coding sequence was cloned in the
BamHI-digested yeast expression plasmid YEP112A1 (Riesmeyer
et al., 1992). YEP112A1, YEP112A1-ZAT-1 sense, and YEP112A1-ZAT-1
antisense were used to transform yeast strain YPH250 (ura3,
lys2, ade2, trp1, his3, and leu2; Sikorski and Hieter, 1989). Transformants were
selected on minimal yeast medium (Zonneveld, 1986) lacking Trp.
For plant expression studies the same 1460-bp ZAT
BamHI/BglII fragment was cloned in two
orientations between the cauliflower mosaic virus 35S promoter and the
octopine synthase terminator in BamHI-digested pART7
(Gleave, 1992), resulting in pART7-ZAT-S (sense) and pART7-ZAT-AS
(antisense). Plasmid pART7-ZAT-S was digested with XbaI and
religated. This resulted in pART7-ZAT-dC, a cDNA insert truncated just
after the sixth TM domain. Expression cassettes present in pART7 were
isolated as NotI fragments and cloned in similarly digested
pART27 (Gleave, 1992) in Escherichia coli strain DH5
(Clontech, Palo Alto, CA). Plasmids with the octopine synthase
terminator of the 35S expression cassette adjacent to the right border
were selected and mobilized to Agrobacterium tumefaciens
strain GV2260 (Deblaere et al., 1985) using a triparental mating
procedure (Ditta et al., 1980). Transconjugants were selected on medium
containing carbenicillin, rifampicin, and spectinomycin (100, 20, and
125 mg/L, respectively).
Transformation of Arabidopsis
The A. tumefaciens strains harboring the binary pART27
plasmid (control) or the pART27 derivatives with ZAT inserts were used for cocultivation experiments with root explants of Arabidopsis ecotype
C24 essentially as described previously (Valvekens et al., 1988).
Transgenic shoots were selected on kanamycin (50 mg/L). Seeds of
T1 plants were germinated on one-half-strength
Murashige-Skoog medium (Murashige and Skoog, 1962; but one-half
strength for macrosalts) supplemented with 10 g/L Suc, 7 g/L agar, and
50 mg/L kanamycin at 21°C under a 16-h light/8-h dark regime.
Kanamycin-resistant seedlings of randomly chosen independent primary
transformants whose progeny segregated 3:1 for kanamycin resistance
were allowed to set seed, and homozygous T3 lines
were selected. For the ZAT-AS and ZAT-dC constructs four randomly
chosen lines were used for further analysis, together with two vector
controls. In a similar manner, two homozygous lines with one T-DNA
locus were obtained for the ZAT-S construct derived from the only two
fertile primary transformants obtained with that construct.
Heavy-Metal-Resistance Assays
Homozygous T3 seeds were sown on plates
containing one-half-strength Murashige-Skoog medium supplemented with
various concentrations of ZnSO4 or
CdCl2 (Analar, British Drug House, Poole,
UK). The standard one-half-strength Murashige-Skoog medium
contains 30 µM Zn2+ and 0.75 mM SO42 . After
4 d at 4°C, seeds were germinated at 21°C under a 16-h light/8-h dark regime.
More detailed analyses of Zn resistance and content were done using the
hydroponic testing system described by Schat and ten Bookum (1992b).
Seeds were sown on moist peat and after cold stratification were
transferred to a growth chamber (20°C/15°C day/night temperatures; 75% RH; 14-h light [250 µmol m2
s1]/10-h dark regime). About 12 d after
germination the plants were transferred to polyethylene pots filled
with 600 mL of continuously aerated nutrient solution (Schat and Kalff,
1992). After 1 week of preculture, during which time the solution was
refreshed once, the plants were exposed to a series of Zn
concentrations. The background solution was identical to the preculture
solution, except for FeNaEDTA, which was omitted to prevent ZnEDTA
complex formation, and the pH was set at 5.25 with 2 mM
Mes/KOH. Before exposure to Zn the roots were stained black using the
charcoal-suspension method (Schat and ten Bookum, 1992b). After 72 h of exposure, the length of the unstained apical segment of the
longest lateral root was measured and the roots were subsequently
desorbed in ice-cold 5 mM
Pb(NO3)2 for 1 h. Then
roots and shoots were harvested separately, frozen in liquid nitrogen,
lyophilized, and stored under vacuum until analysis. Powdered
lyophilized materials were digested in a 1:4 mixture (v/v) of 37% HCl
and 65% HNO3 in closed Teflon bombs at 140°C
for 7 h. The Zn concentration in the digests was measured using
flame atomic absorption spectroscopy (model 1100, Perkin-Elmer).
Root lengths and Zn contents were statistically analyzed using two-way
analysis of variance, after ln transformation of the data.
RNA Isolation and Northern-Blot Analysis
Aliquots of homozygous seeds were surface-sterilized for 1 min in
70% (v/v) ethanol followed by soaking for 10 min in a NaOCl solution
(1% active ingredient supplemented with 0.05% [v/v] Tween 20 [Merck, Darmstadt, Germany]). Seeds were cultured in 250-mL flasks
containing 50 mL of Gamborg B5 medium (Gamborg et al., 1968) on a
gyratory shaker (100 rpm) at 21°C with a 16-h light/8-h dark cycle.
After 14 d approximately 10 g fresh weight of total seedlings
was used for RNA isolation (van Slogteren et al., 1983). Amounts of 30 µg of glyoxylated total RNA were electrophoresed on a 1.5% agarose
gel and capillary blotted onto GeneScreen Plus membranes (NEN-DuPont)
according to standard procedures (Sambrook et al., 1989).
For induction experiments seedlings were grown similarly in
one-half-strength Murashige-Skoog medium (see above). Extra
ZnSO4 was added to the flasks after 11 d of
growth. Incubation in the flasks then continued for 6 or 24 h
before mRNA isolation.
The cDNA insert was labeled by random priming. For generation of
strand-specific RNA probes a 1460-bp BamHI/BglII
fragment containing the complete ZAT coding sequence was cloned in
BamHI-digested pBluescript SK+ plasmid
(Stratagene). The plasmid with insert orientation giving rise to an
antisense RNA probe with T7 RNA polymerase was linearized with
BamHI, and the sense probe was generated from the plasmid with the reverse insert orientation after digestion with
XbaI. Labeling reactions were performed using the TransProbe
T kit (Pharmacia). Prehybridization was performed for 2 h at
42°C in 50% deionized formamide, 5× SSPE (1× SSPE = 180 mM NaCl, 1 mM EDTA, and 10 mM sodium phosphate, pH 6.5), 2.5% (w/v) SDS,
followed by 24 h of hybridization in the same solution after
addition of the probe. Blots were washed at 65°C in 0.1× SSPE, 0.1%
SDS for a total of 1 h, and were then exposed to film (Fuji-RX,
Fuji Photo Film Co. Ltd., Tokyo, Japan).
| |
RESULTS |
ZAT Encodes a Putative Zn Transporter
Sequence analysis of a fortuitously isolated Arabidopsis cDNA (see
``Materials and Methods'' for details) revealed that it encodes a
protein of 398 amino acid residues. Several in-frame stop codons are
present upstream of the ATG initiation codon, and the TAA stop codon is present well before the end of the cDNA sequence (Fig.
1). The cDNA sequence is therefore full
length as far as the coding region is concerned. A BLAST search
(February 24, 1998) with the protein sequence showed that the protein
was related to a wide family of proteins that are (putatively) involved
in the transport of Zn across biological membranes in various
organisms. Except for a partial sequence from potato (accession no.
U60071), which so far has been described only as a putative plant
disease-resistance gene (Leister et al., 1996), no homologous plant
protein sequences were detected, indicating that this type of protein
has thus far not been found or recognized as such in plants. For
reasons described below we have designated this protein ZAT.
View larger version (83K):
[in this window]
[in a new window]
| Figure 1.
The Arabidopsis ZAT nucleotide
sequence is shown with the amino acid sequence below (one letter code).
ATG initiation and TAA termination codons are shown in boldface type.
The cDNA sequence did not end in a poly(A+) tail, but
occurred just after the putative polyadenylation signal fused with a
second cDNA sequence (not shown). The XbaI site used for
the C-terminal deletion just after the sixth TM domain (see Fig. 3) is
indicated in boldface.
|
|
All related proteins are characterized by a structure with six
potential TM regions and a long cytoplasmic C-terminal tail. When
degrees of relatedness are visualized by a dendrogram it becomes clear
that ZAT is most closely related to a subgroup of Zn transporters
recently described for mammalian cells, ZnT-2, ZnT-3, and ZnT-4
(Palmiter et al., 1996a, 1996b; Huang and Gitschier, 1997,
respectively) (Fig. 2). The levels of
amino acid identity/similarity between ZAT and ZnT-2, ZnT-3, and ZnT-4
are 40.8%/52.5%, 37.4%/47.3%, and 35.3%/49.1%, respectively.
These values correspond to 40% to 50% identity at the nucleotide
level. Rat ZnT-2 facilitates Zn transport into an endosomal/lysosomal
compartment and thus protects cells from Zn toxicity (Palmiter et al.,
1996a). By analogy, it was hypothesized that the strongly related ZnT-3
and ZnT-4 proteins are involved in the same process (Huang and
Gitschier, 1997). Three sequences from Caenorhabditis
elegans encoding possible homologs that belong to the same group
of proteins can be found with a BLAST search (only one of them is
included in Fig. 2).
View larger version (12K):
[in this window]
[in a new window]
| Figure 2.
Dendrogram of the amino acid sequences of several
(putative) Zn transporters. CzcD, Alcaligenes eutrophus
(accession no. D67044); YrdO (CzcD), Bacillus subtilis
(accession no. U93876 [U62055]); CzcD-Bst,
Bacillus stearothermophilus
(accession no. D87026); ZnT-2, Rattus norvegicus
(accession no. S70632; Palmiter et al., 1996a); ZnT-3, Homo
sapiens (accession no. U76010; Palmiter et al., 1996b); ZCE,
C. elegans (accession no. T18D3.3 in Z68119); ZnT-4,
H. sapiens (accession no. AF025409; Huang and Gitschier,
1997); ZAT, Arabidopsis (this study); COT1, Saccharomyces
cerevisiae (accession no. P32798; Conklin et al., 1992); ZRC1,
S. cerevisiae (accession no. P20107; Kamizono et al.,
1989); ZRC-Sp, ZRC1 homolog, Schizosaccharomyces pombe
(accession no. D89236); ZnT-1, R. norvegicus (accession
no. S54303; Palmiter et al., 1995); COT-Ce, COT1 homolog in C. elegans (accession no. U23529). The distance between the
branches on the horizontal axis are proportional to the degree of
relatedness according to the program used, whereas the distances along
the vertical axis are without any meaning.
|
|
Two other groups of structurally related proteins can be discerned
(Fig. 2). ZnT-1, the first mammalian Zn transporter reported and found
to be involved in Zn efflux (Palmiter and Findley, 1995), forms
a subgroup together with Zn/Cd and Co transporters from yeast. The
level of amino acid identity between these proteins and ZAT ranges
between 20% and 30%. The partial potato protein sequence mentioned
above seems to belong to this group (data not shown), but this
classification might change when the complete sequence becomes
available. C. elegans also contains a gene encoding a
protein of this group. Metal-transporting proteins from bacteria form a
third subgroup, possessing 30% to 37% amino acid identity with ZAT.
The putative Zn-uptake carriers with eight predicted TM regions, such
as the yeast ZRT genes (Zhao and Eide, 1996a, 1996b)
and Arabidopsis ZIP genes (Grotz et al., 1998), are only distantly related to the genes mentioned and were not included in the
analysis.
Alignment of the amino acid sequence of ZAT with other sequences of its
subgroup showed that ZAT is deviating mainly due to the relatively
large stretch of 81 amino acids between putative TM regions IV and V,
which is more than twice the length of the corresponding region of its
nearest homologs (Fig. 3). The His-rich region between TM regions IV and V is predicted to form a cytoplasmic loop in which the His residues are most likely responsible for the
binding of Zn (Palmiter et al., 1996a, 1996b; Huang and
Gitschier, 1997). ZAT contains 21 His residues in this region, but
apart from the amino acid sequence HSH, there is little sequence
identity with the ZnT-2, ZnT-3, and ZnT-4 proteins in this area. Most
of the conserved amino acids are present within or just around the TM
regions and also in the long cytoplasmic tails of the aligned proteins
(Fig. 3). As far as the length of the putative cytoplasmic loop between
TM regions IV and V is concerned, ZAT is more similar to the group of
proteins constituting mammalian ZnT-1 and the yeast Zn/Cd and Co
transporters, which also have a long loop sequence. However, in terms
of conserved amino acids, the similarity is just as restricted as with
the cytoplasmic loop of ZnT-2, ZnT-3, and ZnT-4 (comparison not shown).
Within the CzcD group of bacterial cation-transport or efflux proteins,
the putative TM regions IV and V are separated by only 12 to 13 amino
acid residues, none of which is a His residue (not shown).
View larger version (85K):
[in this window]
[in a new window]
| Figure 3.
Multiple alignment of representatives of
(putative) Zn transporters most closely related to ZAT. The six
putative TM regions are overlined. Amino acid residues that are
identical in four or five of the sequences are shown in boldface. See
legend to Figure 2 for further details.
|
|
ZAT from Arabidopsis is thus most closely related to the Zn
transporters ZnT-2, ZnT-3, and ZnT-4 from mammals and the putative homologs of these proteins from C. elegans. Since expression
of ZnT-2 was shown to result in enhanced Zn tolerance of cells,
possibly by enhancing vesicular sequestration of Zn (Palmiter et al.,
1996a), we pursued further analysis of ZAT along this line.
Ectopic Expression of ZAT
Expression of sense or antisense ZAT mRNA in the yeast S. cerevisiae under control of the yeast ADH promoter did not lead to
a detectable change in the resistance of transgenic yeast cells toward
Zn, Cd, or Co (data not shown). The yeast strain used was wild type for
Zn, Cd, and Co tolerance and was therefore not as sensitive to changes
in resistance to these metals as certain mutant yeast strains (Conklin
et al., 1994). However, a marked change in Zn tolerance could be
detected in wild-type yeast cells expressing ZnT-4 (Huang and
Gitschier, 1997). Apparently, such a change in sensitivity was not
achieved with ZAT under the conditions used.
A genomic blot of Arabidopsis DNA probed under stringent conditions
with the complete ZAT cDNA sequence exhibited a simple restriction
enzyme digestion pattern, indicative of the presence of a single gene.
Northern blots with RNA from different plant parts showed that mRNA was
expressed throughout the plant at similar but low levels (data not
shown). Treatment of seedlings with increasingly toxic levels of Zn for
6 and 24 h failed to induce ZAT mRNA (Fig. 4). To study the effect of altered levels
of ZAT mRNA in Arabidopsis, we pursued expression of sense and
antisense ZAT mRNA under control of the strong cauliflower mosaic virus
35S promoter in stably transformed Arabidopsis plant lines. In
addition, a construct that should lead to overexpression of a ZAT
protein lacking the C-terminal cytoplasmic domain was transformed in
Arabidopsis. In animal cells it was shown that such a construct can
lead to a toxic phenotype (Palmiter and Findley, 1995). We reproducibly found that the root-transformation protocol that worked very
efficiently for the empty-vector control construct, as well as the
ZAT-AS (antisense) and ZAT-dC (deletion of C-terminal cytoplasmic
domain) constructs, was virtually ineffective for the ZAT-S (sense)
construct. Only a few transgenic calli were obtained with ZAT-S, and
only two independent calli formed shoots that could set seed.
View larger version (32K):
[in this window]
[in a new window]
| Figure 4.
Nothern blots of RNA isolated from seedlings
treated in liquid medium with increasing concentrations of
ZnSO4 for 6 and 24 h. Lanes C, Control seedlings (30 µM Zn already present in the medium); lanes 0.25, 0.50, and 1.00, seedlings with different millimolar concentrations of extra
ZnSO4 added. Exposure was for 2 d at  70°C with an
intensifying screen.
|
|
Homozygous T3 seed lines containing one T-DNA
locus were used for Zn- and Cd-resistance tests. Two control lines,
four ZAT-AS lines, and four ZAT-dC lines were randomly chosen, along
with the only two ZAT-S lines and the nontransformed C24 ecotype. All lines were phenotypically normal in vitro and in the greenhouse. In the
first test, seedlings were grown on plates supplemented or not with
0.25 mM ZnSO4 or 0.1 mM
CdCl2. Only the two ZAT-S lines were nearly
completely resistant to the extra 0.25 mM Zn. No
differences were observed for the Cd treatment. The two ZAT-S lines,
the two control lines, and C24 were tested again on medium containing a
series of added Zn (0.25, 0.5, and 1.0 mM) or Cd (25, 50, and 100 µM) concentrations and compared with growth on the normal medium (containing 30 µM Zn as a trace
element). Again, the two ZAT-S lines were nearly fully resistant to
added 0.25 mM Zn, a concentration that resulted in
chlorosis and slower development in the controls (Fig.
5; note the full expansion of the true
leaves of the ZAT-S line at 0.25 mM extra Zn, whereas in
the control most of the true leaves were smaller than the cotyledons).
View larger version (83K):
[in this window]
[in a new window]
| Figure 5.
Zn-resistance test of representative Arabidopsis
transgenic lines. C, Control; S, sense. Seedlings were germinated and
grown on solid medium (one-half-strength Murashige-Skoog medium). 0.5 MS, Control seedlings (30 µM Zn already present in the
medium); 0.25, 0.50, and 1.00, seedlings with different millimolar
concentrations of extra ZnSO4 added.
|
|
When 0.5 or 1.0 mM extra Zn was added, development of both
the control and the ZAT-S line was severely inhibited. Although the
ZAT-S lines were still growing slightly better than the controls at
these high Zn concentrations (Fig. 5), it was evident that the gain in
Zn resistance of the ZAT-S lines was relatively modest. No differences
in Cd sensitivity were observed, although the concentration range used
now covered the complete spectrum of low (25 µM) to high
(100 µM) toxicity (the latter concentration completely
inhibited root development).
One each of the lines with control, ZAT-AS, ZAT-S, and ZAT-dC was
subjected to more detailed testing for Zn resistance and accumulation
using a hydroponic system. As inferred from the root-growth response
(Fig. 6), only the ZAT-S line exhibited
altered Zn sensitivity, being virtually resistant to a 200 µM concentration of external Zn. At 200 µM
the difference from the other lines was highly significant (P < 0.001). However, a 400 µM concentration of external Zn
inhibited ZAT-S root growth about as severely as in the other lines.
Therefore, the increase in Zn resistance in the ZAT-S line was clear,
but was limited to a rather narrow concentration range. The ZAT-S line
also showed a different pattern of Zn accumulation in the root (Fig.
7). At up to 50 µM external
Zn there were no significant differences between lines, but at higher
concentrations the ZAT-S line showed a significantly higher root Zn
content than the other lines. No significant differences were detected
in shoot Zn content between the different lines (data not shown).
Control, ZAT-AS, and ZAT-dC lines were not significantly different in
the measured parameters. A less-detailed analysis with a second line of
each transgenic type, including the second ZAT-S line, yielded very similar results, confirming the enhanced Zn resistance and root Zn
content due to the ZAT-S construct.
View larger version (17K):
[in this window]
[in a new window]
| Figure 6.
Arabidopsis root growth during 72 h of
exposure to Zn in hydroponic culture.  , Control;  , ZAT-AS;  ,
ZAT-S;  , ZAT-dC. Data points are the means of 15 plants. Vertical
bars represent ±SE.
|
|
View larger version (19K):
[in this window]
[in a new window]
| Figure 7.
Arabidopsis root Zn content after 72 h of
exposure to Zn in hydroponic culture. , Control; , ZAT-AS; ,
ZAT-S; , ZAT-dC. Data points are the means of five pooled samples of
three plants each. Vertical bars represent ±SE. DW, Dry
weight.
|
|
RNA was isolated from all the plant lines and analyzed on northern
blots with strand-specific probes (Fig.
8). The antisense probe detected a high
level of extra sense ZAT mRNA in the two ZAT-S lines and a similarly
high level of the truncated ZAT-dC mRNA in the four ZAT-dC lines at a
lower-molecular-mass position (Fig. 8A). A difference of 264 bases is
expected between the mRNA produced by these constructs. The endogenous
ZAT mRNA ran at a lower-molecular-mass position than the ectopically
expressed ZAT-S mRNA, and apparently was of the same length as the
ZAT-dC mRNA. The cauliflower mosaic virus 35S expression cassette used
therefore leads to a mRNA about 260 bases longer than the endogenous
ZAT mRNA. Although all four ZAT-AS lines produced high levels of
antisense mRNA (Fig. 8B), this did not lead to significant changes in
the level of endogenous ZAT mRNA detected with the antisense probe in
the same plant lines (Fig. 8A).
View larger version (91K):
[in this window]
[in a new window]
| Figure 8.
Northern blots probed with strand-specific RNA
probes. A, Antisense probe (showing sense mRNA). B, Sense probe
(showing antisense RNA). C, Ethidium-bromide-stained total RNA present
on the original gels. Lane C24, Nontransformed C24 ecotype; lane C,
vector-transformed control; lane AS, antisense construct line; lane S,
sense construct line; lane dC, C-terminal deletion construct line.
Exposure was for 1 d without an intensifying screen.
|
|
| |
DISCUSSION |
We have shown that Arabidopsis expresses a gene designated
ZAT that is most closely related to the ZnT-2,
ZnT-3, and ZnT-4 family of Zn-transporter genes
that so far have been described only in mammals and in the nematode
C. elegans. More generally, ZAT has significant
similarity to a variety of genes that encode proteins with six
predicted TM regions and a long C-terminal cytoplasmic domain (see
legend to Fig. 2). This structure is characteristic for a wide family
of proteins that are involved in the transport of certain heavy metals,
mostly Zn, across cellular membranes. In plants such proteins have not
been described previously. A partial sequence from potato that has been
reported as a putative disease-resistance gene (Leister et al., 1996)
does, however, indicate that these proteins are present in more plant
species. It should be noted that the ZAT-related proteins are
structurally different from the ZRT1 and ZRT2 proteins, which have
eight predicted TM regions and mediate Zn uptake in yeast cells (Zhao
and Eide, 1996a, 1996b). Recently, a family of Zn transporters
related to the yeast ZRT genes was identified in
Arabidopsis. These ZIP genes were indeed able to confer
Zn-uptake activities to yeast cells, and for this reason can be
regarded as functional homologs of the yeast ZRT genes
(Grotz et al., 1998).
For the mammalian genes most related to ZAT, experimental
evidence strongly suggests that their encoded proteins are involved in
the facilitation of vesicular sequestration of Zn, which can result in
increased resistance to otherwise toxic levels of extracellular Zn.
Overexpression of ZnT-2 in a Zn-sensitive hamster kidney cell line
resulted in elevated levels of intravesicular Zn and an increase in Zn
tolerance when cells were grown at a high Zn concentration (Palmiter et
al., 1996a). A similar experiment with ZnT-3 did not result in these
effects (Palmiter et al., 1996b), but the ZnT-3 protein was found to be
localized in synaptic vesicle membranes, suggesting that it is also
involved in the vesicular sequestration of Zn in certain cells (Wenzel
et al., 1997). ZnT-4 was able to confer enhanced Zn resistance to yeast
(Huang and Gitschier, 1997).
ZAT mRNA seemed to be expressed constitutively throughout the plant and
was not induced by increasing Zn concentrations. Overexpression of ZAT
mRNA in transgenic Arabidopsis plants resulted in a modest but
significant increase in Zn tolerance and an enhanced accumulation of Zn
in the root at high external Zn concentrations. These latter data
indicate that the ZAT protein performs a function similar to the animal
ZnT-2 protein. The ZAT protein might therefore be involved in the
sequestration of Zn, thereby contributing to Zn resistance. In plants
Zn can be transported into the vacuole, as was found in the Zn-tolerant
species Silene vulgaris. Transport of Zn across the
tonoplast was found to be 2.5 times higher than in Zn-sensitive plants
of the same species (Verkleij et al., 1998). It is tempting to
speculate that this elevated Zn transport is due to a ZAT-like protein,
but at the moment we lack data that ZAT is indeed located in the
tonoplast membrane.
Although it is likely that ZnT/ZAT-like proteins are involved in
transport of Zn, the mechanism by which they do so is far from clear.
The proteins lack an obvious nucleotide-binding domain indicative of an
active transporter. Ion transport across a membrane is thus probably
coupled to some other energetically favorable reaction and therefore
more likely to be some kind of facilitated diffusion (Palmiter et al.,
1996a). Bacterial CzcD proteins are related to ZnT/ZAT proteins (Fig.
2), but their role is still obscure. It has been postulated that the
CzcD protein performs a regulatory role for the metal-transporting,
membrane-bound CzcCBA protein complex (Nies, 1992), whereas the CzcA
protein is the most likely candidate as the central cation-proton
antiporter (Rensing et al., 1997). However, CzcA is without further
similarity to ZnT/ZAT and related proteins. By analogy to the bacterial
CzcCBA system, we therefore speculated that ZnT proteins may be part of
a larger complex in which they determine ion specificity or regulate Zn
transport, rather than being directly involved in transport (Palmiter
et al., 1996a). However, it should be noted, as mentioned in
"Results," that bacterial CzcD proteins lack the His-rich loop
between TM regions IV and V that is present in the other putative
transporters. If such a loop is indeed essential for Zn transport, the
CzcD proteins will necessarily have to operate in a mechanistically
different manner than the eukaryotic Zn transporters or Zn-transporting
systems.
Whatever the exact molecular mechanism, it is clear that Zn resistance
mediated by ZAT overexpression is correlated with increased root Zn
content and is certainly not based on a reduction of the net Zn influx.
When compared at similar degrees of root-growth inhibition, root Zn
contents were about 2-fold higher in the ZAT-S plants than in the other
lines (Figs. 6 and 7). The increase in Zn resistance in ZAT-S plants
can be estimated to be about 2-fold as well. Considering the huge
amount of sense ZAT mRNA in the transgenic ZAT-S plants (Fig. 8A),
these differences are small. The simplest explanation is that the extra
mRNA is poorly translated, but an alternative reason for this
discrepancy has to be considered. When the ZAT protein is part of a
larger protein complex, as discussed above, overexpression of only one
of the components of the complex will have only marginal effects. Only
when the endogenous ZAT concentration is relatively low and the other
components are more abundant will extra ZAT protein lead to more
functional complexes. Considering the difficulties in acquiring
transgenic calli and shoots after transformation with the ZAT-S
construct, it could be that the same situation exists during the
initial stages of callus formation and/or regeneration. Cells may get
intoxicated by an excessive accumulation of Zn in the vacuole.
The C-terminal intracellular domain seems to be important for ZAT
function, because the ZAT-dC construct lacking this domain was not able
to confer Zn resistance or Zn accumulation. A toxic effect of the
C-terminal deletion was not observed, in contrast to a similar
construct made for rat ZnT-1 (Palmiter and Findley, 1995). This could
mean that the inactive C-terminal deletion mutant of ZAT is not able to
intoxicate multimeric protein complexes that contain ZAT or a related
protein. Alternatively, it could mean that intoxication or disruption
of such protein complexes are without effect under the conditions used.
The lack of effect of antisense ZAT mRNA expression on Zn resistance
and accumulation seems to suggest the latter possibility. However, for
unknown reasons, the level of endogenous ZAT mRNA in the ZAT-AS lines appeared to be unaffected by the high level of antisense mRNA, so the
lack of effect has to be interpreted with caution.
The precise role of the ZAT gene in normal, wild-type Zn
homeostasis remains elusive. However, based on the data presented here,
we propose that it is involved in the vesicular/vacuolar sequestration
of Zn, thereby contributing to Zn tolerance in plants. Further study of
ZAT, together with the recently cloned ZIP genes that are likely to be responsible for Zn uptake (Grotz et al., 1998),
will lead to a better understanding of Zn homeostasis and tolerance in
higher plants.
| |
FOOTNOTES |
*
Corresponding author; e-mail zaal{at}rulbim.leidenuniv.nl; fax
31-71-527-4999.
Received October 19, 1998;
accepted December 6, 1998.
| |
ABBREVIATIONS |
Abbreviation:
TM, transmembrane.
| |
ACKNOWLEDGMENTS |
We thank Riet Vooijs for technical assistance, Dr. Frank van
Iren for stimulating discussions, Drs. Werner Pansegrau and Dorien Postma-Haarsma for help with protein alignments, and Bram Wetselaar and
Elly Schrijnemakers for plant care in the greenhouse. We also thank
Peter Hock for preparation of the figures and Adri't Hooft and Peter
van Mulken for photography.
| |
LITERATURE CITED |
Conklin D,
Culbertson MR,
Kung C
(1994)
Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae.
Mol Gen Genet
244:
303-311
[Medline]
Conklin DS,
McMaster JA,
Culbertson MR,
Kung C
(1992)
COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae.
Mol Cell Biol
12:
3678-3688
[Abstract/Free Full Text]
Deblaere R,
Bytebier B,
de Greve H,
Deboeck F,
Schell J,
van Montagu M,
Leemans J
(1985)
Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants.
Nucleic Acids Res
13:
4777-4788
[Abstract/Free Full Text]
Devereux J,
Haeberli P,
Smithies O
(1984)
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res
12:
387-395
Ditta G,
Stanfield S,
Corbin D,
Helinski DR
(1980)
Broad host range DNA cloning system from gram-negative bacteria: construction of a gene bank of Rhizobium meliloti.
Proc Natl Acad Sci USA
77:
7347-7351
[Abstract/Free Full Text]
Gamborg OL,
Miller RA,
Ojima K
(1968)
Nutrient requirements of suspension cultures of soybean root cells.
Exp Cell Res
50:
151-158
[CrossRef][ISI][Medline]
Gleave AP
(1992)
A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.
Plant Mol Biol
20:
1203-1207
[CrossRef][ISI][Medline]
Grotz N,
Fox T,
Connolly E,
Park W,
Guerinot ML,
Eide D
(1998)
Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency.
Proc Natl Acad Sci USA
95:
7220-7224
[Abstract/Free Full Text]
Howden R,
Andersen CR,
Goldsbrough PB,
Cobett CS
(1995a)
A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana.
Plant Physiol
107:
1067-1073
[Abstract]
Howden R,
Goldsbrough PB,
Andersen CR,
Cobett CS
(1995b)
Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.
Plant Physiol
107:
1059-1066
[Abstract]
Huang L,
Gitschier J
(1997)
A novel gene involved in zinc transport is deficient in the lethal milk mouse.
Nat Genet
17:
292-297
[CrossRef][ISI][Medline]
Kamizono A,
Nishizawa M,
Teranishi Y,
Murata K,
Kimura A
(1989)
Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae.
Mol Gen Genet
219:
161-167
[CrossRef][ISI][Medline]
Leister D,
Ballvora A,
Salamini F,
Gebhardt C
(1996)
A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants.
Nat Genet
14:
421-429
[CrossRef][ISI][Medline]
Murashige T,
Skoog F
(1962)
A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiol Plant
15:
473-497
[CrossRef]
Neuteboom LW,
Ng JMY,
Kuyper M,
Clijdesdale OR,
Hooykaas PJJ,
van der Zaal BJ
(1999)
Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation.
Plant Mol Biol
39:
273-287
[CrossRef][ISI][Medline]
Nielsen H,
Engelbrecht J,
Brunak S,
von Heijne G
(1997)
Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.
Protein Eng
10:
1-6
[Abstract/Free Full Text]
Nies DH
(1992)
CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus.
J Bacteriol
174:
8102-8110
[Abstract/Free Full Text]
Palmiter RD,
Cole TB,
Findley SD
(1996a)
ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration.
EMBO J
15:
1784-1791
[ISI][Medline]
Palmiter RD,
Cole TB,
Quaife CJ,
Findley SD
(1996b)
ZnT-3, a putative transporter of zinc into synaptic vesicles.
Proc Natl Acad Sci USA
93:
14934-14939
[Abstract/Free Full Text]
Palmiter RD,
Findley SD
(1995)
Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc.
EMBO J
14:
639-649
[ISI][Medline]
Rensing C,
Pribyl T,
Nies,
DH
(1997)
New functions for the three subunits of the CzcCBA cation-proton antiporter.
J Bacteriol
179:
6871-6879
[Abstract/Free Full Text]
Riesmeier JW,
Willmitzer L,
Frommer WB
(1992)
Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.
EMBO J
11:
4705-4713
[ISI][Medline]
Sambrook L,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual, Ed 2.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sanger F,
Nicklen S,
Coulson AR
(1977)
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA
74:
5463-5467
[Abstract/Free Full Text]
Schat H,
Kalff MMA
(1992)
Are phytochelatins involved in differential metal tolerance or do they merely reflect metal-imposed strain?
Plant Physiol
99:
1475-1480
[Abstract/Free Full Text]
Schat H, ten Bookum WM (1992a) Metal-specificity of heavy metal
tolerance syndromes in higher plants. In AJM Baker, RD
Reeves, J Proctor, eds, The Vegetation of Ultramaffic (Serpentine)
Soils. Intercept, Andover, UK, pp 337-353
Schat H,
ten Bookum WM
(1992b)
Genetic control of copper tolerance in Silene vulgaris.
Heredity
68:
219-229
Schat H, Verkleij JAC (1998) The role of non-woody plants
in phytorestoration: possibilities to exploit adaptive heavy metal
tolerance. In J Vangronsveld, C Cunningham, eds, In Situ
Phytoremediation of Heavy Metals Contaminated Sites. Springer Verlag
and R.G. Landes Co., Austin, TX, pp 51-65
Schat H,
Vooijs R
(1997)
Multiple tolerance and co-tolerance to heavy metals in Silene vulgaris: a co-segragation analysis.
New Phytol
136:
489-496
[CrossRef]
Sikorski RS,
Hieter P
(1989)
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics
122:
19-27
[Abstract/Free Full Text]
Tabata K,
Kashiwagi S,
Mori H,
Ueguchi C,
Mizuno T
(1997)
Cloning of a cDNA encoding a putative metal-transporting P-type ATPase from Arabidopsis thaliana.
Biochim Biophys Acta
1326:
1-6
[Medline]
Valvekens D,
van Montagu M,
van Lijsebettens M
(1988)
Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.
Proc Natl Acad Sci USA
85:
5536-5540
[Abstract/Free Full Text]
van Slogteren GMS,
Hoge JHC,
Hooykaas PJJ,
Schilperoort RA
(1983)
Clonal analysis of heterogenous crown gall tumour tissues induced by wild-type and shooter mutant strains of Agrobacterium tumefaciens: expression of T-DNA genes.
Plant Mol Biol
2:
321-333
[CrossRef]
Verkleij JAC,
Koevoets PLM,
Blake-Kalff MMA,
Chardonnens AN
(1998)
Evidence for an important role of the tonoplast in the mechanism of naturally selected zinc tolerance in Silene vulgaris.
J Plant Physiol
153:
188-191
Wenzel HJ,
Cole TB,
Born DE,
Schwartzkroin PA,
Palmiter RD
(1997)
Ultrastructural localization of Zn transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey.
Proc Natl Acad Sci USA
94:
12676-12681
[Abstract/Free Full Text]
Zhao H,
Eide D
(1996a)
The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation.
Proc Natl Acad Sci USA
93:
2454-2458
[Abstract/Free Full Text]
Zhao H,
Eide D
(1996b)
The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae.
J Biol Chem
271:
23203-23210
[Abstract/Free Full Text]
Zhou J,
Goldsbrough PB
(1994)
Functional homologs of fungal metallothionein genes from Arabidopsis.
Plant Cell
6:
875-884
[Abstract]
Zonneveld BJM
(1986)
Cheap and simple yeast media.
J Microbiol Methods
4:
287-291
[CrossRef]
This article has been cited by other articles:
|
|
|
|
 
M. A. Klein, H. Sekimoto, M. J. Milner, and L. V. Kochian
Investigation of Heavy Metal Hyperaccumulation at the Cellular Level: Development and Characterization of Thlaspi caerulescens Suspension Cell Lines
Plant Physiology,
August 1, 2008;
147(4):
2006 - 2016.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Milner and L. V. Kochian
Investigating Heavy-metal Hyperaccumulation using Thlaspi caerulescens as a Model System
Ann. Bot.,
July 1, 2008;
102(1):
3 - 13.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kawachi, Y. Kobae, T. Mimura, and M. Maeshima
Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity
J. Biol. Chem.,
March 28, 2008;
283(13):
8374 - 8383.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. F. Mills, M. L. Doherty, R. L. Lopez-Marques, T. Weimar, P. Dupree, M. G. Palmgren, J. K. Pittman, and L. E. Williams
ECA3, a Golgi-Localized P2A-Type ATPase, Plays a Crucial Role in Manganese Nutrition in Arabidopsis
Plant Physiology,
January 1, 2008;
146(1):
116 - 128.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Peiter, B. Montanini, A. Gobert, P. Pedas, S. Husted, F. J. M. Maathuis, D. Blaudez, M. Chalot, and D. Sanders
A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance
PNAS,
May 15, 2007;
104(20):
8532 - 8537.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Willems, D. B. Drager, M. Courbot, C. Gode, N. Verbruggen, and P. Saumitou-Laprade
The Genetic Basis of Zinc Tolerance in the Metallophyte Arabidopsis halleri ssp. halleri (Brassicaceae): An Analysis of Quantitative Trait Loci
Genetics,
May 1, 2007;
176(1):
659 - 674.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Haydon and C. S. Cobbett
A Novel Major Facilitator Superfamily Protein at the Tonoplast Influences Zinc Tolerance and Accumulation in Arabidopsis
Plant Physiology,
April 1, 2007;
143(4):
1705 - 1719.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. E. Tsyganov, A. A. Belimov, A. Y. Borisov, V. I. Safronova, M. Georgi, K.-J. Dietz, and I. A. Tikhonovich
A Chemically Induced New Pea (Pisum sativum) Mutant SGECdt with Increased Tolerance to, and Accumulation of, Cadmium
Ann. Bot.,
February 1, 2007;
99(2):
227 - 237.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Martinoia, M. Maeshima, and H. E. Neuhaus
Vacuolar transporters and their essential role in plant metabolism
J. Exp. Bot.,
January 1, 2007;
58(1):
83 - 102.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. van de Mortel, L. Almar Villanueva, H. Schat, J. Kwekkeboom, S. Coughlan, P. D. Moerland, E. Ver Loren van Themaat, M. Koornneef, and M. G.M. Aarts
Large Expression Differences in Genes for Iron and Zinc Homeostasis, Stress Response, and Lignin Biosynthesis Distinguish Roots of Arabidopsis thaliana and the Related Metal Hyperaccumulator Thlaspi caerulescens
Plant Physiology,
November 1, 2006;
142(3):
1127 - 1147.
[Abstract]
[Full Text]
[PDF]
|
|
| |
Top
Abstract
Introduction