Overexpression of a Novel Arabidopsis Gene Related to Putative
Zinc-Transporter Genes from Animals Can Lead to Enhanced Zinc
Resistance and Accumulation |
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Authors: | Bert J van der Zaal Leon W Neuteboom Johan E Pinas Agnes N Chardonnens Henk Schat Jos AC Verkleij and Paul JJ Hooykaas |
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Institution: | 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.);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.) |
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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.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. |
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