Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:141-160:
244
Zeroing in on zinc uptake in yeast and plants
Mary Lou Guerinot* and David Eide†
Zinc is an essential micronutrient. Genes responsible for zinc
uptake have now been identified from yeast and plants. These
genes belong to an extended family of cation transporters called
the ZIP gene family. Zinc efflux genes that belong to another
transporter family, the CDF family, have also been identified in
yeast and Arabidopsis. It is clear that studies in yeast can greatly
aid our understanding of zinc metabolism in plants.
Addresses
*Department of Biological Sciences, 6044 Gilman, Dartmouth
College, Hanover, New Hampshire 03755, USA;
e-mail: Guerinot@Dartmouth.edu
†Nutritional Sciences Program, Department of Biochemistry, 217
Gwynn Hall, University of Missouri, Columbia, Missouri 65211, USA;
e-mail: deide@showme.missouri.edu
Current Opinion in Plant Biology 1999, 2:244–249
http://biomednet.com/elecref/1369526600200244
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
CDF
cation diffusion facilitator
ZIP
ZRT, IRT-like protein
Introduction
Zinc plays an amazing number of critical roles in all organisms — it is an essential component of more than 300
enzymes including RNA polymerase, alkaline phosphatase, alcohol dehydrogenase, Cu/Zn superoxide
dismutase, and carbonic anhydrase. Moreover, greater than
3% of the protein sequences inferred from the two eukaryotic genomes completely sequenced to date, that is the
genomes of S. cerevisiae and C. elegans, contain sequence
motifs characteristic of zinc binding structural domains [1].
These include several motifs found in DNA binding proteins, including the C2H2 zinc finger domain (see Note
added in proof), the C6 zinc cluster domain, and the C4
hormone receptor domain. Because zinc is a hydrophilic,
highly charged species, it cannot cross biological membranes by passive diffusion, but rather it must be
transported. It is taken up from the soil solution as a divalent cation [2]. Once taken up, zinc is neither oxidized nor
reduced; thus, the role of zinc in cells is based on its behavior as a divalent cation that has a strong tendency to form
tetrahedral complexes (for a review see [3]).
Zinc deficiency is probably the most widespread micronutrient deficiency limiting crop production and quality in
cereals [4•]. A global sampling of 190 soils from 25 countries
found that 49% were low in zinc. Unlike other micronutrient deficiencies, zinc deficiency is ubiquitous: it occurs in
cold and warm climates, in drained and flooded soils and in
acid and alkaline soils. This review describes how recent
studies of zinc uptake and its regulation in yeast have now
led to new insight into how plants obtain zinc from soils.
Zinc uptake in yeast
Kinetic studies of zinc uptake by yeast cells grown with different amounts of zinc in the medium suggested the
presence of at least two uptake systems [5•]. One system has
a high affinity for zinc with an estimated apparent Km of
10 nM Zn2+ and is only active in zinc-limited cells [6]. The
second system has a lower affinity for zinc (apparent Km of
100 nM Zn2+) and is detectable in zinc replete cells [7].
The ZRT1 gene encodes the transporter protein of the high
affinity system [6]. The level of ZRT1 mRNA correlates
with activity of the high affinity system; overexpressing
ZRT1 increases high affinity uptake whereas disrupting the
ZRT1 gene eliminates high affinity activity and results in
poor growth of the mutant on zinc-limiting media. In similar studies, it was determined that the ZRT2 gene encodes
the transporter of the low affinity uptake system [7]. More
recently, the ZRT1 protein has been found to be glycosylated and localized to the plasma membrane of the cell
[8••]. Additional, as yet uncharacterized zinc uptake systems are also present in S. cerevisiae as demonstrated by the
observation that the zrt1 zrt2 mutant is viable [7].
The ZRT1 and ZRT2 proteins share 44% sequence identity and 67% similarity. They each contain eight potential
transmembrane domains and have a similar predicted
membrane topology in which the amino- and carboxy-terminal ends of the protein are located on the outside surface
of the plasma membrane (Figure 1). These proteins were
originally identified because of their similarity to the IRT1
transporter from Arabidopsis [9]. The IRT1 gene was cloned
by functional expression in a yeast mutant (fet3 fet4) defective for iron uptake. Our current hypothesis is that IRT1 is
an Fe2+ transporter that takes up iron from the soil, a proposal that is consistent with the observation that yeast
expressing IRT1 possess a novel Fe2+ uptake activity.
Moreover, in Arabidopsis, IRT1 mRNA is expressed in roots
and is induced by iron-limiting growth conditions.
Although IRT1 was originally identified as an iron transporter, we now know from studies in yeast that IRT1 is able
to transport both Mn and Zn in addition to Fe [10]. In addition to sharing sequence similarity and numbers of
potential transmembrane domains, ZRT1, ZRT2, and
IRT1 each have a potential metal-binding domain between
transmembrane domains three and four that is predicted to
be cytoplasmic. For example, in ZRT1, this sequence is
HDHTHDE and in IRT1, this motif is HGHGHGH.
Although the function of this motif is currently unknown,
its conserved location in these three proteins and its potential for metal binding suggests that it plays an important
role in metal ion uptake or its regulation.
Through DNA sequence database comparisons and additional expression cloning studies, it is now clear that these
Zeroing in on zinc uptake in yeast and plants Guerinot and Eide
245
Figure 1
Predicted topology of a ZIP family member.
The protein is predicted to cross the
membrane eight times (TOP-PRED II). There
is a variable region between transmembrane
domains 3 and 4 that is histidine rich and
predicted to lie in the cytoplasm.
N
C
Outside
Inside
Current Opinion in Plant Biology
three metal ion transporters are members of a family of
proteins found in a diverse array of eukaryotic organisms
[11]. This family is referred to collectively as the ZIP family for ‘ZRT, IRT-like protein.’ At this time, 24 ZIP
members have been identified and these genes fall into
two discrete subfamilies based on amino acid similarities
(Figure 2). Subfamily I includes 11 genes in plants (9 from
Arabidopsis, one from pea and one from rice), the two yeast
genes (i.e. ZRT1 and ZRT2), and a gene from the protozoan
T. brucei. Subfamily II includes 8 genes in the nematode C.
elegans and two in humans. There are also two more distantly related family members, one in humans and one in
mouse that are not shown on the dendrogram in Figure 2
[11]. All but two of these proteins contain the putative
metal-binding domain described above.
Zinc uptake in plants
Using a method similar to the one used to isolate IRT1, the
ZIP1, ZIP2, and ZIP3 genes of Arabidopsis were isolated by
functional expression cloning in a zrt1 zrt2 mutant yeast
strain; expression of these genes in yeast restored zinc-limited growth to this mutant [12••]. Biochemical analysis of
metal uptake has demonstrated that these genes encode
zinc transporters. Yeast expressing ZIP1, ZIP2, and ZIP3
each have different time-, temperature-, and concentration-dependent zinc uptake activities with apparent Km
values of 10–100 nM Zn2+. These values are similar to the
levels of free Zn2+ available in the rhizosphere [13].
Moreover, no Fe2+ or Fe3+ uptake activity has been detected with any of these proteins in uptake experiments using
55Fe. We propose that each of these three genes plays a
role in zinc transport in plants and that they represent the
first zinc transporter genes to be cloned from any plant
species. A fourth Arabidopsis ZIP homolog, ZIP4, was identified in the DNA sequence databases, but its expression
did not confer zinc uptake activity in yeast [12••]. This
may not be surprising as ZIP4 is predicted to have a chloroplast targeting sequence and, therefore, may not localize
properly in yeast cells.
Some plants are better able to grow on zinc deficient soils and
hence have been termed zinc efficient. The physiological
mechanisms responsible for differential tolerance to zinc
deficiency among genotypes are not present. Rengel and
Hawkesford [14] have previously reported the presence of a
34 kDa polypeptide that is strongly induced in a zinc efficient variety of wheat grown under zinc deficiency. The
34 kDa polypeptide localized to the plasma membrane of
roots, leading the authors to speculate that it might be a
structural or regulatory component of the plasma membrane
zinc transporter. The ZIP proteins we have identified to
date are predicted to range in size from 36 to 39 kDa, suggesting that the wheat protein may be a related transporter.
A second gene family that could potentially play a role in
zinc transport in plants is the Nramp family. This family of
transport proteins has been identified in plants [15,16] as
well as other eukaryotes [17]. A recent study has demonstrated that one member, DCT1/Nramp2, is capable of
transporting a variety of metal ions including zinc [18].
Although DCT1/Nramp2 is clearly important for iron
uptake in mammals [19,20], evidence for a role of DCT1
in zinc transport in any organism is still lacking.
Do phytosiderophores play a role in
zinc uptake?
Grasses release metal chelators, called phytosiderophores,
both under iron and zinc deficiency [21,22]. Although the
role of phytosiderophores in iron nutrition is well accepted
[23••], whether phytosiderophores play an important role
in zinc nutrition remains an unanswered question. In a
recent study comparing wheat genotypes that differed in
their tolerance to zinc deficiency, the tolerant genotype
released more phytosiderophores, and showed both greater
uptake of zinc by roots and greater transport of zinc to the
shoots than did the genotype sensitive to zinc deficiency
[24]. Although chelate splitting is not a prerequisite for the
uptake of zinc from zinc-phytosiderophores by maize, free
zinc is taken up more readily than chelated zinc [25].
What’s special about zinc hyperaccumulators?
Plant species which are endemic to metalliferous soils are
metal tolerant, either by excluding metals or by virtue of
being able to accumulate and sequester metals. Over 400
246
Physiology and metabolism
Figure 2
Arabidopsis IRT1
Arabidopsis IRT2
Pea RIT1
Dendrogram showing amino acid sequence
relationships among the ZIP family members.
Computer database comparisons were
performed using BLAST and the multiple
sequence alignment was performed using
PILEUP and PRETTY (GCG).
Oryza sativa D49213
Arabidopsis ZIP3
Arabidopsis ZIP5
Arabidopsis ZIP4
Arabidopsis IRT3
Arabidopsis ZIP1
Arabidopsis ZIP6
S. cerevisiae ZRT1
S. cerevisiae ZRT2
T. brucei AA601838
Arabidopsis ZIP2
Homo sapiens H20615
Homo sapiens 998569
C. elegans U92784
C. elegans U42437
C. elegans Z70306
C. elegans U80447
C. elegans AL03268
C. elegans U28944
C. elegans Z81110
C. elegans Z81044
Current Opinion in Plant Biology
metal hyperaccumulating species of plants have been
reported, of which about 16 are zinc hyperaccumulators
(defined as containing more than 10,000 µg zinc g–1 in
shoot tissues [26,27]. Certain populations of Thlaspi
caerulescens can accumulate up to 40,000 µg zinc g–1 tissue
in their shoots whereas optimal zinc concentration for most
plants is between 20 and 100 µg g–1 tissue. There is great
interest in hyperaccumulators because of their potential for
use in extracting metals from soils, either in aid of phytoremediation [28•] or in aid of phytomining [27].
Radiotracer studies with T. caerulescens and a non-hyperaccumulating related species, T. arvense, have shown that the
Vmax for the uptake of zinc was 4.5 fold greater for
T. caerulescens than for the non-hyperaccumulator whereas
their Km values were not significantly different. This suggests
that zinc uptake is controlled by regulating the number of
active transporters in the membrane [29]. A T. caerulescens
gene, ZNT1, has been identified by functional expression
cloning in a zrt1 zrt2 mutant yeast strain [30]. This gene
encodes a presumptive zinc transporter that is 88% similar
to the Arabidopsis ZIP4 transporter. Northern analysis
shows that the ZNT1 transcript is very abundant in roots
and shoots regardless of zinc status. This is especially
interesting in light of the fact that ZIP4 is also expressed
in both roots and shoots of Arabidopsis but only when
plants are starved for zinc [12••]. Constitutive expression
of a zinc transporter gene regardless of zinc status may, in
part, explain the ability of T. caerulecens to accumulate zinc.
The zinc that is taken up by T. caerulescens also appears to be
more readily available for loading into the xylem, as
Zeroing in on zinc uptake in yeast and plants Guerinot and Eide
T. caerulescens xylem sap contained five-fold more zinc than
xylem sap from T. arvense [31••]. Similar conclusions were
reached in a comparison of T. caerulescens and the non-hyperaccumulator T. ochroleucum [32]. Interestingly, T. caerulescens
has a greater requirement for zinc than other plants, suggesting that constitutive sequestration mechanisms exist
which decrease its physiological availability [32]. Once in
the shoot, zinc is sequestered in a soluble form in the vacuoles of leaf cells, preventing the buildup of toxic levels in
the cytoplasm [33]. In the zinc tolerant plant Silene vulgaris,
zinc transport across the tonoplast was about 2.5 times higher than in zinc-sensitive plants of the same species [34].
Regulation by zinc
In all organisms, zinc uptake is tightly controlled to ensure
that adequate levels of the metal are accumulated while
potentially toxic overaccumulation is prevented. In yeast,
this control is exerted at both the transcriptional and posttranslational level. At the transcriptional level, expression of
the ZRT1 and ZRT2 genes is induced more than 10-fold in
zinc-limited cells [6,7]. Regulation of these genes in response
to zinc is mediated by the product of the ZAP1 gene [35,36].
ZAP1 is a zinc-responsive transcriptional activator protein
that senses intracellular zinc levels, perhaps by direct binding of zinc to the protein, and translates that signal into
changes in gene expression. The mechanism of this regulation is currently unknown but may involve direct binding of
zinc to ZAP1 which could inhibit DNA binding or activation
domain activity. Post-translational regulation of ZRT1 occurs
when cells are exposed to high levels of extracellular zinc
[8••]. Under these conditions, ZRT1 uptake activity is rapidly lost and this decrease is due to endocytosis of the ZRT1
protein and its subsequent degradation in the vacuole. Zincinduced endocytosis of ZRT1 is a specific response to zinc
and allows the rapid shutoff of zinc uptake activity thereby
protecting cells from zinc overaccumulation.
In plants, we also have evidence that expression of the zinc
transporters is metal responsive — ZIP1, ZIP3, and ZIP4
mRNAs are all induced in zinc-limited plants.
Furthermore, ZIP1 shows zinc-induced inactivation when
expressed in yeast. That is, ZIP1-dependent zinc uptake is
rapidly lost when cells are exposed to high levels of zinc
(ML Guerinot, D Eide, unpublished data). We are currently testing transgenic plants carrying a CaMV 35S-ZIP1
construct to see if a zinc-induced endocytosis mechanism
like that affecting ZRT1 activity is also operating in plants.
Intracellular zinc transport
Once zinc is transported into a eukaryotic cell, it must be
delivered to intracellular compartments to supply zincdependent proteins within those compartments.
Moreover, it is likely that excess zinc is stored in intracellular compartments such as the vacuole. Two potential
intracellular zinc transporters have been identified in S.
cerevisiae. These transporters are encoded by the ZRC1 and
COT1 genes. ZRC1 was isolated as a suppressor of zinc toxicity, that is overexpression of ZRC1 results in zinc
247
resistance [37]. The COT1 gene was isolated in a similar
fashion to ZRC1, that is as a suppressor of cobalt toxicity,
but was later found to confer zinc resistance as well [38,39].
Disruption of either ZRC1 and COT1 resulted in greater
sensitivity to zinc, further supporting the potential role of
these genes in zinc compartmentalization.
ZRC1 and COT1 are closely related proteins (60% identity) of approximately 440 amino acids and six–seven
potential transmembrane domains. The physiological roles
of these transporters are still unclear. Neither ZRC1 nor
COT1 are essential genes, and a zrc1 cot1 mutant is also
viable. Neither protein appears to catalyze zinc efflux from
the cell. Although COT1 was originally thought to be
localized in the mitochondria [38], both COT1 and ZRC1
have recently been demonstrated to be vacuolar proteins
[40]. These observations suggest that COT1 and ZRC1
play roles in transporting zinc into the vacuole.
ZRC1 and COT1 belong to yet another family of metal
transport proteins, referred to as the CDF (cation diffusion
facilitator) family [41]. In all, over 16 members of this family have been identified in prokaryotes and eukaryotes.
Analyses of other CDF proteins lend support to the role of
the yeast proteins in metal transport. For example, one
member of this family, the CzcD protein of the prokaryote
Alcaligenes eutrophus, confers resistance to zinc as well as
cobalt and cadmium [42]. Four mammalian CDF proteins,
ZnT-1-4, are zinc transporters [43]. ZnT-1 is a zinc efflux
transporter in the plasma membrane [44]. ZnT-2 is found
in the membrane of an acidic endosomal/lysosomal compartment and may play a role in zinc sequestration [45].
ZnT-3 is expressed only in the brain and testis and is most
abundant in the neurons of the hippocampus and the cerebral cortex [46]. ZnT-3 is localized to synaptic vesicle
membranes, suggesting that this protein transports zinc
into this compartment. ZnT-4 has been identified as the
lethal-milk gene and is thus implicated in the deposition of
zinc into the maternal milk supply [47]. The similarity of
these proteins with ZRC1 and COT1 supports the hypothesis that the yeast proteins detoxify excess zinc by
transporting the metal into an intracellular vesicular compartment. In an intriguing point of similarity with ZIP
proteins, the eukaryotic CDF proteins contain a histidinerich region with the sequence (HX)n where n = 3–6. As is
the case of the ZIP proteins, this domain is predicted to be
cytoplasmic and its function is still unknown. There are
currently two CDF Arabidopsis homologs in the database
(accession numbers AC005310 and AC004561). One of
these, now called ZAT1, leads to increased zinc accumulation and increased zinc tolerance when overexpressed in
Arabidopsis [48••]. In these overexpressing lines, zinc accumulates in the roots but not in the shoots. Expression of
ZAT1 in yeast did not lead to increased resistance to zinc.
Conclusions
Why do plants have multiple zinc transporters? One lesson
we have learned from studies in yeast is that any particular
248
Physiology and metabolism
metal will have two or more relatively specific transport
systems: high affinity systems that are active in metal limiting conditions and low affinity systems that function
when substrates are more abundant [5•]. We also know that
zinc, like other metals, is transported from the soil into the
root and then must cross both cellular and organellar membranes as it is distributed throughout the plant. Specific
zinc transporters may play different roles in this distribution process. Various molecular approaches ultimately can
tell us not only in what tissue and cell type certain transporters are expressed but where within a cell each is
expressed. We are also now in a position to identify plant
mutants carrying insertions in particular transporter genes;
this will greatly help in assigning functions. Having cloned
genes in hand is also allowing us to undertake
structure–function studies on the encoded proteins themselves. Finally, moving beyond how any one transporter
functions, we need to keep in mind that we want to understand zinc transport at the whole plant level and to use
such knowledge to create plants with enhanced mineral
content as well as plants that bioaccumulate or exclude this
potentially toxic cation. Such understanding will require
knowledge of how zinc levels are sensed by plants and how
gene expression is controlled by zinc. Studies in yeast are
providing important information about zinc regulation that
we believe will be directly applicable to plants. Over the
next five years, the relationship between high and low
affinity zinc transporters, between transporters responsible
for zinc influx and those responsible for zinc efflux, and
between transporters and the system(s) responsible for
sensing zinc levels in cells should become clearer.
Note added in proof
A new paper on zinc finger proteins in plants has recently
been published [49].
Acknowledgements
We wish to thank members of our laboratories for their contributions. Work
in the Guerinot lab is supported by grants from the National Science
Foundation and the Department of Energy and work in the Eide lab by
grants from the National Science Foundation, the Department of Energy
and the National Institutes of Health.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Clarke ND, Berg JM: Zinc fingers in Caenorhabditis elegans:
finding families and probing pathways. Science 1998, 282:20182022.
2.
Marschner H: Mineral Nutrition of Higher Plants, edn 2. Boston:
Academic Press; 1995.
3.
Berg JM, Shi Y: The galvanization of biology: a growing
appreciation for the roles of zinc. Science 1996, 271:1081-1085.
Ruel MT, Bouis HE: Plant breeding: a long term strategy for the
control of zinc deficiency in vulnerable populations. Amer J Clin
Nutr 1998, 68:488S-494S.
This article describes ongoing plant breeding strategies that could increase
the intake of bioavailable zinc from staple food crops. The three most promising breeding strategies include increasing the concentration of zinc, reducing the amount of phytic acid and raising the concentration of sulfur
4.
•
containing amino acids. Phytic acid decreases zinc absorption in humans,
whereas addition of sulfur-containing amino acids to the diet has been
shown to increase the bioavailability of zinc.
5. Eide DJ: The molecular biology of metal ion transport in
•
Saccharomyces cerevisiae. Annu Rev Nutr 1998, 18:441-469.
This is an overview of the genes involved in transport of the transition metals
Cu, Fe, Mn and Zn in S. cerevisiae.
6.
Zhao H, Eide D: The yeast ZRT1 gene encodes the zinc
transporter of a high affinity uptake system induced by zinc
limitation. Proc Natl Acad Sci USA 1996, 93:2454-2458.
7.
Zhao H, Eide D: The ZRT2 gene encodes the low affinity zinc
transporter in Saccharomyces cerevisiae. J Biol Chem 1996,
271:23203-23210.
Gitan RS, Luo H, Rodgers J, Broderius M, Eide D: Zinc-induced
inactivation of the yeast ZRT1 zinc transporter occurs through
endocytosis and vacuolar degradation. J Biol Chem 1998,
273:28617-28624.
Regulated endocytosis of plasma membrane proteins plays an important role
in controlling nutrient uptake and responses to extracellular signals. The
authors demonstrate that zinc-induced ZRT1 inactivation is a specific regulatory mechanism to shut off zinc uptake in cells exposed to high zinc levels,
thereby preventing overaccumulation of this potentially toxic metal. It is clear
that we will need to understand such feed-back mechanisms if we wish to
design plants that have altered metal content.
8.
••
9.
Eide D, Broderius M, Fett J, Guerinot ML: A novel iron-regulated
metal transporter from plants identified by functional expression
in yeast. Proc Natl Acad Sci USA 1996, 93:5624-5628.
10. Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB: The
IRT1 protein from Arabidopsis thaliana is a metal transporter with
broad specificity. Plant Mol Biol 1999, 40:37-44.
11. Eng BH, Guerinot ML, Eide D, Saier MHJ: Sequence analyses and
phylogenetic characterization of the ZIP family of metal ion
transport proteins. J Membr Biol 1998, 166:1-7.
12. Grotz N, Fox T, Connolly EL, Park W, Guerinot ML, Eide D:
•• Identification of a family of zinc transporter genes from
Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci
USA 1998, 95:7220-7224.
This paper reports the identification of the first zinc transporter genes, ZIP14, cloned from plants. The genes belong to a family of transporters called the
ZIP gene family. ZIP1 and ZIP3 are expressed in roots in response to zinc
deficiency, suggesting that they transport zinc from the soil into the plant.
ZIP4 is induced in both roots and shoots of zinc-limited plants.
13. Norvell WA, Welch RM: Growth and nutrient uptake by barley
(Hordeum vulgare L. cv. Herta): studies using an N-(2hydroxyethyl)ethylenedinitrilotriacetic acid-buffered nutrient
solution technique. I. Zinc ion requirements. Plant Physiol 1993,
101:619-625.
14. Rengel Z, Hawkesford MJ: Biosynthesis of a 34-kDa polypeptide
in the root-cell plasma membrane of a Zn-efficient wheat
genotype increases upon Zn deficiency. Aust J Plant Physiol
1997, 24:307-315.
15. Belouchi A, Cellier M, Kwan T, Saini HS, Leroux G, Gros P: The
macrophage specific membrane protein Nramp controlling
natural resistance in mice has homologues expressed in the root
systems of plants. Plant Mol Biol 1995, 29:1181-1196.
16. Belouchi A, Kwan T, Gros P: Cloning and characterization of the
OsNramp family from Oryza sativa, a new family of membrane
proteins possibly implicated in the transport of metal ions. Plant
Mol Biol 1997, 33:1085-1092.
17.
Cellier M, Privé G, Belouchi A, Kwan T, Rodrigues V, Chia W, Gros P:
Nramp defines a family of membrane proteins. Proc Natl Acad Sci
USA 1995, 92:10089-10093.
18. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF,
Boron WF, Nussberger S, Gollon JL, Hediger MA: Cloning and
characterization of a mammalian proton-coupled metal-ion
transporter. Nature 1997, 388:482-488.
19. Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR, Dietrich WF,
Andrews NC: Microcytic anaemia mice have a mutation in
Nramp2, a candidate iron transporter gene. Nature Genet 1997,
16:383-386.
20. Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD,
Andrews NC: Nramp2 is mutated in the anemic Belgrade (b) rat:
evidence of a role for Nramp2 in endosomal iron transport. Proc
Natl Acad Sci USA 1998, 95:1148-1153.
Zeroing in on zinc uptake in yeast and plants Guerinot and Eide
21. Cakmak I, Gülüt KY, Marschner H, Graham RD: Effect of zinc and
iron deficiency on phytosiderophore release in wheat genotypes
differing in zinc efficiency. J Plant Nutr 1994, 17:1-17.
22. Walter A, Römheld V, Marschner H, Mori S: Is the release of
phytosiderophores in zinc-deficient wheat plants a response to
impaired iron utilization? Physiol Plantarum 1994, 92:493-500.
23. Fox TC, Guerinot ML: Molecular biology of cation transport in
•• plants. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:669-696.
This review summarizes current knowledge about genes whose products
function in the transport of various cationic macronutrients (K, Ca) and
micronutrients (Cu, Fe, Mn and Zn) in plants.
24. Rengel Z, Römheld V, Marschner H: Uptake of zinc and iron by
wheat genotypes differing in tolerance to zinc deficiency. J Plant
Physiol 1998, 152:433-438.
25. von Wiren N, Marschner H, Römheld V: Roots of iron-efficient maize
also absorb phytosiderophore-chelated zinc. Plant Physiol 1996,
111:1119-1125.
26. Baker AJM, Brooks RR: Terrestrial higher plants which
hyperaccumulate metallic elements — a review of their distribution,
ecology and phytochemistry. Biorecovery 1989, 1:81-126.
27.
Brooks RR, Chambers MF, Nicks LJ, Robinson BH: Phytomining.
Trends Plant Sci 1998, 3:359-362.
28. Salt DE, Smith RD, Raskin I: Phytoremediation. Annu Rev Plant
•
Physiol Plant Mol Biol 1998, 49:643-668.
An excellent overview of the current information on using plants to clean up
the environment. Toxic heavy metals and organic pollutants are the major targets for phytoremediation.
29. Lasat MM, Baker AJM, Kochian LV: Physiological characterization of
root Zn2+ absorption and translocation to shoots in Zn
hyperaccumulator and nonaccumulator species of Thlaspi. Plant
Physiol 1996, 112:1715-1722.
30. Pence N, Larsen P, Ebbs S, Garvin D, Eide D, Kochian L: Cloning and
characterization of a heavy metal transporter (ZNT1) from the
Zn/Cd hyperaccumulator Thlaspi caerulescens. Plant Physiol 1998,
[Abstract 656 http://www.sheridan.com/aspp98/abs/45/ 0650.html]
31. Lasat MM, Baker AJM, Kochian LV: Altered Zn compartmentation in
•• the root symplasm and stimulated Zn absorption into the leaf as
mechanisms involved in Zn hyperaccumulation in Thlaspi
caerulescens. Plant Physiol 1998, 118:875-883.
The hyperaccumulator Thlaspi caerulescens differs in a number of respects
from the related non-hyperaccumulating species Thalspi arvense. The
authors show the zinc that is taken up by T. caerulescens is more readily
available for loading into the xylem.
249
of naturally selected zinc tolerance in Silene vulgaris. J Plant
Physiol 1998, in press.
35. Zhao H, Eide D: Zap1p, a metalloregulatory protein involved in
zinc-responsive transcriptional regulation in Saccharomyces
cerevisiae. Mol Cell Biol 1997, 17:5044-5052.
36. Zhao H, Butler E, Rodgers J, Spizzo T, Duesterhoeft S, Eide D:
Regulation of zinc homeostasis in yeast by binding of the ZAP1
transcriptional activator to zinc-responsive promoter elements.
J Biol Chem 1998, 273:28713-28720.
37.
Kamizono A, Nishizawa M, Teranishi Y, Murata K, Kimura A: Identification
of a gene conferring resistance to zinc and cadmium ions in the
yeast Saccharomyces cerevisiae. Mol Gen Genet 1989, 219:161-167
38. Conklin DS, McMaster JA, Culbertson MR, Kung C: COT1, a gene
involved in cobalt accumulation in Saccharomyces cerevisiae. Mol
Cell Biol 1992, 12:3678-3688.
39. Conklin DS, Culbertson MR, Kung C: Interactions between gene
products involved in divalent cation transport in Saccharomyces
cerevisiae. Mol Gen Genet 1994, 244:303-311.
40. Li L, Kaplan J: Defects in the yeast high affinity iron transport
system result in increased metal sensitivity because of the
increased expression of transporters with a broad transition
metal specificity. J Biol Chem 1998, 273:22181-22187.
41. Paulsen LT, Saier MH: A novel family of ubiquitous heavy metal ion
transport proteins. J Membr Biol 1997, 156:99-103.
42. Nies DH: CzcR and CzcD, gene products affecting regulation of
resistance to cobalt, zinc and cadmium (czc system) in
Alcaligenes eutrophus. J Bacteriol 1992, 174:8102-8110.
43. McMahon RJ, Cousins RJ: Mammalian zinc transporters. J Nutr
1998, 128:667-670.
44. Palmiter RD, Findley SD: Cloning and functional characterization of
a mammalian zinc transporter that confers resistance to zinc.
EMBO J 1995, 14:639-649.
45. Palmiter RD, Cole TB, Findley SD: ZnT-2, a mammalian protein that
confers resistance to zinc by facilitating vesicular sequestration.
EMBO J 1996, 15:1784-1791.
46. Palmiter RD, Cole TB, Quaife CJ, Findley SD: ZnT-3, a putative
transporter of zinc into synaptic vesicles. Proc Natl Acad Sci USA
1996, 93:14934-14939.
47.
Huang L, Gitschier J: A novel gene involved in zinc transport is
deficient in the lethal milk mouse. Nature Genetics 1997, 17:292-295.
33. Küpper H, Zhao FJ, McGrath SP: Cellular compartmentation of zinc
in leaves of the hyperaccumulator Thlaspi caerulescens. Plant
Physiol 1999, 119:305-311.
48. van der Zaal EJ, Neuteboom LW, Pinas JE, Schat H, Verkleij J,
•• Hooykaas PJJ: Overexpression of a zinc transporter gene from
Arabidopsis can lead to enhanced zinc resistance and zinc
accumulation. Plant Physiol 1999, 119:1-9.
A homolog of mammalian zinc effluxers has been identified in Arabidopsis.
The authors show that overexpression of the homolog leads to a modest
increase in zinc resistance and to an increase in the zinc content of roots.
They speculate that ZAT1 may be involved in vacuolar sequestration of zinc.
34. Verkleij JAC, Koevoets PLM, Blake-Kalff MMA, Chardonnens AN:
Evidence for an important role of the tonoplast in the mechanism
49. Takatsuji H: Zinc finger proteins: the classic zinc finger emerges in
contemporary plant science. Plant Mol Biol 1999, 39:1073-1078.
32. Shen ZG, Zhao FJ, McGrath P: Uptake and transport of zinc in the
hyperaccumulator Thlaspi caerulescens and the nonhyperaccumulator Thlaspi ochroleucum. Plant Cell Environ 1997,
20:898-906.
Zeroing in on zinc uptake in yeast and plants
Mary Lou Guerinot* and David Eide†
Zinc is an essential micronutrient. Genes responsible for zinc
uptake have now been identified from yeast and plants. These
genes belong to an extended family of cation transporters called
the ZIP gene family. Zinc efflux genes that belong to another
transporter family, the CDF family, have also been identified in
yeast and Arabidopsis. It is clear that studies in yeast can greatly
aid our understanding of zinc metabolism in plants.
Addresses
*Department of Biological Sciences, 6044 Gilman, Dartmouth
College, Hanover, New Hampshire 03755, USA;
e-mail: Guerinot@Dartmouth.edu
†Nutritional Sciences Program, Department of Biochemistry, 217
Gwynn Hall, University of Missouri, Columbia, Missouri 65211, USA;
e-mail: deide@showme.missouri.edu
Current Opinion in Plant Biology 1999, 2:244–249
http://biomednet.com/elecref/1369526600200244
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
CDF
cation diffusion facilitator
ZIP
ZRT, IRT-like protein
Introduction
Zinc plays an amazing number of critical roles in all organisms — it is an essential component of more than 300
enzymes including RNA polymerase, alkaline phosphatase, alcohol dehydrogenase, Cu/Zn superoxide
dismutase, and carbonic anhydrase. Moreover, greater than
3% of the protein sequences inferred from the two eukaryotic genomes completely sequenced to date, that is the
genomes of S. cerevisiae and C. elegans, contain sequence
motifs characteristic of zinc binding structural domains [1].
These include several motifs found in DNA binding proteins, including the C2H2 zinc finger domain (see Note
added in proof), the C6 zinc cluster domain, and the C4
hormone receptor domain. Because zinc is a hydrophilic,
highly charged species, it cannot cross biological membranes by passive diffusion, but rather it must be
transported. It is taken up from the soil solution as a divalent cation [2]. Once taken up, zinc is neither oxidized nor
reduced; thus, the role of zinc in cells is based on its behavior as a divalent cation that has a strong tendency to form
tetrahedral complexes (for a review see [3]).
Zinc deficiency is probably the most widespread micronutrient deficiency limiting crop production and quality in
cereals [4•]. A global sampling of 190 soils from 25 countries
found that 49% were low in zinc. Unlike other micronutrient deficiencies, zinc deficiency is ubiquitous: it occurs in
cold and warm climates, in drained and flooded soils and in
acid and alkaline soils. This review describes how recent
studies of zinc uptake and its regulation in yeast have now
led to new insight into how plants obtain zinc from soils.
Zinc uptake in yeast
Kinetic studies of zinc uptake by yeast cells grown with different amounts of zinc in the medium suggested the
presence of at least two uptake systems [5•]. One system has
a high affinity for zinc with an estimated apparent Km of
10 nM Zn2+ and is only active in zinc-limited cells [6]. The
second system has a lower affinity for zinc (apparent Km of
100 nM Zn2+) and is detectable in zinc replete cells [7].
The ZRT1 gene encodes the transporter protein of the high
affinity system [6]. The level of ZRT1 mRNA correlates
with activity of the high affinity system; overexpressing
ZRT1 increases high affinity uptake whereas disrupting the
ZRT1 gene eliminates high affinity activity and results in
poor growth of the mutant on zinc-limiting media. In similar studies, it was determined that the ZRT2 gene encodes
the transporter of the low affinity uptake system [7]. More
recently, the ZRT1 protein has been found to be glycosylated and localized to the plasma membrane of the cell
[8••]. Additional, as yet uncharacterized zinc uptake systems are also present in S. cerevisiae as demonstrated by the
observation that the zrt1 zrt2 mutant is viable [7].
The ZRT1 and ZRT2 proteins share 44% sequence identity and 67% similarity. They each contain eight potential
transmembrane domains and have a similar predicted
membrane topology in which the amino- and carboxy-terminal ends of the protein are located on the outside surface
of the plasma membrane (Figure 1). These proteins were
originally identified because of their similarity to the IRT1
transporter from Arabidopsis [9]. The IRT1 gene was cloned
by functional expression in a yeast mutant (fet3 fet4) defective for iron uptake. Our current hypothesis is that IRT1 is
an Fe2+ transporter that takes up iron from the soil, a proposal that is consistent with the observation that yeast
expressing IRT1 possess a novel Fe2+ uptake activity.
Moreover, in Arabidopsis, IRT1 mRNA is expressed in roots
and is induced by iron-limiting growth conditions.
Although IRT1 was originally identified as an iron transporter, we now know from studies in yeast that IRT1 is able
to transport both Mn and Zn in addition to Fe [10]. In addition to sharing sequence similarity and numbers of
potential transmembrane domains, ZRT1, ZRT2, and
IRT1 each have a potential metal-binding domain between
transmembrane domains three and four that is predicted to
be cytoplasmic. For example, in ZRT1, this sequence is
HDHTHDE and in IRT1, this motif is HGHGHGH.
Although the function of this motif is currently unknown,
its conserved location in these three proteins and its potential for metal binding suggests that it plays an important
role in metal ion uptake or its regulation.
Through DNA sequence database comparisons and additional expression cloning studies, it is now clear that these
Zeroing in on zinc uptake in yeast and plants Guerinot and Eide
245
Figure 1
Predicted topology of a ZIP family member.
The protein is predicted to cross the
membrane eight times (TOP-PRED II). There
is a variable region between transmembrane
domains 3 and 4 that is histidine rich and
predicted to lie in the cytoplasm.
N
C
Outside
Inside
Current Opinion in Plant Biology
three metal ion transporters are members of a family of
proteins found in a diverse array of eukaryotic organisms
[11]. This family is referred to collectively as the ZIP family for ‘ZRT, IRT-like protein.’ At this time, 24 ZIP
members have been identified and these genes fall into
two discrete subfamilies based on amino acid similarities
(Figure 2). Subfamily I includes 11 genes in plants (9 from
Arabidopsis, one from pea and one from rice), the two yeast
genes (i.e. ZRT1 and ZRT2), and a gene from the protozoan
T. brucei. Subfamily II includes 8 genes in the nematode C.
elegans and two in humans. There are also two more distantly related family members, one in humans and one in
mouse that are not shown on the dendrogram in Figure 2
[11]. All but two of these proteins contain the putative
metal-binding domain described above.
Zinc uptake in plants
Using a method similar to the one used to isolate IRT1, the
ZIP1, ZIP2, and ZIP3 genes of Arabidopsis were isolated by
functional expression cloning in a zrt1 zrt2 mutant yeast
strain; expression of these genes in yeast restored zinc-limited growth to this mutant [12••]. Biochemical analysis of
metal uptake has demonstrated that these genes encode
zinc transporters. Yeast expressing ZIP1, ZIP2, and ZIP3
each have different time-, temperature-, and concentration-dependent zinc uptake activities with apparent Km
values of 10–100 nM Zn2+. These values are similar to the
levels of free Zn2+ available in the rhizosphere [13].
Moreover, no Fe2+ or Fe3+ uptake activity has been detected with any of these proteins in uptake experiments using
55Fe. We propose that each of these three genes plays a
role in zinc transport in plants and that they represent the
first zinc transporter genes to be cloned from any plant
species. A fourth Arabidopsis ZIP homolog, ZIP4, was identified in the DNA sequence databases, but its expression
did not confer zinc uptake activity in yeast [12••]. This
may not be surprising as ZIP4 is predicted to have a chloroplast targeting sequence and, therefore, may not localize
properly in yeast cells.
Some plants are better able to grow on zinc deficient soils and
hence have been termed zinc efficient. The physiological
mechanisms responsible for differential tolerance to zinc
deficiency among genotypes are not present. Rengel and
Hawkesford [14] have previously reported the presence of a
34 kDa polypeptide that is strongly induced in a zinc efficient variety of wheat grown under zinc deficiency. The
34 kDa polypeptide localized to the plasma membrane of
roots, leading the authors to speculate that it might be a
structural or regulatory component of the plasma membrane
zinc transporter. The ZIP proteins we have identified to
date are predicted to range in size from 36 to 39 kDa, suggesting that the wheat protein may be a related transporter.
A second gene family that could potentially play a role in
zinc transport in plants is the Nramp family. This family of
transport proteins has been identified in plants [15,16] as
well as other eukaryotes [17]. A recent study has demonstrated that one member, DCT1/Nramp2, is capable of
transporting a variety of metal ions including zinc [18].
Although DCT1/Nramp2 is clearly important for iron
uptake in mammals [19,20], evidence for a role of DCT1
in zinc transport in any organism is still lacking.
Do phytosiderophores play a role in
zinc uptake?
Grasses release metal chelators, called phytosiderophores,
both under iron and zinc deficiency [21,22]. Although the
role of phytosiderophores in iron nutrition is well accepted
[23••], whether phytosiderophores play an important role
in zinc nutrition remains an unanswered question. In a
recent study comparing wheat genotypes that differed in
their tolerance to zinc deficiency, the tolerant genotype
released more phytosiderophores, and showed both greater
uptake of zinc by roots and greater transport of zinc to the
shoots than did the genotype sensitive to zinc deficiency
[24]. Although chelate splitting is not a prerequisite for the
uptake of zinc from zinc-phytosiderophores by maize, free
zinc is taken up more readily than chelated zinc [25].
What’s special about zinc hyperaccumulators?
Plant species which are endemic to metalliferous soils are
metal tolerant, either by excluding metals or by virtue of
being able to accumulate and sequester metals. Over 400
246
Physiology and metabolism
Figure 2
Arabidopsis IRT1
Arabidopsis IRT2
Pea RIT1
Dendrogram showing amino acid sequence
relationships among the ZIP family members.
Computer database comparisons were
performed using BLAST and the multiple
sequence alignment was performed using
PILEUP and PRETTY (GCG).
Oryza sativa D49213
Arabidopsis ZIP3
Arabidopsis ZIP5
Arabidopsis ZIP4
Arabidopsis IRT3
Arabidopsis ZIP1
Arabidopsis ZIP6
S. cerevisiae ZRT1
S. cerevisiae ZRT2
T. brucei AA601838
Arabidopsis ZIP2
Homo sapiens H20615
Homo sapiens 998569
C. elegans U92784
C. elegans U42437
C. elegans Z70306
C. elegans U80447
C. elegans AL03268
C. elegans U28944
C. elegans Z81110
C. elegans Z81044
Current Opinion in Plant Biology
metal hyperaccumulating species of plants have been
reported, of which about 16 are zinc hyperaccumulators
(defined as containing more than 10,000 µg zinc g–1 in
shoot tissues [26,27]. Certain populations of Thlaspi
caerulescens can accumulate up to 40,000 µg zinc g–1 tissue
in their shoots whereas optimal zinc concentration for most
plants is between 20 and 100 µg g–1 tissue. There is great
interest in hyperaccumulators because of their potential for
use in extracting metals from soils, either in aid of phytoremediation [28•] or in aid of phytomining [27].
Radiotracer studies with T. caerulescens and a non-hyperaccumulating related species, T. arvense, have shown that the
Vmax for the uptake of zinc was 4.5 fold greater for
T. caerulescens than for the non-hyperaccumulator whereas
their Km values were not significantly different. This suggests
that zinc uptake is controlled by regulating the number of
active transporters in the membrane [29]. A T. caerulescens
gene, ZNT1, has been identified by functional expression
cloning in a zrt1 zrt2 mutant yeast strain [30]. This gene
encodes a presumptive zinc transporter that is 88% similar
to the Arabidopsis ZIP4 transporter. Northern analysis
shows that the ZNT1 transcript is very abundant in roots
and shoots regardless of zinc status. This is especially
interesting in light of the fact that ZIP4 is also expressed
in both roots and shoots of Arabidopsis but only when
plants are starved for zinc [12••]. Constitutive expression
of a zinc transporter gene regardless of zinc status may, in
part, explain the ability of T. caerulecens to accumulate zinc.
The zinc that is taken up by T. caerulescens also appears to be
more readily available for loading into the xylem, as
Zeroing in on zinc uptake in yeast and plants Guerinot and Eide
T. caerulescens xylem sap contained five-fold more zinc than
xylem sap from T. arvense [31••]. Similar conclusions were
reached in a comparison of T. caerulescens and the non-hyperaccumulator T. ochroleucum [32]. Interestingly, T. caerulescens
has a greater requirement for zinc than other plants, suggesting that constitutive sequestration mechanisms exist
which decrease its physiological availability [32]. Once in
the shoot, zinc is sequestered in a soluble form in the vacuoles of leaf cells, preventing the buildup of toxic levels in
the cytoplasm [33]. In the zinc tolerant plant Silene vulgaris,
zinc transport across the tonoplast was about 2.5 times higher than in zinc-sensitive plants of the same species [34].
Regulation by zinc
In all organisms, zinc uptake is tightly controlled to ensure
that adequate levels of the metal are accumulated while
potentially toxic overaccumulation is prevented. In yeast,
this control is exerted at both the transcriptional and posttranslational level. At the transcriptional level, expression of
the ZRT1 and ZRT2 genes is induced more than 10-fold in
zinc-limited cells [6,7]. Regulation of these genes in response
to zinc is mediated by the product of the ZAP1 gene [35,36].
ZAP1 is a zinc-responsive transcriptional activator protein
that senses intracellular zinc levels, perhaps by direct binding of zinc to the protein, and translates that signal into
changes in gene expression. The mechanism of this regulation is currently unknown but may involve direct binding of
zinc to ZAP1 which could inhibit DNA binding or activation
domain activity. Post-translational regulation of ZRT1 occurs
when cells are exposed to high levels of extracellular zinc
[8••]. Under these conditions, ZRT1 uptake activity is rapidly lost and this decrease is due to endocytosis of the ZRT1
protein and its subsequent degradation in the vacuole. Zincinduced endocytosis of ZRT1 is a specific response to zinc
and allows the rapid shutoff of zinc uptake activity thereby
protecting cells from zinc overaccumulation.
In plants, we also have evidence that expression of the zinc
transporters is metal responsive — ZIP1, ZIP3, and ZIP4
mRNAs are all induced in zinc-limited plants.
Furthermore, ZIP1 shows zinc-induced inactivation when
expressed in yeast. That is, ZIP1-dependent zinc uptake is
rapidly lost when cells are exposed to high levels of zinc
(ML Guerinot, D Eide, unpublished data). We are currently testing transgenic plants carrying a CaMV 35S-ZIP1
construct to see if a zinc-induced endocytosis mechanism
like that affecting ZRT1 activity is also operating in plants.
Intracellular zinc transport
Once zinc is transported into a eukaryotic cell, it must be
delivered to intracellular compartments to supply zincdependent proteins within those compartments.
Moreover, it is likely that excess zinc is stored in intracellular compartments such as the vacuole. Two potential
intracellular zinc transporters have been identified in S.
cerevisiae. These transporters are encoded by the ZRC1 and
COT1 genes. ZRC1 was isolated as a suppressor of zinc toxicity, that is overexpression of ZRC1 results in zinc
247
resistance [37]. The COT1 gene was isolated in a similar
fashion to ZRC1, that is as a suppressor of cobalt toxicity,
but was later found to confer zinc resistance as well [38,39].
Disruption of either ZRC1 and COT1 resulted in greater
sensitivity to zinc, further supporting the potential role of
these genes in zinc compartmentalization.
ZRC1 and COT1 are closely related proteins (60% identity) of approximately 440 amino acids and six–seven
potential transmembrane domains. The physiological roles
of these transporters are still unclear. Neither ZRC1 nor
COT1 are essential genes, and a zrc1 cot1 mutant is also
viable. Neither protein appears to catalyze zinc efflux from
the cell. Although COT1 was originally thought to be
localized in the mitochondria [38], both COT1 and ZRC1
have recently been demonstrated to be vacuolar proteins
[40]. These observations suggest that COT1 and ZRC1
play roles in transporting zinc into the vacuole.
ZRC1 and COT1 belong to yet another family of metal
transport proteins, referred to as the CDF (cation diffusion
facilitator) family [41]. In all, over 16 members of this family have been identified in prokaryotes and eukaryotes.
Analyses of other CDF proteins lend support to the role of
the yeast proteins in metal transport. For example, one
member of this family, the CzcD protein of the prokaryote
Alcaligenes eutrophus, confers resistance to zinc as well as
cobalt and cadmium [42]. Four mammalian CDF proteins,
ZnT-1-4, are zinc transporters [43]. ZnT-1 is a zinc efflux
transporter in the plasma membrane [44]. ZnT-2 is found
in the membrane of an acidic endosomal/lysosomal compartment and may play a role in zinc sequestration [45].
ZnT-3 is expressed only in the brain and testis and is most
abundant in the neurons of the hippocampus and the cerebral cortex [46]. ZnT-3 is localized to synaptic vesicle
membranes, suggesting that this protein transports zinc
into this compartment. ZnT-4 has been identified as the
lethal-milk gene and is thus implicated in the deposition of
zinc into the maternal milk supply [47]. The similarity of
these proteins with ZRC1 and COT1 supports the hypothesis that the yeast proteins detoxify excess zinc by
transporting the metal into an intracellular vesicular compartment. In an intriguing point of similarity with ZIP
proteins, the eukaryotic CDF proteins contain a histidinerich region with the sequence (HX)n where n = 3–6. As is
the case of the ZIP proteins, this domain is predicted to be
cytoplasmic and its function is still unknown. There are
currently two CDF Arabidopsis homologs in the database
(accession numbers AC005310 and AC004561). One of
these, now called ZAT1, leads to increased zinc accumulation and increased zinc tolerance when overexpressed in
Arabidopsis [48••]. In these overexpressing lines, zinc accumulates in the roots but not in the shoots. Expression of
ZAT1 in yeast did not lead to increased resistance to zinc.
Conclusions
Why do plants have multiple zinc transporters? One lesson
we have learned from studies in yeast is that any particular
248
Physiology and metabolism
metal will have two or more relatively specific transport
systems: high affinity systems that are active in metal limiting conditions and low affinity systems that function
when substrates are more abundant [5•]. We also know that
zinc, like other metals, is transported from the soil into the
root and then must cross both cellular and organellar membranes as it is distributed throughout the plant. Specific
zinc transporters may play different roles in this distribution process. Various molecular approaches ultimately can
tell us not only in what tissue and cell type certain transporters are expressed but where within a cell each is
expressed. We are also now in a position to identify plant
mutants carrying insertions in particular transporter genes;
this will greatly help in assigning functions. Having cloned
genes in hand is also allowing us to undertake
structure–function studies on the encoded proteins themselves. Finally, moving beyond how any one transporter
functions, we need to keep in mind that we want to understand zinc transport at the whole plant level and to use
such knowledge to create plants with enhanced mineral
content as well as plants that bioaccumulate or exclude this
potentially toxic cation. Such understanding will require
knowledge of how zinc levels are sensed by plants and how
gene expression is controlled by zinc. Studies in yeast are
providing important information about zinc regulation that
we believe will be directly applicable to plants. Over the
next five years, the relationship between high and low
affinity zinc transporters, between transporters responsible
for zinc influx and those responsible for zinc efflux, and
between transporters and the system(s) responsible for
sensing zinc levels in cells should become clearer.
Note added in proof
A new paper on zinc finger proteins in plants has recently
been published [49].
Acknowledgements
We wish to thank members of our laboratories for their contributions. Work
in the Guerinot lab is supported by grants from the National Science
Foundation and the Department of Energy and work in the Eide lab by
grants from the National Science Foundation, the Department of Energy
and the National Institutes of Health.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Clarke ND, Berg JM: Zinc fingers in Caenorhabditis elegans:
finding families and probing pathways. Science 1998, 282:20182022.
2.
Marschner H: Mineral Nutrition of Higher Plants, edn 2. Boston:
Academic Press; 1995.
3.
Berg JM, Shi Y: The galvanization of biology: a growing
appreciation for the roles of zinc. Science 1996, 271:1081-1085.
Ruel MT, Bouis HE: Plant breeding: a long term strategy for the
control of zinc deficiency in vulnerable populations. Amer J Clin
Nutr 1998, 68:488S-494S.
This article describes ongoing plant breeding strategies that could increase
the intake of bioavailable zinc from staple food crops. The three most promising breeding strategies include increasing the concentration of zinc, reducing the amount of phytic acid and raising the concentration of sulfur
4.
•
containing amino acids. Phytic acid decreases zinc absorption in humans,
whereas addition of sulfur-containing amino acids to the diet has been
shown to increase the bioavailability of zinc.
5. Eide DJ: The molecular biology of metal ion transport in
•
Saccharomyces cerevisiae. Annu Rev Nutr 1998, 18:441-469.
This is an overview of the genes involved in transport of the transition metals
Cu, Fe, Mn and Zn in S. cerevisiae.
6.
Zhao H, Eide D: The yeast ZRT1 gene encodes the zinc
transporter of a high affinity uptake system induced by zinc
limitation. Proc Natl Acad Sci USA 1996, 93:2454-2458.
7.
Zhao H, Eide D: The ZRT2 gene encodes the low affinity zinc
transporter in Saccharomyces cerevisiae. J Biol Chem 1996,
271:23203-23210.
Gitan RS, Luo H, Rodgers J, Broderius M, Eide D: Zinc-induced
inactivation of the yeast ZRT1 zinc transporter occurs through
endocytosis and vacuolar degradation. J Biol Chem 1998,
273:28617-28624.
Regulated endocytosis of plasma membrane proteins plays an important role
in controlling nutrient uptake and responses to extracellular signals. The
authors demonstrate that zinc-induced ZRT1 inactivation is a specific regulatory mechanism to shut off zinc uptake in cells exposed to high zinc levels,
thereby preventing overaccumulation of this potentially toxic metal. It is clear
that we will need to understand such feed-back mechanisms if we wish to
design plants that have altered metal content.
8.
••
9.
Eide D, Broderius M, Fett J, Guerinot ML: A novel iron-regulated
metal transporter from plants identified by functional expression
in yeast. Proc Natl Acad Sci USA 1996, 93:5624-5628.
10. Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB: The
IRT1 protein from Arabidopsis thaliana is a metal transporter with
broad specificity. Plant Mol Biol 1999, 40:37-44.
11. Eng BH, Guerinot ML, Eide D, Saier MHJ: Sequence analyses and
phylogenetic characterization of the ZIP family of metal ion
transport proteins. J Membr Biol 1998, 166:1-7.
12. Grotz N, Fox T, Connolly EL, Park W, Guerinot ML, Eide D:
•• Identification of a family of zinc transporter genes from
Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci
USA 1998, 95:7220-7224.
This paper reports the identification of the first zinc transporter genes, ZIP14, cloned from plants. The genes belong to a family of transporters called the
ZIP gene family. ZIP1 and ZIP3 are expressed in roots in response to zinc
deficiency, suggesting that they transport zinc from the soil into the plant.
ZIP4 is induced in both roots and shoots of zinc-limited plants.
13. Norvell WA, Welch RM: Growth and nutrient uptake by barley
(Hordeum vulgare L. cv. Herta): studies using an N-(2hydroxyethyl)ethylenedinitrilotriacetic acid-buffered nutrient
solution technique. I. Zinc ion requirements. Plant Physiol 1993,
101:619-625.
14. Rengel Z, Hawkesford MJ: Biosynthesis of a 34-kDa polypeptide
in the root-cell plasma membrane of a Zn-efficient wheat
genotype increases upon Zn deficiency. Aust J Plant Physiol
1997, 24:307-315.
15. Belouchi A, Cellier M, Kwan T, Saini HS, Leroux G, Gros P: The
macrophage specific membrane protein Nramp controlling
natural resistance in mice has homologues expressed in the root
systems of plants. Plant Mol Biol 1995, 29:1181-1196.
16. Belouchi A, Kwan T, Gros P: Cloning and characterization of the
OsNramp family from Oryza sativa, a new family of membrane
proteins possibly implicated in the transport of metal ions. Plant
Mol Biol 1997, 33:1085-1092.
17.
Cellier M, Privé G, Belouchi A, Kwan T, Rodrigues V, Chia W, Gros P:
Nramp defines a family of membrane proteins. Proc Natl Acad Sci
USA 1995, 92:10089-10093.
18. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF,
Boron WF, Nussberger S, Gollon JL, Hediger MA: Cloning and
characterization of a mammalian proton-coupled metal-ion
transporter. Nature 1997, 388:482-488.
19. Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR, Dietrich WF,
Andrews NC: Microcytic anaemia mice have a mutation in
Nramp2, a candidate iron transporter gene. Nature Genet 1997,
16:383-386.
20. Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD,
Andrews NC: Nramp2 is mutated in the anemic Belgrade (b) rat:
evidence of a role for Nramp2 in endosomal iron transport. Proc
Natl Acad Sci USA 1998, 95:1148-1153.
Zeroing in on zinc uptake in yeast and plants Guerinot and Eide
21. Cakmak I, Gülüt KY, Marschner H, Graham RD: Effect of zinc and
iron deficiency on phytosiderophore release in wheat genotypes
differing in zinc efficiency. J Plant Nutr 1994, 17:1-17.
22. Walter A, Römheld V, Marschner H, Mori S: Is the release of
phytosiderophores in zinc-deficient wheat plants a response to
impaired iron utilization? Physiol Plantarum 1994, 92:493-500.
23. Fox TC, Guerinot ML: Molecular biology of cation transport in
•• plants. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:669-696.
This review summarizes current knowledge about genes whose products
function in the transport of various cationic macronutrients (K, Ca) and
micronutrients (Cu, Fe, Mn and Zn) in plants.
24. Rengel Z, Römheld V, Marschner H: Uptake of zinc and iron by
wheat genotypes differing in tolerance to zinc deficiency. J Plant
Physiol 1998, 152:433-438.
25. von Wiren N, Marschner H, Römheld V: Roots of iron-efficient maize
also absorb phytosiderophore-chelated zinc. Plant Physiol 1996,
111:1119-1125.
26. Baker AJM, Brooks RR: Terrestrial higher plants which
hyperaccumulate metallic elements — a review of their distribution,
ecology and phytochemistry. Biorecovery 1989, 1:81-126.
27.
Brooks RR, Chambers MF, Nicks LJ, Robinson BH: Phytomining.
Trends Plant Sci 1998, 3:359-362.
28. Salt DE, Smith RD, Raskin I: Phytoremediation. Annu Rev Plant
•
Physiol Plant Mol Biol 1998, 49:643-668.
An excellent overview of the current information on using plants to clean up
the environment. Toxic heavy metals and organic pollutants are the major targets for phytoremediation.
29. Lasat MM, Baker AJM, Kochian LV: Physiological characterization of
root Zn2+ absorption and translocation to shoots in Zn
hyperaccumulator and nonaccumulator species of Thlaspi. Plant
Physiol 1996, 112:1715-1722.
30. Pence N, Larsen P, Ebbs S, Garvin D, Eide D, Kochian L: Cloning and
characterization of a heavy metal transporter (ZNT1) from the
Zn/Cd hyperaccumulator Thlaspi caerulescens. Plant Physiol 1998,
[Abstract 656 http://www.sheridan.com/aspp98/abs/45/ 0650.html]
31. Lasat MM, Baker AJM, Kochian LV: Altered Zn compartmentation in
•• the root symplasm and stimulated Zn absorption into the leaf as
mechanisms involved in Zn hyperaccumulation in Thlaspi
caerulescens. Plant Physiol 1998, 118:875-883.
The hyperaccumulator Thlaspi caerulescens differs in a number of respects
from the related non-hyperaccumulating species Thalspi arvense. The
authors show the zinc that is taken up by T. caerulescens is more readily
available for loading into the xylem.
249
of naturally selected zinc tolerance in Silene vulgaris. J Plant
Physiol 1998, in press.
35. Zhao H, Eide D: Zap1p, a metalloregulatory protein involved in
zinc-responsive transcriptional regulation in Saccharomyces
cerevisiae. Mol Cell Biol 1997, 17:5044-5052.
36. Zhao H, Butler E, Rodgers J, Spizzo T, Duesterhoeft S, Eide D:
Regulation of zinc homeostasis in yeast by binding of the ZAP1
transcriptional activator to zinc-responsive promoter elements.
J Biol Chem 1998, 273:28713-28720.
37.
Kamizono A, Nishizawa M, Teranishi Y, Murata K, Kimura A: Identification
of a gene conferring resistance to zinc and cadmium ions in the
yeast Saccharomyces cerevisiae. Mol Gen Genet 1989, 219:161-167
38. Conklin DS, McMaster JA, Culbertson MR, Kung C: COT1, a gene
involved in cobalt accumulation in Saccharomyces cerevisiae. Mol
Cell Biol 1992, 12:3678-3688.
39. Conklin DS, Culbertson MR, Kung C: Interactions between gene
products involved in divalent cation transport in Saccharomyces
cerevisiae. Mol Gen Genet 1994, 244:303-311.
40. Li L, Kaplan J: Defects in the yeast high affinity iron transport
system result in increased metal sensitivity because of the
increased expression of transporters with a broad transition
metal specificity. J Biol Chem 1998, 273:22181-22187.
41. Paulsen LT, Saier MH: A novel family of ubiquitous heavy metal ion
transport proteins. J Membr Biol 1997, 156:99-103.
42. Nies DH: CzcR and CzcD, gene products affecting regulation of
resistance to cobalt, zinc and cadmium (czc system) in
Alcaligenes eutrophus. J Bacteriol 1992, 174:8102-8110.
43. McMahon RJ, Cousins RJ: Mammalian zinc transporters. J Nutr
1998, 128:667-670.
44. Palmiter RD, Findley SD: Cloning and functional characterization of
a mammalian zinc transporter that confers resistance to zinc.
EMBO J 1995, 14:639-649.
45. Palmiter RD, Cole TB, Findley SD: ZnT-2, a mammalian protein that
confers resistance to zinc by facilitating vesicular sequestration.
EMBO J 1996, 15:1784-1791.
46. Palmiter RD, Cole TB, Quaife CJ, Findley SD: ZnT-3, a putative
transporter of zinc into synaptic vesicles. Proc Natl Acad Sci USA
1996, 93:14934-14939.
47.
Huang L, Gitschier J: A novel gene involved in zinc transport is
deficient in the lethal milk mouse. Nature Genetics 1997, 17:292-295.
33. Küpper H, Zhao FJ, McGrath SP: Cellular compartmentation of zinc
in leaves of the hyperaccumulator Thlaspi caerulescens. Plant
Physiol 1999, 119:305-311.
48. van der Zaal EJ, Neuteboom LW, Pinas JE, Schat H, Verkleij J,
•• Hooykaas PJJ: Overexpression of a zinc transporter gene from
Arabidopsis can lead to enhanced zinc resistance and zinc
accumulation. Plant Physiol 1999, 119:1-9.
A homolog of mammalian zinc effluxers has been identified in Arabidopsis.
The authors show that overexpression of the homolog leads to a modest
increase in zinc resistance and to an increase in the zinc content of roots.
They speculate that ZAT1 may be involved in vacuolar sequestration of zinc.
34. Verkleij JAC, Koevoets PLM, Blake-Kalff MMA, Chardonnens AN:
Evidence for an important role of the tonoplast in the mechanism
49. Takatsuji H: Zinc finger proteins: the classic zinc finger emerges in
contemporary plant science. Plant Mol Biol 1999, 39:1073-1078.
32. Shen ZG, Zhao FJ, McGrath P: Uptake and transport of zinc in the
hyperaccumulator Thlaspi caerulescens and the nonhyperaccumulator Thlaspi ochroleucum. Plant Cell Environ 1997,
20:898-906.