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Molecular mechanisms of plant metal tolerance and homeostasis

Article in Planta · April 2001

DOI: 10.1007/s004250000458 · Source: PubMed

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Planta (2001) 212: 475±486

Review
Molecular mechanisms of plant metal tolerance and homeostasis
Stephan Clemens
Leibniz Institute of Plant Biochemistry, Department of Stress and Environmental Biology, Weinberg 3, 06120 Halle (Saale), Germany

Received: 14 July 2000 / Accepted: 22 September 2000

Abstract. Transition metals such as copper are essential proteins such as superoxide dismutase, cytochrome c
for many physiological processes yet can be toxic at oxidase and plastocyanin. However, these same metals
elevated levels. Other metals (e.g. lead) are nonessential can be toxic at supraoptimal concentrations. The
and potentially highly toxic. Plants ± like all other reactivity of copper, for instance, can lead to the
organisms ± possess homeostatic mechanisms to main- generation of harmful reactive oxygen species. Further-
tain the correct concentrations of essential metal ions in more, plants encounter metals such as cadmium, lead or
di€erent cellular compartments and to minimize the mercury, which are generally considered nonessential
damage from exposure to nonessential metal ions. A and are potentially highly toxic due to their reactivity
regulated network of metal transport, chelation, traf- with S and N atoms in amino acid side chains. In order
®cking and sequestration activities functions to provide to maintain the concentration of essential metals within
the uptake, distribution and detoxi®cation of metal ions. physiological limits and to minimize the detrimental
Some of the components of this network have now been e€ects of nonessential metals, plants, like all other
identi®ed: a number of uptake transporters have been organisms, have evolved a complex network of homeo-
cloned as well as candidate transporters for the vacuolar static mechanisms that serve to control the uptake,
sequestration of metals. Chelators and chaperones are accumulation, tracking and detoxi®cation of metals.
known, and evidence for intracellular metal tracking is The main components of metal homeostasis are
emerging. This recent progress in the molecular under- transport, chelation and sequestration processes
standing of plant metal homeostasis and tolerance is (Fig. 1). Their regulated activities ensure the proper
reviewed. delivery and distribution of metal ions at the cellular and
at the organismal level, resulting in a basic level of metal
Key words: Chelation ± Hyperaccumulation ± Metal tolerance. Loss of one of these critical processes leads to
homeostasis ± Metal tolerance ± Metal tracking ± hypersensitivity. For example, the phytochelatin-synthe-
Metal transport sis-de®cient Arabidopsis mutant cad1 is more sensitive to
Cd2+ than wild-type plants (Howden et al. 1995; see
below).
Basic metal tolerance is ubiquitous. Current evidence
suggests that plants share several common metal-toler-
Introduction ance mechanisms. Some plant species and genotypes,
however, can grow on soil that naturally, or due to
A number of heavy metals, including copper and zinc, human activities, contains growth-prohibiting concen-
are essential micronutrients required for a wide variety trations of metals. These plants belong to a specialized
of physiological processes. Zn2+, for example, serves as ¯ora that has colonized Ni-rich serpentine soils, or areas
cofactor for many enzymes, and a vast number of polluted by, for instance, Zn smeltering or mining
protein sequences contain Zn2+-binding structural do- activities (Ernst 1974). They possess naturally selected
mains (Clarke and Berg 1998). Copper is a vital higher levels of tolerance (``hypertolerance'', Chaney
component of electron-transfer reactions mediated by et al., 1997). Mostly, this hypertolerance is speci®c for
certain metals (Schat and Vooijs 1997).
Some plants not only tolerate higher levels of metals
Abbreviations: GSH ˆ reduced glutathione; MT ˆ metallothion- but hyperaccumulate them. About 400 di€erent species
ein; PC ˆ phytochelatin belonging to a wide range of taxa have been described as
Correspondence to: S. Clemens; hyperaccumulators (Baker and Brooks 1989). Hyperac-
E-mail: [email protected]; Fax: +49-345-5582164 cumulators generally refer to plants able to accumulate
476 S. Clemens: Plant metal tolerance

homologs exist for many of the molecular components

identi®ed in yeast to date. Thus, studies on yeast metal
homeostasis will be covered in some detail.
A number of important aspects such as the mobili-
zation of metals, the role of mycorrhizal colonization for
tolerance, etc. will not be included due to space
constraints. I apologize to all colleagues whose work
cannot be cited within the scope of this review.

Uptake of metal ions

In recent years we have seen tremendous advances in our

molecular understanding of the entry of both essential
and nonessential metal ions into plant cells. By comple-
mentation of S. cerevisiae uptake mutants, cDNAs
encoding micronutrient transporters have been cloned
(Fig. 2). Arabidopsis COPT1 rescues the high-anity
copper uptake de®ciency of S. cerevisiae mutant ctr1-3
and is hypothesized to represent a copper transporter
Fig. 1. Simpli®ed hypothetical scheme of the cellular plant metal
homeostasis network. Following uptake through transporters, metal (Kampfenkel et al. 1995). However, since the COPT1
ions are bound by chelators and chaperones. Chelators bu€er mRNA is not detectable in root tissue, plant transport-
cytosolic metal concentrations; chaperones are involved in metal ers involved in Cu uptake from the soil remain to be
tracking. Essential metal ions are delivered speci®cally to metal- identi®ed.
requiring cytosolic proteins and to organelles. Uptake into the Uptake Fe2+ and Zn2+ is mediated by a group of
organelles is catalyzed by metal-ion pumps that directly interact with transporters belonging to the ZIP family (for ZRT, IRT
speci®c chaperones. Free chaperones can then bind a metal ion again.
Detoxi®cation and storage of excess metal is achieved by sequestra- related Proteins, Fox and Guerinot 1998; TC 2.A.5
tion in the vacuole. Transporters residing in the tonoplast mediate the according to the classi®cation system for transmem-
passage of metal ions and metal-chelator complexes brane solute transporters, Saier 2000), members of
which can be found in a diverse range of eukaryotes.
First isolated was IRT1 from Arabidopsis (Eide et al.
>0.1% of dry weight of elements such as Ni, Co or 1996). IRT1 suppresses the growth defects of the iron-
Pb. For Zn the limit is >1%, for Cd >0.01% of dry transport-de®cient S. cerevisiae fet3 fet4 mutant under
weight. The hyperaccumulation phenotype is the basis iron-limiting conditions, and IRT1-expressing yeast cells
for the concept of phytoremediation, i.e. the use of
metal-hyperaccumulating plants for the clean-up of
metal-contaminated soil (Chaney et al. 1997). Hyperac-
cumulation is mainly observed with Ni, Zn, Co and Se.
Also, four hyperaccumulators of Pb and one hyperac-
cumulator each for Cd and As have been identi®ed. It is
interesting to note that approximately 75% of the
hyperaccumulators characterized to date have been Ni
hyperaccumulators (Baker and Brooks 1989).
The focus of this review is the recent advances in our
molecular understanding of plant metal homeostasis and
tolerance mechanisms. This will include a discussion of
the transport of metal ions into the cell, since metal-
uptake rates directly in¯uence metal tolerance and
sensitivity. The metals mainly considered are those that
have been studied most intensively with regard to
tolerance: Zn, Cu, Ni and Cd. The metal ions are
relevant for organisms. However, redox-active metals
such as Cu occur in di€erent oxidation states in
biological systems. Thus, copper will be mentioned in
the elemental form. For biologically redox-inert metals
(e.g. Zn, Cd) the ionic forms will be used in the context Fig. 2 Plant metal transporters identi®ed to date. Plant proteins that
of experiments where the oxidation state is clear. have been shown in heterologous systems to be involved in the uptake,
tracking or sequestration of metal ions are shown with their
Much of the fundamental insight into eukaryotic respective substrates. The localization is depicted as hypothesized for
metal homeostasis has come from experiments invol- these transporters. HMT1 is an ABC-type transporter from Schizo-
ving Saccharomyces cerevisiae, and to some degree saccharomyces pombe, of which no plant homolog has been identi®ed
Schizosaccharomyces pombe. Mammalian and plant to date
S. Clemens: Plant metal tolerance 477

exhibit a novel Fe(II)-uptake activity. IRT1 transcrip- ethylene insensitivity in Arabidopsis (EIN2, Alonso et al.
tion is induced in Arabidopsis roots by iron starvation, 1999) and abnormal taste behaviour in Drosophila
which makes this transporter a likely candidate for (MVL, Rodrigues et al. 1995). Metal-transport activity
mediating Fe2+ uptake from the soil. Additional studies has been demonstrated for the rat DCT1 ( ˆ Nramp2) as
in yeast showed that IRT1 has a broad substrate range well as for SMF1 from S. cerevisiae (Gunshin et al.
and also transports Mn2+, Zn2+ and possibly Cd2+ 1997; Supek et al. 1996). Recently, Nramp metal trans-
(Korshunova et al. 1999). porters from Arabidopsis and rice have been character-
The cloning of IRT1 has led to the discovery of the ized. Based on sequence comparison, this family of
related ZIP family. Saccharomyces cerevisae ZRT1 and proteins can be divided into two classes, AtNramp1 and
ZRT2 were identi®ed based on sequence similarity to OsNramp1 belonging to one, AtNramp2±5 and OsN-
IRT1 and shown to represent high-anity and low- ramp2 to the other (Curie et al. 2000). AtNramp1
anity Zn2+ transporters, respectively (Zhao and Eide complements the fet3 fet4 mutant and is up-regulated
1996a,b). A zrt1 zrt2 yeast mutant, which requires under iron-limiting conditions. Therefore, it is likely to
elevated zinc levels in the medium for growth, was then play a role in iron homeostasis (Curie et al. 2000).
used to clone plant Zn2+ transporters. Arabidopsis ZIP Similar results were obtained for AtNramp3 and AtN-
transporters 1±3 confer Zn2+ uptake activity with an ramp4 (Thomine et al. 2000). Furthermore, analysis of
apparent Km in the nanomolar range to yeast cells an AtNramp3 insertion mutant and AtNramp3-overex-
(Grotz et al. 1998; Guerinot and Eide 1999). ZIP1 and pressing plants indicated that AtNramp3 is also involved
ZIP3 are expressed mainly in roots and are induced in Cd2+ uptake. Disruption of the gene slightly enhanc-
under zinc-deplete conditions, suggesting a role in Zn2+ es Cd2+ tolerance, whereas overexpression causes Cd2+
uptake from the soil. A fourth gene, ZIP4, was identi®ed hypersensitivity.
as a genomic sequence. The ZIP4 transcript level is With the exception of the recently described Cd-
elevated in both root and shoot tissues of zinc-starved carbonic anhydrase of marine diatoms (Lane and Morel
plants. The assumed role of ZIP transporters in zinc 2000), no biological function is known to date for the
nutrition is supported by the recently published ®ndings potentially highly toxic heavy metals Pb and Cd. Thus,
on ZIP homologs in di€erent Thlaspi species. Physio- it is unlikely that transporters with speci®cities for the
logical studies had demonstrated that Zn2+ uptake into respective metal cations exist. Instead, these nonessen-
roots of the Zn2+ hyperaccumulator T. caerulescens tial metals are likely to enter cells through cation
shows a similar Km when compared with uptake by the transporters with a broad substrate speci®city. It is well
non-hyperaccumulating relative T. arvense, but a 4.5- documented that iron-de®ciency leads to enhanced
fold higher Vmax (Lasat et al. 1996). A Zn2+ transporter uptake of other metal ions (Cohen et al. 1998). This is
(ZNT1) was then cloned from T. caerulescens by attributable to the induction of such cation transporters
complementation of zrt1 zrt2 and found to be homol- under low-iron conditions. As mentioned above, direct
ogous to ZIP4 from Arabidopsis (Pence et al. 2000). The evidence has now been obtained for Cd2+-transport
Km of ZNT1-mediated Zn2+ uptake in yeast cells lies in activity of IRT1, ZNT1 and AtNramp3 (Korshunova
the range of the Km of Zn2+ uptake into T. caerulescens. et al. 1999; Pence et al. 2000; Thomine et al. 2000). A
Interestingly, the higher Vmax of Zn2+ uptake in search among putative cation transporter cDNAs for
T. caerulescens correlates with a signi®cantly higher e€ects on growth in the presence of Cd2+ upon
expression of ZNT1 in these plants, indicating that expression in yeast led to the observation, that wheat
increased transporter expression could be one factor LCT1 (TC 9.A.20) renders yeast cells more Cd2+
determining metal accumulation rates. sensitive (Clemens et al. 1998). LCT1 had originally
Most recent data on a third ZIP homolog from been cloned by complementation of the K+ high-anity
S. cerevisae, ZRT3, demonstrated that members of this uptake-de®cient yeast mutant CY162 and had been
family are not only involved in the uptake of metal ions. shown to also mediate Na+ in¯ux (Schachtman et al.
ZRT3 is proposed to function in the mobilization of 1997). Additional studies revealed that LCT1 expression
accessibly stored zinc from the vacuole (MacDiarmid leads to increased Cd2+ and Ca2+ uptake activity in S.
et al. 2000). The protein localizes to the vacuolar cerevisiae (Clemens et al. 1998).
membrane and ZRT3 expression is induced under The ®rst example of a plant transporter possibly
zinc-deplete conditions. Also, using a bioassay for the mediating Pb2+ uptake has been described by Arazi
cytosolic labile zinc pool it was found that even when it et al. (1999). They overexpressed a calmodulin-binding
had excess zinc a zrt3 mutant was unable to mobilize cyclic nucleotide-gated channel (NtCBP4), which was
that store. found to localize to the plasma membrane, in tobacco.
Another family of proteins is emerging as a major This resulted in an increased sensitivity toward Pb2+
factor involved in plant metal uptake, Nramp and correlated with enhanced Pb2+ accumulation.
(=Natural resistance associated macrophage proteins, Interestingly, NtCBP4 overexpressors at the same time
TC 2.A.55) metal transporters. Nramp1 was originally are more Ni2+ tolerant. Possible explanations for this
identi®ed in mouse as a gene conferring bacterial observation are, as suggested by the authors, interaction
resistance (Vidal et al. 1993). Homologous sequences of NtCBP4 with Ni2+, which attenuates uptake, or the
have since been identi®ed in bacteria, fungi, plants and suppression of other, more Ni2+-selective channels, by
animals. Mutations in these Nramp-related genes are NtCBP4 overexpression. Uptake studies with NtCBP4
implicated in a diverse range of phenotypes such as will help our understanding of the reported phenotypes.
478 S. Clemens: Plant metal tolerance

From the data gathered to date about plant metal type that is GSH-dependent and correlates with the
transporters it is obvious that multiple pathways exist synthesis of PCs. Puri®ed recombinant PCS proteins
for most metal ions. Genetic studies will be needed to from Arabidopsis and S. pombe catalyze the formation of
test which transporters contribute most to the uptake of PCs from GSH (Vatamaniuk et al. 1999; Clemens et al.
which metal. 1999). The cloning of PCS genes and their regulation
has also been reviewed recently (Cobbett 2000a,b).
Chelation The Arabidopsis cad1-3 mutant is highly sensitive to
Cd2+ and AsO24 compared to the wild type and also
The reactivity and limited solubility of most metal ions displays slightly elevated sensitivities towards Cu, Hg
require constant chelation once they are taken up into and Ag (Ha et al. 1999). An S. pombe SpPCS-knock-out
the cell. This has most clearly been demonstrated for is Cd2+, AsO24 and to a lesser extent Cu hypersensitive
copper in yeast cells (Rae et al. 1999). It was shown that (Ha et al. 1999; Clemens et al. 1999). The overexpres-
yeast cells contain less than one ``free'' copper atom per sion of AtPCS1 in S. cerevisiae leads to an elevation of
cell (see below). The metal ions are bound by chelators Cd2+, Hg and arsenate tolerance (Vatamaniuk et al.
and chaperones. Chelators contribute to metal detoxi®- 1999). However, sensitivity towards Zn2+ and Ni2+ is
cation by bu€ering cytosolic metal concentrations. not altered, and e€ects on Cu sensitivity are small. These
Chaperones speci®cally deliver metal ions to organelles data underscore the importance of PCs for the detoxi-
and metal-requiring proteins. In plants the principal ®cation of a range of metals and metalloids. They also
classes of known metal chelators include phytochelatins, demonstrate that PC formation cannot represent the
metallothioneins, organic acids and amino acids. only mechanism of metal tolerance in plants. Further-
more, PCs appear not to be involved in the generation of
naturally selected metal tolerance (see below).
Phytochelatins

Phytochelatins (PCs) (=cadystins in S. pombe) are Metallothioneins

small, metal-binding peptides of the general structure
(c-Glu-Cys)n-Gly (n ˆ 2±11) (Kondo et al. 1984; Grill Metallothioneins (MTs) are ubiquitous low-molecular-
et al. 1985; Jackson et al. 1987). They have been found weight, cysteine-rich proteins, which bind metal ions in
in virtually all plants tested and certain fungi (Gekeler metal-thiolate clusters (Hamer 1986). The archetypical
et al. 1989). PCs are synthesized non-translationally class-I MTs from mammalian cells are known to confer
from reduced glutathione (GSH) in a transpeptidation Cd2+ tolerance (Masters et al. 1994). The metal pre-
reaction (Grill et al. 1989). The extensive literature on dominantly bound is Zn2+, which is why mammalian
structure and synthesis of PCs and related molecules MTs are hypothesized to be involved in Zn2+ bu€ering.
such as (c-Glu-Cys)n-Ser, (c-Glu-Cys)n-b-Ala, (c-Glu- Yeast MTs, which belong to class II (i.e. the alignment
Cys)n-Glu and (c-Glu-Cys)n has been reviewed recently of Cys residues is divergent from that of class-I MTs),
(Rauser 1995, 1999) and will not be discussed here. appear to bind mainly Cu. They constitute one of the
Phytochelatin synthesis, catalyzed by the enzyme PC cytosolic Cu stores. The copper-inducible S. cerevisiae
synthase (Grill et al. 1989), is induced within minutes MT CUP1 contributes to copper detoxi®cation (Hamer
following exposure to a variety of metals or metalloids. et al. 1985). A disruption of cup1 causes Cu hypersen-
Interestingly, some inducers do not seem to serve as sitivity; the overexpression of cup1 enhances Cu toler-
substrates for chelation. Low-molecular-weight cytosolic ance. As with class-I MTs, Cd2+ binding could also be
PC-Cd complexes, which are transported into the detected. MT-Cd complexes are formed in S. pombe cells
vacuole where high-molecular-weight complexes are expressing a S. cerevisiae MT (Yu et al. 1994).
formed under incorporation of S2) (see below), are well The ®rst MT identi®ed in plants was the wheat Ec (for
characterized. Other metals that could be detected in early Cys-labelled) protein. It was isolated from mature
complexes with PCs include Ag, Cu (Maitani et al. 1996) embryos and shown to bind Zn2+ (Lane et al. 1987). To
and As (SchmoÈger et al. 2000). The signi®cance of PC- date, more than 50 MT-like sequences have been found
Cd complex formation for the detoxi®cation of Cd2+ in in various plants (Rauser 1999). They di€er considerably
plants was supported by the isolation of the Arabidopsis from the mammalian and fungal MTs. Most of the genes
cad1 mutant, which contains wild-type levels of GSH, are also diverse from the MT-II-like wheat Ec sequence
yet is PC-de®cient and Cd2+ hypersensititve (Howden and have been categorized into di€erent types based
et al. 1995). The CAD1 gene has been cloned recently by predominantly on the Cys arrangements (Robinson
positional cloning and was found to encode a PC et al. 1993). It has been hypothesized that the di€erences
synthase (the CAD1 gene is therefore now also called in Cys motifs (e.g. type 1: exclusively C-X-C; type 2:
AtPCS1) as shown by detection of GSH- and metal- combination of C-X-C, C-C and C-X-X-C) could
dependent PC synthesis in Escherichia coli cells express- account for di€erences in metal speci®cities (Robinson
ing AtPCS1 (Ha et al. 1999). Independently, AtPCS1 et al. 1993). In Arabidopsis, at least three type-1
and TaPCS1 from wheat were isolated in screens for sequences, two type-2 sequences, 2 Ec-type sequences
plant cDNAs conferring Cd2+ tolerance (Vatamaniuk and at least two sequences of a di€erent type, designated
et al. 1999; Clemens et al. 1999). S. cerevisiae cells MT3, have been found (Zhou and Goldsbrough 1995;
expressing these genes display a Cd2+-tolerance pheno- Murphy et al. 1997).
S. Clemens: Plant metal tolerance 479

Numerous studies have been published on the range of processes, including di€erential metal tolerance,
expression of plant MT-like genes. Evidence for devel- metal transport through the xylem and vacuolar metal
opmental regulation was found as well as responsiveness sequestration (reviewed in Rauser 1999). Citric acid, for
to a variety of stimuli including metal exposure, instance, has been hypothesized to be a major Cd2+
hormone treatments, cold, osmotic stress or heat stress ligand at low Cd2+ concentrations (Wagner 1993), has
(reviewed and listed by Rauser 1999). A detailed been shown to form complexes with Ni2+ in Ni-
investigation of Arabidopsis MT1a and MT2a expres- hyperaccumulating plants (Sagner et al. 1998) and has
sion using reverse transcription-polymerase chain reac- been suggested to contribute to Zn2+ accumulation and
tion (RT-PCR) and in-situ hybridization revealed tolerance (Godbold et al. 1984). Similarly, malate was
distinct patterns for the two genes (Garcia-Hernandez proposed as a cytosolic zinc chelator in zinc-tolerant
et al. 1998). While both mRNA species were detected in plants (Mathys 1977). However, with the exception of Al
root maturation zones and leaf trichomes, only MT1a tolerance (Delhaize and Ryan 1995), unequivocal evi-
appeared to be expressed also in vascular tissue and dence for a function of organic acids in plant metal
mesophyll cells. The induction of MT2a by Cu treat- tolerance has been dicult to obtain. Clear correlations
ment, which was described earlier and was found to between the concentration of a particular organic acid
correlate with Cu tolerance of ten Arabidopsis ecotypes and the degree of exposure to a particular metal ± which
(Murphy and Taiz 1995), was restricted to the cotyle- have to be postulated in the light of the metal speci®city
dons. of most homeostatic and tolerance mechanisms ± have
Understanding the role of MT-like genes in plants not been observed. Genetic data are missing and
has been hampered by the lack of protein data. Attempts powerful non-invasive analytical tools such as X-ray
to purify MTs from plant tissues have been unsuccessful absorption spectroscopy are restricted to information on
with the exception of wheat Ec and, more recently, the ligand environment of a metal ion. Modelling of
Arabidopsis MT1 and MT2 (Murphy et al. 1997). Only metal speciation (Rauser 1999) is limited by the lack of
for wheat Ec, has association of the protein with metals knowledge about additional potential metal ligands and
been demonstrated. The diculty in obtaining puri®ed their respective association constants. Moreover, ligands
MTs has been attributed to the vulnerability of the MTs for a metal ion change depending on, for instance,
to proteolysis, particularly within the long linker region compartmentalization, availability of other ions and pH.
typical of plant MT-like sequences, that is found When Cd2+ binding was analyzed in Brassica juncea by
between the Cys domains (Kille et al. 1991). X-ray absorption spectroscopy, interaction with O or N
Functional data have been obtained from heterolo- ligands was shown for the xylem, while in root tissue an
gous systems. The copper-sensitivity of the S. cerevisiae S ligand (PCs) was detected (Salt et al. 1995).
cup1D strain can be suppressed by expression of The one example known to date for a signi®cant and
Arabidopsis MT1 and MT2 (Zhou and Goldsbrough proportional change in amino acid or organic acid
1994). Also, these genes confer an increase in Cd2+ concentration, elicited by a change in metal exposure, is
tolerance. Arabidopsis MT2 partially rescues the Zn2+ the so-called histidine response (KraÈmer et al. 1996). A
hypersensitivity of a Synechococcus mutant de®cient in comparative analysis of two Alyssum (Brassicaceae)
the Zn2+ MT smtA (Robinson et al. 1996). Escherichia species, the Ni-hyperaccumulating A. lesbiacum and
coli cells expressing a Pisum sativum type-1 MT as a the non-hyperaccumulating A. montanum, revealed sig-
glutathione S-transferase (GST) fusion accumulated ni®cantly higher Ni tolerance and shoot Ni content, but
more Cu; a similar experiment using fava bean type-1 not root Ni content, in A. lesbiacum. This ®nding
and type-2 MTs yielded binding of Cu and Cd2+. In indicated di€erences in root-to-shoot translocation as a
both cases, binding of Zn2+ was less pronounced (Evans potential factor determining tolerance and accumulation
et al. 1992; Foley et al. 1997). rate. Indeed, exposure of A. lesbiacum to Ni caused an
In summary, these data suggest a role of MT-like increase in amino acid content of xylem sap, which could
genes and their respective products in plant metal not be observed in A. montanum. This was almost
homeostasis. Proposed functions include detoxi®cation exclusively attributable to an enormous increase in
of metals (copper in particular), cytosolic zinc bu€ering, histidine levels. A linear relationship between xylem Ni
scavenging of metals during leaf senescence or involve- and histidine concentrations was found over a wide
ment in metal secretion via leaf trichomes (Robinson range of Ni exposures. The assumed role of histidine for
et al. 1996; Garcia-Hernandez et al. 1998; Rauser 1999). the chelation of Ni was con®rmed by extended X-ray
Genetic and biochemical data will help to ®nd unequiv- absorption ®ne-structure analysis of A. lesbiacum xylem
ocal evidence for MT function ± which even for the more sap, which produced evidence for coordination of Ni
extensively studied class I-MTs of mammals is still with histidine.
considered ``enigmatic'' (Palmiter 1998). Other Ni-hyperaccumulating Alyssum species,
A. murale and A. bertolonii, showed a rise in histidine
levels proportional to Ni exposure similar to A. lesbia-
Organic acids, amino acids cum (KraÈmer et al. 1996). However, recent evidence
suggests that the His response might not be the general
Due to the reactivity of metal ions with S, N and O, Ni-tolerance mechanism of plants. When the Ni-hyper-
carboxylic and amino acids represent potential ligands. accumulator Thlaspi goesingense was analyzed, no such
Citrate, malate and oxalate have been implicated in a histidine response was found (Persans et al. 1999).
480 S. Clemens: Plant metal tolerance

Histidine concentrations in xylem sap and shoots together, these data demonstrate that copper chaperones
remained basically unchanged after transfer of plants are essential in providing the copper atoms for several
to high Ni medium while root histidine concentrations Cu-requiring proteins, because they can compete for Cu
actually declined. When compared to the non-hyperac- with other chelators and metal-sequestrating processes
cumulator T. arvense, no signi®cant di€erences in at the extremely low cytosolic concentrations of free Cu
histidine concentrations in xylem sap, roots and shoots ions. Based on the number of Cu atoms and SOD
were found during Ni exposure. Also, three cDNAs molecules, the ratio of active to apo-SOD1 and the
encoding enzymes involved in histidine biosynthesis association constant for Cu binding to apo-SOD, Rae
were cloned from T. goesingense. No changes in expres- et al. (1999) estimated the concentration of ``free'' Cu in
sion were observed upon Ni treatment. a yeast cell to be less than 10)18 M, which corresponds
Nicotianamine is a non-proteinaceous amino acid, to less than one ``free'' copper atom per cell.
synthesized from three molecules of S-adenosyl methio- In addition to yCCS two other cytosolic copper
nine. It is a precursor of the phytosiderophore mugeneic chaperones are known from S. cerevisiae. COX17
acid, but unlike mugeneic acid it is not extruded by delivers Cu to the cytochrome c oxidase complex
roots and it has been found in all plants investigated (Glerum et al. 1996). ATX1 is involved in copper
(Stephan and Scholz 1993). Nicotianamine is a chelator transfer to post-Golgi vesicles via interaction with
of Fe2+ and other divalent metal ions as well as Fe3+ CCC2, a Cu-pumping P-type ATPase residing in the
(von WireÂn et al. 1999). Its physiological role has been membrane of post-Golgi vesicles (Fu et al. 1995; Pufahl
mainly studied in the nicotianamine-de®cient tomato et al. 1997). Homologs of ATX1 have been found in
mutant chloronerva, which exhibits severe growth humans (HAH1, Klomp et al. 1997) and in Arabidopsis
impairment and intercostal chlorosis. This phenotype is (CCH, Himelblau et al. 1998). HAH1 directly interacts
attributed to disturbances of the internal transport of Fe with closely related P-type ATPases, the Wilson and
and other metals, Cu in particular, through the phloem Menkes' disease proteins (WDP, MNK)(Hamza et al.
(Stephan and Scholz 1993; Pich and Scholz 1996). 1999). Wilson and Menkes' disease are genetic Cu
Nicotianamine synthase genes have now been identi®ed. transport disorders caused by mutations in the respec-
The chloronerva gene has been isolated by map-based tive ATPases (Bull and Cox 1994). Mutations in the
cloning (Ling et al. 1999), and nicotianamine synthases transporters associated with the genetic defects impair
from barley have been puri®ed and the corresponding Cu delivery to the ATPase. Interestingly, MNK was
genes cloned (Herbik et al. 1999; Higuchi et al. 1999). shown to re-localize from the Golgi to the plasma
membrane in medium with elevated Cu concentrations.
Under these conditions MNK is thought to remove
Chaperones and metal tracking excess Cu by pumping it out of the cell (Petris et al.
1996). This observation illustrates the close relationship
With the recent identi®cation of Cu chaperones and Cu of metal tracking and detoxi®cation.
transporters from S. cerevisiae and humans, intracellular Recently, an Arabidopsis gene (RAN1) with signi®-
metal tracking pathways are now emerging (reviewed cant similarity to the Menkes' and Wilson disease genes
by O'Halloran and Culotta 2000). The yeast copper was identi®ed, that is required for the ethylene signaling
chaperone, yCCS, encoded by LYS7, is essential for pathway (Hirayama et al. 1999). ran1 mutants show
expression of the active, Cu-containing form of yeast ethylene phenotypes in response to an ethylene antag-
superoxide dismutase (ySOD1) (Culotta et al. 1997). onist. RAN1 protein functions as a Cu-ATPase. Ex-
When the function of yCCS was studied in more detail, pression in S. cerevisiae rescues ccc2D cells. In analogy
it was found that the number of ySOD1 molecules and to CCC2, RAN1 is hypothesized to reside in post-Golgi
the number of Cu atoms per cell are not a€ected by the vesicles and to be involved in delivery of copper into
absence of yCCS in a knock-out strain, indicating that proteins of the secretory pathway. Among those are the
yCCS is not involved in Cu uptake or in stabilizing members of the ETR1 family of ethylene receptors,
ySOD1. However, the ratio of active copper-bound form which are known to require Cu for ethylene binding
of the enzyme to the inactive apo form was drastically (Rodriguez et al. 1999). RAN1 appears to play a role in
lowered. Hence, yCCS appeared more likely to directly several Cu-dependent processes. This is indicated by the
activate ySOD1, and Cu delivery to puri®ed denatured rosette-lethal phenotype of the strong ran1-3 loss-of-
apo-SOD1 was tested in vitro. It was found that yCCS, function mutant (Woeste and Kieber 2000), which is not
Cu(I)-GSH and copper salts reactivated ySOD1. In the suppressed by ethylene-insensitive mutants.
presence of Cu-scavenging molecules such as batho- The identi®cation of RAN1 and the Arabidopsis
cuproine sulfonate, however, reactivation was seen only ATX1 homolog CCH1 suggests that in plant cells a Cu
with yCCS. From this observation it was concluded, tracking network exists which is analogous to the one
that yCCS directly activates ySOD by metal insertion described in yeast and human cells (Himelblau and
and that yCCS is essential only at negligibly low Amasino 2000; Woeste and Kieber 2000). Following
cytosolic concentrations of Cu. Accordingly, the re- uptake through high-anity transporters such as
quirement for yCCS could be overcome by raising the COPT1, Cu ions are chelated by speci®c Cu chaperones
intracellular Cu concentration. SOD activity increased and delivered to Cu pumps for transport into organelles
when medium Cu concentrations were elevated and in or directly to cytosolic Cu-dependent proteins. Both Cu
the absence of the MTs CUP1 and CRS5. Taken chelation and Cu pumping activity are not only required
S. Clemens: Plant metal tolerance 481

for Cu acquisition but also for other processes. For related sequences have been found in Arabidopsis (Lu
instance, during leaf senescence Cu and other metal ions et al. 1997, 1998; Tommasini et al. 1998). Because of the
are mobilized to growing parts of the plant in order to apparent lack of an HMT1 homolog in plants, the
minimize loss of valuable nutrients. CCH1 and RAN1 AtMRPs are considered likely candidates as mediators
are up-regulated in senescent leaves and therefore might of PC-Cd transport across the tonoplast (Rea et al.
be contributing to the scavenging of Cu ions which is 1998). Although AtMRP3 partially complements a
required for this mobilization (Himelblau and Amasino ycf1D strain (Tommasini et al. 1998) no data have been
2000). published showing PC-Cd transport activity in vacuoles
Other speci®c chelator proteins involved in tracking of S. cerevisiae cells expressing either of the known
of essential metal ions await identi®cation in plants. AtMRPs. This has led to the speculation that a
From the emerging similarity of the Cu-tracking chaperone-like co-reactant, absent from the in vitro
networks in eukaryotes it appears likely that homologs assays, might be required for AtMRP3-dependent
for the other known Cu chaperones will be found in transport of bis(glutathionato)cadmium or PC-Cd com-
plants. Furthermore, it remains to be seen in plants, as plexes (Rea et al. 1998).
well as in other organisms, which analogous mechanisms
exist for less reactive essential metal ions such as Zn2+.
Cation di€usion facilitators

Intracellular sequestration The ®rst members of the Cation Di€usion Facilitator

(CDF) family of metal transporters (TC 2.A.4) were
Excess metal ions have to be removed from the cytosol. found in bacteria (Nies 1992) and have now been
Principally this can be achieved by e‚ux or by identi®ed in yeast, animals and plants (Paulsen and Saier
compartmentalization. The main storage compartment 1997; van der Zaal et al. 1999). The main substrates for
for toxic compounds in plant cells is the vacuole and these mechanistically poorly understood transporters
there is evidence for vacuolar sequestration of metal ions apparently are Zn2+ and Cd2+ . In Ralstonia eutropha,
in plants (VoÈgeli-Lange and Wagner 1990). Transporters CzcD mediates Zn2+ e‚ux (Anton et al. 1999), and the
potentially mediating this process have been identi®ed in S. cerevisiae proteins COT1 and ZRC1 confer cobalt
S. cerevisae, S. pombe and plants (Fig. 2). and zinc/cadmium tolerance, respectively, when overex-
pressed (Kamizono et al. 1989; Conklin et al. 1992).
Both proteins localize to the vacuolar membrane,
ABC-type transporters indicating an involvement in metal sequestration (Li
and Kaplan 1998). S. pombe cells constitutively express a
Phytochelatins are found in low-molecular-weight ZRC1 homolog that contributes to Zn2+ tolerance, as
(LMW) and high-molecular-weight (HMW) Cd-binding disruption of this gene renders S. pombe cells Zn2+
complexes in plant and S. pombe cells (reviewed in hypersensitive (Clemens, unpublished). From mammals,
Rauser 1995). It is generally assumed that LMW four Zn2+ transporters that belong to the CDF family
complexes are formed in the cytosol and subsequently are known. Rat ZnT1 complemented the Zn2+ sensitiv-
transported into the vacuole where more Cd2+ and ity of a hamster cell line. Since ZnT1 protein was
sul®de are incorporated to produce the HMW complex, detected in the plasma membrane, this phenotype was
which represents the main storage form of Cd. The ®rst attributed to a Zn2+ e‚ux activity (Palmiter and
molecular insight into the vacuolar sequestration of Cd Findley 1995). For two other ZnTs, a function in
came from the cloning of HMT1, which complemented a Zn2+ transport out of the cytosol into di€erent com-
S. pombe mutant de®cient in the formation of the HMW partments was suggested (Palmiter et al. 1996, 1998).
complex (Ortiz et al. 1992). HMT1 is a protein with One CDF has been studied in Arabidopsis to date, at
similarity to ABC-type transporters (TC 3.A.1). It least two additional sequences can be found in the
localizes to the vacuolar membrane and mediates the genome (accession numbers AL353032 and AC004561).
MgATP-energized, vanadate-inhibitable transport of An Arabidopsis cDNA, named ZAT, with homology to
PC-Cd complexes and apo-PCs (Ortiz et al. 1995). A the mammalian ZnTs was introduced into Arabidopsis in
similar transport activity was detected in the tonoplast sense and antisense orientation under 35S control (van
of oat root cells, indicating the operation of an HMT1- der Zaal et al. 1999). Transgenic plants obtained with
like mechanism in plant cells (Salt and Rauser 1995). No the sense construct showed higher growth rates than
HMT1 homolog has been identi®ed in plants, however. control plants on medium supplemented with 250 lM
In S. cerevisae, the ABC-type transporter YCF1 ZnCl2. Cadmium sensitivity was not altered. The Zn2+-
mediates Cd2+ tolerance. YCF1 shares sequence ho- tolerance phenotype was accompanied by an increase in
mology with the human multi-drug resistance-associated root Zn2+ accumulation when plants were analyzed in
protein (MRP) and was demonstrated to function as a hydroponic culture. Shoot zinc content was not di€erent
glutathione S-conjugate pump (Szczypka et al. 1994; Li from that of control plants. These ®ndings were inter-
et al. 1996). Substrate for YCF1 is bis(glutathiona- preted by the authors as an indication of ZAT involve-
to)cadmium as determined by transport studies using ment in Zn2+ sequestration. It remains to be determined
vacuolar membrane vesicles isolated from a ycf1D strain whether the ZAT protein resides in the tonoplast as
and an isogenic wild-type strain (Li et al. 1997). MRP- suggested.
482 S. Clemens: Plant metal tolerance

Other vacuolar transporters external Cd2+ concentrations (de Knecht et al. 1994). PC
composition and degree of sul®de incorporation into
As mentioned earlier, more Cd2+ is incorporated into HMW complexes were the same (de Knecht et al. 1994).
PC-Cd complexes in the course of HMW-complex When exposed to their respective no-e€ect or IC50 Cu
formation in the vacuole. This additional Cd2+ has concentrations, Cu-sensititve and Cu-tolerant ecotypes
been hypothesized to reach the vacuole via a direct showed equal PC synthesis (Schat and Kal€ 1992).
transport of Cd2+ ions (Rauser 1995). A candidate Similarly, Zn tolerant S. vulgaris plants produced less
pathway is the Cd2+/H+ antiport activity detected in PCs than Zn-sensitive plants both following exposure to
tonoplast-enriched vesicles derived from oat root cells the same Zn concentration and to equal-e€ect Zn
(Salt and Wagner 1993). The pH-dependent saturable concentrations (Harmens et al. 1993).
Cd2+ uptake displayed an apparent Km of 5.5 lM, As described earlier, Ni hypertolerance is correlated
which the authors estimated to lie in the range of with the histidine response in at least some species. It
cytosolic Cd2+ concentrations in plants growing on Cd- was demonstrated that spraying of plants with histidine
polluted soil. It was suggested that molecularly the same can result in an increase in Ni tolerance (KraÈmer et al.
transporter might account for this Cd2+/H+ antiport 1996). For Ni hypertolerant Thlaspi goesingense, en-
and the vacuolar Ca2+/H+ antiport (Salt and Wagner hanced Ni tolerance of leaf protoplasts was observed in
1993). Plant genes encoding transporters with the latter comparison with T. arvense (KraÈmer et al. 1997), indi-
activity have been cloned. Arabidopsis CAX1 and CAX2 cating the operation of a detoxi®cation mechanism at
(belonging to transporter family 2.A.19) rescue the the cellular level. Sequestration of Ni in the vacuole
S. cerevisiae double mutant vcx1 pmc1, defective in appears to contribute to this (KraÈmer et al. 2000).
vacuolar Ca2+ uptake (Hirschi et al. 1996). Both Enhanced vacuolar sequestration has also been im-
proteins resemble microbial Ca2+/H+ antiporters. In plicated in di€erential Zn and Cd tolerance (Chardonn-
vacuolar membrane-enriched vesicles from S. cerevisiae ens et al. 1998; Verkleij et al. 1998). The most direct
mutant cells expressing either CAX1 or CAX2, a DpH- evidence was obtained from S. vulgaris. A Zn-tolerant
dependent Ca2+ uptake was detectable, which was ecotype displayed a 2.5- to 3-fold enhancement of
absent from control vesicles. CAX1 and CAX2 were MgATP-energized transport of Zn2+ across the tonop-
found to di€er signi®cantly in anities for Ca2+. CAX1 last when compared to a Zn-sensitive ecotype. In crosses
was characterized as high-anity (Km of 13.1 lM), of those ecotypes the enhanced Zn uptake into the
CAX2 as low-anity (Km of >100 lM). The Km of vacuole co-segregated with higher Zn tolerance (Char-
CAX2 was considered too high to be of physiological donnens et al. 1999). Future work will show whether
relevance for maintaining the Ca2+ resting state. this is a correlation found generally in Zn-hypertolerant
Instead, based on preliminary selectivity experiments plants ± and possibly Ni- or Cd-hypertolerant plants ±
the authors proposed for CAX2 a function as a high- and which transporters, e.g. ZAT homologs (van der
anity, high-capacity H+/heavy metal cation antiporter Zaal et al. 1999), are involved.
(Hirschi et al. 1996). This would render CAX2 a
candidate for the Cd2+/H+ antiport. However, no
additional data adding support to this hypothesis have Conclusions
been published.
From the work on Cu homeostasis in human and yeast
cells, in particular, it has become apparent that a
Naturally selected tolerance (hypertolerance) complex regulated network controls the tracking of
essential metal ions. Transport, chelation and seques-
``Metallophytes'', i.e. plants that are able to grow on soils tration processes balance the intracellular amounts of
enriched in heavy metals such as zinc, copper, nickel, Cu, Zn and other essential metals. Several genetic
cadmium or lead, have evolved increased levels of disorders and mutant phenotypes have been linked to
tolerance, which are usually metal-speci®c (Verkleij and disturbances of metal homeostasis, highlighting the
Schat 1990). Genetically this hypertolerance is believed physiological importance of this network.
to be controlled by a small number of genes (Macnair In recent years we have seen the identi®cation of a
1993). The molecular mechanisms accounting for hyper- number of molecular components involved in metal
tolerance are little understood. Numerous studies have homeostasis from various eukaryotic systems. In plants,
been conducted with Silene vulgaris, which has many uptake transporters for Fe2+, Zn2+, Cu, and Mn ions
ecotypes di€ering in tolerance of Cu, Cd or Zn (Ernst have been cloned and possible entry pathways for Cd2+
1974). All evidence available from comparative studies and Pb2+ have been determined. Phytochelatin synthase
on sensitive and tolerant ecotypes of S. vulgaris suggests genes have been isolated, as well as Cu chaperone genes.
that the typical plant response to metal exposure, the RAN1 was characterized as a potential target protein
formation of phytochelatins ± which clearly is involved in for chaperones, and candidate vacuolar transporters for
the detoxi®cation of a range of metals (see above) ± is not metal ions or metal complexes have been identi®ed.
responsible for di€erential metal tolerance to Cd, Cu Histidine response, enhanced vacuolar metal uptake and
and Zn. The analysis of Cd-sensitive and Cd-tolerant increased uptake transporter expression have been
S. vulgaris revealed that the sensitive plants produced found as determinants of plant metal hypertolerance
PCs more rapidly and in higher amounts at the same and hyperaccumulation.
S. Clemens: Plant metal tolerance 483

Still, we are only beginning to understand the tolerance and Pb2+ hypersensitivity in transgenic plants. Plant
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