Excess heavy metal ions are removed from the cytosol by sequestration. The compartment mainly involved in this process in yeast and plant cells is the vacuole. Mediators of sequestration are transporters in the respective membrane. The actual substrates for most of these transporters are as yet unknown.

1. Roles of ABC-type Transporters in Sequestration

Hmtl from S. pombe was the first gene identified that encodes a protein involved in vacuolar sequestration of heavy metals (66). Hmtl was cloned on the basis of the complementation of a Cd-hypersensitive S. pombe strain lacking vacuolar sulfide-containing PC-metal complexes. The corresponding protein belongs to the large family of ABC-type transporters. It localizes to the vacuolar membrane and transports PC-Cd complexes and apo-PCs (67). S. pombe cells overexpressing hmtl accumulate more Cd2+ and are more Cd2+ tolerant. This indicates the potential of corresponding experiments in plants. However, no transgenic plants expressing hmtl have been described yet. Also, the respective ABC transporters that account for the transport of PC-metal complexes in plants are not yet known. The Arabidopsis MRPs 1-4 were shown to mediate the vacuolar sequestration of various xenobiot-ics conjugated to glutathione but lack PC transport activity (68). This led to the speculation that additional factors might be involved in the sequestration of Cd ions as PC complexes in plants (68).

In S. cerevisiae, the transporter YCF1 is required for Cd tolerance of yeast cells (69). YCF1-deficient cells are Cd hypersensitive. From transport studies with vacuolar membrane vesicles purified from wild-type and ycf ~ S. cerevisiae cells it is known that YCF1 mediates the Mg-ATP dcpcndenl transport of bis(giutathionato)cadmium into vacuoles (70). YCF1 apparently also represents one of two pathways in S. cerevisiae for arsenite detoxification (71). A ycfl-knockout strain is As(III) hypersensitive and deficient in MgATP- and glutathione-dependent vacuolar uptake of "As(III).

2. Cation Diffusion Facilitators (CDFs)

The cation diffusion facilitator family constitutes another group of proteins involved in metal ion transport. First found in bacteria (72), members of this family have now been described in yeast, animals, and plants (73,74). The S. cerevisiae proteins COT1 and ZRC1 confer tolerance of cobalt (COT1) (75) and zinc/cadmium (ZRC1) (76) when overexpressed. The observation that both proteins localize to the vacuolar membrane (77) suggests a role in metal transport into the vacuole. S. pombe expresses one ZRC homologue that appears to be involved in Zn accumulation and Zn tolerance as a knockout strain is Zn hypersensitive and accumulates less Zn (Clemens, S., Nies, D. H., unpublished).

Four mammalian zinc transporters (ZnT 1-4) of the CDF family are known. ZnT-1 was cloned as a rat cDNA complementing the zinc sensitivity of a hamster cell line (78). The protein was detected in the plasma membrane and proposed to mediate Zn efflux. Both ZnT-2 and ZnT-3 reside in endo-membranes, suggesting a function in zinc transport into the respective compartments: lysosomes (ZnT-2) (79) and synaptic vesicles (ZnT-3) (80). To date, one CDF has been studied in Arabidopsis, at least two additional sequences can be found in the genome (Accession numbers AL353032, AC004561). The Arabidopsis cDNA ZAT was isolated and, because sequence alignments indicated potential significance for metal transport, introduced into Arabidopsis in sense and antisense orientation fused to the 35S promoter (74). The sense lines showed a slight Zn tolerance phenotype when grown alongside control plants on medium containing toxic Zn concentrations. No differences were observed in Cd sensitivity. When Zn accumulation was measured in a hydroponic culture, the sense line contained significantly more Zn in the roots than the control line, whereas shoot contents were similar for both lines. From these results the authors speculate that the ZAT protein might be a vacuolar transporter involved in the sequestration of Zn.

3. Other Transporters

Direct transport of Cd2+ ions could represent a pathway of Cd sequestration in addition to the transport of PC-Cd complexes. In tonoplast-enriched vesicles from oat roots, Salt and Wagner (81) detected a Cd2+/H+ antiport activity. They demonstrated saturable ApH-dependent uptake of Cd2+ with an apparent KM of 5.5 /xM. The authors estimated this KM to lie in the range of cytoplasmic Cd2+ concentration for plants growing in Cd-contaminated soil, meaning that this antiport activity could play a role in Cd accumulation in the vacuole. Molecularly, the Cd2VH' antiport might be attributable to the same transporter as the Ca2+/H+ antiport in the vacuolar membrane. This was suggested by Salt and Wagner and others (82). Ca2+/H+ antiporters CAX1 and CAX2 have been cloned from Arabidopsis on the basis of the complementation of a Ca-hypersensitive S. cerevisiae mutant (83). CAX1 was shown to mediate high-affinity Ca2+/H+ exchange when expressed in yeast, and the KM of CAX2 for Ca2+ is too high to be of any physiological relevance. Instead, the authors mention for CAX2 high-affinity and high-capacity H+/heavy metal cation antiporter activity. This would make CAX2 another target for engineering higher Cd accumulation in plants. No reports have been published yet on CAX2 overexpressors.

C. Efflux

The pumping of toxic heavy metal ions out of the cell represents the main tolerance mechanism in bacteria. Chromosomally or plasmid-encoded efflux systems have been found in all eubacterial groups studied so far (84). The metals for which specific transporters exist include copper, cadmium, zinc, silver, lead, and arsenite. The respective transporter genes are generally part of metal tolerance operons also containing regulatory genes and genes coding for metal-binding proteins. Most of the bacterial metal transporters belong to the family of CPx-type ATPases, a subclass of the P-type ATPases (85). Well-studied examples are the Cd2+-specific pump CadA from Staphylococcus aureus and the Cu pumps CopA and CopB from Enterococcus hirae (86,87). Copper-transporting CPx-type ATPases have now also been characterized in eukaryotes. The human Wilson and Menkes disease proteins as well as the S. cerevisiae CCC2 protein are involved in transport of Cu ions into a post-Golgi compartment (88-90). Defects in these systems lead to deficiencies in copper-dependent iron uptake systems. In addition to functioning in the intracellular Cu distribution, eukaryotic CPx-type ATPases can mediate Cu tolerance in a fashion similar to that in the bacterial systems. In Candida albicans a Cu pump was identified that is essential for Cu tolerance and localizes primarily to the plasma membrane, suggesting an efflux pump activity (91).

The one functionally characterized CPx-type ATPase from plants, RANI from Arabidopsis, probably serves a function similar to CCC2 and the Wilson and Menkes disease proteins. It is proposed to be involved in supplying Cu to the ethylene receptor (54). However, there are numerous other sequences in the Arabidopsis genome containing the signature motifs of CPx-type ATPases, such as the putative metal-binding CxxC elements in the amino terminus. At least two of the protein sequences show homology to the Cd2+ pump CadA from S. aureus. Thus, there could well exist efflux systems in plants conferring heavy metal tolerance. To date, no such activity has been described physiologically in plants and the functional data have not been published for any of the other putative plant metal pumps. It is conceivable that these pumps, as well as the bacterial pumps, can be exploited for enhancing the tolerance of plants to Cu, Cd, and possibly other metals.

D. Engineering Metal Uptake Systems

We have seen tremendous advances in our molecular understanding of plant metal uptake. By complementation of respective yeast mutants, uptake transporters have been cloned for the micronutrients Fe, Cu, and Zn. Potential pathways for the entry of the nonessential metals Pb and Cd have been identified. The cloning of transporters and the thorough characterization with regard to expression levels, tissue distribution, cellular localization, and biophysical parameters will make it possible to enhance plant metal tolerance or plant metal accumulation by altering the properties of plant metal uptake. Conceivable strategies include the overexpression or the down-regulation of particular transporters as well as the expression of transporters with altered affinities and specificities.

1. The ZIP Family of Transporters

The ZIP family of metal ion transporters (for ZRT-like, IRT-like proteins) (10) comprises proteins from eukaryotic organisms as diverse as trypano-somes and humans. The first member isolated was IRT1 from Arabidopsis. It complements the iron uptake deficiency of the S. cerevisiae fet3fet4 mutant and encodes an Fe2+ transporter (92). This activity and the expression in roots, which is inducible by iron limitation, indicate that IRT1 mediates uptake of Fe2+ from the soil. Additional experiments in yeast showed that IRT1 has a broad substrate range and also transports Mn2+, Zn2+, and, judging from competition experiments, Cd2+ (93). The yeast homologues ZRT1 and ZRT2 were identified on the basis of sequence similarities to IRT1. They were subsequently shown to mediate high-affinity and low-affinity Zn2+ uptake, respectively (94,95). A zrtl/zrt2 mutant was then used to clone plant Zn uptake transporters. Three Arabidopsis cDNAs were found that complement the zinc uptake deficiency, designated ZIP 1-3 (96). They encode transporters with Km values for Zn2+ in the nanomolar range, suggesting a role in plant Zn2+ uptake.

Expression levels of ZIP transporters could represent one factor determining metal ion uptake and accumulation. When the Zn hyperaccumulator Thlaspi caerulescens and its nonaccumulating relative T. arvense were analyzed, it was found that Zn uptake into T. caerulescens roots shows a KM similar to that of the uptake in T. arvense but a 4.5-fold higher Vmax (97). Subsequently, a Zn2+ transporter was cloned from T. caerulescens that complemented the Zn uptake-deficient yeast strain zhy3 (98) and displays kinetic properties matching the data for Zn uptake into T. caerulescens roots. According to Northern analysis, this Zn transporter, termed ZNT1, is expressed at very high levels in T. caerulescens irrespective of the Zn status whereas in T. arvense the ZNT1 homologue mRNA is of low abundance in Zn-sufficient media and up-regulated under Zn-limiting conditions. These observations suggest that by altering the expression levels of uptake systems it is possible to engineer metal accumulation in plants.

2. Nramp Metal Transporters

Genes encoding members of the Nramp family of integral membrane proteins were identified through very diverse genetic screens (99). Nrampl de termines sensitivity to mycobacterium infection such as tuberculosis or leprosy and gave its name, natural resistance-associated macrophage protein, to the gene family (100). Nramp homologous sequences have now been identified in bacteria, fungi, plants, and animals. It has been shown that the Nramp homologues SMF1 in yeast and DCT1/Nramp2 in mammals mediate the uptake of a broad range of metals (101-104). In plants, the ethylene insensitivity gene EIN2, which functions in signal transduction, contains an Nramp homologous domain but has no demonstrated metal transport function (105). Arabidopsis homologues of Nramp genes have been cloned and their function as metal transporters has been shown both in the yeast heterologous expression system and in planta (19,106).

Disruption of the AtNramp3 gene leads to an increase in Cd2+ resistance, whereas overexpression of this gene confers increased Cd2+ sensitivity in Arabidopsis (19). These results point to a role of AtNramps in physiological Cd2+ transport and Cd2+ sensitivity in plants. AtNramp3, AtNramp4, and to some extent AtNrampl complement the phenotype offet3fet4, a yeast mutant deficient in Fe uptake (see earlier). This result indicates that two families of Fe transporters, IRTs and AtNramps may contribute to Fe homeostasis in plants (19,106). The role of AtNramps in Fe transport in planta is supported by both the induction of AtNramps upon Fe starvation and the observation that AtNramp3-overexpressing plants can accumulate higher levels of Fe upon Cd2+ treatment (19), whereas /I/Mywm/5/-overexpressing plants confer resistance to toxic levels of Fe.

Thus, the broad specificity of Nramp transporters may render them good target genes to engineer metal nutrient uptake and transport in plants as well as toxic metal sensitivity and accumulation in plants.

3. The Uptake of Nonessential Metal Ions

There is no known biological function for the highly toxic metals Pb and Cd. Thus, it is assumed that no transporters specific for these metals exist. Instead, Pb2+ and Cd2+ ions most likely enter the cytosol of plant cells via nonselective cation transporters. Candidate proteins have been described. The screening for wheat cDNAs that complement a K+ uptake deficiency of S. cerevisiae strain CY162 led to the isolation of a low-affinity cation transporter named LCT1 (107). Testing of effects on Cd2+ sensitivity of expressing yeast cells resulted in the observation that LCT1 mediates Cd2+ uptake (17). Further studies revealed that the physiological substrates for LCT1 are probably Ca2+ and perhaps Na+ (17,107). Other potential pathways include some of the Nramp and ZIP transporters (see earlier). The IRT1-mediated Fe2+, Mn2+, and Zn2+ uptake is inhibited by Cd2+. For ZNT1, low-affinity Cd2+ uptake activity has been demonstrated directly (98).

A cyclic nucleotide-gated channel from tobacco (NtCBP4) has been described as a first example of a plant transporter mediating Pb2+ uptake (18). Originally identified as a calmodulin-binding protein, this channel was found to localize to the plasma membrane. NtCBP4-overexpressing tobacco plants exhibit increased sensitivity toward Pb2+, which correlates with enhanced Pb2+ accumulation. Interestingly, NtCBP4 overexpressors at the same time are more Ni2+ tolerant. Possible explanations for this effect are, as suggested by the authors, interaction of NtCBP4 with Ni2+, which attenuates uptake, or the suppression of other, more Ni2+ selective channels, by NtCBP4 overexpression.

The dual effect described in this study provides another example of the potential of engineered changes in the expression of metal ion uptake systems for enhancing plant metal tolerance or accumulation. This approach will certainly be developed further with a more detailed understanding of the molecular structure of transporter proteins. Elucidation of specificity and affinity determinants, for instance, will allow the expression of mutated proteins that exhibit fewer undesired activities. A more indirect way of potentially minimizing toxic metal uptake is related to iron nutrition. S. cerevisiae cells are rendered more metal sensitive by defects in the high-affinity Fe uptake system. This is due to the Fe deficiency—induced expression of low-affinity uptake transporters such as FET3, which are less selective and facilitate entry of other heavy metal ions (77). Similarly, Cd2+ uptake into root cells of Fe-deficient pea seedlings is sevenfold higher than into Fe-sufficient pea seedlings (108). Because the kinetic properties are not affected by the iron status, this difference is attributable to the induction of iron-regulated IRTl-like transporters. Thus, enhancing the iron efficiency of plants may lead to a reduction in undesired uptake activities.

E. Biotransformation and Direct Removal

Mercury is one of the most hazardous heavy metal contaminants worldwide and has caused numerous ecological disasters, e.g., the poisoning of Minamata Bay in Japan in the 1950s and 1960s. Toward the goal of developing plants suitable for the phytoremediation of Hg-contaminated sites, genes of the mer operon of gram-negative bacteria have been very successfully used. MerA is a mercuric ion reductase catalyzing the production of elemental volatile Hg° from Hg2+. Following extensive modification of the coding region for plant-optimized codon usage and insertion of a consensus plant translation signal into the 5' upstream region, the mer A gene was first introduced into Arabidopsis under 35S control. The resulting transgenic lines grew and developed in the presence of Hg2+ concentrations (50-100 pM) that wild-type plants cannot tolerate (109). In accordance with the obser vation that E. coli expressing merA are more resistant to Au3+ ions, the transgenic Arabidopsis lines also grew better than the wild type on Au3+-containing growth medium. When Hg° evolution was assayed in several lines, it was found to correlate well with merA mRNA levels and mercury resistance.

In order to evaluate the feasibility of this approach for phytoremedia-tion, merA was also expressed in the forest tree yellow poplar (Liriodendron tulipifera) (110). Three constructs differing in the extent of modification of the coding region were used to transform proembryonic masses by particle bombardment. Again, merA expression led to the generation of Hg2+ resistance. Transgenic colonies, embryos, and plantlets were able to grow in the presence of normally toxic HgCl2 concentrations. The most extensively altered construct produced the highest number of HgR colonies. The Hg2+ resistance correlated with the presence of the MerA protein and Hg° release.

The principal form of mercury that biomagnifies, i.e., accumulates in ecosystems, and caused the poisoning of Minamata Bay is methyl mercury (CH3Hg+), which arises through bacterial methylation of Hg(II). It is about 100-fold more toxic than Hg(Il), probably because it can easily diffuse through biological membranes. The mer operon that was identified in bacteria isolated from mercury-contaminated environments consists of merA, a few other genes encoding Hg transporters and regulatory factors, and merB, which codes for an organomercurial lyase. This enzyme catalyzes the formation of Hg(II) from CH3Hg+ via protonolysis of the C—Hg bond. In order to generate plants suitable for phytoremediation of methyl mercury, the merB gene was modified and expressed in Arabidopsis, analogous to merA (111). Transgenic plants transcribing merB mRNA and synthesizing MerB protein were tested for growth on phenylmercuric acetate and mono-methylmercuric chloride. At concentrations (0.5-2 /xM) that are growth prohibiting for wild-type plants, the merB expressors germinated and grew well. The accumulation of Hg2+, which has to be assumed for plants containing an organomercurial lyase and growing in the presence of methyl mercury, is apparently tolerable because Hg(II) is far less toxic and phytochelatins are likely to sequester some of it. Nonetheless, plants showing both the lyase and the reductase activity would represent even more efficient tools for the removal of mercury from contaminated sites. Consequently, the group of Richard Meagher crossed Arabidopsis merA and merB lines (112). The respective plants were found to be up to 10-fold more tolerant to methyl mercury than transgenics expressing merB alone, demonstrating the potential of this approach.

Selenium is an essential element that can be toxic when high levels are reached, for instance, in wetlands as a consequence of irrigation. Because selenium is similar to sulfur, selenocysteine can be formed, which, when incorporated into proteins, may lead to dysfunction (113). Two principal pathways for detoxification can be found in plants, chemical reduction and incorporation into organic compounds. The uptake of Se occurs mainly as selenate via high-affinity sulfate transporters. Because the reduction appeared to be the main factor limiting accumulation, the Arabidopsis ATP sulfurylase (APS), hypothesized to be the enzyme catalyzing selenate reduction, was overexpressed in Brassica júncea (114). The transgenic lines showed twofold higher APS activity and increases in selenate reduction, selenate accumulation, and Se tolerance.

F. Repair Mechanisms

One component of metal tolerance could be the repair of metal-induced damage. Copper is known to cause plasma membrane leakage resulting in K+ efflux (115). A correlation was found in differentially Cu-sensitive Arabidopsis ecotypes between Cu sensitivity and the ability to reverse the K+ efflux following Cu exposure (116). Also, lipid metabolism genes, possibly involved in membrane repair, are induced in Arabidopsis by Cu treatment (Murphy and Taiz, cited in Ref. 117).

Cadmium can denature proteins by interacting with SH groups and by replacing Zn. That the removal of abnormal proteins formed upon Cd exposure is of importance for the basic cellular cadmium tolerance was demonstrated for S. cerevisiae (118). Yeast cells with defects in either specific ubiquitin-conjugating enzymes or the proteasome are Cd hypersensitive. Moreover, expression of the ubiquitin system is induced by Cd treatment. This finding could not be confirmed for the Arabidopsis homologues (119). Thus, it remains unclear whether the UBCs play a similar role in plants.

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