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Plant Physiology 137:428-446 (2005) © 2005 American Society of Plant Biologists A Comparative Inventory of Metal Transporters in the Green Alga Chlamydomonas reinhardtii and the Red Alga Cyanidioschizon merolae1,[w]Metal Homeostasis Group, Max Planck Institute for Plant Molecular Physiology, 14476 Golm, Germany (M.H., U.K.); and Algology, Mycology and Experimental Systematics, Department of Life Sciences, University of Liege, Sart Tilman, 4000 Liege, Belgium (V.D., D.B.)
As in all organisms, metal cations are crucial for nutrition in plants. Several metals, such as copper, iron, zinc, and manganese, act as important cofactors for many enzymes and are essential for both mitochondrial and chloroplast functions. However, when supplied in excess, these essential cations can become toxic, like heavy metals with no generally established function, such as cadmium, lead, or mercury. To maintain micronutrient metal homeostasis and to cope with the deleterious effects of nonessential heavy metals, plants have developed a complex network of metal uptake, chelation, trafficking, and storage processes. Metal transporters are required to maintain metal homeostasis and thus constitute important components of this network (Clemens, 2001
In recent years, a number of membrane transport protein families have been implicated in metal homeostasis in plants. These include the cation diffusion facilitators (CDF), the Zrt-, Irt-like proteins (ZIP), the cation exchangers (CAX), the copper transporters (COPT), the heavy-metal P-type ATPases (HMA), the natural resistance-associated macrophage proteins (NRAMP), and the ATP-binding cassette (ABC) transporters (Williams et al., 2000 In this article, we present an overview of our current knowledge of the metal transport function in metal homeostasis and tolerance in eukaryotes, with a special emphasis on plants. We also provide a timely inventory of putative metal transporters in two unicellular algal models, the green alga Chlamydomonas reinhardtii and the red alga Cyanidioschizon merolae. These new data should facilitate functional genomics and molecular analysis of metal homeostasis and tolerance in photosynthetic organisms. Moreover, the comparison of metal transporters from species belonging to the red and green algae with those of the land plant Arabidopsis, as well as with their human and yeast homologs, allows some light to be shed on the molecular evolution of metal homeostasis and tolerance systems.
The use of a simple model organism is often helpful to dissect the functions and the interactions of various transport systems. The yeasts Saccharomyces cerevisiae and, to a lesser extent, Schizosaccharomyces pombe have been successfully used to elucidate the molecular basis of cellular metal homeostasis (Clemens and Simm, 2003  ; De Freitas et al., 2003; Eide, 2003). However, investigation of the essential chloroplast metal metabolism is not accessible in yeast models.
The green alga Chlamydomonas reinhardtii Dangeard is a well-known model of a photosynthetic cell. This unicellular eukaryote is widely used for studies of a number of physiological processes, such as photosynthesis, respiration, nitrogen assimilation, flagella motility, and basal body function (Rochaix et al., 1998
C. reinhardtii has also been used in the past to study metal tolerance (for review, see Hanikenne, 2003
The red alga Cyanidioschyzon merolae De Luca, Taddei, and Varano is a blue-green-colored unicellular alga that is found in sulfur-rich acidic hot springs. Its ultrastructure and other cytological and biochemical features suggest that it is one of the most primitive algae. C. merolae has no rigid cell wall and contains one nucleus, one mitochondrion, one chloroplast with a centrally located nucleoid, one Golgi body, and one microbody (Seckbach, 1994
According to many phylogenetic studies, red algae and green plants (including green algae) are considered to be sister groups (Cavalier-Smith, 1982
We used a high-throughput semiautomated approach to identify metal transporters in the recently released genomic sequences of C. reinhardtii (U.S. Department of Energy Joint Genome Institute [JGI; http://www.jgi.doe.gov]) and C. merolae (Matsuzaki et al., 2004 ). Briefly, numerous BLAST searches were conducted on both algal genomes, using all members of the human, yeast, and Arabidopsis CDF, ZIP, CAX, COPT, HMA, yellow-stripe 1-like (YSL), Fe transporter (FTR), NRAMP, and iron-regulated 1 (IREG1)-like (sub)families, as well as multidrug resistance-associated protein (MRP) and ABC transporter of the mitochondria (ATM)/heavy-metal tolerance (HMT) subfamilies of ABC transporters (see Supplemental Table I). This custom strategy allowed us to identify, respectively, 41 and 25 putative metal transporters in C. reinhardtii and C. merolae. Their distribution among the different transporter families, compared to the three reference organisms, is given in Table I. The identified proteins were first subjected to topology and targeting predictions, then used to mine available EST databases (Tables II and III; Supplemental Tables II–VII). Finally, evolutionary relationships within each transporter family were examined using robust phylogenetic trees (Figs. 1–7 ). A detailed description of the whole procedure is given in Supplemental Figure 1.
The CDFs form a family of ubiquitous transporters involved in metal homeostasis and tolerance. These proteins catalyze the efflux of transition metal cations, like Zn2+, Cd2+, Co2+, Ni2+, or Mn2+, from the cytoplasm to the outside of the cell or into subcellular compartments. Most CDF proteins possess six putative transmembrane domains, with the N and C termini predicted to be cytoplasmic, and exhibit a His-rich loop region between transmembrane domains IV and V, a signature sequence between transmembrane domains I and II, and a cation efflux domain comprising transmembrane domains I to VI (Paulsen and Saier, 1997 ; Gaither and Eide, 2001). The plant members of the CDF family are named MTPs.
We have identified, respectively, five and three MTPs in the genome sequences of C. reinhardtii and C. merolae (Tables II and III). CrMTP1 and CmMTP1 are related to zinc transporters of higher plants, humans, and, more distantly, yeasts (Fig. 1 ). AtMTP1 (formerly ZAT) confers zinc resistance to Arabidopsis when overexpressed (van der Zaal et al., 1999
CrMTP2 to 4 and CmMTP2 cluster with AtMTP8 to 11 and with ShMTP1, a subgroup of putative manganese transporters. The ShMTP1 protein of the manganese-tolerant legume Stylosanthes hamata confers manganese resistance to yeast and Arabidopsis when overexpressed. It is localized in the plant vacuolar membrane and was proposed to be involved in manganese sequestration in this organelle (Delhaize et al., 2003
CrMTP5 groups with AtMTP7 and HsZnT9, two proteins of unknown function, whereas the remaining CmMTP3 is distantly related to a few other CDF proteins. It is, however, worth mentioning that, similar to ScMSC2, AtMTP12, and HsZnT5 (Fig. 1), CmMTP3 possesses a long N-terminal extension with 6 predicted additional transmembrane domains that share no homology with other CDFs. HsZnT5 is believed to act in the sequestration of zinc in the Golgi complex of different tissues (Palmiter and Huang, 2004
Finally, neither C. reinhardtii nor C. merolae seem to possess a homolog of the yeast ScMMT1 and ScMMT2 (Fig. 1), two mitochondrial proteins believed to play a role in iron homeostasis (Li and Kaplan, 1997
The ZIP protein family forms another ubiquitous transporter family involved in metal homeostasis, generally mediating the influx of metal cations, like zinc, iron, cadmium, or manganese, from outside the cell or from a subcellular compartment into the cytoplasm. ZIPs are predicted to have eight transmembrane domains, with the N and C termini being extracytoplasmic. As a common feature, ZIPs possess a long cytoplasmic loop (the so-called variable region) of variable length and sequence between transmembrane domains III and IV. The variable region very often contains a probable metal-binding His-rich domain. Transmembrane domains IV and V are amphipathic and believed to form a polar cavity required for the cation metal transport, while the loop between transmembrane domains II and III could be the site of initial binding of the substrate (Guerinot and Eide, 1999 ; Guerinot, 2000; Gaither and Eide, 2001).
Based on sequence conservation, Gaither and Eide (2001) We have identified, respectively, 14 and 4 ZIPs in the genomes of C. reinhardtii and C. merolae and, altogether, our findings suggest that these algal ZIPs are mainly localized in the vacuolar or the plasma membrane (Tables II and III).
CrZIP1 to 5 and CmZIP1 cluster with subfamily I, which contains most of the Arabidopsis and S. cerevisae ZIPs (Fig. 2
). Interestingly, the proteins of each of the four species form distinct groups within the subfamily. The protein diversification probably occurred independently in the different groups through duplications of an ancestral gene. In that respect, it is noteworthy that CrZIP4 and 5 are found in tandem on the same genome scaffold (Supplemental Table II). The algal proteins seem to branch shortly before the ScZRT1 and ScZRT2 proteins, the yeast high- and low-affinity zinc uptake systems, respectively, located in the plasma membrane (Zhao and Eide, 1996a
A few Arabidopsis subfamily I ZIPs have been characterized through yeast complementation and expression analyses. AtZIP1 to 4 play a role in cellular zinc uptake, AtZIP1, 3, and 4 being induced under zinc-limiting conditions at the transcriptional level (Guerinot, 2000
CrZIP6 belongs to ZIP subfamily II, together with CmZIP2 and HsZIP1 to 3 (Fig. 2). While HsZIP3 has not been characterized, HsZIP1 and 2 are involved in zinc uptake across the plasma membrane (Eide, 2004
Finally, a LIV1-like protein is found in C. merolae but not in C. reinhardtii. CmZIP4/LIV1 contains a highly conserved motif [(H,E) E(L,F) P(H,Q,A) E(L,I,V,M)(G,S) D(F,L,V)(M,A,V,G)XL(L,I,V), defined by Taylor and Nicholson (2003) It should be mentioned that the CrZIP13 and 14 proteins are only distantly related to the ZIP family. Due to difficulties in aligning them properly with other ZIPs, their positions in the tree vary according to the phylogenetic method used (data not shown). These proteins may be based on inaccurate gene models and should be considered as possibly distant, putative ZIPs. Nonetheless, our findings surprisingly reveal the presence of a very high number of putative zinc transporters of the ZIP family in the C. reinhardtii genome compared to yeast and C. merolae. The unicellular C. reinhardtii possesses as many ZIPs as the complex multicellular eukaryotes Arabidopsis and humans, although the ZIP diversification occurred in different subfamilies in each of these organisms (within subfamily I for Arabidopsis, LIV1 for humans, and GUFA and subfamily I for C. reinhardtii; Fig. 2) from possibly four ancestral genes still present in C. merolae.
The CAX proteins are divalent cation/H+ antiporters generally containing 10 to 14 transmembrane domains. The AtCAX1 and AtCAX2 proteins have been identified by functional complementation of a S. cerevisiae mutant defective in vacuolar calcium accumulation. Both having 11 putative transmembrane domains, AtCAX1 is a vacuolar high-affinity Ca2+/H+ antiporter, while AtCAX2 displays a lower affinity for Ca2+ (Hirschi et al., 1996 ) and has been proposed to transport Mn2+ and Cd2+ across the tonoplast of plant cells (Hirschi et al., 2000). Nine other CAX proteins have been identified in the Arabidopsis genome, but have not been functionally characterized yet (Maser et al., 2001). An additional member of this family is the AtMHX1 protein, which is an H+-coupled antiporter that can transport Mg2+ and Zn2+ across the vacuole membrane (Shaul et al., 1999).
We have found, respectively, four and two CAXs in the genomes of C. reinhardtii and C. merolae (Tables II and III). CrCAX1 to 2 and CmCAX1 are related to the yeast ScVCX1 and AtCAX1 to 6 (Fig. 3
). CrCAX3 to 4 and CmCAX2 are spread among the other CAXs. Maser et al. (2001)
Finally, it is interesting to note that neither C. reinhardtii nor C. merolae seem to possess a close AtMHX1 homolog, suggesting that this function is lacking in algae. In Arabidopsis, AtMHX1 is mainly expressed in vascular tissues and believed to participate in Mg2+ and Zn2+ partitioning between plant organs (Shaul et al., 1999
The COPT proteins form a eukaryotic family of copper transporters (Eide, 1998 ). COPTs share common topology features, including (1) the presence of three transmembrane domains with an extracellular N terminus and a cytoplasmic C terminus; (2) the presence of two Met-rich regions in the N terminus that may act as a copper scavenger in the extracellular milieu; and (3) an additional Met-rich region in transmembrane domain II, probably involved in copper coordination during transmembrane transport (Petris, 2004). In S. cerevisiae, high-affinity copper uptake, like iron uptake (see below), requires plasma membrane reductases to reduce Cu(II) to Cu(I), the reduction step being mediated by the same reductases that are involved in iron uptake. Cu(I) is then transported by the redundant high-affinity copper transporters ScCTR1 and ScCTR3, via a Cu(I)/2 K(I) antiport mechanism that is highly specific for Cu(I) over other metal ions (Eide, 1998). S. cerevisiae possesses an additional CTR-related protein (ScCTR2). Located in the vacuole, the function of ScCTR2 remains unknown (Eide, 1998; Petris, 2004). Two CTR homologs are found in humans, but only HsCTR1 has been functionally characterized. It is a plasma membrane protein involved in copper uptake in various cell types (Zhou and Gitschier, 1997; Petris, 2004). Sancenon et al. (2003) have recently identified 5 COPT proteins (AtCOPT1 to 5) in Arabidopsis and showed that AtCOPT1, 2, 3, and 5 restore copper uptake in a ctr1 ctr3 yeast mutant. Antisense AtCOPT1 transgenic lines display reduced copper uptake together with increased root length and defects in pollen development (Sancenon et al., 2004). AtCOPT1 thus plays an important role in copper acquisition in Arabidopsis. We have identified a single COPT protein in the genomes of both C. reinhardtii and C. merolae (Tables II and III). CrCOPT1 is more related to the Arabidopsis COPTs than to the human and yeast transporters, while CmCOPT1 is only distantly related to the other COPTs (data not shown). It should also be mentioned that CmCOPT1 is substantially larger than the plant and human COPTs. CrCOPT1 might be located in the plasma membrane (Supplemental Table VI), while CmCOPT1 is predicted to be localized in the vacuolar membrane (Supplemental Table VII).
P-type ATPases are transporters characterized by the formation of a phosphorylated intermediate in the reaction cycle. These proteins typically contain 8 to 12 transmembrane domains and a large cytoplasmic loop, including ATP-binding and phosphorylation sites. P-type ATPases transport a broad range of small cations, and possibly phospholipids, and have been classified into five subfamilies according to their predicted substrate specificities and phylogenies. The type 1B subfamily proteins (HMAs or CPx ATPases) are involved in heavy-metal transport. HMAs possess eight transmembrane domains, the sixth of which contains a conserved Cys-Pro-Cys/His/Ser motif (CPx motif) believed to be involved in metal cation translocation across the membrane. Identified in a wide range of organisms, HMAs can be divided into two main groups with different substrate specificities (monovalent Cu+/Ag+ cations or divalent Zn2+/Co2+/Cd2+/Pb2+ cations; Axelsen and Palmgren, 2001 ; Cobbett et al., 2003).
We have identified three and two HMAs in the genomes of C. reinhardtii and C. merolae, respectively (Tables II and III). CrHMA1 and CmHMA1 are related to the divalent cation transporters of bacteria (EcZntA, SaCadA) and Arabidopsis (AtHMA1 to 4), especially to AtHMA1 (Fig. 4
). EcZntA and SaCadA are involved in zinc or cadmium excretion and tolerance in Escherichia coli and Staphylococcus aureus, respectively (Nies, 2003
Although widespread in prokaryotes, the occurrence of these divalent cation-transporting HMAs in eukaryotes is apparently limited to photosynthetic organisms (Fig. 4). Cobbett et al. (2003)
CrHMA2 and 3 and CmHMA2 cluster with the monovalent cation transporters of the P1B-ATPases and are most probably copper transporters (Fig. 4). In particular, CrHMA2 and CmHMA2 are closely related to yeast (ScCCC2), plant (AtRAN1), and human (HsATP7A and B) transporters known to deliver copper to proteins in the trans-Golgi network (Cobbett et al., 2003
CrHMA3 clusters with AtPAA1 and AtHMA6 (Fig. 4). AtPAA1 is a chloroplastic protein mediating the transport of copper into the chloroplast. Arabidopsis paa mutants have a lower chloroplast copper content, accumulate reduced levels of holoplastocyanin, and display reduced chloroplastic copper/zinc superoxide dismutase activity (Shikanai et al., 2003
ABC transporters are ubiquitous transporters involved in a large number of physiological processes. This family is one of the largest protein families with 29, 128, and 48 members in S. cerevisiae, Arabidopsis, and humans, respectively. Typical ABC transporters (the so-called full-size transporters) possess two conserved nucleotide-binding folds responsible for ATP hydrolysis alternating with two highly hydrophobic domains (containing 4–6 transmembrane spans) that specify the substrates to be transported. The half-size ABC transporters possess a single copy of each domain and are assumed to function as homo- or heterodimers (Holland et al., 2003 ).
Based on structural similarities, ABC transporters can be classified in several subfamilies (Decottignies and Goffeau, 1997
MRPs are full-size ABC transporters mainly acting as glutathione S-conjugate pumps (Rea et al., 1998 We have found seven and two MRPs in the genome sequences of C. reinhardtii and C. merolae, respectively (Tables II and III). Although it is obviously a MRP transporter, protein model 155613 is too small (428 amino acid residues) compared to the other members of the family. With a TBLASTN search, we have identified additional ABC transporter domains in close proximity to the gene model on scaffold 142 (Table II; Supplemental Table II). Using three gene prediction software programs (GreenGenie, GeneMark, and GENSCAN), we determined an alternative gene model (encoding CrMRP6, 1,524 amino acid residues), which, albeit imperfect, probably much better reflects the real structure of the gene (data not shown; available upon request). The cloning of the corresponding cDNA will nevertheless be necessary to fully elucidate the gene structure.
CrMRP1 and 2 group with human (HsABCC4, 5, 11, and 12), plant (AtMRP11 and 15), and yeast (ScYOR1) proteins (Fig. 5
), all of which lack the N-terminal domain characteristic of the members of the family (Dean et al., 2003
ATM/HMTs are half-size transporters located either in the mitochondrial or vacuolar membranes. Mitochondrial transporters (HsABCB6 and 7, AtATM1–3, and ScATM1) are involved in the export of iron/sulfur clusters from the mitochondrial matrix to the cytoplasm (Kispal et al., 1997
The vacuolar SpHMT1 of the fission yeast S. pombe is involved in the transport of cadmium-phytochelatin complexes from the cytoplasm into the vacuole. A mutant strain lacking this transporter is unable to accumulate high-Mr cadmium-phytochelatin complexes in the vacuoles and displays hypersensitivity to cadmium (Ortiz et al., 1992 We have identified two and three ATM/HMTs in the genomes of C. reinhardtii and C. merolae, respectively (Tables II and III). Intriguingly, we could not find any protein model corresponding to CrCds1 in the JGI sequence data but have found by TBLASTN that it falls within scaffold 122 (Table II; Supplemental Table II).
Two subclusters can be distinguished within the ATM/HMT subfamily (Fig. 6
). On the one hand, subcluster I, which contains CrATM/HMT-2, CmATM/HMT-1 and -2, only includes mitochondrial transporters (AtATM1–3, ScATM1, HsABCB7) possessing 5 or 6 conserved transmembrane-spanning regions. Since they share structural and targeting predictions with ScATM1 (Supplemental Tables VI and VII), the 3 algal proteins are likely functional homologs of the yeast transporter and might have a role in iron homeostasis of mitochondria. On the other hand, subcluster II, which contains CrCds1, CrATM/HMT-3, and CmATM/HMT-3, includes both mitochondrial (HsABCB6) and vacuolar (SpHMT1) transporters possessing 5 additional transmembrane-spanning segments at the N-terminal end of the protein. Phytochelatins are the main intracellular chelators for cadmium and accumulate in the vacuole in C. reinhardtii (Howe and Merchant, 1992
Two strategies are known for iron uptake in higher plants. Strategy I (or reduction strategy) occurs in all plants, except graminaceous monocots, and involves 3 steps: (1) soil acidification by H+ ATPases to solubilize iron; (2) reduction of ferric iron [Fe(III)] by plasma membrane ferric chelate reductases; and (3) uptake of ferrous iron [Fe(II)] by AtIRT1, a member of the ZIP family (Guerinot and Yi, 1994 ; Connolly and Guerinot, 2002). We have already mentioned above that neither C. reinhardtii nor C. merolae seem to possess any homologs of IRT1 (Fig. 2). Strategy II (or chelation strategy) is found in graminaceous monocots and involves the release of Fe(III) chelators called phytosiderophores, coupled to the induction of a specific Fe(III) chelate transporter (Guerinot and Yi, 1994; Connolly and Guerinot, 2002). In maize, this transporter, ZmYS1, has recently been cloned (Curie et al., 2001). ZmYS1 belongs to the oligopeptide transporter family and is induced under iron deficiency both at transcriptional and translational levels. It has been shown to cotransport protons and phytosiderophore- or nicotianamine-chelated iron. It is interesting to mention that, although ZmYS1 displays a broad metal specificity (iron, zinc, nickel, copper, and cobalt) in Xenopus oocytes and yeast, it seems to be regulated only by iron availability in planta (Roberts et al., 2004; Schaaf et al., 2004). Moreover, the rice OsYSL2 has been shown to transport iron and manganese chelated by nicotianamine (Koike et al., 2004). Phytosiderophores are not synthesized in Arabidopsis, a strategy I species, but eight YS1 homologs (AtYSL1–8) are found in the plant genome. The AtYSL2 protein has been shown to transport iron and copper chelated by nicotianamine, a precursor of phytosiderophores (DiDonato et al., 2004).
To our knowledge, phytosiderophores or nicotianamine are not found in algae. Nevertheless, we searched for YS1-like proteins in the C. reinhardtii and C. merolae genome sequence but could not identify any homolog. This function may thus have evolved after the emergence of land plants. As soon as genome sequence data become available, it would be interesting to analyze green algae that belong to the evolutionary lineage of higher plants (Charophyta, sensu lato; van den Hoek et al., 1995
In S. cerevisiae, the first step of iron uptake involves, as for the plant strategy I, the reduction of Fe(III) to Fe(II) by ferric reductases. Then occurs high-affinity iron uptake mediated by (1) a multicopper oxidase (ScFET3) that reoxidizes Fe(II) to Fe(III) and (2) an iron permease (ScFTR1) transporting Fe(III) into the cell. ScFET3 and ScFTR1 are induced under iron deficiency and the corresponding proteins form a complex at the plasma membrane. In iron-sufficient conditions, iron uptake is driven by the low-affinity transporter ScFET4 (Radisky and Kaplan, 1999
Recently, La Fontaine et al. (2002)
Members of the NRAMP family are also known to transport Fe(II) ions. These transporters use the transmembrane proton gradient to facilitate transport of divalent cations and iron in particular. NRAMPs are ubiquitous proteins that possess common structural features, including the presence of 12 transmembrane domains, 2 conserved His residues in transmembrane domain 6, and a transport motif in the intracellular loop between transmembrane domains 8 and 9 (Forbes and Gros, 2001
Six NRAMP proteins are encoded in the genome of Arabidopsis (Maser et al., 2001
Three NRAMP homologs, named ScSMF1 to 3, are found in S. cerevisiae. These proteins transport a broad range of divalent cations, but have been more specifically implicated in manganese, copper, and, more marginally, iron homeostasis (Radisky and Kaplan, 1999
We have identified three NRAMPs in the genomes of both C. reinhardtii and C. merolae (Tables II and III). In a previous report, Rosakis and Koster (2004)
Finally, in humans, the HsIREG1 protein (or ferroportin 1) mediates the transport of iron at the basolateral surface of the enterocytes into the blood for delivery to other organs (McKie et al., 2000
Using a custom analysis pipeline (Supplemental Fig. 1), we have identified members of 10 families or subfamilies of metal transporters in the unicellular algae C. reinhardtii and C. merolae (Table I). Most of the identified proteins have never been described in these organisms. The identification of all members in a protein family may become crucial when characterizing single members of this family. This indeed allows speculation as to possible functional redundancies within multigene families. For example, the AtHMA2 and 4 proteins are closely related to each other in Arabidopsis (Cobbett et al., 2003 ). Individual mutants exhibit no apparent phenotype and athma2 athma4 double mutants have been more useful to determine the function of these transporters in metal homeostasis (Hussain et al., 2004).
Rosakis and Koster (2004)
Nevertheless, our work partly suffers from the limitations of all in silico analyses. Although state-of-the-art modeling methods have been used by JGI to predict C. reinhardtii gene structures, these models are of overall poorer quality and reliability than those obtained for C. merolae. Most probably, the presence of many short introns and exons in C. reinhardtii nuclear genes (Silflow, 1998
Another limitation concerns the reliability of the topology and subcellular localization prediction software itself. First of all, although the best program available was used to predict transmembrane domains (TMHMM, according to Krogh et al., 2001 For most of the genes described here, we have identified corresponding ESTs (Supplemental Tables IV and V), indicating that these genes are expressed. As already mentioned, the analysis of these EST data is useful to generate working hypotheses concerning potential gene regulation. On the other hand, genes lacking ESTs could be weakly transcribed in the conditions used for all cDNA library constructions. Moreover, some rare transcripts might have been lost during the normalization procedure. Alternatively, these genes might correspond to pseudogenes where the promoter is no longer functional, as no stop codon interrupting the coding sequence was found in these genes.
Our phylogenetic analyses clearly show that an ancestral gene for almost all families and subfamilies was already present early in eukaryote evolution, with the notable exception of YSL proteins. Therefore, the transition to multicellularity was generally associated with the diversification of existing functions rather than with the appearance of novel gene families. In most cases, it is thus legitimate to use unicellular models to gain a fundamental understanding of cellular metal tolerance and homeostasis.
Two intriguing topics are the considerable diversification among the ZIP protein family in C. reinhardtii and the FTRs in C. merolae (see above), but, as a rule, C. reinhardtii generally appears to be more complex than C. merolae with respect to the number of different metal homeostasis-related transporters (Table I). As a flagellate organism found in water and soils, C. reinhardtii lives under fluctuating environmental conditions and therefore needs the flexibility to adapt. Moreover, C. reinhardtii has a relatively sophisticated life cycle with complicated vegetative and sexual stages, for which specialized functions may have evolved (Harris, 1989
In this update on metal homeostasis and tolerance systems encountered in eukaryotes, we present an inventory of metal transporters found in two unicellular algae, along with topology and targeting predictions, as well as searches of the available EST collections. These data were produced through a carefully designed semiautomated in silico mining strategy that might be useful for the research community, while uncovering the pitfalls of such an approach. Although we mostly speculate on the functional and evolutionary implications unveiled by our findings, this work should provide a basis for further experimental molecular and genomic studies of heavy-metal homeostasis and tolerance in photosynthetic organisms.
The C. reinhardtii sequence data were produced by the U.S. Department of Energy Joint Genome Institute (http://www.jgi.doe.gov) and are provided for use in this publication/correspondence only. Received October 1, 2004; returned for revision November 16, 2004; accepted November 18, 2004.
1 This work was supported by the European Union Research Training Network Metalhome (contract no. HPRN–CT–2002–00243 to M.H., U.K.) and by the German Federal Ministry of Education and Research (grant no. 0311877 to U.K.). D.B. is a postdoctoral researcher of the Fonds National de la Recherche Scientifique (Belgium). 
[w] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.104.054189. * Corresponding author; e-mail hanikenne{at}mpimp-golm.mpg.de; fax 49–331–5678250.
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