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Between a rock and a hard place: Trace element nutrition in Chlamydomonas


Sabeeha S. Merchant , Michael D. Allen, Janette Kropat, Jeffrey L. Moseley 1 , Joanne C. Long, Stephen Tottey 2 , Aimee M. Terauchi
Department of Chemistry and Biochemistry, Box 951569, UCLA, Los Angeles, CA 90095-1569, USA Received 12 January 2006; received in revised form 6 April 2006; accepted 6 April 2006 Available online 26 April 2006

Abstract Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganic nutrients owing to the use of metal cofactor-dependent enzymes in photosynthesis, respiration, inorganic nitrogen and sulfur assimilation. Chlamydomonas reinhardtii is a photosynthetic eukaryotic model organism for the study of trace metal homeostasis. Chlamydomonas spp. are widely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity for acclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas represent a blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomic analysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) as well as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficient Chlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting from compromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Upregulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmed degradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occur only with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surface reductase and a Cu+ transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of back-up enzymes for plastocyanin in photosynthesis and the ferroxidase in iron assimilation plus activation of alternative oxidase to handle the electron overflow resulting from reduced cytochrome oxidase function. Oxygen-dependent enzymes in the tetrapyrrole pathway (coproporphyrinogen oxidase and aerobic oxidative cyclase) are also increased in expression and activity by as much as 10-fold but the connection between copper nutrition and tetrapyrroles is not understood. The copper-deficiency responses are mediated by copper response elements that are defined by a GTAC core sequence and a novel metalloregulator, Crr1, which uses a zinc-dependent SBP domain to bind to the CuRE. The Chlamydomonas model is ideal for future investigation of nutritional manganese deficiency and selenoenzyme function. It is also suited for studies of trace nutrient interactions, nutrition-dependent metabolic changes, the relationship between photo-oxidative stress and metal homeostasis, and the important questions of differential allocation of limiting metal nutrients (e.g., to respiration vs. photosynthesis). 2006 Elsevier B.V. All rights reserved.
Keywords: Copper; Iron; Chloroplast; Algae; Oxidative stress

1. Introduction 1.1. The evolution of trace element homeostasis


Corresponding author. Tel.: +1 310 825 8300; fax: +1 310 206 1035. E-mail address: merchant@chem.ucla.edu (S.S. Merchant). 1 Present address: Department of Plant Biology, 260 Panama St., Carnegie Institution of Washington, Stanford, CA 94305, USA. 2 Present address: Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK. 0167-4889/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2006.04.007

Catalysis of organic transformation, which is the basis of life, is dependent on the inorganic elements, most of which are present in an organism in trace quantities (4 to 6 orders of magnitude lower) relative to carbon, oxygen and hydrogen ([1], Fig. 1). These elements open up a catalytic repertoire in biology owing to their specific chemical properties. For instance, Fe,

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Fig. 1. Elements associated with life [15]. The elements that are the major constituents of organic macromolecules are colored pale blue. The macronutrients (green) vs. the micronutrients (yellow or orange) are defined for vertebrate organisms based on the abundance of the element in the animal. For the micronutrients, the colors distinguish those elements where the biochemical target underlying the requirement is known (orange) vs. those elements that have been shown to be beneficial for the animal but whose targets are unknown (yellow). The beige elements represent examples of requirements restricted to particular microorganisms. The figure was generated from an existing electronic source [144].

Cu, Mn, Mo, and V can exist in multiple stable oxidation states in an aqueous, neutral reaction environment and this facilitates redox chemistry, which is at the heart of biochemistry. Other elements like Zn and Co provide catalysts with chemical properties (such as electrophiles) that are not available in the functional groups found on amino acid side chains, and this facilitates hydrolytic and hydration/dehydration reactions central to metabolism. Because these elements are essential for biochemistry, they must be acquired as nutrients by cells to sustain life. The operation of micronutrient (especially iron) acquisition and sequestration pathways can be determinants of successful outcomes for competing organisms in particular environmental niches or in the establishment of infection in a hostpathogen interaction. It is well accepted that life originated in an aqueous environment and accordingly the inorganic elements that became essential for life (Fig. 1) appear to be represented in organisms based on their availability in sea water [2]. Thus, abundant elements like Si, Al, Ti are not well represented in biology owing to the insolubility of the corresponding oxides or hydroxides, while Mo and V are quite soluble as oxyanions and are accordingly important cofactors in enzymes of S and N metabolism that handle intermediates with various oxidation states. Photosynthetic organisms are among the early life forms on earth and their biochemistry is dependent on a wide range of inorganic nutrients. Besides the cofactor-rich photosynthetic apparatus [3], these organisms also use metal cofactor-dependent enzymes for nitrogen fixation and/or for assimilation of inorganic nitrogen and sulfur into organic compounds [4,5]. The photosynthetic microorganisms evolved mechanisms therefore to assimilate and distribute inorganic nutrients to various metabolic pathways and

the fundamental principles of trace element acquisition of interest in today's multicellular complex organisms have an evolutionary basis in the mechanisms operating in progenitor microbes. Two events that changed the landscape of life are critical determinants of micronutrient homeostasis today. The advent of oxygen on the planet as a byproduct pollutant of oxygenic photosynthesis shifted the oxidation state of the mineral trace elements and hence their bio availability. For iron, the change in the ratio of ferrous (soluble) to ferric (insoluble) ions necessitated the evolution of intricate mechanisms for iron acquisition in aerobic organisms. A survey of iron acquisition mechanisms operating in bacteria, protists, plants or humans reveals the key role of pH and redox chemistry for mobilization of this nutrient. Although the prevalence of iron in enzymes [6] gives evidence of the importance of this element in the establishment of life in the oceans billions of years ago, iron is a limiting nutrient in large portions of the oceans today [79]. While the advent of oxygen decreased iron availability, copper became more available owing to the increased solubility of cupric salts, and the evolution of cupredoxins appears to be co-incident with the appearance of oxygenic photosynthesis [10]. There are many examples of copper- vs. iron-containing enzymes that catalyze similar reactions (Table 1). In some cases, the use of one catalyst over the other is determined by micronutrient (copper or iron) availability [11,12]. The second important landmark was the movement of life from the oceans to land, which changed nutrient availability from a gradient continuum in water to a feast or famine situation on land (Fig. 2). The same chemical properties that make the inorganic elements so important for life also give them the potential to

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Table 1 Copper and iron have similar catalytic potential Function Oxygen transport Hydroxylation Iron Hemoglobin Hemerythrin Diiron MMO Cytochrome P450 Catechol dioxygenase Cytochrome c6 Diiron alternative oxidase PDB 1A3N 1HR3 1MMO 1AMO 2AZQ 1CTJ Copper Hemocyanin Particulate MMO Mononuclear tyrosinase Dinuclear catechol oxidase Plastocyanin CuACuB cytochrome oxidase CuACuZ N2O reductase CuZnSOD Cu nitrite reductase PDB 1JS8 1YEW 1C7G 1BUG 2PLT 1OCC 1FXW 1ESO 1OE2

1.2. Chlamydomonas as an experimental model Chlamydomonas spp. were originally classified among the Volvocales algae in the plant kingdom [16]. Recently, a new taxonomy for eukaryotes was introduced where Chlamydomonas is placed in the cluster (equivalent to Kingdom) Archaeplastida [17]. This cluster includes the green algae and plants and the name replaces the terms Viridiplantae and Chlorobionta. Within this cluster, Chlamydomonas spp. are classified within the first rank Chloroplastids, second rank Chlorophyta. Other special experimental models among the Chlorophyta are Volvox carterii (for cell differentiation and development) and Dunaliella spp. (for acclimation to high light, carotenoid and glycerol production). Chlamydomonas spp. are found world-wide and occupy various environmental niches, including soil (the source of the popular laboratory strain C. reinhardtii), snow fields and glaciers (C. nivalis also known as snow algae), acid mines, peat bogs and sewage ponds. Distinct metal bioavailability is expected in each niche owing to variation in metal content, pH and oxygenation of the habitat and competition with other organisms, suggesting that the genus must have significant capacity for acclimation to micronutrient availability. Because of the convergence of photo-oxidative stress [18] and metal toxicity stress (cited above) on reactive oxygen species, the wide variation in light habitats intimates the existence of highly developed intracellular metal homeostasis mechanisms (reviewed by [19]). C. reinhardtii is the best developed laboratory model for biochemical, molecular and genomic approaches to biological questions in algae [20]. The organism is maintained in the laboratory in liquid culture as motile, haploid vegetative cells, which can be differentiated in the laboratory into gametes (plus or minus depending on the allele at the mating type locus) by nutrient (especially nitrogen) starvation. The existence of sexual reproduction and the possibility of recovering the products of meiosis from a zygote (referred to as tetrad analysis) allow the

Oxidation Electron transfer Terminal oxidase

Anti-oxidant Nitrite reduction

FeSOD Heme nitrite reductase

1COJ 2AKJ

Examples of similar chemistries catalyzed by either an Fe-containing or a Cucontaining active site are listed. SOD, superoxide dismutase; MMO, methane mono-oxygenase. Where available, the PDB coordinates for a representative structure of each enzyme are shown.

cause cellular damage; thus, an organism finds itself between a rock and a hard placefaced with balancing two difficult situations. For some elements this potential for toxicity is exacerbated in the presence of oxygen [13,14]. In the case of iron and selenium, the difference between the highest recommended daily intake (for humans) vs. the estimated minimal toxic daily dose is only about 5- to 6-fold, indicating the need for homeostatic mechanisms [15]. Finally, nutrient acquisition mechanisms operate in the context of deficiencies in some elements and excesses of other (sometimes chemically related) elements and interactions between individual trace element uptake pathways must be considered for a complete understanding of the natural physiology of trace element metabolism.

Fig. 2. Chlamydomonas reinhardtii. Nutrient manipulation in a simple salt-containing medium reveals Fe-deficiency chlorosis (top, left). Wild-type (strain 4A+) was grown in TAP medium containing iron-EDTA supplement as indicated (M). Mutant strains defective in Fe-metabolism can be identified visually (top, right). Mutants defective in photosynthesis are easily identified by fluorescence (bottom, left). Spores from a cross of the crd1 mutant, which lacks photosystem I in copper-deficient medium, were analyzed visually and by fluorescence. Mutants are pale green on copper-deficient medium but dark green on copper-replete medium. Fluorescence rise and decay kinetics shows a defect in the decay phase for copper-deficient crd1 strains [120]. This can be visualized by false color display (purple for crd1 vs. orange for wild-type). The chloroplast is a major iron-utilizing organelle. Light microscopy (bottom, middle) and phase contrast microscopy (bottom, right) show that it occupies about half the volume of the cell.

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application of classical genetic approaches to dissect biological processes. Today, there is a draft sequence of the Chlamydomonas genome [21] and the full repertoire of genomic and proteomic methodologies can therefore be exploited [2225]. Chlamydomonas3 cells contain a single large chloroplast that is biochemically and structurally similar to the ones in vascular plants especially with respect to the photosynthetic apparatus, which is rich in metalloproteins. Wild-type cells can therefore be grown phototrophically (light, CO2 plus inorganic nutrients). The cells also have protist-like mitochondria with respiratory activity and hence it is possible to grow the organism heterotrophically in the dark on acetate as a source of reduced carbon [26,27]. In this situation, the respiratory complexes another set of metal-rich proteins proliferate [28]. Nevertheless, unlike vascular plants that need light to develop a photosynthetic apparatus, Chlamydomonas synthesizes and maintains a photosynthetic electron transfer chain even when it is grown in the dark, but the stoichiometry of components on a per cell basis is several fold reduced. Chlamydomonas can also use multiple different nitrogen sourcesoxidized forms like nitrate, reduced nitrogen in the form of ammonium or organic nitrogen in the form of urea, purines or ureides. The metabolic flexibility with respect to carbon source allows us to address questions relating to intracellular allocation of metals in response to metabolic demand in distinct compartments of the cell (chloroplast vs. mitochondria) and the flexibility for nitrogen source opened the door to the study of molybdenum metabolism [29]. It is also possible to initiate particular biogenesis pathways in Chlamydomonas, such as flagellar regeneration or growth of cell walls (e.g., [25,30]), so that the role of particular metalloenzymes or metal transporters and metallochaperones in those processes can be assessed. The cells can be easily synchronized in the laboratory [31], which allows one to address questions related to the timing of metal assimilation. The ultimate advantage of the Chlamydomonas experimental system for studies of metal homeostasis is the possibility to tightly control metal supply because of the absence of multiple metal chelating/buffering groups in the growth medium. (Supplementation with amino acids or serum is not necessary.) As pointed out earlier, some of the mechanisms in operation in Chlamydomonas are likely to have evolved from the progenitor photosynthetic cyanobacterial ancestor of the chloroplast, and their discovery would give us a picture of the fundamental principles underlying trace nutrient distribution to metabolic pathways. It is worth mentioning that while Chlamydomonas is grouped with plants in the Archaeplastida cluster, researchers often refer to it as half beast-half plant because of the strong sequence relationships of some pathways (e.g., chloroplast based ones) to analogous ones in vascular plants and others (e.g., ciliary biogenesis and function) to analogous ones in animals [32,33]. Analysis of the genome indicates that there are also strong relationships to proteins restricted to microorganisms as well. A good example of this is the candidate ferric ion transporter, Ftr1, which is related to the Ftr1 proteins of fungi and the multicopper
3 Chlamydomonas will be used as a proper noun in this article to refer to the common laboratory strain, Chlamydomonas reinhardtii. Other species will be named specifically.

oxidase, Fox1, whose copper binding domains are arranged like the ones in MnxG in a Bacillus strain [34,35]. 1.3. Metal requirement for Chlamydomonas Although there were some early studies on optimization of medium for provision of trace metals to C. mundana, the trace element solution used in most laboratories for C. reinhardtii is based on a generic solution developed about 50 years ago for growth of bacteria [3638]. Accordingly, the solution has not been optimized for the growth or metabolism of Chlamydomonas strains. For instance, although cobalt is provided in the medium, Chlamydomonas, like other eukaryotes, does not have a pathway for Vitamin B12 synthesis and relies on bacteria for provision of this cofactor or can use B12-independent enzymes for methionine biosynthesis [39]. Thus, cobalt is not necessary for growth of Chlamydomonas and its presence in the medium may complicate the analysis of copper signaling [40]. A survey of the micronutrient content of various media used for growth of Chlamydomonas shows a wide range of metal concentrations used [16], but none appears to be optimal based on our work on copper and iron nutrition. The SagerGranick medium is clearly deficient in copper while the Eversole medium is likely to be deficient in iron under respiratory growth conditions and TAP medium contains excessive amounts of zinc and manganese (data not shown). The impact of pH [41], which changes as the acetate in the medium is metabolized, is generally not considered. Owing to cross-talk between metal assimilation pathways [42] and the occurrence of low selectivity divalent cation transporters in many organisms, it is critical that there be a clear reference point for experimental analysis of trace metal homeostasis. One approach to defining the appetite for trace minerals in the growth medium is to measure the metal content of a population of cells grown under different metabolic conditions and to calculate the amount that must be provided in the medium to permit growth of a culture to 2 107 cells per ml (Fig. 3). Mutant strains that make smaller or larger cells were also included in the analysis [43]. The analysis shows that molybdenum, selenium and cobalt are present at ultra trace levels. Cobalt is not required for growth and is presumably found associated with the cell due to adventitious uptake. Molybdenum is important for synthesis of the molybdocofactor and selenium is used in about a dozen selenoenzymes; the amounts required in the medium for synthesis of the relevant enzymes have been tested to be in the sub-micromolar range and this is consistent with the ultralow Mo and Se content [29,44,45]. The transition metals Cu, Fe, Zn and Mn are present at much higher levels and as is the case for animals, Cu is found at lower levels relative to Fe and Zn. The high Mn content is attributed to the abundance of Mn in the photosynthetic apparatus. This has the useful experimental consequence of facilitating studies of Mndeficiency in Chlamydomonas [46]. The low level of Mn in animal cells makes nutritional deficiency much harder to achieve. 2. Recent achievements There are three basic approaches that have been used to discover genes and pathways required for acclimation of

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Fig. 3. Micronutrient content of Chlamydomonas. Wild-type (strains 2137, 21gr, CC425, 4A+, 17D) or mutant (mat34, smt8) Chlamydomonas cells were grown under various conditions (5500 mol per m2 per s light intensity, CO2 vs. acetate as carbon source, 220% oxygen in the atmosphere above the liquid culture, and nitrate vs. ammonium as a nitrogen source) and collected by centrifugation. The cell pellet was washed with 1 mM EDTA to remove extracellular bound metal and dissolved in 30% nitric acid by incubation for 2 days at 65 C. The metal content was measured at the ICP-MS Laboratory at the University of California in Davis. The y-axis plots the range of metal content on a per cell basis under various growth conditions or for different strains. Chlamydomonas cells have a higher Fe content relative to Cu, as is the case also for humans. The metal content of the cells can be used to estimate a minimal supply of the micronutrient that would support a density of 2 107 cells per ml in a typical stationary phase culture.

Chlamydomonas to macro- and micro-nutrients [47]. Two of these, involving the identification of differentially expressed proteins and genes or loss of function mutants that are unable to adapt to low supply of the nutrient of interest, have the most potential for discovery of new components and connections. The third approach of finding homologs of known genes has been facilitated recently by the deposition of ESTs (including from libraries derived from RNAs isolated from nutritionallydepleted cultures) and the advent of a draft sequence of the Chlamydomonas genome [21,48,49]. 2.1. Iron 2.1.1. Iron assimilation 2.1.1.1. Ferroxidase/Ftr1. Buckhout and co-workers separated proteins of purified plasma membranes from iron replete vs. iron-deficient Chlamydomonas cells and noted the differential expression of a number of polypeptides, especially a 150 kDa protein that was identified as a ferroxidase and named FLP based on the similarity of peptide sequences to hephaestin [50,51]. This protein was also discovered in parallel as the product of the FOX1 gene, cDNAs of which were identified among ESTs on the basis of its multicopper oxidase motif [34]. The Chlamydomonas ferroxidase shows highest sequence similarity to the mammalian ceruloplasmins and hephaestins. Taken together with the increased abundance of the polypeptide and of FOX1 mRNA in iron-deficiency, a ferroxidase function in iron assimi-

lation was suggested by both groups. A copper requirement for accumulation of the protein was also noted in both works. Nevertheless, in one work, copper-deficiency was noted to impact iron assimilation while in the other, the lack of an impact of copper-deficiency on iron nutrition suggested the existence of copper-independent pathway(s) for high affinity iron uptake. It is possible that there are genetic differences between the two wild-type strains used in each work. The FOX1 gene encodes an integral, presumed N-terminally anchored, membrane protein with 1142 amino acids and a predicted molecular weight of 130 kDa. The slower migration of the polypeptide on denaturing gels may result from glycosylation (consistent with its plasma membrane location and presumed routing through the secretory pathway). The multicopper oxidases have 3-fold symmetry and are thought to have evolved by triplication of an ancient copper binding motif [52]. La Fontaine et al. noted that the arrangement of the copper binding sites of the Chlamydomonas Fox1 relative to the position in the primary sequence is unconventional and suggests a divergent evolutionary path of this widely distributed type of protein. A Fox1 paralog (55% identity), named Fox2, was identified in the genome, but the corresponding gene is not regulated by metal nutrition and hence a function for Fox2 is obscure. Fox2 has the same arrangement of copper binding motifs as does Fox1 but Fox2 appears to lack the N-terminal hydrophobic membrane-anchor of Fox1, suggesting a distinct sub-cellular location. La Fontaine et al. suggested that the mechanism for copper loading of Chlamydomonas Fox1 is analogous to the one for Fet3p in Saccharomyces cerevisiae [53] or for ceruloplasmin in mammalian cells [54], based on the identification of a functional Atx1 as well as sequences related to the distributive Ptype ATPases, prototyped by Ccc2, MNK and WND [34,53, 55,56]. A more recent analysis of the draft genome sequence indicates multiple candidate genes encoding copper-transporting P-type ATPases and these are named CTP1, CTP2 and CTP3 (Table 2). A fourth gene model is called HMA1 [57]. One of the CTP family members is expected to be involved in loading copper-proteins that mature through the secretory pathway (e.g., Fox1, various amine oxidases and extracellular enzymes) while the other two are likely to be functionally analogous to Arabidopsis PAA1 and PAA2 and cyanobacterial CtaA and PacS that distribute copper across the plastid envelope (or cell membrane in the case of cyanobacteria) and thylakoid membrane, respectively [5862]. The multicopper oxidases are generally associated with a permease or transporter component for delivery of iron across the membrane. The identity of this partner in Chlamydomonas is not known, but a good candidate is Ftr1, whose expression is up-regulated coordinately with Fox1 during Fe-deficiency [34]. The FTR1 cDNA was identified among the EST database with a poor expect value but was determined to be of interest because the region of sequence similarity includes the ironbinding RExxE motif. The full-length cDNA encodes a 541 residue polytopic integral membrane protein with two such motifs and shows greatest relationship to fungal Ftr1 homologs. A physical interaction between Ftr1 and Fox1 in Chlamydomonas has not been demonstrated nor has co-localization of the

S.S. Merchant et al. / Biochimica et Biophysica Acta 1763 (2006) 578594 Table 2 Genes involved in metal metabolism in Chlamydomonas reinhardtii Name Gene model a C_390017 C_600100 C_210127 C_80200 C_980004 C_380039 C_30108 C_380040 C_190162 C_100025 C_1670018 C_210155 C_119152 C_980003 C_670027 C_110062 C_110067 C_110212 C_290110 C_1080007 C_1470022 C_650007 C_260147 C_1430020 C_10220 C_930057 C_60152 C_360043 ATX1 FOX1 C_380052 C_30271 C_960033 C_170137 C_570027 C_440074 C_540039 C_40004 C_30094 C_30095 C_70187 C_70186 C_700044 C_210055 Gene family ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP ZIP CDF CDF CDF CDF CDF Other names CrZIP14 CrZIP7 CrZIP13 CrZIP12 CrZIP5 CrZIP10 CrZIP8 CrZIP11 CrZIP6 CrZIP1 CrZIP2 CrZIP13 CrZIP9 CrZIP4 CrMTP1 CrMTP2 CrMTP3 CrMTP4 CrMTP5 Reference [56], [56], [56], [56], [56], [56], [56], [56], [56], [56], [56], [56], [56], [56], [56] [56] [56] [56] [56] a a a a a a a a a a a a a a

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IRT1 IRT2 ZIP1 ZIP2 ZIP3 ZIP4 ZIP6 ZRT1 ZRT2 ZRT3 ZRT4 ZRT5 MTP1 MTP2 MTP3 MTP4 MTP5 CutC PCS1 CTP1 CTP2 CTP3 HMA1 CTR1 CTR2 COPT1 ATX1 FOX1 FOX2 FTR1 FTX1 NRAMP1 NRAMP2 RET1 CBR1 FRE1 RBOL1 RBOL2 FEA1 FEA2 FER1 FER2

had documented the involvement of ferrireductases in Chlamydomonas iron assimilation but a specific protein or gene was not associated with the activity [64,65]. Examination of the Chlamydomonas genome revealed 4 candidate genes (Table 2) based on sequence similarity to known fungal (FRE gene products) or mammalian ferrireductases (Dcytb) but the genes have not yet been functionally dissected by reverse genetic approaches. The pattern of expression in metal-deficient medium suggests that one or more may correspond to the measured, inducible ferrireductase activity (M.D.A., J.K. and S.M. unpublished). Fre1 is the best candidate based on magnitude of regulation and sequence relationship to plant ferrireductases (FRO1/ FRO2) and algal Hcr2 [66,67]. 2.1.1.3. Extracellular proteins. Chlamydomonas cells have an extracellular space between the wall and the plasma membrane that houses molecules involved in signaling pathways as well as proteins involved in nutrient assimilation [6870]. A comparative analysis of proteins secreted by cell wall deficient mutants of Chlamydomonas grown in iron-replete vs. iron-deficient medium revealed two related proteins, named Fea1 and Fea2 (M.D.A. and S.M., unpublished). The FEA2 gene is physically linked to FEA1. Fea2 is less abundant than Fea1 and this is recapitulated at the mRNA level as assessed from the EST record. Reverse genetic analysis is required to assess whether these proteins possess unique functions. Sayre and co-workers had identified Fea1 already as the product of the H43 gene in a screen for RNAs that are differentially expressed in cadmium-treated cells [71]. In that work, they showed also that the abundance of H43 mRNA was increased in iron-deficient cells and further that expression of H43 could allow increased growth of a yeast fet3fet4 mutant strain, indicating a function of the protein in iron assimilation. There are no homologs of Fea1/H43 in organisms other than green algae. A related protein, Hcr1, was identified in Chlorococcum littorale as being induced under growth in high CO2, which are conditions that induce also ferric reductase activity and iron uptake [66,72]. A multiple sequence alignment of Fea1, Fea2 and Hcr1 reveals a number of conserved residues that could serve as iron binding ligands. A possible function of this protein might simply be to concentrate iron in the periplasmic space in the vicinity of the Fox1/Ftr1 transporter. HCR1 mRNAs are increased in abundance in iron-deficient cells along with HCR2, which encodes a putative ferrireductase [66]. The closest homolog of HCR2 in Chlamydomonas is FRE1. 2.1.1.4. Other transporters. Genetic analysis implicates the involvement of an additional iron transporter in Chlamydomonas. Assuming that Fox1-dependent iron uptake was the sole pathway for high affinity iron transport, copper depletion should result in iron starvation owing to loss of Fox1 activity, and this should lead to chlorosis.4 However, this is not the case [34]. Additionally, the crd2 mutant of Chlamydomonas shows conditional high iron
4 The term chlorosis in plants refers to reduced chlorophyll content and hence a pale green coloring, which is familiar to anyone who has attempted gardening or maintained house plants without the benefit of fertilizer or plant food.

HMA HMA HMA HMA CTR CTR CTR

CrHMA2 CrHMA3 CrHMA1

CrCOPT1 FLP1 CrFTR1

[56,68] [56,68] a [56], a b b [56], b [34] [34,50] a [34,56] [56,70], a [56], a [56], a a a a a [65], a a c c

Nramp Nramp

CrNRAMP1 DMT1 CrNRAMP2 CrNRAMP3

H43 Ferritin Ferritin

Genes are grouped by family or function. Other names indicate alternate gene names in use. a, MDA, JK, SM unpublished; b, M. Dudley Page, JK, SM unpublished; c, JCL SM unpublished. ZIP, Zrt-/Irt-like proteins; CDF, cation diffusion facilitators; HMA, heavy metal (CPx-type) ATPase; CTR, copper transporters; Nramp, natural resistance-associated macrophage protein. a Gene models are from version 2 of the Chlamydomonas genome <http:// genome.jgi-psf.org/chlre2/chlre2.home.html>.

two proteins in the same membrane system, but other candidate transporters with trivalent cation selectivity have not been proposed. Ftr1-type transporters are not generally found in plants, but several unique ESTs encoding moss proteins with good sequence relationship to Chlamydomonas Ftr1, including the RExxE motif, have been retrieved (Blast search in 12/05). 2.1.1.2. Ferrireductases. Iron reduction at the cell surface is a key step in mobilizing iron for uptake [63]. Several earlier works

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requirement in copper-deficient but not copper replete medium [73]. One hypothesis is that the locus represents a component of a back up copper-independent pathway for iron assimilation. This pathway would be induced under copper-deficient growth conditions and its operation would account for the absence of an irondeficiency chlorotic phenotype in copper-deficient Chlamydomonas cells [34]. An ABC-type half-transporter belonging to the ATM subfamily of half-size transporters is encoded by the CDS1 locus and localizes to Chlamydomonas mitochondria [74]. The CDS1 gene is induced strongly by cadmium and loss of its function results in cadmium sensitivity. Although a function in cadmium detoxification is most likely, the iron sensitivity of the mutant strains raises also the question of Cds1 function in iron metabolism. Genes encoding other related transporters of this family have been annotated as HMT-2 and HMT-3 but analysis of the EST dataset suggests that they are expressed at much lower levels than is CDS1 [57]. The HMT-2 gene product is predicted to be organelle targeted. A survey of the Chlamydomonas genome by BLAST search identified over a dozen transporters of the ZIP family (Table 2, our unpublished results, and [57]) that are predicted to be plasma membrane or vacuolar membrane localized. Experimental work is necessary to distinguish their subcellular location, pattern of expression, metal selectivity, affinity, and role in iron vs. zinc (or other divalent cation) assimilation or distribution. Two Nramplike proteins, Nramp1 and Nramp2, were also identified in the Chlamydomonas genome.5 The expression pattern of one of these, named DMT1, was tested to assess a role in metal homeostasis but no metal-responsive pattern of expression was noted [75]. When expressed in S. cerevisiae, the protein could restore growth to smf1 mutants (indicative of manganese transport capability). The resulting strain also showed slightly increased sensitivity to a number of divalent cations, including iron, but not zinc. Nevertheless, the differences are slight and the question of Nramp1/2 function with respect to assimilation vs. distribution and metal selectivity is open. 2.1.2. Metabolic adaptation In a nutrient deficient environment, the activation of assimilation pathways is logically a first line of defense. Indeed, FOX1 and FTR1 expression is increased substantially (and even maximally for FOX1) when the iron content of the medium is reduced from 18 to 1 M, but iron-deficiency symptoms (such as chlorosis and inhibition of cell division) are not evident until the iron content is reduced to sub-micromolar concentrations, at which point iron supply is insufficient for the maintenance of all iron-containing enzymes [34]. Given the central role of iron in essential metabolic pathways, there must be adaptive responses that affect intracellular iron utilization in addition to those impacting assimilation. The best-documented metabolic adaptation is the replacement of an abundant FeS protein, ferredoxin, with an iron-independent flavodoxin in cyanobacteria in iron5 A third NRAMP homolog has been re-named Ret1 (for related to EIN2) because a revised gene model shows sequence relationship to EIN2 of Arabidopsis.

deficient laboratory medium and, more importantly, in an irondeficient marine environment also [76,77]. In cyanobacteria, this is accomplished by transcriptional responses mediated by the well-known independent Fur repressor [78]. With the application of microarray methodologies, it is now becoming clear that metabolic adaptations probably occur in all organisms (e.g., [79,80]). Eukaryotic photosynthetic organisms have two major iron-utilizing pathways, photosynthesis and respiration, that are separated in two distinct organelles, chloroplasts and mitochondria. Compromised function of the redox enzymes in these pathways has the potential to generate reactive oxygen species and cause oxidative damage. Thus, mechanisms for anticipating this danger or dealing with the oxidative stress might be induced. It is also likely that marginal iron-deficiency could activate short-term adaptive responses while sustained and severe deficiency would activate long-term acclimation pathways. We therefore defined 3 levels of iron-nutrition in Chlamydomonas: iron replete, where the iron assimilation pathway is expressed at a low basal level and there is more iron in the medium than required for the maintenance of intracellular iron enzymes; iron-deficient, where the iron assimilation pathways are turned on and the iron supplied in the medium is just enough or slightly less than the amount required to saturate the demand for biosynthesis of ironcontaining proteins; and iron-limited, where the symptoms of poor iron nutrition are visually evident as chlorosis and there is an impact on cell division [81]. 2.1.2.1. Photosystem I. The photosynthetic apparatus uses redox active metal centers and chlorophyll pigments to generate an extraordinarily strong oxidant (Em +1.1 V) in Photosystem II (PSII) that can be reduced by the weak reductant water and a powerful reductant in Photosystem I (Em 1.0 V) that can reduce ferredoxin for biosynthetic metabolism [3]. PSII, composed of about 2 dozen different polypeptides, houses a Mn4Ca cluster and is a target of manganese-deficiency. Photosystem I (PSI) is made up of over a dozen different polypeptides and three Fe4S4 centers as electron acceptors. These account for about half the iron in the photosynthetic apparatus, and accordingly PSI is a prime target of iron deficiency. When the function of PSI acceptors is compromised, the highly reducing species generated by photochemistry in PSI can reduce oxygen to superoxide (Em,7 0.3 V) in the socalled Mehler reaction. Mutants with defects in PSI therefore experience photo-oxidative stress and are light sensitive under aerobic growth conditions [82,83]. The existence of protective mechanisms is critical for surviving iron-deficiency in nature. Chlamydomonas displays progressive modification of PSI as cells adapt from an iron replete to iron-deficient to iron-limited state [81]. PSI (which houses the cofactors involved in electron transfer plus antenna chlorophylls that collect the light) is physically and functionally associated with light harvesting protein complexes (called LHCI). LHCI consists of individual subunits Lhca1, Lhca2, Lhca3 and Lhca4 that are chlorophyll binding proteins. Energy migration from LHCI to PSI occurs through specific paths/routes in the PSILHCI super-complex that depend on particular subunitsubunit interactions [84]. In an iron-replete cell, the LHCI complex is well coupled to PSI

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so that the energy of the photons absorbed by chlorophyll bound to Lhca subunits migrates effectively to the special pigments in PSI where photochemistry occurs. In the iron-deficient state, the structure of PSI is modified so that part of the LHCI pigments are no longer effectively connected. In particular, a subunit called PsaK (which is known to function as an LHCIPSI connector) is dissociated from the PSI complex, resulting in reduced energy input and hence reduced electron flow through this enzyme. This modification is viewed as a pre-emptive move to avoid photo-oxidative stress from damaged/lost FeS clusters. Under iron limitation, Chlamydomonas cells initiate sequential degradation of the LHCI subunits and both photosystems [81]. Interestingly, the light harvesting chlorophyll protein complex LHCII is retained, although with some post-translational modifications of particular subunits. The LHCII in the ironlimited cell accounts for most of the chlorophyll remaining in the iron-limited cell, and this chlorophyllprotein complex is viewed as a reservoir of chlorophyll when de novo synthesis of new photosystems and LHCI subunits is re-initiated by iron supply. The signal transduction pathways for these responses are completely unknown, but we can deduce that they directly sense iron nutrition rather than damaged PSI because transfer to iron deficient conditions can save PSI-defective mutants that are light sensitive in iron replete medium [81]. Mixing experiments indicate also that soluble extracts from iron-deficient cells will degrade thylakoid membrane proteins isolated from iron-replete cells while extracts from iron-replete cells will not degrade thylakoid membrane proteins isolated from iron-deficient cells, again indicating that the signal is iron nutrition rather than damage to the photosynthetic apparatus. 2.1.2.2. Ferredoxins. Ferredoxin is probably the most abundant iron protein in soluble extracts of iron-replete Chlamydomonas cells. This ironsulfur protein accepts electrons from PSI and uses these electrons for almost all the reductive biosynthetic reactions in the chloroplast, including generation of NADPH for the Calvin cycle, nitrogen and sulfur metabolism, thioredoxin-dependent regulation, fatty acid desaturation, and others [85]. The replacement of ferredoxin by flavodoxin in the marine environment (cited above) is therefore a logical means of conserving iron. The genome of Chlamydomonas appears not to encode a flavodoxin, but instead encodes six different ferredoxins, one of which corresponds to the molecule purified from laboratory grown iron replete cells [86]. Given the central role of ferredoxin as a reductant for biosynthetic metabolism, it is tempting to speculate that the changes in the ferredoxin profile as a function of iron nutrition may be a means of prioritized electron distribution to metabolic pathways. Indeed, the expression of a novel ferredoxin has been observed in iron-deficient Chlamydomonas (N. Fischer and J.-D. Rochaix, personal communication; A.T. and S.M., unpublished). 2.1.2.3. Ferritin. Ferritin accounts for stored iron in a plant and is a source of iron for chloroplast development or during nodulation for the synthesis of leghemoglobin and respiratory components in the bacteroid [87]. Plant ferritin, which consists of a single subunit, is localized to the plastid. The corresponding genes encode pre-proteins with N-terminal targeting sequences.

The accumulation of ferritin is determined at transcriptional and post-transcriptional levels by multiple signals including iron nutrition status, physiological iron demand depending on developmental status, and the potential for toxicity under conditions of oxidative stress [5,88]. For a full picture of ferritin regulation, it is important to measure the RNA (which gives a picture of the potential for ferritin action), protein (as a measure of the iron chelation capacity) and the amount of iron in the core (for the actual level of mineralized iron). When soybean ferritin was over-expressed in tobacco plants, the transgenic over-expressing plants displayed symptoms of deficiency even though total iron content in the plant was increased [89]. This finding emphasized the role of plastid ferritin as a component of iron homeostasis. In Chlamydomonas, FER1 gene expression is counter-intuitively increased in iron-deficient/iron-limited cells relative to iron-replete cells [34]. One possibility is that the increase is important to chelate the iron that is released from the programmed degradation of PSI and ferredoxin in iron-limited cells (discussed above). This idea is precedented by the finding that ferritin expression is increased also in senescing leaves engaged in programmed destruction of the photosynthetic apparatus [90]. It may also be important to increase the iron chelating potential of the chloroplast in an iron-limited situation because of the potential for generating reactive oxygen species in a membrane with a compromised electron transfer chain. In this situation, ferritin would be providing a cytoprotective anti-oxidant role. While there are enzymes to de-toxify hydrogen peroxide and superoxide, the only defense mechanism to combat hydroxyl radical is removal of reactive iron. A second ferritin-encoding gene FER2 was found in the Chlamydomonas genome, but the gene model may be incomplete because of a gap in the present version of the sequence. 2.1.2.4. Mn-superoxide dismutase. Plants contain 3 types of superoxide dismutase, CuZnSOD in the cytosol, plastid and peroxisome, MnSOD in the mitochondria and FeSOD in the plastid [91]. While Arabidopsis encodes only a single MnSOD, Chlamydomonas has five candidate MSD genes. In E. coli and cyanobacteria, the expression of the manganese- vs. iron-containing SODs is dependent upon the iron nutrition status through the Fur regulator (reviewed by [92,93]). Given the connection between iron-deficiency and photo-oxidative stress in Chlamydomonas, it is possible that one of the MSD genes serves to replace FSD1 function in an iron-deficient situation. 2.2. Copper Three abundant copper enzymes are known in Chlamydomonascytochrome oxidase in the respiratory chain in the mitochondrion, plastocyanin in the photosynthetic electron transfer chain in the chloroplast, and the previously mentioned multicopper oxidase involved in iron assimilation. Accordingly, copper is an essential nutrient for Chlamydomonas. In addition, there are other copper enzymes whose contribution to the cellular copper requirement (at least of vegetatively grown laboratory cultures) is likely to be less significant based on representation in

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the EST database. Among these are matrix metalloproteinases with an atypical QExxH site that shows 10-fold in vitro selectivity for copper over zinc in terms of activity, amine oxidases (peroxisomal with PTS1 targetting motifs, amiloride-sensitive and plant like), blue copper proteins, ascorbate-dependent monooxygenases, and various multicopper oxidases, including Fox2 [94 97]. The specific function of these enzymes is not known and it is likely that the copper-requirement of a Chlamydomonas cell might be greater in situations where one or more of these enzymes is more highly expressed. 2.2.1. Copper uptake The standard Chlamydomonas medium contains 6 M cupric ions complexed with EDTA, and this is considered to be a replete medium, since 500 nM chelated copper is sufficient to support the synthesis of plastocyanin (accounting for at least half the copper in a photosynthetic cell) at its usual stoichiometry (2 per PSI) in the photosynthetic apparatus [98,99]. Leaving out the copper sulfate in Hutner's trace element solution reduces the copper content of the medium to the point of deficiency (with respect to plastocyanin synthesis), which makes it possible to monitor copper-nutrition dependent pathways of copper utilization [11,100]. For reproducible results, it is critical that (1) the purity of the salts used for preparation of media be monitored, (2) the glassware be acid washed immediately prior to use, and (3) the water be purified [101]. With care, the concentration of copper in the medium can be reduced to <3 nM. Since copper is a nutrient that is toxic in excess, organisms regulate assimilation to avoid toxicity. Measurement of copper uptake in copper-deficient vs. copper-replete Chlamydomonas cells indicated about 20-fold greater activity in the former population of cells [102]. The increased activity was attributed to a Vmax effect with a Km of about 2 102 nM calculated for both samples of cells, suggesting that the same transporter(s) operate(s) in replete and deficient conditions but the number of transporter molecules is greater in a copper-deficient cell. Cupric reductase activity was also increased (2-fold) in a copper-deficient cell suggesting that cupric ions were reduced to cuprous prior to transport of Cu(I) ions. In earlier work, we had noted that copper-deficient cells were more sensitive to Ag(I) toxicity relative to copper-replete cells and this was attributed to the increased capacity for Ag(I) uptake in the former owing to increased expression of copper uptake components [103]. Taken together, a pathway for copper uptake consisting of a cell surface reductase and cuprous transporter was proposed, analogous to the system operating in other organisms [104,105]. Whether the reductase is copper-specific or a broad substrate specificity ferrireductase (Table 2) remains to be determined [106]. Since the effect of copper nutrition on silver toxicity as well as on the synthesis of a silver-stress-induced peptide depended on the time of exposure to copper, we concluded that increased enzyme synthesis contributed to regulation of the copper assimilation pathway in Chlamydomonas [103]. Examination of the draft genome reveals three copper transporters of the COPT1/CTR family that are selective for Cu (I) ion transport [107] (Table 2). It should be possible to distinguish whether one or more of these is a target of the nutritional

copper sensor, Crr1 (see below), which would make it a good candidate for an assimilatory transporter. 2.2.2. Back up enzymes 2.2.2.1. CYC6. Plastocyanin, a blue copper protein, is an essential component of the photosynthetic apparatus owing to its function in the transfer of electrons from the cytochrome b6 f complex to PSI [3]. The importance of plastocyanin in photosynthesis was deduced from the phenotype of a plastocyanin-deficient Chlamydomonas strain that resulted from a frame-shift mutation at the PCY1 locus encoding the polypeptide [108 110]. Yet, copper-deficient Chlamydomonas strains that contain no immunodetectable or spectroscopically detectable plastocyanin [101,111] can grow photoautotrophically (Fig. 4, bottom). The answer to this conundrum became evident with the discovery of Cyt c6 as a copper-independent/heme-containing substitute for plastocyanin in photosynthetic electron transfer [111,112]. The CYC6 gene encoding this back up protein is regulated by copper nutrition: Cyt c6 is not present in a copper replete cell and the pcy1 mutant strain shows a photosynthesisminus phenotype, but in copper-deficiency CYC6 is transcriptionally activated and the back up, Cyt c6, compensates for the loss of plastocyanin function [113]. The equivalence of Cyt c6 was further established in suppressor strains of pcy1-ac208 where presumed loss of function of an assimilatory copper

Fig. 4. Copper-independent phototrophic growth. Wild type (strain CC125) can grow photosynthetically in copper-deficient growth medium (top). The pcy1 mutant (strain AP6, pcy1-1 arg7) cannot grow photosynthetically because it lacks the copper protein plastocyanin that functions in photosynthetic electron transfer (bottom). However, in copper-deficient medium, the strain does not exhibit a phenotype because the gene for the back up protein, Cyt c6, is induced. This established the role of Cyt c6 as a back-up and further indicated that CYC6 expression is regulated by copper nutrition [112].

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transporter allowed Cyt c6 synthesis in copper replete medium [112]. Comparative biochemistry of the two proteins from a variety of cyanobacteria shows that the isoelectric points of the two proteins appear to co-vary within a wide range from 4 and 9, indicating co-evolution, which is consistent with the proposed parallel function of the two proteins [114]. The replacement of plastocyanin with Cyt c6 occurs in many algae and cyanobacteria, suggesting that these organisms do experience copper-deficiency in nature and execute the switch in order to conserve copper [115]. As for Chlamydomonas, the petJ gene encoding cyanobacterial Cyt c6 is also transcriptionally regulated [116,117]. Plastocyanin abundance is decreased in copper-deficiency but the mechanism of down-regulation of plastocyanin varies. In Chlamydomonas, the protein is degraded by copper-deficiency-induced proteolysis whereas in Scenedesmus obliquus PCY1 mRNA abundance is copper-responsive [110,118]. In Synechocystis sp. 6803 and Anabaena, the petE gene encoding cyanobacterial plastocyanin also responds to copper at the level of mRNA abundance probably by mechanisms operating through transcription initiation [116,119,120]. 2.2.2.2. CRD2. A multicopper oxidase is presumed to function in a high affinity iron assimilation pathway in Chlamydomonas based on its pattern of expression in iron-deficient cells and its coregulation with FTR1 and ATX1 (see above); yet, copper-deficient Chlamydomonas cells do not show symptoms of iron-deficiency (Fig. 4, top) as do yeast and mammalian cells [121]. Even under iron-limiting conditions, copper-deficiency does not exacerbate the iron-deficiency phenotype, suggesting that another (copperindependent) iron uptake pathway must operate in this situation [34]. This idea was solidified with the isolation of a Chlamydomonas mutant crd2-1 that showed a copper-deficiency-conditional requirement for high iron nutrition [73]. The crd2-1 strain is indistinguishable from wild-type when it is grown in standard copper replete medium (6 M chelated copper) except that the accumulation of FOX1, FTR1 and FEA1 mRNAs may be slightly increased, but when copper is eliminated from the medium, the strain is distinctly chlorotic and growth-inhibited even in standard iron-replete (18 M iron) medium. The FOX1, FTR1 and FEA1 mRNAs show progressively greater expression as the copper content of the medium is decreased, indicative of internal iron deficiency in response to poor copper nutrition. The copper-nutrition conditional iron-deficiency phenotype of crd2 can be rescued by increasing the iron content of the medium. The product of the CRD2 locus is not yet known. It is possible that it encodes a copper-independent ferroxidase that could partner with Ftr1 or it might encode a component of a completely independent high affinity iron transporter. Iron regulation is very tightly regulated owing to the toxicity of excess iron. Therefore, CRD2 should be expressed only when its function is necessary that is, under conditions of copper-deficiency and this could be exploited for the identification of Crd2 (and associated components). Another possibility is that CRD2 encodes a system for prioritized allocation of copper to the ferroxidase (discussed in [73]). 2.2.2.3. AOX. Plants and fungi express an enzyme called alternative oxidase that functions as a terminal oxidase in a situation

when the ubiquinone pool is over-reduced [122]. When cytochrome oxidase function is compromised in heterotrophicallygrown copper-deficient Chlamydomonas cells, the abundance of alternative oxidase increases 2-fold [123]. Chlamydomonas has two genes AOX1 and AOX2 encoding enzyme isoforms and it is not known whether one or both forms is/are increased in copperdeficiency. Nor is it known whether the increase responds directly to copper nutrition or to the level of oxidation of the ubiquinol pool. Alternative oxidase catalyzes the direct oxidation of ubiquinol by molecular oxygen (bypassing complexes III and IV in respiration) without concomitant proton translocation; hence it is not an energy-conserving pathway. Thus, there is no back-up for copper function in respiration as there is for photosynthesis and iron assimilation. 2.2.3. Metabolic adaptation The increased expression of copper assimilation components and back up enzymes has a physiological rationale. Three other Chlamydomonas genes also show changed expression in copper deficiency but the underlying metabolic rationale is obscure. 2.2.3.1. CPX1. A 35-kDa soluble protein was purified from Chlamydomonas cells as a protein of interest for understanding nutritional copper deficiency because it showed increased accumulation in copper-deficient vs. copper-replete medium [124]. Sequence analysis of internal tryptic peptides identified the protein as coproporphyrinogen III oxidase. The corresponding gene is transcriptionally activated in copper-deficiency and soluble extracts of copper-deficient Chlamydomonas cells display more coproporphyrinogen oxidase activity relative to extracts from copper-replete cells. The protein, which shows greatest sequence relationship to plant coproporphyrinogen III oxidase, is targeted to the chloroplast where the tetrapyrrole biosynthetic pathway is localized [125]. The Chlamydomonas genome encodes a second isoform, named Cpx2, which is more closely related to the animal mitochondrial enzyme. This gene is not regulated by copper nutrition at least at the level of mRNA accumulation (J.K, M.D. Page and S.M., unpublished). The subcellular location of Cpx2 is not known. In the tetrapyrrole pathway, the enzyme coproporphyrinogen oxidase is known to be rate limiting after the synthesis of -aminolevulinic acid. Perhaps tetrapyrrole metabolism is compromised or there is an increased demand for tetrapyrrole precursors in copper-deficient cells. We know that copper-deficient cells do synthesize an abundant heme protein (Cyt c6), but this would constitute a small draw from the tetrapyrrole precursor pool relative to the chlorophyll proteins. The CPX1 gene is also up-regulated by oxygen-deficiency. Interestingly, this response requires the copper-response element (CuRE) as well as a related hypoxia-responsive element (see below). 2.2.3.2. CRD1/CTH1. Two other genes that show differential expression in copper-deficient Chlamydomonas cells, CRD1 (renamed CHL27A) and CTH1 (renamed CHL27B) were identified genetically [126,127]. They encode isoforms of another enzyme in the tetrapyrrole pathway: the aerobic oxidative cyclase. This enzyme uses a di-iron active site to catalyze

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the oxygen-dependent formation of Ring V in chlorophyll from the methyl-esterified propionate side chain of Ring III [128]. In Arabidopsis, the protein (encoded by a single copy gene CHL27) is localized to both the inner envelope membrane of the chloroplast as well as the thylakoid membrane. The distribution of the two isoforms of the Chlamydomonas enzyme is not known, but it is possible that they may be differently localized. Their pattern of expression is reciprocal as a function of copper nutrition or oxygen supply [127]. Crd1/Chl27A predominates in copperdeficient or hypoxic cells while Cth1/Chl27B predominates in copper-replete or well-oxygenated cells. Loss-of-function crd1 mutants show copper-nutrition-conditional loss of a subset of chlorophyll proteins and are pale green or chlorotic. Although Cth1/Chl27B regulation is normal in these mutants, the amount of total enzyme (Crd1 + Cth1) at this step must clearly be ratelimiting for the pathway in copper-starved cells. Interestingly, crd1 mutants show progressive loss of particular chlorophyll proteins depending on the severity of copperdeficiency, metabolic physiology (phototrophic vs. heterotrophic growth), the nature of the mutation or penetrance of the allele [126]. Photosystem I and its associated light harvesting proteins, LHCIs, are essentially completely absent while LHCII proteins are reduced in abundance. This suggests that there may be some level of metabolic compartmentation/channeling in the tetrapyrrole pathway with the two isoforms directing precursors to distinct chlorophyll-proteins. The alternate hypothesis of hierarchical chlorophyll allocation to photosystems and accessory antennae is not favoured because other mutants with comparable reduction in total chlorophyll do not display the hierarchy. It is worth noting that the cyanobacteria, which also switch between plastocyanin and Cyt c6 based on copper nutrition, encode multiple isoforms of CHL27. The pattern of expression of CRD1/ CHL27A and CTH1/CHL27B in hypoxia has also not been rationalized, but hypoxic crd1 strains show the same chlorotic phenotype with loss of PSI and LHCI as do the copper-deficient crd1 strains, which argues that CRD1 expression and function in hypoxic cells is physiologically relevant. A metabolite profile of tetrapyrrole precursors as a function of iron and copper nutrition in wild-type vs. mutant Chlamydomonas strains may be informative. Coprangen oxidase and Chl27 catalyze rate-limiting, oxygen-dependent steps in the tetrapyrrole pathway. Substantial research effort is dedicated to the understanding of regulatory mechanisms in this pathway but the impact of micronutrients has not been examined systematically [129,130]. The discovery of copper-deficiency regulated expression of key enzymes in heme and chlorophyll biosynthesis may be hinting at an additional layer of regulatory complexity in this ancient pathway. 2.2.4. Intracellular copper distribution The prototypical pathway for copperprotein assembly suggests that proteins that are processed through the secretory pathway (e.g., extracellular and plasma membrane enzymes) are loaded with copper in a compartment in that pathway before they reach their final destination, while the mitochondrial and chloroplast enzymes are loaded with copper at their site of action (e.g., mitochondrial membrane for cytochrome oxidase, thylakoid lumen for plastocyanin). Upon entry into cells (via a

CTR-type transporter), copper is picked up by the copper chaperone Atx1 (also called Atox1/Hah1 in animals or CCH in plants). Atx1 transfers copper to a copper-transporting P-type ATPase in the secretory pathway (Ccc2p in S. cerevisiae, RAN1 in plants and ATP7A/B in animals). The energy of ATP hydrolysis is used to concentrate copper in a vesicular compartment for loading the active sites of plasma membrane or secreted proteins like ceruloplasmin, hephaestin, or the ethylene receptor. Analogous copper-transporting P-type ATPases, PAA1 and PAA2, function at the chloroplast envelope and thylakoid membranes for delivery of copper to the stroma where chloroplast CuZnSOD is assembled and thylakoid lumen where plastocyanin is assembled [59]. The copper chaperones Cox17 and Cox19 and assembly factors Cox11 and Sco1/2 are required for loading the CuA and CuB sites of cytochrome oxidase [131]. Chlamydomonas encodes homologues of each of these intracellular copper-distributing molecules [21,55,57,73]. A role for Chlamydomonas Atx1 in the secretory pathway has been established on the basis of pattern of expression in response to iron nutrition and its ability to rescue the corresponding S. cerevisiae mutant [34]. However, the three copper-transporting P-type ATPases, named Ctp1, Ctp2 and Ctp3 (Table 2), need to be distinguished on the basis of sub-cellular localization and functional tests. The above-mentioned pathways for intracellular copper distribution are unidirectional. A new area of interest is the question of how copper in one compartment can be transferred to a different compartment. This is particularly relevant in the situation of nutritional copper deficiency when it may be necessary to recycle copper from abundant but less physiologically important cuproproteins. We have established that plastocyanin is actively degraded in copper-deficient Chlamydomonas [100,110] and suggested that the underlying purpose must be to save copper for other important copper enzymes such as cytochrome oxidase. The question of how copper might leave the chloroplast has not been addressed yet. The existence of multiple CTR-type transporters suggests that one or more of these could be important for intracellular re-allocation in copper recycling pathways. 2.2.5. Copper-responsive signal transduction 2.2.5.1. CuREs. The involvement of transcriptional regulation in nutritional copper signaling in Chlamydomonas was established through reporter gene constructs in which the 5 flanking sequences upstream of the 5 end of the CYC6 and CPX1 transcripts were fused to a reporter gene [113,132]. When tested in the context of a minimal promoter, the 5 flanking sequences activated transcription in copper-deficiency rather than repressing transcription in copper replete cells, indicating that the CuREs served as targets for a transcriptional activator. In contrast, when a constitutive promoter drove synthesis of CYC6 mRNA, the mRNA accumulated even in copper-replete cells, indicating that post-transcriptional processes did not play a significant role in copper-responsive gene expression. This is consistent with a measured in vivo half-life of 4560 min for the CYC6 mRNA independent of the copper content of the

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medium [133]. Promoter dissection revealed that the CYC6 promoter houses two CuREs, each of which is sufficient on its own to confer copper-responsiveness to a reporter gene. The CPX1 gene is associated with one additional CuRE that is required also for the response of the gene to hypoxia. Mutational analysis of the CuREs indicated that a core sequence, GTAC, was absolutely critical (but not sufficient) for CuRE activity. There are GTAC sequences upstream of the CRD1 gene as well but these have not been tested functionally. 2.2.5.2. Crr1. A screen for Chlamydomonas mutants that displayed copper-deficiency-conditional phenotypes revealed one regulatory locus, CRR1 [73]. The crr1 mutants are characterized by very poor growth in copper-deficient medium. Restoration of growth requires excess copper in the medium because Crr1 is probably required for expression of a copper assimilation pathway. CYC6 is not expressed in copper-deficient crr1 cells, and CPX1 and CRD1/CHL27A are expressed only at basal +Cu levels, consistent with loss of function of a transcriptional activator. The crr1 strains also accumulate apoplastocyanin, indicating that the gene for the plastocyanin-degrading protease is another target of Crr1. It is likely that Crr1 is required also for expression of the copper-independent backup pathway for iron assimilation (defined by the CRD2 locus) because irondeficiency exacerbates the crr1 phenotype while excess iron provides relief. A gene corresponding to CRR1 was cloned by complementation [134]. It encodes a large protein (1232 residues) with a 76-residue Zn-dependent DNA binding domain named SBP for the founding member of this plant-specific domain [135], a putative nuclear localization signal, ankyrin repeats and a Cterminal Cys-rich region that resembles metallothionein. The Crr1-SBP shows sequence specific binding to the CuREs in the CYC6 and CPX1 genes: mutations that block CuRE activity in vivo in reporter gene constructs also make less effective competitors in electrophoretic mobility shift assays of the Crr1-SBP on authentic CuREs [134]. The CuRE in Chlamydomonas is unrelated to CuREs of other organisms, and Crr1 is likewise unrelated in sequence to other metalloregulatory transcription factors [136]. It is not known yet whether Crr1 is the copper sensor or whether it interacts with the copper sensor (through the ankyrin repeats). At present, the former possibility is favoured because of the presence of two candidate metal binding domains in Crr1the SBP and the C-terminal Cys-rich region. In vitro and in vivo mutational analysis should distinguish between the different models presented for metal sensing by Crr1 [134]. Each of the target genes, CYC6, CPX1 and CRD1, can be artificially activated in vivo in the presence of excess copper in the medium by nickel and cobalt ions, and this response is dependent on the CuREs and Crr1 [40]. One explanation for this artificial response is that nickel and cobalt are antagonistic at the copper-sensing site in this signal transduction pathway. Alternatively, the presence of two histidine residues in the SBP domain may allow it to accommodate various transition metals with different geometries and hence different gene expression activity.

2.2.5.3. Down-regulation of Cth1/Chl27B and plastocyanin in copper-deficiency. Crr1 is required also for down-regulating the abundance of Cth1/Chl27B [127]. The mechanism of downregulation relies on transcriptional activation upstream of the CTH1 locus by Crr1 through putative CuREs. In copper-replete cells, the downstream promoter produces a 2 kb mRNA that encodes Cth1/Chl27B, but in copper-deficient cells, Crr1 activity produces a 5 extended 3 kb mRNA with a long leader sequence encoding multiple small ORFs in each frame. At the same time, promoter occlusion inhibits production of the 2 kb mRNA. Since the 3 kb mRNA cannot be translated (M.A. and S.M. unpublished), the CTH1 locus produces much less protein in a copper-deficient cell compared to a copper replete cell. In parallel, the CRD1 locus is transcriptionally activated in copperdeficiency by Crr1 so that more Crd1/Chl27A is synthesized assuring a perfectly reciprocal pattern of expression. A reciprocal pattern of expression was noted already for copper-dependent plastocyanin and Cyt c6 accumulation in Chlamydomonas [137]. However, for plastocyanin, pulse-chase experiments established that down-regulation occurs by induced proteolysis [100,110]. Nevertheless, this is still Crr1-dependent (discussed above). We conclude that Crr1 is a master regulator of nutritional copper homeostasis: it up-regulates one set of genes through CuREs associated with those genes and down-regulates another set of genes either through the transcriptional activation of specifically-target proteases or through intergenic upstream transcriptional activation that represses the production of downstream functional mRNAs, thereby assuring perfectly coordinated reciprocal patterns of expression. 3. Outlook 3.1. Functional analysis of candidate genes 3.1.1. Transporters The draft genome of Chlamydomonas has revealed a number of candidate metal transporters of the HMA, MTP and ZIP families whose function in terms of metal selectivity and intracellular location is not yet evident. The dominance of transcriptional regulation in determining the pattern of expression of assimilatory transporters coupled with the increased expression of mitochondrial proteins during the transition from phototrophic to heterotrophic growth should facilitate functional assignments of these transporters. Furthermore, in the study of a unicellular eukaryote, complexities arising from specialized physiology and function of organs or particular stages of development are avoided. The technology for knock-down of individual genes is expected to improve in the next few years as are the development of compartment-specific markers, and these would enhance the repertoire of experimental approaches. 3.1.2. Metalloenzymes The Chlamydomonas genome encodes a number of copper enzymes like amine oxidases, matrix metalloproteases and multicopper oxidases that seem to be poorly expressed in laboratory cultures as assessed from the distribution of ESTs. These enzymes are not yet assigned to a particular pathway although the matrix

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metalloproteinases are thought to be important in sexual reproduction. It is possible to use real time PCR methods to monitor the expression of candidate copper enzymes under various stress situations or at different stages of the cell cycle or sexual reproduction cycle to assess the importance of these enzymes and deduce a window for phenotypic assessment of knock-down strains. This approach should also be powerful for other metalloenzymes of interest. 3.2. Other nutrients 3.2.1. Mn and Se Manganese and Selenium are required at such low levels for most organisms that it is experimentally challenging to generate strong phenotypes of deficiency. Chlamydomonas has a high manganese requirement relative to non-photosynthetic organisms and this makes the imposition of deficiency easier. As noted for other micronutrients, the strength of the deficiency phenotype is dependent on the metabolic requirements of the cell. Phototrophically grown cells are strictly dependent on manganese nutrition whereas heterotrophically grown cells show a milder impact (Fig. 5), but this can be exacerbated in the presence of exogenous peroxides (S.T. and S.M., unpublished). In contrast to S. cerevisiae and A. thaliana, Chlamydomonas uses selenoenzymes and the repertoire is almost comparable to that in mammalian models [44]. Although selenium supplementation appears to have only a marginal beneficial effect on Chlamydomonas, it is possible that more dramatic phenotypes could be noted under situations of photo-oxidative or metal toxicity stress when intracellular glutathione pools are depleted [18,103]. 3.2.2. Variable metal requirement The relationship between metabolism and metal requirement is an aspect that needs greater appreciation and consideration in experimental design. The impact of pH, ionic strength and inorganic anions (like phosphate) also needs to be considered, since bioavailability of metal micronutrients is dependent on chemical speciation. In this respect, a microorganism that can be maintained in a chemostat offers a unique experimental oppor-

tunity. In particular, the fact that Chlamydomonas does not require organic nutrient supplementation might simplify the design of experiments aimed at dissecting the complex interactions between metal nutrients. 3.3. Special aspects 3.3.1. Chloroplast vs. mitochondria The chloroplast and mitochondria are rich in metalloproteins and the question of how metal allocation to one organelle vs. the other is prioritized has not been addressed. The availability of mutants affected specifically in photosynthesis (acetate-requiring strains) or respiration (dark-diers) and the possibility to grow wild-type cells either phototrophically, heterotrophically or mixotrophically makes Chlamydomonas a unique system for addressing these questions [26,138]. The possibility to compare metabolite profiles as a function of metal and carbon-source nutrition is value-added [139]. 3.3.2. Phytoremediation and oxidative stress There is historical interest on the capacity and mechanisms of Chlamydomonas spp. to handle toxic metals like cadmium, lead and mercury [71,103,140143]. While a pathway for glutathione and phytochelatin-dependent detoxification is evident, there is no indication that metallothionein-like molecules exist in the Chlamydomonas genome. But it should be noted that the gene models are generally biased against small proteins with low complexity sequences that are not represented in the EST database, and they may well show up when tiling microarrays are applied to this question. Excess light generates oxidative stress in chloroplasts when the absorbed light energy is greater than the capacity of metabolism to use the reducing equivalents that are generated. While there is substantial research on the protective (such as activation of genes in anti-oxidant pathways) and preventative mechanisms (such as movement away, remodeling of antennae) used by Chlamydomonas to avoid oxidative injury in this situation, the question of how metal homeostasis and re-allocation responds to light intensity is another unexplored area.

Fig. 5. Manganese-dependent photosynthetic growth. Wild type strain CC425 was grown either heterotrophically (orange) or phototrophically (black) in medium supplemented with no (dots), 0.1 (dash) or 1 (solid) M chelated manganese salts. Growth was estimated by counting cells in a hemocytometer.

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Acknowledgments We are grateful to the National Institutes of Health, the Department of Agriculture and the Department of Energy for supporting research projects in the Merchant laboratory, and we thank the Joint Genome Institute for the draft sequence of the Chlamydomonas genome. MDA and AMT are supported by Institutional Kirschstein Fellowships, GM07185 and GM07104, respectively, and ST was supported by the UC Toxic Substances Research and Teaching Program. Several colleagues and coworkers have contributed ideas and expertise to the project, including James Gober, M. Dudley Page, William Schopf, Frederik Sommer and Vinzenz Unger: we thank them for help and stimulating discussions. References
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