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Biochimica et Biophysica Ac

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

Abstract

Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganicnutrients 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. arewidely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity foracclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas representa blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomicanalysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) aswell as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficientChlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting fromcompromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Up-regulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmeddegradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occuronly with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surfacereductase and a Cu+ transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of “back-up” enzymes forplastocyanin 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 andaerobic oxidative cyclase) are also increased in expression and activity by as much as 10-fold but the connection between copper nutrition andtetrapyrroles is not understood. The copper-deficiency responses are mediated by copper response elements that are defined by a GTAC coresequence and a novel metalloregulator, Crr1, which uses a zinc-dependent SBP domain to bind to the CuRE. The Chlamydomonas model is idealfor 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 ofdifferential 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

⁎ Corresponding author. Tel.: +1 310 825 8300; fax: +1 310 206 1035.E-mail address: [email protected] (S.S. Merchant).

1 Present address: Department of Plant Biology, 260 Panama St., CarnegieInstitution 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

1. Introduction

1.1. The evolution of trace element homeostasis

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

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http://dx.doi.org/10.1016/j.bbamcr.2006.04.007

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 colorsdistinguish those elements where the biochemical target underlying the requirement is known (orange) vs. those elements that have been shown to be beneficial for theanimal but whose targets are unknown (yellow). The beige elements represent examples of requirements restricted to particular microorganisms. The figure wasgenerated from an existing electronic source [144].

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Cu, Mn, Mo, and V can exist in multiple stable oxidation statesin an aqueous, neutral reaction environment and this facilitatesredox chemistry, which is at the heart of biochemistry. Otherelements like Zn and Co provide catalysts with chemicalproperties (such as electrophiles) that are not available in thefunctional groups found on amino acid side chains, and thisfacilitates hydrolytic and hydration/dehydration reactionscentral to metabolism. Because these elements are essentialfor biochemistry, they must be acquired as “nutrients” by cellsto sustain life. The operation of micronutrient (especially iron)acquisition and sequestration pathways can be determinants ofsuccessful outcomes for competing organisms in particularenvironmental niches or in the establishment of infection in ahost–pathogen interaction.

It is well accepted that life originated in an aqueous environ-ment and accordingly the inorganic elements that became essen-tial for life (Fig. 1) appear to be represented in organisms basedon their availability in sea water [2]. Thus, abundant elementslike Si, Al, Ti are not well represented in biology owing to theinsolubility of the corresponding oxides or hydroxides, whileMo and V are quite soluble as oxyanions and are accordinglyimportant cofactors in enzymes of S and N metabolism thathandle intermediates with various oxidation states. Photosyn-thetic organisms are among the early life forms on earth and theirbiochemistry is dependent on a wide range of inorganic nut-rients. Besides the cofactor-rich photosynthetic apparatus [3],these organisms also use metal cofactor-dependent enzymes fornitrogen fixation and/or for assimilation of inorganic nitrogenand sulfur into organic compounds [4,5]. The photosyntheticmicroorganisms evolvedmechanisms therefore to assimilate anddistribute inorganic nutrients to various metabolic pathways and

the fundamental principles of trace element acquisition ofinterest in today's multicellular complex organisms have anevolutionary basis in the mechanisms operating in progenitormicrobes.

Two events that changed the landscape of life are criticaldeterminants of micronutrient homeostasis today. The advent ofoxygen on the planet as a byproduct pollutant of oxygenicphotosynthesis shifted the oxidation state of the mineral traceelements and hence their “bio”availability. For iron, the changein the ratio of ferrous (soluble) to ferric (insoluble) ions neces-sitated the evolution of intricate mechanisms for iron acquisitionin aerobic organisms. A survey of iron acquisition mechanismsoperating in bacteria, protists, plants or humans reveals the keyrole of pH and redox chemistry for mobilization of this nutrient.Although the prevalence of iron in enzymes [6] gives evidenceof the importance of this element in the establishment of life inthe oceans billions of years ago, iron is a limiting nutrient in largeportions of the oceans today [7–9]. While the advent of oxygendecreased iron availability, copper becamemore available owingto the increased solubility of cupric salts, and the evolution ofcupredoxins appears to be co-incident with the appearance ofoxygenic photosynthesis [10]. There are many examples ofcopper- vs. iron-containing enzymes that catalyze similar reac-tions (Table 1). In some cases, the use of one catalyst over theother is determined bymicronutrient (copper or iron) availability[11,12]. The second important landmark was the movement oflife from the oceans to land, which changed nutrient availabilityfrom a gradient continuum in water to a feast or famine situationon land (Fig. 2).

The same chemical properties that make the inorganicelements so important for life also give them the potential to

Table 1Copper and iron have similar catalytic potential

Function Iron PDB Copper PDB

Oxygentransport

Hemoglobin 1A3N Hemocyanin 1JS8Hemerythrin 1HR3

Hydroxylation Diiron MMO 1MMO Particulate MMO 1YEWCytochrome P450 1AMO Mononuclear

tyrosinase1C7G

Oxidation Catecholdioxygenase

2AZQ Dinuclear catecholoxidase

1BUG

Electrontransfer

Cytochrome c6 1CTJ Plastocyanin 2PLT

Terminaloxidase

Diiron alternativeoxidase

CuACuB cytochromeoxidase

1OCC

CuACuZ N2Oreductase

1FXW

Anti-oxidant FeSOD 1COJ CuZnSOD 1ESONitrite

reductionHeme nitritereductase

2AKJ Cu nitrite reductase 1OE2

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

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cause cellular damage; thus, an organism finds itself between arock and a hard place — faced with balancing two difficultsituations. For some elements this potential for toxicity is exa-cerbated in the presence of oxygen [13,14]. In the case of ironand selenium, the difference between the highest recommendeddaily intake (for humans) vs. the estimated minimal toxic dailydose is only about 5- to 6-fold, indicating the need for homeo-static mechanisms [15]. Finally, nutrient acquisition mechan-isms operate in the context of deficiencies in some elements andexcesses of other (sometimes chemically related) elements andinteractions between individual trace element uptake pathwaysmust be considered for a complete understanding of the naturalphysiology of trace element metabolism.

Fig. 2. Chlamydomonas reinhardtii.Nutrient manipulation in a simple salt-containinggrown in TAP medium containing iron-EDTA supplement as indicated (μM). Mutantdefective in photosynthesis are easily identified by fluorescence (bottom, left). Sporemedium, were analyzed visually and by fluorescence. Mutants are pale green on coppand decay kinetics shows a defect in the decay phase for copper-deficient crd1 strainswild-type). The chloroplast is a major iron-utilizing organelle. Light microscopy (bottabout half the volume of the cell.

1.2. Chlamydomonas as an experimental model

Chlamydomonas spp. were originally classified among theVolvocales algae in the plant kingdom [16]. Recently, a newtaxonomy for eukaryotes was introduced where Chlamydomo-nas is placed in the cluster (equivalent to Kingdom) Archae-plastida [17]. This cluster includes the green algae and plantsand the name replaces the terms Viridiplantae and Chlorobionta.Within this cluster, Chlamydomonas spp. are classified withinthe first rank Chloroplastids, second rank Chlorophyta. Otherspecial experimental models among the Chlorophyta are Volvoxcarterii (for cell differentiation and development) and Duna-liella spp. (for acclimation to high light, carotenoid and glycerolproduction). Chlamydomonas spp. are found world-wide andoccupy various environmental niches, including soil (the sourceof the popular laboratory strain C. reinhardtii), snow fields andglaciers (C. nivalis also known as snow algae), acid mines, peatbogs and sewage ponds. Distinct metal bioavailability is expec-ted in each niche owing to variation in metal content, pH andoxygenation of the habitat and competition with other orga-nisms, suggesting that the genus must have significant capacityfor acclimation to micronutrient availability. Because of theconvergence of photo-oxidative stress [18] and metal toxicitystress (cited above) on reactive oxygen species, the wide variationin light habitats intimates the existence of highly developedintracellular metal homeostasis mechanisms (reviewed by [19]).

C. reinhardtii is the best developed laboratory model forbiochemical, molecular and genomic approaches to biologicalquestions in algae [20]. The organism is maintained in thelaboratory in liquid culture as motile, haploid vegetative cells,which can be differentiated in the laboratory into gametes (plusor minus depending on the allele at the mating type locus) bynutrient (especially nitrogen) starvation. The existence of sexualreproduction and the possibility of recovering the products ofmeiosis from a zygote (referred to as tetrad analysis) allow the

medium reveals Fe-deficiency “chlorosis” (top, left). Wild-type (strain 4A+) wasstrains defective in Fe-metabolism can be identified visually (top, right). Mutantss from a cross of the crd1 mutant, which lacks photosystem I in copper-deficienter-deficient medium but dark green on copper-replete medium. Fluorescence rise[120]. This can be visualized by false color display (purple for crd1 vs. orange forom, middle) and phase contrast microscopy (bottom, right) show that it occupies


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application of classical genetic approaches to dissect biologicalprocesses. Today, there is a draft sequence of the Chlamydo-monas genome [21] and the full repertoire of genomic andproteomic methodologies can therefore be exploited [22–25].

Chlamydomonas3 cells contain a single large chloroplast thatis biochemically and structurally similar to the ones in vascularplants especially with respect to the photosynthetic apparatus,which is rich in metalloproteins. Wild-type cells can therefore begrown phototrophically (light, CO2 plus inorganic nutrients). Thecells also have protist-like mitochondria with respiratory activityand hence it is possible to grow the organism heterotrophically inthe dark on acetate as a source of reduced carbon [26,27]. In thissituation, the respiratory complexes – another set of metal-richproteins – proliferate [28]. Nevertheless, unlike vascular plantsthat need light to develop a photosynthetic apparatus, Chlamy-domonas synthesizes and maintains a photosynthetic electrontransfer chain even when it is grown in the dark, but the stoichio-metry of components on a per cell basis is several fold reduced.Chlamydomonas can also use multiple different nitrogensources — oxidized forms like nitrate, reduced nitrogen in theform of ammonium or organic nitrogen in the form of urea,purines or ureides. The metabolic flexibility with respect to car-bon source allows us to address questions relating to intracellularallocation of metals in response to metabolic demand in distinctcompartments of the cell (chloroplast vs. mitochondria) and theflexibility for nitrogen source opened the door to the study ofmolybdenum metabolism [29]. It is also possible to initiate par-ticular biogenesis pathways in Chlamydomonas, such as flagellarregeneration or growth of cell walls (e.g., [25,30]), so that the roleof particular metalloenzymes or metal transporters and metallo-chaperones in those processes can be assessed. The cells can beeasily synchronized in the laboratory [31], which allows one toaddress questions related to the timing of metal assimilation. Theultimate advantage of the Chlamydomonas experimental systemfor studies of metal homeostasis is the possibility to tightly controlmetal supply because of the absence of multiple metal chelating/buffering groups in the growth medium. (Supplementation withamino acids or serum is not necessary.) As pointed out earlier,some of the mechanisms in operation in Chlamydomonas arelikely to have evolved from the progenitor photosyntheticcyanobacterial ancestor of the chloroplast, and their discoverywould give us a picture of the fundamental principles underlyingtrace nutrient distribution to metabolic pathways.

It is worth mentioning that while Chlamydomonas is groupedwith plants in the Archaeplastida cluster, researchers often referto it as “half beast-half plant” because of the strong sequencerelationships of some pathways (e.g., chloroplast based ones) toanalogous ones in vascular plants and others (e.g., ciliary bio-genesis and function) to analogous ones in animals [32,33].Analysis of the genome indicates that there are also strong rela-tionships to proteins restricted to microorganisms as well. Agood 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 thecommon laboratory strain, Chlamydomonas reinhardtii. Other species will benamed specifically.

oxidase, Fox1, whose copper binding domains are arranged likethe ones in MnxG in a Bacillus strain [34,35].

1.3. Metal requirement for Chlamydomonas

Although there were some early studies on optimization ofmedium for provision of trace metals to C. mundana, the traceelement solution used in most laboratories for C. reinhardtii isbased on a “generic” solution developed about 50 years ago forgrowth of bacteria [36–38]. Accordingly, the solution has notbeen optimized for the growth ormetabolism ofChlamydomonasstrains. For instance, although cobalt is provided in the medium,Chlamydomonas, like other eukaryotes, does not have a pathwayfor Vitamin B12 synthesis and relies on bacteria for provision ofthis cofactor or can use B12-independent enzymes for methio-nine biosynthesis [39]. Thus, cobalt is not necessary for growthof Chlamydomonas and its presence in the medium may com-plicate the analysis of copper signaling [40]. A survey of themicronutrient content of various media used for growth of Chla-mydomonas shows a wide range of metal concentrations used[16], but none appears to be optimal based on our work on copperand iron nutrition. The Sager–Granick medium is clearly defi-cient in copper while the Eversole medium is likely to bedeficient in iron under respiratory growth conditions and TAPmedium contains excessive amounts of zinc andmanganese (datanot shown). The impact of pH [41], which changes as the acetatein themedium ismetabolized, is generally not considered. Owingto cross-talk between metal assimilation pathways [42] and theoccurrence of low selectivity divalent cation transporters inmanyorganisms, it is critical that there be a clear reference point forexperimental analysis of trace metal homeostasis.

One approach to defining the appetite for trace minerals in thegrowth medium is to measure the metal content of a population ofcells grown under different metabolic conditions and to calculatethe amount that must be provided in the medium to permit growthof a culture to 2 × 107 cells per ml (Fig. 3). Mutant strains thatmake smaller or larger cells were also included in the analysis [43].The analysis shows that molybdenum, selenium and cobalt arepresent at ultra trace levels. Cobalt is not required for growth and ispresumably found associated with the cell due to adventitiousuptake. Molybdenum is important for synthesis of the molybdo-cofactor and selenium is used in about a dozen selenoenzymes; theamounts required in the medium for synthesis of the relevantenzymes have been tested to be in the sub-micromolar range andthis is consistent with the ultralow Mo and Se content [29,44,45].The transition metals Cu, Fe, Zn and Mn are present at muchhigher levels and as is the case for animals, Cu is found at lowerlevels relative to Fe and Zn. The high Mn content is attributed tothe abundance ofMn in the photosynthetic apparatus. This has theuseful experimental consequence of facilitating studies of Mn-deficiency in Chlamydomonas [46]. The low level of Mn inanimal cells makes nutritional deficiency much harder to achieve.

2. Recent achievements

There are three basic approaches that have been used todiscover genes and pathways required for acclimation of

Fig. 3. Micronutrient content of Chlamydomonas. Wild-type (strains 2137, 21gr,CC425, 4A+, 17D−) or mutant (mat3–4, smt8) Chlamydomonas cells weregrown under various conditions (5–500 μmol per m2 per s light intensity, CO2

vs. acetate as carbon source, 2–20% oxygen in the atmosphere above the liquidculture, and nitrate vs. ammonium as a nitrogen source) and collected bycentrifugation. The cell pellet was washed with 1 mM EDTA to removeextracellular bound metal and dissolved in 30% nitric acid by incubation for2 days at 65 °C. The metal content was measured at the ICP-MS Laboratory atthe University of California in Davis. The y-axis plots the range of metal contenton 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 alsofor humans. The metal content of the cells can be used to estimate a minimalsupply of the micronutrient that would support a density of 2 × 107 cells per mlin a typical stationary phase culture.


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Chlamydomonas to macro- and micro-nutrients [47]. Two ofthese, involving the identification of differentially expressedproteins and genes or loss of function mutants that are unable toadapt to low supply of the nutrient of interest, have the mostpotential for discovery of new components and connections.The third approach of finding homologs of known genes hasbeen facilitated recently by the deposition of ESTs (includingfrom libraries derived from RNAs isolated from nutritionally-depleted cultures) and the advent of a draft sequence of theChlamydomonas genome [21,48,49].

2.1. Iron

2.1.1. Iron assimilation

2.1.1.1. Ferroxidase/Ftr1. Buckhout and co-workers separat-ed proteins of purified plasma membranes from iron replete vs.iron-deficient Chlamydomonas cells and noted the differentialexpression of a number of polypeptides, especially a 150 kDaprotein that was identified as a ferroxidase and named FLP basedon the similarity of peptide sequences to hephaestin [50,51].This protein was also discovered in parallel as the product of theFOX1 gene, cDNAs of which were identified among ESTs onthe basis of its multicopper oxidase motif [34]. The Chlamydo-monas ferroxidase shows highest sequence similarity to themammalian ceruloplasmins and hephaestins. Taken togetherwith the increased abundance of the polypeptide and of FOX1mRNA in iron-deficiency, a ferroxidase function in iron assimi-

lation was suggested by both groups. A copper requirementfor accumulation of the protein was also noted in both works.Nevertheless, in one work, copper-deficiency was noted to im-pact iron assimilation while in the other, the lack of an impact ofcopper-deficiency on iron nutrition suggested the existence ofcopper-independent pathway(s) for high affinity iron uptake. Itis possible that there are genetic differences between the two“wild-type” strains used in each work. The FOX1 gene encodesan integral, presumed N-terminally anchored, membrane proteinwith 1142 amino acids and a predicted molecular weight of130 kDa. The slower migration of the polypeptide on denaturinggels may result from glycosylation (consistent with its plasmamembrane location and presumed routing through the secretorypathway). The multicopper oxidases have 3-fold symmetry andare thought to have evolved by triplication of an ancient copperbinding motif [52]. La Fontaine et al. noted that the arrangementof the copper binding sites of the Chlamydomonas Fox1 relativeto the position in the primary sequence is unconventional andsuggests a divergent evolutionary path of this widely distributedtype of protein. A Fox1 paralog (55% identity), named Fox2,was identified in the genome, but the corresponding gene is notregulated by metal nutrition and hence a function for Fox2is obscure. Fox2 has the same arrangement of copper bindingmotifs as does Fox1 but Fox2 appears to lack the N-terminalhydrophobic membrane-anchor of Fox1, suggesting a distinctsub-cellular location.

La Fontaine et al. suggested that the mechanism for copperloading of Chlamydomonas Fox1 is analogous to the one forFet3p in Saccharomyces cerevisiae [53] or for ceruloplasmin inmammalian cells [54], based on the identification of a func-tional Atx1 as well as sequences related to the distributive P-type ATPases, prototyped by Ccc2, MNK and WND [34,53,55,56]. A more recent analysis of the draft genome sequenceindicates multiple candidate genes encoding copper-transport-ing P-type ATPases and these are named CTP1, CTP2 andCTP3 (Table 2). A fourth gene model is called HMA1 [57]. Oneof the CTP family members is expected to be involved inloading copper-proteins that mature through the secretory path-way (e.g., Fox1, various amine oxidases and extracellular en-zymes) while the other two are likely to be functionally analo-gous to Arabidopsis PAA1 and PAA2 and cyanobacterial CtaAand PacS that distribute copper across the plastid envelope (orcell membrane in the case of cyanobacteria) and thylakoidmembrane, respectively [58–62].

The multicopper oxidases are generally associated with apermease or transporter component for delivery of iron acrossthe membrane. The identity of this partner in Chlamydomonasis not known, but a good candidate is Ftr1, whose expression isup-regulated coordinately with Fox1 during Fe-deficiency [34].The FTR1 cDNA was identified among the EST database witha poor “expect” value but was determined to be of interestbecause the region of sequence similarity includes the iron-binding RExxE motif. The full-length cDNA encodes a 541residue polytopic integral membrane protein with two suchmotifs and shows greatest relationship to fungal Ftr1 homologs.A physical interaction between Ftr1 and Fox1 in Chlamydo-monas has not been demonstrated nor has co-localization of the

Table 2Genes involved in metal metabolism in Chlamydomonas reinhardtii

Name Gene modela Gene family Other names Reference

C_390017 ZIP CrZIP14 [56], aC_600100 ZIP CrZIP7 [56], a

IRT1 C_210127 ZIP CrZIP13 [56], aIRT2 C_80200 ZIP CrZIP12 [56], aZIP1 C_980004 ZIP CrZIP5 [56], aZIP2 C_380039 ZIP CrZIP10 [56], aZIP3 C_30108 ZIP CrZIP8 [56], aZIP4 C_380040 ZIP CrZIP11 [56], aZIP6 C_190162 ZIP CrZIP6 [56], aZRT1 C_100025 ZIP CrZIP1 [56], aZRT2 C_1670018 ZIP CrZIP2 [56], aZRT3 C_210155 ZIP CrZIP13 [56], aZRT4 C_119152 ZIP CrZIP9 [56], aZRT5 C_980003 ZIP CrZIP4 [56], aMTP1 C_670027 CDF CrMTP1 [56]MTP2 C_110062 CDF CrMTP2 [56]MTP3 C_110067 CDF CrMTP3 [56]MTP4 C_110212 CDF CrMTP4 [56]MTP5 C_290110 CDF CrMTP5 [56]CutC C_1080007PCS1 C_1470022CTP1 C_650007 HMA CrHMA2 [56, 68]CTP2 C_260147 HMA CrHMA3 [56, 68]CTP3 C_1430020 HMA aHMA1 C_10220 HMA CrHMA1 [56], aCTR1 C_930057 CTR bCTR2 C_60152 CTR bCOPT1 C_360043 CTR CrCOPT1 [56], bATX1 ATX1 [34]FOX1 FOX1 FLP1 [34,50]FOX2 C_380052 aFTR1 C_30271 CrFTR1 [34,56]FTX1 C_960033NRAMP1 C_170137 Nramp CrNRAMP1 DMT1 [56,70], aNRAMP2 C_570027 Nramp CrNRAMP2 [56], aRET1 C_440074 CrNRAMP3 [56], aCBR1 C_540039 aFRE1 C_40004 aRBOL1 C_30094 aRBOL2 C_30095 aFEA1 C_70187 H43 [65], aFEA2 C_70186 aFER1 C_700044 Ferritin cFER2 C_210055 Ferritin c

Genes are grouped by family or function. Other names indicate alternate genenames in use. a, MDA, JK, SM unpublished; b, M. Dudley Page, JK, SMunpublished; c, JCL SM unpublished. ZIP, Zrt-/Irt-like proteins; CDF, cationdiffusion facilitators; HMA, heavy metal (CPx-type) ATPase; CTR, coppertransporters; 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>.

4 The term “chlorosis” in plants refers to reduced chlorophyll content and hencea pale green coloring, which is familiar to anyone who has attempted gardeningor maintained house plants without the benefit of fertilizer or “plant food”.


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two proteins in the same membrane system, but other candidatetransporters with trivalent cation selectivity have not beenproposed. Ftr1-type transporters are not generally found inplants, but several unique ESTs encoding moss proteins withgood sequence relationship to Chlamydomonas Ftr1, includingthe RExxE motif, have been retrieved (Blast search in 12/05).

2.1.1.2. Ferrireductases. Iron reduction at the cell surface is akey step in mobilizing iron for uptake [63]. Several earlier works

had documented the involvement of ferrireductases in Chlamy-domonas iron assimilation but a specific protein or gene was notassociated with the activity [64,65]. Examination of theChlamydomonas genome revealed 4 candidate genes (Table 2)based on sequence similarity to known fungal (FRE geneproducts) or mammalian ferrireductases (Dcytb) but the geneshave not yet been functionally dissected by reverse genetic ap-proaches. The pattern of expression in metal-deficient mediumsuggests that one or more may correspond to the measured,inducible ferrireductase activity (M.D.A., J.K. and S.M. unpub-lished). Fre1 is the best candidate based on magnitude of regu-lation and sequence relationship to plant ferrireductases (FRO1/FRO2) and algal Hcr2 [66,67].

2.1.1.3. Extracellular proteins. Chlamydomonas cells have anextracellular space between the wall and the plasma membranethat houses molecules involved in signaling pathways as well asproteins involved in nutrient assimilation [68–70]. A comparativeanalysis of proteins secreted by cell wall deficient mutants ofChlamydomonas grown in iron-replete vs. iron-deficient mediumrevealed two related proteins, named Fea1 and Fea2 (M.D.A. andS.M., unpublished). The FEA2 gene is physically linked to FEA1.Fea2 is less abundant than Fea1 and this is recapitulated at themRNA level as assessed from the EST record. Reverse geneticanalysis is required to assess whether these proteins possessunique functions.

Sayre and co-workers had identified Fea1 already as theproduct of the H43 gene in a screen for RNAs that are differen-tially expressed in cadmium-treated cells [71]. In that work, theyshowed also that the abundance of H43mRNAwas increased iniron-deficient cells and further that expression of H43 couldallow increased growth of a yeast fet3fet4 mutant strain, indica-ting a function of the protein in iron assimilation. There are nohomologs of Fea1/H43 in organisms other than green algae. Arelated protein, Hcr1, was identified in Chlorococcum littoraleas being induced under growth in high CO2, which are condi-tions that induce also ferric reductase activity and iron uptake[66,72]. A multiple sequence alignment of Fea1, Fea2 and Hcr1reveals a number of conserved residues that could serve as ironbinding ligands. A possible function of this protein might simplybe to concentrate iron in the periplasmic space in the vicinity ofthe Fox1/Ftr1 transporter. HCR1 mRNAs are increased inabundance in iron-deficient cells along with HCR2, whichencodes a putative ferrireductase [66]. The closest homolog ofHCR2 in Chlamydomonas is FRE1.

2.1.1.4. Other transporters. Genetic analysis implicates theinvolvement of an additional iron transporter in Chlamydomonas.Assuming that Fox1-dependent iron uptake was the sole pathwayfor high affinity iron transport, copper depletion should result iniron starvation owing to loss of Fox1 activity, and this should leadto chlorosis.4 However, this is not the case [34]. Additionally, thecrd2 mutant of Chlamydomonas shows conditional high iron

http://genome.jgi%1Epsf.org/chlre2/chlre2.home.html

http://genome.jgi%1Epsf.org/chlre2/chlre2.home.html


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requirement in copper-deficient but not copper replete medium[73]. One hypothesis is that the locus represents a component of aback up copper-independent pathway for iron assimilation. Thispathway would be induced under copper-deficient growth con-ditions and its operation would account for the absence of an iron-deficiency chlorotic phenotype in copper-deficient Chlamydo-monas cells [34].

An ABC-type “half”-transporter belonging to the ATM sub-family of half-size transporters is encoded by the CDS1 locusand localizes to Chlamydomonas mitochondria [74]. The CDS1gene is induced strongly by cadmium and loss of its functionresults in cadmium sensitivity. Although a function in cadmiumdetoxification is most likely, the iron sensitivity of the mutantstrains raises also the question of Cds1 function in iron meta-bolism. Genes encoding other related transporters of this familyhave been annotated as HMT-2 and HMT-3 but analysis of theEST dataset suggests that they are expressed at much lowerlevels than is CDS1 [57]. The HMT-2 gene product is predictedto be organelle targeted.

A survey of the Chlamydomonas genome by BLAST searchidentified over a dozen transporters of the “ZIP” family (Table 2,our unpublished results, and [57]) that are predicted to be plasmamembrane or vacuolar membrane localized. Experimental workis necessary to distinguish their subcellular location, pattern ofexpression, metal selectivity, affinity, and role in iron vs. zinc (orother divalent cation) assimilation or distribution. Two Nramp-like proteins, Nramp1 and Nramp2, were also identified in theChlamydomonas genome.5 The expression pattern of one ofthese, named DMT1, was tested to assess a role in metal homeo-stasis but no metal-responsive pattern of expression was noted[75]. When expressed in S. cerevisiae, the protein could restoregrowth to smf1 mutants (indicative of manganese transportcapability). The resulting strain also showed slightly increasedsensitivity to a number of divalent cations, including iron, butnot zinc. Nevertheless, the differences are slight and the questionof Nramp1/2 function with respect to assimilation vs. distribu-tion and metal selectivity is open.

2.1.2. Metabolic adaptationIn a nutrient deficient environment, the activation of assimi-

lation pathways is logically a first line of defense. Indeed, FOX1and FTR1 expression is increased substantially (and evenmaximally for FOX1) when the iron content of the medium isreduced from 18 to 1 μM, but iron-deficiency symptoms (suchas chlorosis and inhibition of cell division) are not evident untilthe iron content is reduced to sub-micromolar concentrations, atwhich point iron supply is insufficient for the maintenance of alliron-containing enzymes [34]. Given the central role of iron inessential metabolic pathways, there must be adaptive responsesthat affect intracellular iron utilization in addition to thoseimpacting assimilation. The best-documented metabolic adap-tation is the replacement of an abundant FeS protein, ferredoxin,with an iron-independent flavodoxin in cyanobacteria in iron-

5 A third NRAMP homolog has been re-named Ret1 (for related to EIN2)because a revised gene model shows sequence relationship to EIN2 ofArabidopsis.

deficient laboratory medium and, more importantly, in an iron-deficient marine environment also [76,77]. In cyanobacteria,this is accomplished by transcriptional responses mediated bythe well-known independent Fur repressor [78]. With the appli-cation of microarray methodologies, it is now becoming clearthat metabolic adaptations probably occur in all organisms (e.g.,[79,80]). Eukaryotic photosynthetic organisms have two majoriron-utilizing pathways, photosynthesis and respiration, that areseparated in two distinct organelles, chloroplasts and mito-chondria. Compromised function of the redox enzymes in thesepathways has the potential to generate reactive oxygen speciesand cause oxidative damage. Thus, mechanisms for anticipa-ting this danger or dealing with the oxidative stress might beinduced.

It is also likely that marginal iron-deficiency could activateshort-term adaptive responses while sustained and severe defi-ciency would activate long-term acclimation pathways. We there-fore defined 3 levels of iron-nutrition in Chlamydomonas: ironreplete, where the iron assimilation pathway is expressed at a lowbasal level and there is more iron in the medium than requiredfor the maintenance of intracellular iron enzymes; iron-deficient,where the iron assimilation pathways are turned on and the ironsupplied in the medium is just enough or slightly less than theamount required to saturate the demand for biosynthesis of iron-containing proteins; and iron-limited, where the symptoms of pooriron nutrition are visually evident as chlorosis and there is animpact on cell division [81].

2.1.2.1. Photosystem I. The photosynthetic apparatus usesredox activemetal centers and chlorophyll pigments to generate anextraordinarily strong oxidant (Em ∼ +1.1 V) in Photosystem II(PSII) that can be reduced by the weak reductant water and apowerful reductant in Photosystem I (Em∼−1.0V) that can reduceferredoxin for biosynthetic metabolism [3]. PSII, composed ofabout 2 dozen different polypeptides, houses a Mn4Ca cluster andis a target of manganese-deficiency. Photosystem I (PSI) is madeup of over a dozen different polypeptides and three Fe4S4 centersas electron acceptors. These account for about half the iron in thephotosynthetic apparatus, and accordingly PSI is a prime target ofiron deficiency. When the function of PSI acceptors is compro-mised, the highly reducing species generated by photochemistry inPSI can reduce oxygen to superoxide (Em,7 ∼ − 0.3 V) in the so-called Mehler reaction. Mutants with defects in PSI thereforeexperience photo-oxidative stress and are light sensitive underaerobic growth conditions [82,83]. The existence of protectivemechanisms is critical for surviving iron-deficiency in nature.

Chlamydomonas displays progressive modification of PSI ascells adapt from an iron replete to iron-deficient to iron-limitedstate [81]. PSI (which houses the cofactors involved in electrontransfer plus antenna chlorophylls that collect the light) isphysically and functionally associated with light harvestingprotein complexes (called LHCI). LHCI consists of individualsubunits Lhca1, Lhca2, Lhca3 and Lhca4 that are chlorophyllbinding proteins. Energy migration from LHCI to PSI occursthrough specific paths/routes in the PSI–LHCI super-complexthat depend on particular subunit–subunit interactions [84]. Inan 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 boundto Lhca subunits migrates effectively to the special pigments inPSI where photochemistry occurs. In the iron-deficient state, thestructure of PSI is modified so that part of the LHCI pigmentsare no longer effectively connected. In particular, a subunitcalled PsaK (which is known to function as an LHCI–PSI con-nector) is dissociated from the PSI complex, resulting in reducedenergy input and hence reduced electron flow through this en-zyme. Thismodification is viewed as a pre-emptivemove to avoidphoto-oxidative stress from damaged/lost FeS clusters.

Under iron limitation, Chlamydomonas cells initiate sequen-tial degradation of the LHCI subunits and both photosystems[81]. Interestingly, the light harvesting chlorophyll proteincomplex LHCII is retained, although with some post-translation-al modifications of particular subunits. The LHCII in the iron-limited cell accounts for most of the chlorophyll remaining in theiron-limited cell, and this chlorophyll–protein complex is viewedas a reservoir of chlorophyll when de novo synthesis of newphotosystems and LHCI subunits is re-initiated by iron supply.

The signal transduction pathways for these responses arecompletely unknown, but we can deduce that they directly senseiron nutrition rather than damaged PSI because transfer to irondeficient conditions can save PSI-defective mutants that arelight sensitive in iron replete medium [81]. Mixing experimentsindicate also that soluble extracts from iron-deficient cells willdegrade thylakoid membrane proteins isolated from iron-repletecells while extracts from iron-replete cells will not degradethylakoid membrane proteins isolated from iron-deficient cells,again indicating that the signal is iron nutrition rather thandamage to the photosynthetic apparatus.

2.1.2.2. Ferredoxins. Ferredoxin is probably the most abun-dant iron protein in soluble extracts of iron-replete Chlamydo-monas cells. This iron–sulfur protein accepts electrons from PSIand uses these electrons for almost all the reductive biosyntheticreactions in the chloroplast, including generation of NADPH forthe Calvin cycle, nitrogen and sulfur metabolism, thioredoxin-dependent regulation, fatty acid desaturation, and others [85]. Thereplacement of ferredoxin by flavodoxin in the marine environ-ment (cited above) is therefore a logical means of conserving iron.The genome of Chlamydomonas appears not to encode a flavo-doxin, but instead encodes six different ferredoxins, one of whichcorresponds to the molecule purified from laboratory grown ironreplete cells [86]. Given the central role of ferredoxin as a reduc-tant for biosynthetic metabolism, it is tempting to speculate thatthe changes in the ferredoxin profile as a function of iron nutritionmay be a means of prioritized electron distribution to metabolicpathways. Indeed, the expression of a novel ferredoxin has beenobserved 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 plantand is a source of iron for chloroplast development or duringnodulation for the synthesis of leghemoglobin and respiratorycomponents in the bacteroid [87]. Plant ferritin, which consistsof a single subunit, is localized to the plastid. The correspondinggenes encode pre-proteins with N-terminal targeting sequences.

The accumulation of ferritin is determined at transcriptional andpost-transcriptional levels by multiple signals including ironnutrition status, physiological iron demand depending on deve-lopmental status, and the potential for toxicity under conditionsof oxidative stress [5,88]. For a full picture of ferritin regulation,it is important to measure the RNA (which gives a picture of thepotential for ferritin action), protein (as a measure of the ironchelation capacity) and the amount of iron in the core (forthe actual level of mineralized iron). When soybean ferritin wasover-expressed in tobacco plants, the transgenic over-expres-sing plants displayed symptoms of deficiency even thoughtotal iron content in the plant was increased [89]. This findingemphasized the role of plastid ferritin as a component of ironhomeostasis.

In Chlamydomonas, FER1 gene expression is counter-intui-tively increased in iron-deficient/iron-limited cells relative toiron-replete cells [34]. One possibility is that the increase isimportant to chelate the iron that is released from the prog-rammed degradation of PSI and ferredoxin in iron-limited cells(discussed above). This idea is precedented by the finding thatferritin expression is increased also in senescing leaves engagedin programmed destruction of the photosynthetic apparatus [90].It may also be important to increase the iron chelating potentialof the chloroplast in an iron-limited situation because of thepotential for generating reactive oxygen species in a membranewith 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 andsuperoxide, the only defense mechanism to combat hydroxylradical is removal of reactive iron. A second ferritin-encodinggene – FER2 – was found in the Chlamydomonas genome, butthe gene model may be incomplete because of a gap in thepresent version of the sequence.

2.1.2.4. Mn-superoxide dismutase. Plants contain 3 types ofsuperoxide dismutase, CuZnSOD in the cytosol, plastid andperoxisome, MnSOD in the mitochondria and FeSOD in theplastid [91]. While Arabidopsis encodes only a single MnSOD,Chlamydomonas has five candidate MSD genes. In E. coli andcyanobacteria, the expression of the manganese- vs. iron-contai-ning SODs is dependent upon the iron nutrition status through theFur regulator (reviewed by [92,93]). Given the connection be-tween iron-deficiency and photo-oxidative stress in Chlamydo-monas, it is possible that one of theMSD genes serves to replaceFSD1 function in an iron-deficient situation.

2.2. Copper

Three abundant copper enzymes are known in Chlamydo-monas — cytochrome oxidase in the respiratory chain in themitochondrion, plastocyanin in the photosynthetic electrontransfer chain in the chloroplast, and the previously mentionedmulticopper oxidase involved in iron assimilation. Accordingly,copper is an essential nutrient for Chlamydomonas. In addition,there are other copper enzymeswhose contribution to the cellularcopper requirement (at least of vegetatively grown laboratorycultures) is likely to be less significant based on representation in

Fig. 4. Copper-independent phototrophic growth.Wild type (strain CC125) cangrow photosynthetically in copper-deficient growth medium (top). The pcy1mutant (strain AP6, pcy1-1 arg7) cannot grow photosynthetically because itlacks the copper protein plastocyanin that functions in photosynthetic electrontransfer (bottom). However, in copper-deficient medium, the strain does notexhibit 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 CYC6expression is regulated by copper nutrition [112].


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the EST database. Among these are matrix metalloproteinaseswith an atypical QExxH site that shows 10-fold in vitro selec-tivity for copper over zinc in terms of activity, amine oxidases(peroxisomal with PTS1 targettingmotifs, amiloride-sensitive andplant like), blue copper proteins, ascorbate-dependent monoox-ygenases, and various multicopper oxidases, including Fox2 [94–97]. The specific function of these enzymes is not known and it islikely that the copper-requirement of aChlamydomonas cell mightbe greater in situations where one or more of these enzymes ismore highly expressed.

2.2.1. Copper uptakeThe standard Chlamydomonas medium contains 6 μMcupric

ions complexed with EDTA, and this is considered to be a repletemedium, since 500 nM chelated copper is sufficient to supportthe synthesis of plastocyanin (accounting for at least half thecopper in a photosynthetic cell) at its usual stoichiometry (∼2per PSI) in the photosynthetic apparatus [98,99]. Leaving out thecopper sulfate in Hutner's trace element solution reduces thecopper content of the medium to the point of deficiency (withrespect to plastocyanin synthesis), which makes it possible tomonitor copper-nutrition dependent pathways of copper utiliza-tion [11,100]. For reproducible results, it is critical that (1) thepurity of the salts used for preparation ofmedia bemonitored, (2)the glassware be acid washed immediately prior to use, and 3)the water be purified [101]. With care, the concentration ofcopper in the medium can be reduced to <3 nM.

Since copper is a nutrient that is toxic in excess, organismsregulate assimilation to avoid toxicity. Measurement of copperuptake in copper-deficient vs. copper-replete Chlamydomonascells indicated about 20-fold greater activity in the formerpopulation of cells [102]. The increased activity was attributedto a Vmax effect with a Km of about 2 × 102 nM calculated forboth samples of cells, suggesting that the same transporter(s)operate(s) in replete and deficient conditions but the numberof transporter molecules is greater in a copper-deficient cell.Cupric reductase activity was also increased (2-fold) in a cop-per-deficient cell suggesting that cupric ions were reduced tocuprous prior to transport of Cu(I) ions. In earlier work, we hadnoted that copper-deficient cells were more sensitive to Ag(I)toxicity relative to copper-replete cells and this was attributed tothe increased capacity for Ag(I) uptake in the former owing toincreased expression of copper uptake components [103]. Takentogether, a pathway for copper uptake consisting of a cell surfacereductase and cuprous transporter was proposed, analogous tothe system operating in other organisms [104,105]. Whether thereductase is copper-specific or a broad substrate specificityferrireductase (Table 2) remains to be determined [106]. Sincethe effect of copper nutrition on silver toxicity as well as on thesynthesis of a silver-stress-induced peptide depended on the timeof exposure to copper, we concluded that increased enzymesynthesis contributed to regulation of the copper assimilationpathway in Chlamydomonas [103].

Examination of the draft genome reveals three coppertransporters of the COPT1/CTR family that are selective for Cu(I) ion transport [107] (Table 2). It should be possible to distin-guish whether one or more of these is a target of the nutritional

copper sensor, Crr1 (see below), which would make it a goodcandidate for an assimilatory transporter.

2.2.2. Back up enzymes

2.2.2.1. CYC6. Plastocyanin, a blue copper protein, is an es-sential component of the photosynthetic apparatus owing to itsfunction in the transfer of electrons from the cytochrome b6 fcomplex to PSI [3]. The importance of plastocyanin in photosyn-thesis was deduced from the phenotype of a plastocyanin-defi-cient Chlamydomonas strain that resulted from a frame-shiftmutation at the PCY1 locus encoding the polypeptide [108–110]. Yet, copper-deficient Chlamydomonas strains that containno immunodetectable or spectroscopically detectable plastocy-anin [101,111] can grow photoautotrophically (Fig. 4, bottom).The answer to this conundrum became evident with the dis-covery of Cyt c6 as a copper-independent/heme-containingsubstitute for plastocyanin in photosynthetic electron transfer[111,112]. The CYC6 gene encoding this “back up” protein isregulated by copper nutrition: Cyt c6 is not present in a copperreplete cell and the pcy1 mutant strain shows a photosynthesis-minus phenotype, but in copper-deficiency CYC6 is transcrip-tionally activated and the back up, Cyt c6, compensates for theloss of plastocyanin function [113]. The equivalence of Cyt c6was further established in suppressor strains of pcy1-ac208where presumed loss of function of an assimilatory copper


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transporter allowed Cyt c6 synthesis in copper replete medium[112]. Comparative biochemistry of the two proteins from avariety of cyanobacteria shows that the isoelectric points of thetwo proteins appear to co-vary within a wide range from 4 and 9,indicating co-evolution, which is consistent with the proposedparallel function of the two proteins [114].

The replacement of plastocyanin with Cyt c6 occurs in manyalgae and cyanobacteria, suggesting that these organisms doexperience copper-deficiency in nature and execute the switch inorder to conserve copper [115]. As for Chlamydomonas, the petJgene encoding cyanobacterial Cyt c6 is also transcriptionallyregulated [116,117]. Plastocyanin abundance is decreased incopper-deficiency but the mechanism of down-regulation ofplastocyanin varies. In Chlamydomonas, the protein is degradedby copper-deficiency-induced proteolysis whereas in Scenedes-mus obliquus PCY1 mRNA abundance is copper-responsive[110,118]. In Synechocystis sp. 6803 and Anabaena, the petEgene encoding cyanobacterial plastocyanin also responds tocopper at the level of mRNA abundance probably bymechanismsoperating through transcription initiation [116,119,120].

2.2.2.2. CRD2. Amulticopper oxidase is presumed to functionin a high affinity iron assimilation pathway in Chlamydomonasbased on its pattern of expression in iron-deficient cells and its co-regulationwithFTR1 andATX1 (see above); yet, copper-deficientChlamydomonas cells do not show symptoms of iron-deficiency(Fig. 4, top) as do yeast and mammalian cells [121]. Even underiron-limiting conditions, copper-deficiency does not exacerbatethe iron-deficiency phenotype, suggesting that another (copper-independent) iron uptake pathway must operate in this situation[34]. This idea was solidified with the isolation of a Chlamydo-monas mutant crd2-1 that showed a copper-deficiency-condi-tional requirement for high iron nutrition [73]. The crd2-1 strain isindistinguishable from wild-type when it is grown in standardcopper replete medium (6 μM chelated copper) except that theaccumulation of FOX1, FTR1 and FEA1mRNAsmay be slightlyincreased, but when copper is eliminated from the medium, thestrain is distinctly chlorotic and growth-inhibited even in standardiron-replete (18 μM iron) medium. The FOX1, FTR1 and FEA1mRNAs show progressively greater expression as the coppercontent of the medium is decreased, indicative of internal irondeficiency in response to poor copper nutrition. The copper-nu-trition conditional iron-deficiency phenotype of crd2 can berescued by increasing the iron content of the medium.

The product of the CRD2 locus is not yet known. It is possiblethat it encodes a copper-independent ferroxidase that could partnerwith Ftr1 or it might encode a component of a completely inde-pendent high affinity iron transporter. Iron regulation is very tightlyregulated owing to the toxicity of excess iron. Therefore, CRD2should be expressed only when its function is necessary – that is,under conditions of copper-deficiency – and this could be ex-ploited for the identification of Crd2 (and associated components).Another possibility is that CRD2 encodes a system for prioritizedallocation of copper to the ferroxidase (discussed in [73]).

2.2.2.3. AOX. Plants and fungi express an enzyme called alter-native oxidase that functions as a terminal oxidase in a situation

when the ubiquinone pool is over-reduced [122]. When cyto-chrome oxidase function is compromised in heterotrophically-grown copper-deficient Chlamydomonas cells, the abundance ofalternative oxidase increases 2-fold [123]. Chlamydomonas hastwo genes AOX1 and AOX2 encoding enzyme isoforms and it isnot known whether one or both forms is/are increased in copper-deficiency. Nor is it known whether the increase responds directlyto copper nutrition or to the level of oxidation of the ubiquinolpool. Alternative oxidase catalyzes the direct oxidation of ubi-quinol by molecular oxygen (bypassing complexes III and IV inrespiration) without concomitant proton translocation; hence it isnot an energy-conserving pathway. Thus, there is no back-up forcopper function in respiration as there is for photosynthesis andiron assimilation.

2.2.3. Metabolic adaptationThe increased expression of copper assimilation components

and back up enzymes has a physiological rationale. Three otherChlamydomonas genes also show changed expression in copperdeficiency but the underlying metabolic rationale is obscure.

2.2.3.1. CPX1. A 35-kDa soluble protein was purified fromChlamydomonas cells as a protein of interest for understandingnutritional copper deficiency because it showed increased accu-mulation in copper-deficient vs. copper-replete medium [124].Sequence analysis of internal tryptic peptides identified theprotein as coproporphyrinogen III oxidase. The correspondinggene is transcriptionally activated in copper-deficiency andsoluble extracts of copper-deficient Chlamydomonas cells displaymore coproporphyrinogen oxidase activity relative to extractsfrom copper-replete cells. The protein, which shows greatestsequence relationship to plant coproporphyrinogen III oxidase, istargeted to the chloroplast where the tetrapyrrole biosyntheticpathway is localized [125]. The Chlamydomonas genome en-codes a second isoform, named Cpx2, which is more closelyrelated to the animal mitochondrial enzyme. This gene is notregulated by copper nutrition at least at the level of mRNAaccumulation (J.K, M.D. Page and S.M., unpublished). The sub-cellular location of Cpx2 is not known. In the tetrapyrrolepathway, the enzyme coproporphyrinogen oxidase is known to berate limiting after the synthesis of δ-aminolevulinic acid. Perhapstetrapyrrole metabolism is compromised or there is an increaseddemand for tetrapyrrole precursors in copper-deficient cells. Weknow that copper-deficient cells do synthesize an abundant hemeprotein (Cyt c6), but this would constitute a small draw from thetetrapyrrole 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 (seebelow).

2.2.3.2. CRD1/CTH1. Two other genes that show differentialexpression in copper-deficient Chlamydomonas cells, CRD1(renamed CHL27A) and CTH1 (renamed CHL27B) wereidentified genetically [126,127]. They encode isoforms ofanother enzyme in the tetrapyrrole pathway: the aerobic oxi-dative cyclase. This enzyme uses a di-iron active site to catalyze


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the oxygen-dependent formation of Ring V in chlorophyll fromthe methyl-esterified propionate side chain of Ring III [128]. InArabidopsis, the protein (encoded by a single copy geneCHL27)is localized to both the inner envelope membrane of the chloro-plast as well as the thylakoid membrane. The distribution of thetwo isoforms of the Chlamydomonas enzyme is not known, butit is possible that they may be differently localized. Their patternof expression is reciprocal as a function of copper nutrition oroxygen supply [127]. Crd1/Chl27A predominates in copper-deficient or hypoxic cells while Cth1/Chl27B predominates incopper-replete or well-oxygenated cells. Loss-of-function crd1mutants show copper-nutrition-conditional loss of a subset ofchlorophyll proteins and are pale green or chlorotic. AlthoughCth1/Chl27B regulation is normal in these mutants, the amountof total enzyme (Crd1 + Cth1) at this step must clearly be rate-limiting for the pathway in copper-starved cells.

Interestingly, crd1 mutants show progressive loss of partic-ular chlorophyll proteins depending on the severity of copper-deficiency, metabolic physiology (phototrophic vs. heterotro-phic growth), the nature of the mutation or penetrance of theallele [126]. Photosystem I and its associated light harvestingproteins, LHCIs, are essentially completely absent while LHCIIproteins are reduced in abundance. This suggests that there maybe some level of metabolic compartmentation/channeling in thetetrapyrrole pathway with the two isoforms directing precursorsto distinct chlorophyll-proteins. The alternate hypothesis ofhierarchical chlorophyll allocation to photosystems and acces-sory antennae is not favoured because other mutants with com-parable reduction in total chlorophyll do not display the hierarchy.It is worth noting that the cyanobacteria, which also switch be-tween plastocyanin and Cyt c6 based on copper nutrition, encodemultiple isoforms of CHL27. The pattern of expression of CRD1/CHL27A and CTH1/CHL27B in hypoxia has also not beenrationalized, but hypoxic crd1 strains show the same chloroticphenotype with loss of PSI and LHCI as do the copper-deficientcrd1 strains, which argues that CRD1 expression and function inhypoxic cells is physiologically relevant. A metabolite profile oftetrapyrrole precursors as a function of iron and copper nutrition inwild-type vs. mutant Chlamydomonas strainsmay be informative.

Coprangen oxidase and Chl27 catalyze rate-limiting, oxy-gen-dependent steps in the tetrapyrrole pathway. Substantialresearch effort is dedicated to the understanding of regulatorymechanisms in this pathway but the impact of micronutrients hasnot been examined systematically [129,130]. The discovery ofcopper-deficiency regulated expression of key enzymes in hemeand chlorophyll biosynthesis may be hinting at an additionallayer of regulatory complexity in this ancient pathway.

2.2.4. Intracellular copper distributionThe prototypical pathway for copper–protein assembly

suggests that proteins that are processed through the secretorypathway (e.g., extracellular and plasma membrane enzymes) areloaded with copper in a compartment in that pathway beforethey reach their final destination, while the mitochondrial andchloroplast enzymes are loaded with copper at their site ofaction (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 copperchaperone Atx1 (also called Atox1/Hah1 in animals or CCH inplants). Atx1 transfers copper to a copper-transporting P-typeATPase in the secretory pathway (Ccc2p in S. cerevisiae, RAN1in plants and ATP7A/B in animals). The energy of ATP hy-drolysis is used to concentrate copper in a vesicular com-partment for loading the active sites of plasma membrane orsecreted proteins like ceruloplasmin, hephaestin, or the ethylenereceptor. Analogous copper-transporting P-type ATPases,PAA1 and PAA2, function at the chloroplast envelope andthylakoid membranes for delivery of copper to the stroma wherechloroplast CuZnSOD is assembled and thylakoid lumen whereplastocyanin is assembled [59]. The copper chaperones Cox17and Cox19 and assembly factors Cox11 and Sco1/2 are requiredfor loading the CuA and CuB sites of cytochrome oxidase [131].Chlamydomonas encodes homologues of each of these intracel-lular copper-distributing molecules [21,55,57,73]. A role forChlamydomonas Atx1 in the secretory pathway has been esta-blished on the basis of pattern of expression in response to ironnutrition and its ability to rescue the corresponding S. cerevisiaemutant [34]. However, the three copper-transporting P-typeATPases, named Ctp1, Ctp2 and Ctp3 (Table 2), need to bedistinguished on the basis of sub-cellular localization and func-tional tests.

The above-mentioned pathways for intracellular copperdistribution are unidirectional. A new area of interest is thequestion of how copper in one compartment can be transferred toa different compartment. This is particularly relevant in thesituation of nutritional copper deficiency when it may be ne-cessary to “recycle” copper from abundant but less physiolo-gically important cuproproteins. We have established thatplastocyanin is actively degraded in copper-deficient Chlamy-domonas [100,110] and suggested that the underlying purposemust be to “save” copper for other important copper enzymessuch as cytochrome oxidase. The question of how copper mightleave the chloroplast has not been addressed yet. The existenceof multiple CTR-type transporters suggests that one or more ofthese could be important for intracellular re-allocation in copperrecycling pathways.

2.2.5. Copper-responsive signal transduction

2.2.5.1. CuREs. The involvement of transcriptional regulationin nutritional copper signaling in Chlamydomonas was esta-blished through reporter gene constructs in which the 5′ flank-ing sequences upstream of the 5′ end of the CYC6 and CPX1transcripts were fused to a reporter gene [113,132]. When testedin the context of a minimal promoter, the 5′ flanking sequencesactivated transcription in copper-deficiency rather than repres-sing transcription in copper replete cells, indicating that theCuREs served as targets for a transcriptional activator. In con-trast, when a constitutive promoter drove synthesis of CYC6mRNA, the mRNA accumulated even in copper-replete cells,indicating that post-transcriptional processes did not play asignificant role in copper-responsive gene expression. This isconsistent with a measured in vivo half-life of 45–60 min forthe CYC6 mRNA independent of the copper content of the


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medium [133]. Promoter dissection revealed that the CYC6promoter houses two CuREs, each of which is sufficient on itsown to confer copper-responsiveness to a reporter gene. TheCPX1 gene is associated with one additional CuRE that isrequired also for the response of the gene to hypoxia. Muta-tional analysis of the CuREs indicated that a core sequence,GTAC, was absolutely critical (but not sufficient) for CuREactivity. There are GTAC sequences upstream of the CRD1 geneas well but these have not been tested functionally.

2.2.5.2. Crr1. A screen for Chlamydomonas mutants thatdisplayed copper-deficiency-conditional phenotypes revealedone regulatory locus, CRR1 [73]. The crr1 mutants are charac-terized by very poor growth in copper-deficient medium. Res-toration of growth requires excess copper in the medium becauseCrr1 is probably required for expression of a copper assimilationpathway. CYC6 is not expressed in copper-deficient crr1 cells,and CPX1 and CRD1/CHL27A are expressed only at basal +Culevels, consistent with loss of function of a transcriptional acti-vator. The crr1 strains also accumulate apoplastocyanin, indi-cating that the gene for the plastocyanin-degrading protease isanother target of Crr1. It is likely that Crr1 is required also forexpression of the copper-independent backup pathway for ironassimilation (defined by the CRD2 locus) because iron-deficiency exacerbates the crr1 phenotype while excess ironprovides relief.

A gene corresponding to CRR1 was cloned by complemen-tation [134]. It encodes a large protein (1232 residues) with a76-residue Zn-dependent DNA binding domain named “SBP”for the founding member of this plant-specific domain [135], aputative nuclear localization signal, ankyrin repeats and a C-terminal Cys-rich region that resembles metallothionein. TheCrr1-SBP shows sequence specific binding to the CuREs in theCYC6 and CPX1 genes: mutations that block CuRE activity invivo in reporter gene constructs also make less effective com-petitors in electrophoretic mobility shift assays of the Crr1-SBPon authentic CuREs [134]. The CuRE in Chlamydomonas isunrelated to CuREs of other organisms, and Crr1 is likewiseunrelated in sequence to other metalloregulatory transcriptionfactors [136]. It is not known yet whether Crr1 is the coppersensor or whether it interacts with the copper sensor (throughthe ankyrin repeats). At present, the former possibility is fa-voured because of the presence of two candidate metal bindingdomains in Crr1— the SBP and the C-terminal Cys-rich region.In vitro and in vivo mutational analysis should distinguish be-tween the different models presented for metal sensing by Crr1[134].

Each of the target genes, CYC6, CPX1 and CRD1, can beartificially activated in vivo in the presence of excess copper inthe medium by nickel and cobalt ions, and this response isdependent on the CuREs and Crr1 [40]. One explanation for thisartificial response is that nickel and cobalt are antagonistic at thecopper-sensing site in this signal transduction pathway. Alterna-tively, the presence of two histidine residues in the SBP do-main may allow it to accommodate various transition metalswith different geometries and hence different gene expressionactivity.

2.2.5.3. Down-regulation of Cth1/Chl27B and plastocyanin incopper-deficiency. Crr1 is required also for down-regulatingthe abundance of Cth1/Chl27B [127]. The mechanism of down-regulation relies on transcriptional activation upstream of theCTH1 locus by Crr1 through putative CuREs. In copper-repletecells, the downstream promoter produces a 2 kb mRNA thatencodes Cth1/Chl27B, but in copper-deficient cells, Crr1 acti-vity produces a 5′ extended 3 kb mRNA with a long leadersequence encoding multiple small ORFs in each frame. At thesame time, promoter occlusion inhibits production of the 2 kbmRNA. Since the 3 kb mRNA cannot be translated (M.A. andS.M. unpublished), the CTH1 locus produces much less proteinin a copper-deficient cell compared to a copper replete cell. Inparallel, the CRD1 locus is transcriptionally activated in copper-deficiency by Crr1 so that more Crd1/Chl27A is synthesized—assuring a perfectly reciprocal pattern of expression.

A reciprocal pattern of expression was noted already forcopper-dependent plastocyanin and Cyt c6 accumulation inChlamydomonas [137]. However, for plastocyanin, pulse-chaseexperiments established that down-regulation occurs by inducedproteolysis [100,110]. Nevertheless, this is still Crr1-dependent(discussed above).We conclude that Crr1 is a master regulator ofnutritional copper homeostasis: it up-regulates one set of genesthrough CuREs associated with those genes and down-regulatesanother set of genes either through the transcriptional activationof specifically-target proteases or through intergenic upstreamtranscriptional activation that represses the production ofdownstream functional mRNAs, thereby assuring perfectlycoordinated reciprocal patterns of expression.

3. Outlook

3.1. Functional analysis of candidate genes

3.1.1. TransportersThe draft genome of Chlamydomonas has revealed a number

of candidate metal transporters of the HMA, MTP and ZIPfamilies whose function in terms of metal selectivity andintracellular location is not yet evident. The dominance oftranscriptional regulation in determining the pattern of expres-sion of assimilatory transporters coupled with the increasedexpression of mitochondrial proteins during the transition fromphototrophic to heterotrophic growth should facilitate functionalassignments of these transporters. Furthermore, in the study of aunicellular eukaryote, complexities arising from specializedphysiology and function of organs or particular stages ofdevelopment are avoided. The technology for “knock-down” ofindividual genes is expected to improve in the next few years asare the development of compartment-specific markers, and thesewould enhance the repertoire of experimental approaches.

3.1.2. MetalloenzymesThe Chlamydomonas genome encodes a number of copper

enzymes like amine oxidases, matrix metalloproteases and multi-copper oxidases that seem to be poorly expressed in laboratorycultures as assessed from the distribution of ESTs. These enzymesare not yet assigned to a particular pathway although the matrix


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metalloproteinases are thought to be important in sexual re-production. It is possible to use real time PCR methods to moni-tor the expression of candidate copper enzymes under variousstress situations or at different stages of the cell cycle or sexualreproduction cycle to assess the importance of these enzymes anddeduce a window for phenotypic assessment of knock-downstrains. This approach should also be powerful for other metallo-enzymes of interest.

3.2. Other nutrients

3.2.1. Mn and SeManganese and Selenium are required at such low levels for

most organisms that it is experimentally challenging to generatestrong phenotypes of deficiency. Chlamydomonas has a highmanganese requirement relative to non-photosynthetic organismsand this makes the imposition of deficiency easier. As noted forother micronutrients, the strength of the deficiency phenotype isdependent on the metabolic requirements of the cell. Phototro-phically grown cells are strictly dependent onmanganese nutritionwhereas heterotrophically grown cells show a milder impact(Fig. 5), but this can be exacerbated in the presence of exogenousperoxides (S.T. and S.M., unpublished).

In contrast to S. cerevisiae and A. thaliana, Chlamydomonasuses selenoenzymes and the repertoire is almost comparable to thatin mammalian models [44]. Although selenium supplementationappears to have only a marginal beneficial effect on Chlamydo-monas, it is possible thatmore dramatic phenotypes could be notedunder situations of photo-oxidative or metal toxicity stress whenintracellular glutathione pools are depleted [18,103].

3.2.2. Variable metal requirementThe relationship between metabolism and metal requirement

is an aspect that needs greater appreciation and consideration inexperimental design. The impact of pH, ionic strength andinorganic anions (like phosphate) also needs to be considered,since bioavailability of metal micronutrients is dependent onchemical speciation. In this respect, a microorganism that can bemaintained in a chemostat offers a unique experimental oppor-

Fig. 5. Manganese-dependent photosynthetic growth. Wild type strain CC425 wassupplemented with no (dots), 0.1 (dash) or 1 (solid) μM chelated manganese salts. G

tunity. In particular, the fact that Chlamydomonas does not requireorganic nutrient supplementation might simplify the design ofexperiments aimed at dissecting the complex interactions betweenmetal nutrients.

3.3. Special aspects

3.3.1. Chloroplast vs. mitochondriaThe chloroplast and mitochondria are rich in metalloproteins

and the question of how metal allocation to one organelle vs. theother is prioritized has not been addressed. The availability ofmutants affected specifically in photosynthesis (acetate-requir-ing strains) or respiration (dark-diers) and the possibility togrow wild-type cells either phototrophically, heterotrophicallyor mixotrophically makes Chlamydomonas a unique system foraddressing these questions [26,138]. The possibility to comparemetabolite profiles as a function of metal and carbon-sourcenutrition is value-added [139].

3.3.2. Phytoremediation and oxidative stressThere is historical interest on the capacity and mechanisms of

Chlamydomonas spp. to handle toxic metals like cadmium, leadand mercury [71,103,140–143]. While a pathway for glutathioneand phytochelatin-dependent detoxification is evident, there is noindication that metallothionein-like molecules exist in the Chla-mydomonas genome. But it should be noted that the gene modelsare generally biased against small proteins with low complexitysequences that are not represented in the EST database, and theymay well show up when tiling microarrays are applied to thisquestion.

Excess light generates oxidative stress in chloroplasts when theabsorbed light energy is greater than the capacity of metabolism touse the reducing equivalents that are generated. While there issubstantial research on the protective (such as activation of genesin anti-oxidant pathways) and preventative mechanisms (such asmovement away, remodeling of antennae) used by Chlamydo-monas to avoid oxidative injury in this situation, the question ofhow metal homeostasis and re-allocation responds to lightintensity is another unexplored area.

grown either heterotrophically (orange) or phototrophically (black) in mediumrowth was estimated by counting cells in a hemocytometer.


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Acknowledgments

We are grateful to the National Institutes of Health, theDepartment of Agriculture and the Department of Energy forsupporting research projects in the Merchant laboratory, and wethank the Joint Genome Institute for the draft sequence of theChlamydomonas genome. MDA and AMT are supported byInstitutional Kirschstein Fellowships, GM07185 and GM07104,respectively, and STwas supported by the UC Toxic SubstancesResearch and Teaching Program. Several colleagues and co-workers 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 helpand stimulating discussions.

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