Abstract. The green alga Chlamydomonas reinhardtiiDangeard CW-15 exhibited very low rates of plasma-membrane Fe(III) reductase activity when grown underFe-su�cient conditions. After switching the medium toan Fe-free formulation, both ferricyanide reductase andferric chelate reductase activities rapidly increased,reaching a maximum after 3 d under iron-free condi-tions. Both of the Fe(III) reductase activities increased inparallel over time, they exhibited similar Km values(approximately 10 lM) with respect to Fe(III), displayedthe same pH pro®le of activity, and both exhibited thesame degree of light stimulation which could beinhibited by 3-(3¢,4¢-dichlorophenyl)-1,1-dimethylurea(DCMU). Furthermore, ferricyanide competitively in-hibited ferric chelate reduction by iron-limited cells.These results indicate that both Fe(III) reductase activ-ities were mediated by the same iron-limitation-inducedplasma-membrane reductase. No evidence was found forthe presence of Fe(III)-reducing substances in the culturemedium, or for the involvement of active oxygen speciesin the process of Fe(III) reduction. Chlamydomonasreinhardtii appears to respond to iron limitation in amanner similar to Strategy I higher plants.

Key words: Chlamydomonas (Fe limitation) ± Ferricchelate reductase ± Ferricyanide reductase ± Ironlimitation

Introduction

Iron in aerobic soils and waters is found mostly in theform of insoluble Fe(III) oxides, while Fe(II) is typically

not found in high concentrations in the environmentunder aerobic conditions (Lindsay 1979). The Fe(III)oxides are accessed by organisms via an iron-limitation-inducible suite of physiological and biochemical mech-anisms. These mechanisms have been classi®ed into twodistinct strategies for accessing extracellular iron. Dicots,non-graminaceous monocots and yeast, utilize a strategythat involves solubilization of Fe(III) by extracellularacidi®cation, reduction of Fe(III) to Fe(II) by a plasma-membrane redox system, followed by uptake of Fe(II)by a speci®c transporter (Guerinot and Yi 1994). Thismechanism involves the obligatory reduction of extra-cellular ferric chelates, leading to chelate splitting andthe subsequent uptake of the released Fe(II) (Chaneyet al. 1972; RoÈ mheld and Marschner 1983). Gram-inaceous plant species, bacteria, and various fungi,utilize a di�erent strategy that involves the export ofiron-chelating organic compounds (siderophores orphytosiderophores), followed by uptake of the Fe(III)-chelator complex across the plasma membrane. Inplants, these two mechanisms have been designated asStrategy I and Strategy II respectively (Guerinot and Yi1994; Marschner and RoÈ mheld 1994).

The presence of at least two distinct plasma-mem-brane Fe(III)-reducing systems has been postulated toexist for Strategy I plant cells (Bienfait and LuÈ ttge 1988):the constitutive ``standard'' reductase is capable ofreducing extracellular ferricyanide (impermeant non-physiological electron acceptor) at high rates (ferricya-nide reductase activity; FeCN-R), but exhibits only lowrates of ferric chelate reductase activity (FC-R). Ironlimitation has been suggested to increase synthesis and/or activity of the ``turbo'' reductase, capable of reducingboth ferric chelates and ferricyanide (Moog and BruÈ g-gemann 1994). The situation is made more complicatedby the fact that plant plasma membranes typicallydisplay multiple electron transport activities (Jones et al.1987; Luster and Buckhout 1988; Rubinstein and Luster1993; LuÈ thje et al. 1997), and that extracellular, cell wall-associated, peroxidase activity may also be capable ofreducing Fe(III) to Fe(II) (MacrõÁ et al. 1992). Moreover,there is mounting evidence which indicates that plant

Planta (1998) 204: 360±365

Iron limitation results in induction of ferricyanide reductase and ferricchelate reductase activities in Chlamydomonas reinhardtii

Jaret A. Lynnes*, Tina L.M. Derzaph, Harold G. Weger

Department. of Biology, University of Regina, Regina, SK, S4S 0A2, Canada

Received: 24 June 1997 /Accepted: 2 August 1997

*Present address: Biolin Research Inc., Saskatoon, SK, S7N 3R2,Canada

Abbreviations: BPDS = bathophenanthroline disulphonate;DCMU = 3-(3¢,4¢-dichlorophenyl)-1,1-dimethylurea; EDDHA =ethylenediamine di(o-hydroxyphenylacetic acid; FC-R = ferricchelate reductase; FeCN-R = ferricyanide reductase

Correspondence to: H.G. Weger; E-mail: harold.weger@uregina.ca;Fax: 1 (306) 585 4894; Tel.: 1 (306) 585 4479

plasma membranes may possess nitrate reductase activ-ity (Tischner et al. 1989; Ward et al. 1989), an enzymewhich also exhibits the capacity for Fe(III) reduction(Redinbaugh and Campbell 1983).

In terrestrial systems, iron limitation of plant pro-ductivity is most frequently encountered in areas ofcalcareous soils, which further limit the solubility ofFe(III) (Lindsay 1979). Additionally, there is now strongevidence that iron limitation controls algal productivityin some parts of the open ocean (e.g. Martin et al. 1994;Coale et al. 1996). Among algae, the cyanobacteriaappear to utilize siderophores to access extracellulariron, perhaps in a manner similar to Strategy II higherplants (Wilhelm and Trick 1994). The mechanism(s) bywhich eukaryotic algae access extracellular iron is lessclear. Iron-limited cells of the marine diatom Thalassios-ira weis¯ogii reduce Fe(III)-EDTA to Fe(II) (Andersonand Morel 1980), while the marine diatom Phaeodacty-lum tricornutum can utilize both ferrioxamine B andferrioxamine E as iron sources for growth (Soria-Denggand Horstmann 1995). However, in the latter species,evidence indicates that the two tested iron sources wereutilized by di�erent mechanisms; uptake of iron fromferrioxamine B resembles higher-plant strategy I, whileuptake from ferrioxamine E resembles strategy II (Soria-Dengg and Horstmann 1995).

Among the green algae (Chlorophyta), Benderlievand Ivanova (1994) provided evidence that Scenedesmusincrassalatulus utilizes an iron-limitation-inducible Strat-egy II mechanism for uptake of extracellular iron. Ironlimitation resulted in release of siderophores, while thecapacity for uptake of the Fe(III)-siderophore complexappeared to be constitutive. There was no evidence forreductive splitting of extracellular ferric chelates inS. incrassalatulus. Allnutt and Bonner (1984, 1987a,b)provided the ®rst evidence that iron de®ciency results inincreased FC-R activity in a green alga (Chlorellavulgaris), but the possibility of saturation kinetics wasnot investigated. The most e�ciently reduced ferricchelates were those of the bacterial siderophores fer-rioxamine B and ferrirhodotorulate, while ferric citratewas reduced at a much lower rate (Allnutt and Bonner1987a). Conversely, C. reinhardtii is unable to utilizeferrioxamine B as an iron source for growth (Bailey andTaub 1980). An iron-limitation-induced increase in bothFeCN-R and FC-R activities have been demonstratedfor Chlorella pyrenoidosa, but the activities did notexhibit saturation kinetics and increased linearly withsubstrate concentration (Moog and BruÈ ggemann 1994),leading to the suggestion that extracellular ferric chelatemay act simply as a non-speci®c electron trap for plasmamembrane redox systems in that organism. Iron starva-tion also resulted in a 50% increase in FeCN-R activityby the green alga Monoraphidium braunii (Corzo et al.1991), but again, the possibility of saturation kineticswas not investigated.

In this paper we investigate the response of plasma-membrane redox systems of the green alga Chlamydo-monas reinhardtii to iron limitation. We provide evidencethat redox activity greatly increases over several daysafter switching to an iron-free medium. Furthermore, we

provide evidence that only one redox system predomi-nately responds to iron limitation in this organism.

Materials and methods

Cell culture. The cell wall-less mutant Chlamydomonas reinhardtiiDangeard CW-15 was obtained from Dr. R.G. Smith (UniversityCollege of the Cariboo, Kamloops, B.C., Canada). A cell wall-lessstrain of C. reinhardtii was chosen for the experiments in order tominimize possible binding of iron to the cell wall (Hudson andMorel 1989), and to minimize the potential confounding e�ects ofthe wall-associated peroxidase activity (Torkelson et al. 1995).

Cells were grown in semi-continuous culture, in 1.4-L water-jacketted, glass chemostat vessels at a temperature of 20 °C and aphoton ¯uence rate of 200 lmol quanta á m)2 á s)1 (Weger et al.1996). Light was provided by a bank of 12 cool-white, high-output¯uorescent lights (F48T12/CW/HO; Philips Electronics, Scarbor-ough, Ont., Canada). Cultures were aerated with 1% CO2 in airand continuously stirred. The medium was a modi®cation of thatformulated by Hughes (Hughes et al. 1958), and contained 300 lMK2HPO4 and 6 mM NH4Cl; Na2CO3 and NaSiO3 were omitted,and minor elements were added as in Allen (1968). The mediumwas bu�ered at pH 7.5 with 15 mM Hepes-KOH; acidi®cation bythe cells resulted in a ®nal pH of approximately 6.5. In order tomaintain exponential growth, medium was continuously pumpedinto the chemostat vessels, and cells were drained from the vesselsonce per day. The dilution rate was 1.3 d)1.

Fe-su�cient cells were generated by including iron in themedium. The iron stock contained Na2-EDTA (0.4 g á L)1), citricacid (4.8 g á L)1) and FeSO4 á 7H2O (2.4 g á L)1); 2.5 mL was usedper litre of medium, to provide a ®nal iron concentration of21.6 lM. Iron-limited cells were generated by switching theincoming medium to an iron-free modi®ed Hughes formulation(identical to that described above, except minus iron). Afterswitching to iron-free medium, the dilution rate was maintainedat 1.3 á d)1.

Fe(III)-reduction experiments. Cells were harvested by centrifu-gation (2000 g for 4 min) and resuspended in bu�er (15 mMHepes-KOH, pH 6.5). For determining the e�ects of extracellularpH on the rate of Fe(III) reduction, two bu�ers were used:15 mM Mes-KOH for pH 5.0±6.5, and 15 mM Hepes-KOH forpH 6.5±8.0. Resuspended cells were placed in water-jackettedreaction vessels (20 °C) in the dark, with gentle stirring andaeration.

For measurement of ferricyanide reductase (FeCN-R) activity,K-ferricyanide (100 mM stock solution) was added to the resus-pended cells. Subsamples of 800 lL were harvested by rapidcentrifugation (19 000 g) every 4 min, and the supernatant wasused for the spectrophotometric determination of ferricyanidereduction (A420±A650; e420 = 1.0 á mM)1). The FeCN-R activitywas calculated by linear regression over a 28-min time course.

Ferric chelate reductase (FC-R) activity was determined usingFe(III)-EDTA (added from a 100 mM stock solution) or Fe(III)-EDDHA (added from a 25 mM stock solution), and 500 lMbathophenanthroline disulphonate (BPDS) to trap and quantify theFe(II) produced from Fe(III) reduction. Concentrations of Fe(II)-BPDS3 were determined spectrophotometrically (A536±A750).Molar absorptivities (e536) for Fe(II)-BPDS3 were a�ected by thenature of the iron chelator (EDTA versus EDDHA), and weredetermined at each of the examined pH values. When using Fe(III)-EDTA, for the range pH 5.0±6.5, a e536 of 21 221 á M)1 á cm)1 wasemployed. At pH 7.0, e536 was determined to be 19 948 á M)1 ácm)1, while at pH 8.0 e536 was 11 035 á M)1 á cm)1. For Fe(III)-EDDHA, e536 was independent of pH, and was determined to be21 201 á M)1 á cm)1. The increase in absorbance (A536±A750) withincreasing Fe(II)-BPDS3 concentrations was linear to approximate-ly 100 lM. The FC-R activity was calculated by linear regressionover a 28 min course.

J.A. Lynnes et al.: Iron limitation in Chlamydomonas 361

Quanti®cation of total extracellular iron [Fe(II) and Fe(III)] inthe cell suspension was accomplished by adding 10 lL of asaturated Na-dithionite solution (thereby reducing the remainingFe(III) to Fe(II)) to a 1.0-mL sample containing 500 lM BPDS.Preliminary experiments indicated that the size of the totalextracellular iron pool did not measurably decrease over the courseof the experiments, indicating that cellular uptake of iron wasminimal in the presence of BPDS.

Competitive inhibition of Fe(III) reduction between ferricya-nide and Fe(III)-EDTA was examined using Fe-limited cellsresuspended in 15 mM Hepes-KOH (pH 6.5). Potassium-ferricya-nide was added at a concentration of either 0 or 250 lM, andFe(III)-EDTA was added at 0, 25, 50 or 100 lM. Bathophenan-throline disulfonate was added at a concentration of 500 lM. TheFe(II)-BPDS3 concentration was quanti®ed spectrophotometricallyas described above (A536±A750). Ferricyanide concentrations werealso determined spectrophotometrically, using empirically derivedsimultaneous equations to correct for the absorbance of the Fe(II)-BPDS3 complex at 420 nm.

Competition between ferricyanide and BPDS for Fe(II). In order toassess the ability of BPDS to chelate and stabilize Fe(II), and thusprevent the Fe(II)-dependent reduction of ferricyanide to ferrocy-anide, cell-free experiments were carried out in 15 mM Hepes-KOH (pH 6.5). The BPDS was added to the bu�er at a ®nalconcentration of 500 lM, and ferricyanide was added at either 100or 250 lM. Subsequently, 50 or 100 lM of freshly dissolvedFe(II)SO4 (chelator-free) was added to the bu�er, and the relativeconcentrations of ferricyanide and Fe(II)-BPDS3 were determinedspectrophotometrically (as described above).

Other methods. Oxygen consumption was measured using anoxygen electrode (Hansatech, King's Lynn, UK), using cells thathad been harvested by centrifugation and resuspended in 15 mMHepes-KOH (pH 6.5). Cell density was determined using a hema-cytometer. Chlorophyll was quanti®ed spectrophotometrically afterextraction in 100% methanol (Porra et al. 1989). All spectropho-tometric work was carried out using a diode array spectrophotom-eter (HP8452A; Hewlett-Packard, Palo Alto, Calif., USA).Anaerobic conditions were achieved by a combination of bubblingthe cell suspension with N2 gas, and addition of 10 mM glucose,100 lg á mL)1 glucose oxidase and 20 lg á mL)1 catalase. TheFe(III)-EDDHA was prepared by dissolving EDDHA in a fourfoldmolar excess of KOH, followed by addition of an equimolaramount of FeCl3 (Chaney and Bell 1987). Glucose oxidase wasfrom Boehringer Mannheim Canada (Montreal, Quebec, Canada),and Hepes was from Research Organics (Cleveland, Ohio, USA).All other reagents and enzymes were from Sigma Chemical Co.(St. Louis, Mo., USA).

Results and discussion

Switching the culture to iron-free medium, while main-taining the dilution rate at 1.3 á d)1, did not result indetectable changes in cell density or chlorophyll contentfor the ®rst day (Fig. 1A). At day 2, chlorophyll levelsbegan to decrease, while cell density declined beginningat day 4 (Fig. 1A). However, plasma-membrane ironreductase activity (both FeCN-R and FC-R) began toincrease by day 1 in iron-free medium, and reached amaximum by day 3 of iron limitation (Fig. 1B). Rates ofboth FeCN-R and FC-R changed in parallel (Fig. 1B),indicating that they may represent the same activity orenzyme.

Both reductases also exhibited similar kinetics withrespect to substrate concentration, with the Km forFe(III) reduction at approximately 10 lM (Fig. 2). As

well, both FeCN-R and FC-R activities were insensitiveto pH over the range 5±8 (Fig. 3), further indicating thatthey represent the same enzyme. The chelator EDDHAwas tested in addition to EDTA for the pH pro®le ofreductase activity (Fig. 3) because the stability of themetal-EDDHA complex is much less a�ected by highpH than is the stability of the metal-EDTA complex

Fig. 1A,B. Changes in cell culture characteristics (A) and ferricreductase activities (B) ofC. reinhardtii as a result of switching to iron-free medium (at day 0). A Cell density (s) and chlorophyll content(d). Data represent the means from two separate experiments (threedeterminations per experiment). The SE bars are smaller than thesymbols. B FeCN-R activity (s) and FC-R activity (d). Datarepresent the means from two separate experiments (three determi-nations per experiment). Bars represent SE (n = 6)

Fig. 2. Rate of Fe(III) reduction by iron-limitedC. reinhardtii cells asa function of substrate concentration: FeCN-R activity (s) and FC-Ractivity (d). Bars represent SE (n = 6)

362 J.A. Lynnes et al.: Iron limitation in Chlamydomonas

(Lindsay 1979). Both Fe(III)-EDTA and Fe(III)-ED-DHA provided similar results (Fig. 3).

Attempts to extend the pH range below 5.0 resultedin cell lysis of this wall-less C. reinhardtii strain; lysis wasrapid at pH 3.0, and somewhat slower at pH 4.0.Resuspension of the cells in pH 9.0 bu�er also resultedin slow cell lysis.

The Km value for Fe(III) EDTA reduction (approx.10 lM, Fig. 3) by C. reinhardtii, as well as thepH pro®les for FC-R activity (Fig. 3), are di�erent thanhave been commonly reported for iron-limited higher-plant tissue. Reported Km values for higher-plant FC-Ractivity range from 45 to 800 lM at optimum pH (e.g.Schmidt and Janiesch 1991; Moog et al. 1995), and thereis usually a clearly optimum pH, that is typically lessthan 6.5 (e.g. RoÈ mheld and Marschner 1983; Schmidtand Janiesch 1991).

Further evidence that FC-R and FeCN-R activitiesrepresent the same reductase was obtained from com-petitive inhibition experiments (Fig. 4). Similar to resultsfrom experiments with iron-limited cells of the bacteri-um Vibrio anguillarum (Mazoy and Lemos 1996),ferricyanide was preferentially reduced by iron-limitedcells compared with ferric chelate. Addition of 250 lMferricyanide prevented the reduction of Fe(III)-EDTAuntil most of the ferricyanide had been reduced (Fig. 4).While we were unable to discern a di�erence in the Km

values for FeCN-R and FC-R activities (Fig. 2), the factthat Vmax for FeCN-R activity is clearly slightly higherthan for FC-R activity (Fig. 1B) may explain thepreferential reduction of ferricyanide versus Fe(III)-EDTA.

We also addressed the possibility that the apparentpreference for ferricyanide reduction compared withreduction of ferric chelate (Fig. 4) was due to non-enzymatic reduction of ferricyanide by Fe(II) producedvia FC-R activity, before the Fe(II) could be complexedby BPDS (it is well-established that non-chelated Fe(II)

has the ability to donate an electron to ferricyanide,thereby producing ferrocyanide). The data shown inFig. 5 indicate that addition of non-chelated Fe(II) tobu�er containing BPDS and ferricyanide will result inthe formation of both Fe(II)-BPDS3 (Fig. 5) and ferro-cyanide (not shown), but that a greater proportion of

Fig. 3. The e�ect of pH on FeCN-R activity (s, d) and FC-Ractivity. The FC-R activity was measured using Fe(III)-EDTA (,, .)and Fe(III)-EDDHA (h, j). Assays were performed in 15 mMMes-KOH (open symbols) or in 15 mM Hepes-KOH (solid symbols). Ratesare expressed as the percent of the control rate measured in 15 mMHepes-KOH, pH 6.5. Bars represent SE (n = 6)

Fig. 4A,B. Inhibition of Fe(III)-EDTA reduction by ferricyanide.A Reduction of various concentrations of added Fe(III)-EDTA (FC )as a�ected by the presence or absence of 250 lM ferricyanide (FeCN).B Ferricyanide reduction in the same experiments as inA. Data fromindividual experiments, with the same batch of cells, are presented.Experiments were performed in 15 mM Hepes-KOH (pH 6.5)

Fig. 5. Competition between BPDS and FeCN for Fe(III), added asFeSO4. The y-axis represents Fe(II) recovered as the Fe(II)-BPDS3complex. BPDS was added at a concentration of 500 lM, and FeCNwas added at 100 (d) or 250 lm (s). Experiments were performed in15 mM Hepes-KOH (pH 6.5); Fe(III) chelators were absent. Barsrepresent SE (n = 4)

J.A. Lynnes et al.: Iron limitation in Chlamydomonas 363

Fe(II) is complexed by BPDS (Fig. 5). Furthermore, at aferricyanide concentration of 100 lM, greater than 80%of the Fe(II) is complexed by BPDS (Fig. 5). Moreover,the data presented in Fig. 4 clearly indicate that in thepresence of cells, there is no detectable Fe(II)-BPDS3formation until ferricyanide levels are far below 100 lM.Given the fact that the presence of EDTA furtherdecreases the ability of Fe(II) to reduce ferricyanide(iron was added as Fe(III)-EDTA in experiments ofFig. 4), and that the Fe(II)-BPDS3 complex does notmeasurably catalyze the reduction of ferricyanide (datanot shown), we conclude that the apparent preferencefor ferricyanide reduction is not due to non-enzymaticreduction by Fe(II).

Based upon the data presented in Figs. 4 and 5, wesuggest that ferricyanide competes for cellular reductantwith ferric chelates. However, the data do not permit usto distinguish between the possibilities that ferricyanideand ferric chelates are reduced by the same enzyme onthe plasma membrane, or that ferricyanide acceptselectrons at a prior site on the plasma-membraneelectron-transport system. Nonetheless, we suggest thatat least portions of the electron transport system(s)responsible for FeCN-R and FC-R activities must havecommon components.

The energetic requirements for extracellular Fe(III)reduction were demonstrated by the light-stimulation ofboth FeCN-R and FC-R activities (Table 1). Additionof the photosynthetic electron-transport chain inhibitor3-(3¢,4¢-dichlorophenyl)-1,1-dimethylurea (DCMU) de-creased the reduction almost to the level of the darkcontrol (Table 1). Anaerobic conditions have beendemonstrated to stimulate FC-R activity by plasma-membrane vesicles from barley roots (BruÈ ggemann andMoog 1989), presumably by diverting reducing poweraway from O2-consuming reactions (similar to thesituation in which anaerobic conditions result in anincrease in the rate of nitrate reduction by nitratereductase, e.g. Canvin and Woo 1979). In contrast,anaerobiosis decreased the rates of both ferricyanide andFe(III)-EDTA reduction by C. reinhardtii (Table 1).

Contrary to some reports from higher plants(Cakmak et al. 1987; BruÈ ggemann et al. 1990), additionof exogenous superoxide dismutase did not decrease therate of Fe(III) reduction, nor did the addition ofexogenous NADH stimulate iron reduction (Table 1).It has been suggested that NADH-dependent peroxidaseactivity is able to catalyze reduction of extracellularFe(III) to Fe(II), in a process that also results in astimulation of O2 consumption and is sensitive toinhibition by superoxide dismutase (MacrõÁ et al. 1992).Furthermore, exogenous NADH increases the ferricya-nide reduction rate by protoplasts (Lin 1982) and intactroots (Rubinstein et al. 1984). We have previouslydemonstrated that addition of exogenous NADH stim-ulates peroxidase-mediated O2 consumption byC. reinhardtii, which can be inhibited by subsequentaddition of superoxide dismutase (Torkelson et al.1995). Thus, the results presented in Table 1 provideevidence that neither extracellular peroxidase nor reac-tive oxygen species are responsible for the observed ironreduction by iron-limited C. reinhardtii.

Using an oxygen electrode, we examined the e�ects ofaddition of 500 lM ferricyanide or Fe(III)-EDTA toiron-limited cells. Contrary to the suggestion thatoxygen may compete with ferricyanide for electrons(Bienfait and LuÈ ttge 1988; BoÈ ttger and Hilgendorf 1988;NesÏ purkova et al. 1989), we found no detectable e�ecton the rate of cellular O2 consumption (data not shown)with either Fe(III) source. Thus, despite the high FeCN-R activity displayed by iron-limited cells, O2 is appar-ently not a physiological electron acceptor. The lack ofe�ect on the O2 consumption also provides furtherevidence against the participation of reactive oxygenspecies in the process of Fe(III) reduction.

Lastly, while there is some evidence supporting theidea of reduction of Fe(III) by root exudates (e.g. Hetheret al. 1984; Vempati et al. 1995) or compounds exudedby eukaryotic algae (Anderson and Morel 1980), wefound no evidence for the excretion of Fe(III)-reducingsubstances into the medium; no Fe(III) reductase activ-ity was detectable in culture medium in which the cellshad been removed by centrifugation (Table 1), i.e.reduction of Fe(III) required the presence of cells.

In summary, the results presented in this paperindicate that C. reinhardtii possesses an iron-limitation-inducible higher-plant-type Strategy I mechanism forreducing extracellular iron. However, these results donot rule out the possibility that C. reinhardtii alsoexcretes Fe(III)-chelating siderophores into the sur-roundings, followed by uptake of the Fe(III)-sideroph-ore complex (i.e. higher-plant Strategy II). Furthermore,we have provided evidence that no extracellular reducingsubstances are produced by iron-limited C. reinhardtiicells. Lastly, we provide evidence that under iron-su�cient conditions, Fe(III) reducing capacity is verylow, and that induction of the FeCN-R and FC-Ractivities are likely mediated by the same reductase.

This work was supported by the Natural Sciences and EngineeringResearch Council of Canada. The comments of two anonymousreviewers were used to improve the manuscript.

Table 1. Activities of FeCN-R and FC-R in cell suspensions ofC. reinhardtii under di�erent conditions. Except for the experimentexamining Fe(III) reduction by culture medium (cells removed), allassays were carried out in 15 mM Hepes-KOH (pH 6.5). For ex-periments in the light (photon ¯uence rate = 250 lmol quanta ám)2 á s)1), background rates of light-stimulated Fe(III) reduction,in the absence of cells, were subtracted prior to calculating percentof dark control. Rates are expressed as percent of aerobic darkcontrol � SE (n). SOD, superoxide dismutase

Reductase activity (% of aerobic darkcontrol)

Ferricyanide Ferric chelate

Light 257 � 9 (6) 234 � 8 (6)Light + 5 lM DCMU 122 � 5 (6) 122 � 4 (6)+SOD (100 U á mL±1) 99 � 2 (3) 102 � 3 (3)+NADH (1 mM) 101 � 4 (3) 102 � 2 (3))Oxygen 61 � 4 (3) 58 � 7 (3)Cell-free culture medium 0 (3) 0 (3)

364 J.A. Lynnes et al.: Iron limitation in Chlamydomonas

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