Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I higher plants1,2

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1 Journal of Experimental Botany, Vol. 49, No. 24, pp , July 1998 Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I higher plants1,2 Ulrich Eckhardt and Thomas J. Buckhout Angewandte Botanik, Humboldt Universität zu Berlin, Invalidenstrabe 42, D Berlin, Germany Recieved November 1997; Accepted 16 February 1998 Abstract Key words: Ferric chelate reduction, iron assimilation, iron The mechanism of adaptation to Fe-deficiency stress uptake, unicellular green algae, Chlamydomonas. was investigated in the unicellular green alga, Chlamydomonas reinhardtii. Upon removal of nutri- Introduction tional Fe, the activity of a cell surface Fe(III)-chelate For higher plants Fe is an essential nutritional element reductase was increased by at least 15-fold within that is required in micromolar concentrations. In spite of 24 h. This increase was negatively correlated with the its widespread abundance in the Earth s crust, Fe is Fe concentration in the growth media. Incubation poorly available in an oxidizing environment, which of cells in the presence of the Fe2+-specific chelafavours the formation of poorly soluble Fe( III)-oxides tor, bathophenanthrolinedisulphonic acid, led to an and -hydroxides at ph values above neutral. Nevertheless, increased Fe+ reductase activity, even when suffiexcess Fe is toxic to the cells due to its ability to catalyse cient Fe was present. Growth of cells in Cu-free media the formation of reactive oxygen species such as superfor 48 h led to no statistically significant increase in oxide anions and hydroxyl radicals ( Halliwell and Fe+ reductase activity. The Fe(III)-chelate reductase Gutteridge, 1989). activity in Fe-starved cells was saturable with an In higher plants, two basic strategies for iron acquisiapparent K of 1 mm and was inhibited by uncouplers m tion have been observed (reviewed in Guerinot and Yi, of the transmembrane proton gradient but not by 1994). In grasses (Strategy II plants), Fe is taken up as SH-specific reagents. a Fe( III)-phytosiderophore complex, and presumably in Fe uptake was only observed in Fe-deficient cells. all other plants, a second mechanism (Strategy I) is Uptake was specific for Fe in that a 100-fold excess employed, which involves soil acidification, formation of of a number of metal ions in the transport assay did lateral roots and specific transfer cells in the rhizodermis not inhibit uptake activity. However, a 100-fold excess as well as induction of an Fe( III)-chelate reductase and of Cu resulted in a 87% inhibition of Fe uptake. The of Fe2+ transporters (Moog and Brüggemann, 1994). In V for Fe+ reduction activity was 250-fold greater max the rhizosphere of Strategy I plants, extracellular Fe(III)- than for Fe uptake; although the K values for both m chelates are reduced to Fe2+ which is subsequently transprocesses differed by only 10-fold. Thus, the rate limit- ported into the root cells (Chaney et al., 1972) either by ing step in Fe assimilation was transport and not iron-specific or non-specific divalent cation transporters reduction. These results indicate that Fe assimilation ( Eide et al., 1996). in C. reinhardtii involves a reductive step and thus The Fe( III)-chelate reductase ( EC ) in higher resembles the mechanism of Fe uptake in Strategy I plants is located on the root cell plasma membrane higher plants. (Buckhout et al., 1989). Attempts to purify the enzyme 1 We dedicate this work to the memory of Dr William J VanDerWoude. 2 A portion of this report was presented at the 9th Symposium on Iron Nutrition and Interaction in Plants. To whom correspondence should be addressed. Fax: Thomas=Buckhout@biologie.hu-berlin.de Abbreviations: BCDS, bathocuproinedisulphonic acid; BPDS, bathophenanthrolinedisulphonic acid; Cu-EDTA, CuSO 4, chelated with equimolar EDTA; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; Fe-HEDTA, FeCl chelated with equimolar HEDTA; HEDTA, N-hydroxyethyl-ethylenediaminetriacetic acid, PCMBS, p-chloromercuriphenylsulphonic acid. Oxford University Press 1998

2 1220 Eckhardt and Buckhout from tomato roots have resulted in a high enrichment Materials and methods of enzyme activity ( Holden et al., 1994) and recently Algal strains and culture conditions two Fe( III)-citrate reductases have been purified to Chlamydomonas reinhardtii wild-type strains 11 2b (mt+), homogeneity (Bagnaresi et al., 1997). Genetic attempts 11 2c (mt ) and the cell wall-deficient mutant 8.81 (cw15 to characterize the Fe( III)-chelate reductase at the mt+) were obtained from the Sammlung von Algenkulturen, molecular level have been undertaken (Briat and Göttingen, FRG. The CF CF -deficient mutant CC 980 (F Lobréaux, 1997), and recently, several mutants have mt+) was obtained from Dr Matthias Kuhn, Max-Volmerbeen characterized in Arabidopsis thaliana that are either Institut, Technische Universität Berlin. Cells were grown mixotrophically at room temperature (25 27 C) in TAP defective or show constitutively high levels of Fe( III)- medium (Harris, 1988) with moderate shaking ( rpm) chelate reductase activity (Delhaize, 1996; Yi and and constant illumination of about 100 mmol s 1 m2 photosynthetic active radiation in Erlenmeyer flasks. TAP medium Guerinot, 1996). In Saccharomyces cerevisiae the mode of Fe assimilation contained approximately 17 mm Fe, and the ph was adjusted has been investigated in detail. Similar to Strategy I to 7.0. Cell density was estimated by measuring the absorbance at 750 nm and using a calibration curve (Harris, 1988). plants, Fe uptake in yeast requires an Fe+ reductase (Lesuisse et al., 1987) and a mutation in the FRE1 gene abolished this reductase activity (Dancis et al., 1990, Ferric reductase assay 1992). The FRE1 gene product has been shown to be a For analysis of effects of Fe deficiency on Fe assimilation, all flavo-cytochrome (Shatwell et al., 1996) that presumably glassware was rinsed thoroughly with 2% (w/v) EDTA, ph 8.0, and twice with double-distilled water before autoclaving. Cells catalyses the NAD(P)H-dependent single electron trans- from exponentially growing cultures were collected by centrifufer to Fe+. However, the FRE1 gene product appears to gation at 2500 g for min, resuspended in TAP medium without be only one component of an enzyme complex consisting trace elements, and the cell density was determined. Cells were of multiple subunits (Lesuisse et al., 1996). incubated at 0 C in the presence of 200 mm Fe-HEDTA and 600 mm BPDS. After 5 and 10 or 8 and 16 min., the cells were Relatively little is known about Fe assimilation in green sedimented and the absorbance at 55 nm of the supernatant algae. Alnutt and Bonner (1987a, b) characterized the was measured. Reduction rates were calculated assuming an uptake of radiolabelled Fe from a number of siderophore- e of cm2 mmol and chelator-complexes as well as the disappearance of the electron paramagnetic resonance signal of Fe+. They Cupric reductase assay demonstrated that in Chlorella vulgaris the uptake of Reduction of Cu2+ was measured essentially as described by siderophore-bound iron occurred reductively; although, Hill et al. (1996). In brief, cells were harvested and resuspended Moog and Brüggemann (1994) did not find substrate in TAP medium without trace elements. Two 1 ml samples saturation for the Fe( III)-chelate reduction in Chlorella were incubated at 0 C with 100 mm Cu-EDTA (ph 6.5) and pyrenoidosa and concluded that Fe( III)-chelate reduction 200 mm BCDS for 15 and 0 min, respectively. The cells were sedimented and the absorbance of the supernatant at 48 nm was a non-specific reaction in Chlorella. The mechanism was measured. Reduction rates were calculated by using a of Fe accumulation in Scenedesmus was shown to calibration curve. The formation of Cu+ was linear for at resemble Strategy II of higher plants (Benderliev and least 0 min. Ivanova, 1994). More recently, Hill et al. (1996) reported that Cu Iron uptake assay uptake in Chlamydomonas reinhardtii required reduction Exponentially growing cells were washed in TAP medium of Cu2+ to Cu+ presumably by a membrane-bound without trace elements, collected by centrifugation, resuspended reductase. This reductase as well as the putative Cu in uptake buffer (100 mm MES, 20 mm Na-citrate, 2 mm transporter were strongly induced upon Cu starvation, K-acetate, ph 6.0) to an OD of 5 6 and kept on ice for at 750 and it was demonstrated that the apparent K for the Cu least 1 h. Five hundred ml of the cell suspension, maintained at m 0 C, were supplemented with 100 ml of 6mM Fe-HEDTA, uptake reaction in Cu-starved and -replete cells were containing 2000 cpm ml 1 55FeCl (Dupont-NEN, Bad similar, while the V was strongly increased in max Homburg, FRG) or 1000 cpm ml 1 59FeCl (Amersham, Cu-depleted cells. These findings indicated that the same Braunschweig, FRG) in uptake buffer. At 0.5, 10, 20, 0, and enzyme was active under both nutritional conditions (Hill 40 min, 100 ml of the suspension were pipetted into 10 ml of ice-cold quench solution (50 mm MES, 20 mm EDTA, 20 mm et al., 1996). Na -citrate, 2 mm CaCl, ph 6.4), and vacuum filtered through In order to determine whether unicellular algae could 2 nitrocellulose filters, and the filters were washed twice with 5 ml serve as model organisms for Fe assimilation in higher of quench solution. The activity remaining on the filters was plants, the mode of Fe assimilation in the green measured by liquid scintillation counting. To correct for non- alga, Chlamydomonas reinhardtii, was investigated. It specifically bound Fe, uptake experiments were performed on ice, and the control values obtained were subtracted from the is reported that the mechanism of adaptation to uptake rates measured at 0 C. When Fe2+ uptake was Fe-deficiency stress by Chlamydomonas shares many fea- analysed, the assay contained 1 mm FeCl,1mM HEDTA, 167 tures typical of Strategy I. 2 cpm ml 1 59FeCl, and 16.7 mm ascorbic acid in uptake buffer.

3 Results Induction of the Fe(III)-chelate reductase activity under Fe deficiency deficiency stress. Interestingly, the reductase in the chloroplast ATPase-deficient mutant CC 980 was not altered by the Fe concentration in the medium (Fig. 1). Growth of Chlamydomonas was not influenced by Fe supply within the duration of the experiments ( Fig. A). The pattern of induction of activity was investigated over a period of 1 h of growth in Fe-deficient medium. A slight increase in reductase activity was observed between 6 8 h after the removal of Fe, and the activity increased rapidly between approximately 12 and 18 h of Fe-deficient growth ( Fig. B). Induction of the reductase activity occurred only in Fe-deficient cells and was complete after h ( Fig. B). Upon refeeding the iron- starved cells with 10 mm FeCl, the activity decreased within 4 h to control levels. When cells approached late exponential or stationary growth phase, the reductase activity typically decreased slightly. The response to Fe-deficiency of the cell wall-less mutant 8.81 has also been investigated with similar results to those of the wild type shown in Figs 1 (data not shown). Strategy I plants respond to Fe deficiency by the induction of a Fe( III)-chelate reductase. In order to determine the mechanism of Fe assimilation in Chlamydomonas reinhardtii, wild-type cells were grown in TAP medium for 4 d in the presence or absence of Fe. Fe deficiency led to an increase of greater than 15-fold in Fe( III)-chelate reductase activity ( Fig. 1), and this increase in activity was inversely correlated with the Fe concentration in the growth medium ( Fig. 2). Half-maximal stimulation was observed at approximately 0.7 mm Fe after 2 d of Fe Reductive iron assimilation in Chlamydomonas 1221 Physiology of the C. reinhardtii Fe(III)-chelate reductase In Fe-deficient C. reinhardtii cells, the Fe( III)-chelate reductase activity had a broad optimum at ph (data not shown) and exhibited Michaelis Menten-like kinetics towards Fe(III)-HEDTA with an apparent K m of 1.5 (±2.) mm and V of 11 nmol 10 6 cells h 1 max Fig. 1. Analysis of the Fe(III)-chelate reduction in Chlamydomonas. Chlamydomonas reinhardtii wild-type cells and the CF 0 CF 1 -ATPasedeficient mutant, CC 980, were grown for 4 d in TAP medium with or without Fe. Cells were collected by centrifugation, and the Fe(III)- chelate reductase activity was determined as described in the Materials and methods. The results are averages from three experiments with the standard errors shown. Fig. 2. The effect of Fe+ concentration in the growth medium on the activity of the Fe(III)-chelate reductase. C. reinhardtii wild-type cells were grown in TAP medium to late exponential phase, washed in Fe-free medium and resuspended in TAP medium containing the indicated FeCl concentrations. After 2 d, cells were harvested by centrifugation and the Fe+ reductase activity was measured. Data represent the mean of three experiments with the standard errors indicated. Fig.. Kinetic analysis of the induction of Fe(III)-chelate reductase in Chlamydomonas. C. reinhardtii wild-type cells were grown in TAP medium with (triangles) and without (diamonds) Fe. (A) Cell density was estimated spectrophotometrically at 750 nm. (B) Fe(III)-chelate reductase activity was measured every h as indicated in the Materials and methods. After 24 h the Fe-deficient cells were re-fed FeCl to a final concentration of 10 mm (squares). Results are typical of two experiments.

4 1222 Eckhardt and Buckhout ( Fig. 4). When a similar experiment was performed with Table 1. Influence of specific inhibitors on the Fe(III)-chelate Fe-sufficient cells, the kinetic behaviour of the reductase reductase activity in Fe-deficient Chlamydomonas cells with respect to substrate concentration exhibited an Wild-type Chlamydomonas cells were harvested in mid-exponential phase and re-suspended in TAP medium without Fe. After 2 d, cells apparently biphasic kinetic. One component was saturable were harvested and resuspended in TAP medium lacking trace elements. (K $0 mm) and a second was not saturated at 00 mm The effect of ionophores and inhibitors on the Fe(III)-chelate reductase m (Fig. 4). activity was tested following a 5 min pre-incubation as described in the Material and methods. Data shown represent mean of two experiments. To obtain a first insight into the nature of the Activity of 100% corresponds to 18.4±2.7 nmol Fe cells h 1. C. reinhardtii Fe( III)-chelate reductase, the influence of inhibitors and ionophores was investigated. Inhibitors of Inhibitor Concentration Reductase activity photosynthetic (DCMU ) and respiratory electron transport (mm) (%, control ) ( KCN), SH-specific reagents (PCMBS), uncouplers Control 100 of the membrane potential (gramicidin, valinomycin) FCCP FCCP or the transmembrane proton gradient ( FCCP and Gramicidin ,4-DNP) as well as a platinum salt ( K PtCl ) and 2 4 2,4-DNP diphenyleneiodoniumchloride (DPI), were supplied 5 min PCMBS KCN before measuring the Fe( III)-chelate reductase in DCMU Fe-deficient algae. These latter two substances were previ- K PtCl ously shown to inhibit the ferric reductase, Fre1p, in DPI Valinomycin yeast. Table 1 summarizes the results of these experiments. Of all the substances supplied, FCCP and valinomycin clearly inhibited Fe(III)-chelate reduction. No effect was without 17 mm FeCl in the absence or presence of observed with PCMBS, and only small effects were 200 mm of the Fe2+-specific chelator, BPDS. It was observed for gramicidin and DPI. At relatively high assumed that in the presence of BPDS, Fe2+ would be concentrations of K PtCl, 2,4-DNP and DCMU only a 2 4 bound and, thus, unavailable for uptake (Chaney et al., slight inhibition was observed ( Table 1). 1972). The growth kinetics and Fe( III)-chelate reductase In Strategy I plants, Fe2+ is the ionic species taken up activity were monitored. Cell growth over a period of in roots (Chaney et al., 1972). In order to determine 40 h was only slightly affected by Fe deficiency ( Fig. 5A). whether C. reinhardtii cells possess a similar uptake mechanism, wild-type algae were grown for 2 d with or Fig. 4. The effect of substrate concentration on the Fe(III)-chelate reductase activity. Chlamydomonas reinhardtii wild-type cells in the exponential growth phase were harvested by centrifugation, resuspended in fresh TAP medium and grown for 2 d in the absence (filled squares) or presence (open squares) of FeCl. The reductase activity was measured using Fe-HEDTA as substrate at the concentrations indicated. Fig. 5. Effect of Fe-deficiency and the Fe2+ chelator, BPDS, on cell The data represent mean of four independent experiments with standard growth (A) and Fe(III)-chelate reductase activity (B). C. reinhardtii deviations indicated. The insert shows the double reciprocal cells were grown in TAP medium with or without Fe and 0.2 mm (Lineweaver Burk) plot of the data obtained from the Fe-deficient BPDS for 2 d. (A) Cell density was determined as turbidity at 750 nm. cultures. The apparent K was 1.5 mm Fe-HEDTA and V was For comparison, the absorbance of all cultures was set equal to 1 at m max 11 nmol 10 6 cells h 1 for Fe-deficient cells. For Fe-sufficient cells, the beginning of the experiment. (B) After 17 and 27 h, Fe(III)-chelate biphasic kinetics were observed. The apparent K for the saturable reductase activity was measured. The data shown represent the average m component was approximately 0 mm Fe-HEDTA. of the values at 17 and 27 h (n=2).

5 Reductive iron assimilation in Chlamydomonas 122 Addition of 200 mm BPDS further retarded growth in Table 2. Comparison of uptake rates of Fe+ and Fe2+ and cultures growing in both Fe-sufficient and -deficient Fe(III)-chelate reductase activity in C. reinhardtii cells media. In contrast to the marginal effects of BPDS on Chlamydomonas wild-type cells were grown in TAP medium with or without Fe. After 2 d, Fe(III)-chelate reductase activity and Fe uptake cell growth, the induction of Fe( III)-chelate reductase were measured. As a substrate in the uptake assays, 1 mm radiolabelled activity in the same cultures measured after 17 and 27 h Fe was supplied as Fe-HEDTA or as Fe2+ in the presence of 16.7 mm was dramatic ( Fig. 5B). The presence of BPDS in the ascorbic acid. The uptake results shown are from a single experiment. growth media enhanced the effect of Fe deficiency and Growth Reductase activity Fe uptake led to Fe deficiency symptoms, even when sufficient Fe conditions (nmol 10 6 cells h 1) (pmol 10 6 cells h 1) was present. FeCl FeCl 2 Uptake of radiolabelled Fe +Fe 0.9± In an attempt to correlate increases in Fe( III)-chelate reductase activity with Fe uptake in C. reinhardtii, uptake Fe 14.9± of 55Fe or 59Fe in Fe-deficient and -sufficient 8.2±5.2 pmol 10 6 cells h 1 (data not shown). Uptake Chlamydomonas cells was investigated. In Fe-sufficient of Fe2+ was completely abolished by 20 mm FCCP, when cells, only low rates of Fe uptake were observed relative added 5 min before the uptake experiments, and as was to background, whereas Fe-deficient cells showed linear found for the Fe( III)-chelate reductase, PCMBS had no uptake rates for at least 20 min (Fig. 6). The uptake rates effect on Fe transport (data not shown). Increasing the in Fe-deficient cells were approximately pmol ph from 6.0 to 7.0 by addition of TRIS resulted in an 10 6 cells h 1. This uptake activity corresponded to a approximately 20% inhibition of Fe uptake (data not c. 100-fold concentration increase of Fe from the medium shown). To analyse the substrate specificity of the transwithin 15 min, assuming an average, symplastic cell porter, the influence of metal cations on uptake was volume of 50 mm ( Harris, 1988). tested. Fe2+ uptake was not greatly inhibited by a 100-fold To determine which species of Fe was transported, excess of Mg2+, Mn2+, Ni2+, Co2+ or Zn2+, but a radiolabelled FeCl was maintained in the reduced form fold excess of Cu2+ in the transport assay inhibited with ascorbate, and Fe2+ uptake was determined in Fe2+ uptake by 87% (Fig. 7). Cadmium and Ca2+ had a Fe-deficient cells ( Table 2). In Fe-sufficient cells, uptake stimulatory effect on Fe2+ uptake. rates were increased by approximately 2-fold when Fe was supplied as Fe2+ compared to Fe+, and in Interaction of Fe and Cu deficiency in Chlamydomonas Fe-deficient cells the uptake rate was increased by 64%. To exclude effects of the reductase on Fe uptake, Fe was Cu has been shown to inhibit Fe uptake in Fe-deficient supplied as FeCl in the presence of ascorbate in all Chlamydomonas cell ( Fig. 7). In an attempt to determine 2 subsequent experiments. whether growth of Chlamydomonas in Cu-deficient media The uptake of Fe2+ was concentration dependent with would lead to increased Fe( III)-chelate reductase activity, an apparent K of.75±0.65 mm and a V of Cu2+ reduction was measured in response to Fe limitation m max as well as Fe+ reduction in response to Cu limitation. The results are summarized in Table. The data show Fig. 7. The effect of metal ions on the rate of Fe uptake. Uptake of Fig. 6. Uptake of radiolabelled Fe in C. reinhardtii. Cells were grown radiolabelled Fe2+ was assayed in Fe-deficient Chlamydomonas wildtype in TAP medium with or without Fe for 2 d. Following treatment, cells cells in the presence or absence of a 100-fold molar excess of the were collected by centrifugation, and Fe uptake was determined as metal ions indicated. The ions were added to cells immediately prior to described in the Materials and methods with Fe-HEDTA as substrate. the analysis of Fe2+ uptake. Results are an average of three experiments The results are the average of three experiments with the standard with the standard errors indicated. The control uptake was set 100% errors shown. and represented 19.±2.8 pmol 10 6 cells h 1.

6 1224 Eckhardt and Buckhout Table. Analysis of the effects of Cu and Fe deficiency on the rate of Fe+ and Cu2+ reduction in both Fe-and Cu-deficient C. reinhardtii C. reinhardtii cultures were grown to a late exponential growth phase, harvested, washed and resuspended in TAP medium with or without Fe or Cu. After 2 or d, Fe+ and Cu2+ reductase activities were measured. Data represent mean and standard deviations as indicated (n=). Values within a column that are followed by the same letter are not statistically significant at the 95% confidence interval as determined by an analysis of variance. Treatment Cu2+ reduction % Control Fe+ reduction % Control (nmol 10 6 cells h 1) (nmol 10 6 cells h 1) +Fe +Cu 1.2±0.4 a ±0.1 a 100 Fe +Cu 1.9±0.2 a,c ±9.8 b Fe Cu 1.4±0.1 a,c 12 1.±0.4 a 118 Fe Cu 2.±0.4 c ±5.1 b 1768 Arabidopsis was recently identified by functional expression in a yeast mutant (Eide et al., 1996), and Arabidopsis mutants have been described, one of which was defective in the induction of the Fe( III)-chelate reductase, but which properly induced the H+-ATPase under Fe defi- ciency ( Yi and Guerinot, 1996). For Chlamydomonas, the physiological data presented above clearly demonstrate that Fe uptake occurs reductively. Upon Fe starvation, the in vivo Fe( III)-chelate reductase was induced by greater than 15-fold. In cells grown in sufficient Fe, a residual Fe+ reductase was detected with an activity of between nmol 10 6 cells h 1. The kinetic behavi- our of this constitutive activity with respect to substrate concentration did not conform to a Michaelis Mententype kinetic. Rather, the kinetics were biphasic with both a saturable and a non-saturable component. This activity may be analogous to a constitutive redox system on the plant plasma membrane, designated as the Standard reductase (Lüthje et al., 1997). The Fe( III)-chelate reductase in Chlamydomonas was energy-dependent, since a CF CF -deficient mutant 0 1 showed no induction, and the uncoupler FCCP strongly inhibited Fe+ reduction; although, gramicidin was with- out effect. It is proposed that the higher mobility of FCCP depleted not only the plasma membrane potential but also the energy-supplying membrane potentials in the chloroplast and the mitochondria. In contrast to the findings in higher plants (Moog and Brüggemann, 1994), no sulphydryl groups appeared to be involved in the Fe( III)-chelate reductase activity in Chlamydomonas. The induction of the Fe( III)-chelate reductase activity under Fe deficiency occurred in parallel with the induction of Fe transport activity. The V of the Fe(III)-chelate max reductase in Fe-starved cells (9 11 nmol 10 6 cells h 1) was approximately 250 times greater than the V for max Fe uptake (8 pmol 10 6 cells h 1), while the K values m differed by only 10-fold. This indicated that the ratelimiting step in Fe assimilation was transport rather than Fe+ reduction. Because of the inability specifically to inhibit the Fe( III)-chelate reductase, it was not possible to exclude the fact that Fe+ may also have been transported across the plasma membrane. To distinguish that after 2 d of growth, Fe(III)-chelate reductase activity was increased by approximately 20%. The reduction of Cu2+, however, increased about 60% in response to Fe deficiency. These effects were not statistically significant at the 95% confidence interval. The greatest increase in Cu2+ reduction of nearly 100%, which was statistically significant, was observed, when cells were grown under both Fe and Cu deficiency (Table ). The Fe2+ uptake rates under Cu deficiency were not significantly changed from those under Cu sufficient conditions ( Fig. 8). Thus, under the experimental conditions employed, no dependence of the Cu nutritional status on Fe2+ uptake was found. Discussion The response to Fe deficiency in higher plants with the exception of grasses involves acidification of the rhizo- sphere, an increase in trans-plasma membrane electron transport, the subsequent extracellular reduction of Fe( III)-chelates and the uptake of Fe2+ by Fe-regulated transporters (Guerinot and Yi, 1994). On a molecular level, however, the characterization of these processes has only begun. An Fe-regulated cation transporter from Fig. 8. Analysis of the effect of Fe and Cu deficiency on the rate of Fe uptake in Chlamydomonas. Cells were grown in TAP medium with or without Fe or Cu for d. Uptake experiments were performed using Fe2+ (hatched bars) or Fe+ (open bars) as substrate. Results are an average of three experiments with the standard error indicated.

7 Reductive iron assimilation in Chlamydomonas 1225 conclusively between the uptake of Fe2+ and Fe+, reduc- upon Fe deficiency, have been observed in higher plants, tase mutants or specific reductase inhibitors will be yeast and unicellular algae. However, in Chlamydom- needed, or chelate-buffered solutions of Fe2+ with, for onas, several differences were observed. For example, example, Ferrozine need to be employed in further uptake no involvement of SH-groups was observed in studies ( Fox et al., 1996). Chlamydomonas neither for Fe+ reduction nor for Fe It has been observed in higher plants, unicellular algae transport. Cadmium inhibited the Fe transport activity and in yeast, that Cu and Fe metabolism were tightly of the Arabidopsis Fe-regulated transporter, but stimulated linked ( Welch et al., 199; Klausner and Dancis, 1994; Fe transport in Chlamydomonas. The rate-limiting Holden et al., 1995; Hill et al., 1996). In yeast and in C. component in Chlamydomonas was clearly Fe2+ transport reinhardtii, both Cu2+ and Fe+ chelates have been rather than Fe+ reduction. Thus, it was concluded that reported to require reduction to be substrates for trans- Fe uptake in Chlamydomonas occurred by a Strategy port (Hassett and Kosman, 1995; Hill et al., 1996). In I-like mechanism; although, details of the uptake and yeast, Cu was an essential part of the functional oxidase- adaptation processes may be unique. permease complex ( Klausner and Dancis, 1994; Stearman et al., 1996), which was required for high affinity Fe uptake. Therefore, Cu deficiency in baker s yeast indirectly Acknowledgements led to Fe deficiency symptoms. This work was supported by grant from the Deutsche In higher plants, it has been hypothesized that the Forschungsgemeinschaft to TJB. We are indebted to Drs Fe( III)-chelate reductase played a general role in cation Reinhard Gebner (Humboldt University -Virchow Clinic, assimilation and, thus, exhibited rather unspecific sub- Berlin) and Thomas Eitinger (Microbiology, Humboldt strate preferences ( Welch et al., 199). During partial University, Berlin) for providing access to their isotope laboratory facilities. purification, both the Fe+ and the Cu2+ reductases co-purified; althought, the enzymatic properties of the Cu2+ reductase with respect to detergent latency, substrate specificity and electron donor preference were dis- References tinct from the Fe(III)-chelate reductase (Holden et al., Alnutt FCT, Bonner Jr WD. 1987a. Characterization of iron 1995). It would be chemically advantageous for organisms uptake from Ferrioxamine B by Chlorella vulgaris. Plant Physiology 85, to reduce Fe+ but not Cu2+, since Cu+ in the absence Alnutt FCT, Bonner Jr WD. 1987b. Evaluation of reductive of suitable chelators tends to form less soluble salts or to release as a mechanism for iron uptake from Ferrioxamine B disproportionate into Cu0 and Cu2+, whereas Fe2+ is by Chlorella vulgaris. Plant Physiology 85, more soluble than Fe+. Thus, reduction of Cu2+ would Bagnaresi P, Basso B, Pupillo P The NADH-dependent provide no increased availability of Cu. In pea roots, Cu Fe+-chelate reductases of tomato roots. Planta 202, Benderliev KM, Ivanova NI High-affinity siderophoredeficiency led to increased Fe( III)-chelate reductase activmediated iron-transport system in the green alga Scenedesmus ity ( Welch et al., 199). However, only an 18% increase incrassatulus. Planta 19, in Fe+ reduction was observed in Chlamydomonas grown Briat J-F, Lobréaux S Iron transport and storage in under Cu deficiency, and a 60% increase in Cu2+ reduc- plants. Trends in Plant Science 2, tion under Fe deficiency (Table ). These findings support Buckhout TJ, Bell PF, Luster DG, Chaney RL Iron-stress induced redox activity in tomato (Lycopersicon esculentum the view, that the Fe+ reductase, which was induced Mill.) is localized on the plasma membrane. Plant Physiology greater than 15-fold in Fe-deficient cells, exhibited low 90, substrate specificity and thus could also reduce, to a Chaney RL, Brown JC, Tiffin LO Obligatory reduction limited extend, Cu2+. of ferric chelates in iron uptake by soybeans. Plant Physiology In S. cerevisiae cells that expressed the recently isolated 50, Dancis A, Klausner RD, Hinnebusch AG, Barriocanal JG Arabidopsis cdna clone, IRT1, Fe uptake strongly pre- Genetic evidence that ferric reductase is required in iron ferred Fe2+ over Fe+, and uptake of Fe was inhibited uptake in Saccharomyces cerevisiae. Molecular and Cellular by an excess of Zn2+, Mn2+, Co2+, and Cd2+ ( Eide Biology 10, et al., 1996). In Chlamydomonas, Fe2+ was the preferred Dancis A, Roman DG, Anderson GJ, Hinnebusch AG, Klausner substrate for uptake compared to Fe+, and both Ca and RD Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcrip- Cd stimulated Fe2+ uptake, whereas Mg, Zn, Mn, Ni, tional control by iron. Proceedings of the National Academy and Co had no significant effects. Interestingly, Cu2+ of Sciences, USA 89, clearly inhibited Fe uptake when supplied in a 100-fold Delhaize E A metal-accumulator mutant of Arabidopsis excess concentration. Whether this inhibition was compet- thaliana. Plant Physiology 111, itive or not, remains to be determined. Eide D, Broderius M, Fett J, Guerinot M-L A novel iron- regulated metal transporter from plants identified by func- The basic features of Strategy I-like Fe assimilation, tional expression in yeast. 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