OPEN ACCESS ARTICLE

This Article Free via Open Access: OA OA Abstract Full Text (PDF) Supplemental Data OA All Versions of this Article: 144/3/1407    most recent pp.107.100644v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Paz, Y. Articles by Pick, U. Search for Related Content PubMed PubMed Citation Articles by Paz, Y. Articles by Pick, U. Agricola Articles by Paz, Y. Articles by Pick, U.

ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Effects of Iron Deficiency on Iron Binding and Internalization into Acidic Vacuoles in Dunaliella salina^1,[W],[OA]

Department of Biological Chemistry (Y.P., M.W., U.P.) and Electron Microscopy Unit (E.S.), Weizmann Institute of Science, Rehovot 76100, Israel

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe³⁺ ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.

In this work, we studied the regulation of iron binding and uptake. Specifically, we characterized the kinetic changes in iron binding and uptake in response to iron deprivation or to saturation of intracellular iron stores. We show that iron deprivation or overaccumulation leads to large changes in iron binding, suggesting that this is the critical parameter in regulation of iron uptake in D. salina. We also localized the site of iron internalization within acidic vacuoles. The physiological significance of dynamic changes in iron binding is discussed.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Dunaliella Cells Contain Externally Bound Iron

The time course of iron uptake in D. salina reveals an initial time lag of 15 to 20 min. This time lag was apparent in control cells, which have been cultured with iron, but was absent in iron-deficient cells (Fig. 1A ). We found that preincubation of control D. salina cells for 30 to 60 min in iron-deficient medium almost completely eliminated this apparent time lag (Fig. 1B). Conversely, preincubation of iron-deficient cells with iron introduced a similar time lag in the onset of iron uptake (Fig. 1C). These results suggest that the apparent time lag in iron uptake in D. salina may result from prebound iron.

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Prebound iron causes the time lag in Fe uptake. A, Time lag in Fe uptake in D. salina cells. Samples of 5 x 107 iron-deficient or iron-sufficient cells were incubated with 59Fe3+ citrate at room temperature for the indicated time points and analyzed for internalized iron content. Arrow indicates the time lag in iron-sufficient cells. B, Time lag in Fe-sufficient cells. Samples of 5 x 107 cells were either not treated (Con.) or preincubated for 1 h in the light in Fe-deficient medium, for unloading occupied iron-binding sites (PI) or for 20 min on ice with saturating Fe citrate to upload unoccupied iron-binding sites followed by washing (Fe-Cit.). After these treatments, cells were incubated with 59Fe3+ citrate at room temperature for the indicated times and analyzed for internalized iron content. C, No time lag in Fe-deficient cells. Treatments as in B. Averages of 3 (A) or 2 (B and C) separate experiments.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Fe binding in D. salina cells is inhibited by prebound Fe. Cultures of iron-sufficient (+Fe; white symbols) or iron-deficient (–Fe; black symbols) cells were either not treated (circles) or preincubated (PI; squares) to unload bound Fe or preincubated with Fe citrate (Fe-Cit.; triangles) as in Figure 1. Samples were then washed and assayed for Fe binding at 4°C with 59Fe3+ citrate for the indicated times. Representative experiment of four repetitions.

The observation that D. salina cells contain prebound iron that is not removed by extensive washing is consistent with the high binding affinity of D. salina TTf for Fe³⁺ ions that we reported in previous studies (Fisher et al., 1998; Schwarz et al., 2003b). To find out whether iron is irreversibly bound or exchanges with medium Fe³⁺ ions, we added an excess of natural (nonradioactive) isotope mixture of Fe³⁺, as Fe citrate, after binding ⁵⁹Fe³⁺ to control or iron-deficient D. salina cells. As shown in Figure 3 and Table III , there is less than 10% exchange within 1 h at 4°C. The bound iron can be stripped off effectively by EDTA and, to a lesser extent, at acidic pH. These results explain why cells that were precultured with iron do not bind ⁵⁹Fe³⁺ even after extensive washing because all iron-binding sites are occupied with tightly bound iron.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Dissociation and exchange of bound iron. Iron-deficient D. salina cell cultures were incubated with 59Fe3+ citrate as in Figure 2. After 20 min at 4°C, they were supplemented either with 200 µM Fe3+ citrate (natural isotope mixture) or with 5 mM EDTA. Samples were withdrawn at the indicated times, washed twice in Fe-binding stop solution, and assayed for bound 59Fe. Representative experiment of three repetitions.

In a previous study, we reported that bound iron in D. salina cells is internalized subsequent to binding in a temperature-dependent process (Fisher et al., 1998). Because in iron-deprived cells we found a large increase in number and changes in kinetic parameters of iron-binding sites, it seemed of interest to test how much of the bound iron is internalized in these cells. To test this, cells were first preincubated with ⁵⁹Fe³⁺ at 4°C to saturate the iron-binding sites, followed by washing and internalization in the presence of the excess natural isotope mixture Fe citrate, to exclude internalization of nonbound iron. As shown in Figure 4 , practically all the bound iron was internalized in both control and iron-deficient cells. The amount of internalized iron is largely enhanced in iron-deficient cells, corresponding to the much higher binding, but the process is slower, with a t_1/2 of about 30 min compared to about 10 min in control cells. These results suggest that the massive amount of bound iron in iron-deficient cells is committed for internalization. About 30% of the bound iron in iron-deficient cells is not internalized after 1 h of incubation without iron, but can be internalized by extended incubation (data not shown). Because excess iron may be toxic to cells, we wished to test whether D. salina cells can regulate their iron-binding capacity to avoid overaccumulation of iron. To test this, cells were allowed to accumulate iron for 1.5 or 4 h and tested for iron-binding and uptake activities. To dislodge external binding sites from prebound iron, cells were exposed to two different treatments: EDTA washes, which strip off bound iron, or the 1-h unloading treatment to allow internalization of prebound iron. As is shown in Table IV , these treatments gave different results: After 4 h of iron accumulation, EDTA stripping recovered about 90% of the original activity, whereas the unloading treatment recovered only 10% to 15%. These results suggest that, as the internal iron stores fill up, internalization is inhibited and more iron remains externally bound. This may represent a feedback mechanism to avoid iron overloading.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Bound iron is committed for internalization. Iron-sufficient (+Fe) or iron-deficient (–Fe) cell cultures were incubated for 30 min with 59Fe3+ citrate on ice. Cells were washed twice in EDTA-free stop solution and 200 µM Fe citrate (natural isotope mixture) were added. Then cells were transferred to room temperature and illuminated; samples were taken at the indicated time points. Total, cell-associated (EDTA resistant plus EDTA sensitive), bound (EDTA sensitive), and internalized (EDTA resistant). Representative experiment of five repetitions.

In mammalian cells, iron is internalized via the transferrin/transferrin receptor system into acidic vacuoles. To test whether in D. salina iron is also internalized into acidic vacuoles, we tested the effects of vacuolar inhibitors on iron uptake. In previous studies, we have identified and characterized acidic vacuoles in D. salina by staining with a fluorescent indicator quinacrine (Weiss and Pick, 1991) and by localization of intravacuolar polyphosphates, which act as a pH-buffering system (Pick et al., 1991; Pick and Weiss, 1991). We found that amines can induce alkaline stress, which is counterbalanced by massive polyphosphate hydrolysis within the vacuoles combined with amine accumulation. Thus, amines at alkaline pH can disrupt vacuolar activities in D. salina. As shown in Table V , the vacuolar H⁺-ATPase inhibitor, bafilomycin, as well as ammonia at alkaline pH, suppressed iron uptake, but only partially inhibited iron binding. Under these conditions, bafilomycin did not decrease cellular ATP nor did it inhibit uptake of phosphate (data not shown), indicating that the inhibition is specific to vacuolar activities. To visualize internalized iron, we stained D. salina cells with acid ferrocyanide (Prussian blue), a stain utilized for subcellular localization of bound Fe³⁺ ions in different organisms, including plants (Parmley et al., 1978; Kim et al., 2003; Vasconcelos et al., 2003; Arbab et al., 2006; Tallheden et al., 2006) and monitored stained cells by electron microscopy. Bound iron particles were clearly visualized at the extracellullar cell surface (Fig. 5A , inset). The remarkable difference before and after internalization was the dense iron staining within vacuolar-like structures (compare Fig. 5, A and B). At high magnification, iron within some of the vacuoles appears to be associated with a delicate membrane-like reticulum (Fig. 5, E and F) and, in others, is aggregated in dense plaques (Fig. 5, C and D). This stained iron particles resemble in size and general appearance early ultrastructural localizations of iron within phagosomes of macrophage cells (Parmley et al., 1978). The significance of the different patterns of vacuolar iron staining is not clear. They may represent different stages in vacuolar maturation after endocytosis; possibly, the reticular structures may represent residual plasma membrane structures in immature vacuoles, shortly after endocytosis, which later mature to yield aggregated iron morphology. To further characterize these iron-accumulating structures, we treated D. salina cells with the fluorescent indicator LysoSensor Green, which specifically stains acidic vacuoles. As shown in Figure 6A , the stain accumulates in vacuolar structures similar in size and localization to the iron-accumulating vacuoles. To further characterize these vacuoles, we immunolabeled D. salina cells with polyclonal antibodies against a vacuolar marker previously reported to label acidic vacuoles in the green alga C. reinhardtii, the H⁺-transporting pyrophosphatase (Ruiz et al., 2001). Antibodies reacted with a single component on western immunoanalysis corresponding to its predicted M_r (data not shown). As shown in Figure 6B, anti H⁺-pyrophosphatase labeled structures similar in size and localization to acidic vacuoles. Unfortunately, it is not possible to carry out colocalization of the iron stain with either immunolocalization or LysoSensor Green staining because of the different required preparation procedures (acid treatment, fixation, and detergent permeabilization or vital stain, respectively). To test whether iron internalization indeed results from endocytosis of iron-binding proteins, we tagged membrane surface proteins in iron-deficient cells with a membrane-impermeable biotin derivative and monitored biotin-tagged proteins labeled with the fluorescein avidin with a confocal microscope. We found that, in iron-deficient cells, biotin tags almost exclusively three protein bands that were identified as the major iron deprivation-induced plasma membrane proteins (Paz et al., 2007): two transferrin-like proteins, TTf (Fisher et al., 1997, 1998) and DTf (Schwarz et al., 2003a), and a 130-kD band that was found to contain two proteins, a multicopper ferroxidase, termed DFox, and a second unknown protein. In stained cells that were kept at 4°C, biotin-tagged label was confined to the outer cell surface (Fig. 6C). However, after internalization of bound iron for 1 h at 24°C, tagged vacuolar-like structures appeared at the apical side of the cells (Fig. 6D). Preincubation with the vacuolar inhibitor bafilomycin prevented the appearance of biotin-tagged vacuoles, but induced structural deformation that appears like thickening of the membrane surface at the apical side of the cells (Fig. 6E). Taken together, these results suggest that iron is internalized into acidic vacuoles by endocytosis of iron-binding proteins and that the process is inhibited by bafilomycin.

View larger version (158K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Fe localization with acid ferrocyanide staining. A, Cells before internalization. Inset, Magnified outer surface sections. Black arrowheads indicate iron particles. B, Cells after internalization. C to F, Magnified Fe-loaded vacuoles after internalization. Arrows, Iron-loaded vacuoles. Scale, 1 µm in A and B; 0.5 µm in A (inset) and C to F.

View larger version (54K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Localization of acidic vacuoles and endocytosis of biotin-tagged surface proteins in D. salina cells. A, Staining of acidic vacuoles with LysoSensor Green. Cells were preincubated for 10 min with the fluorescent probe. Acidic vacuoles are stained in green (arrow) and chlorophyll, which marks the cup-shaped chloroplast, is stained in red. B, Immunolocalization of acidic vacuoles with antibodies against V-H+-PPase. Cells were fixed, permeabilized, and incubated for 12 h with antibodies against C. reinhardtii V-H+-PPase and next with fluorescein goat anti-rabbit antibodies. Fluorescein indicating V-H+-PPase is stained in green (arrow) and chlorophyll is stained in red. C to E, Biotin tagging of surface proteins. Cells were labeled with biotin, washed, and either fixed immediately (C) or treated to bind and internalize iron in the absence (D) or presence (E) of 5 µM bafilomycin. Fixed cells were permeabilized and incubated with fluorescein avidin. Fluorescein, representing surface labeled proteins, is stained in green (arrow) and chlorophyll is stained in red.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The simplest kinetic model to describe iron uptake in D. salina according to our results is the following (see Supplemental Model S1):

Where Fe³⁺ represents free ferric ions, Tf-iron-binding proteins [Tf-Fe] bound iron and [Fe-V] internalized iron. k_b and k_int represent the rate constants for iron binding and internalization, respectively. k_b is much larger than k_int; therefore, the overall rate of Fe uptake is dominated by the rate of internalization, except at limiting Fe concentration. Overaccumulation of iron, according to this model, is predicted to inhibit k_int, leading to occupation of all iron-binding sites (accumulation of [Fe-Tf]) and to inhibition of Fe uptake. Iron deprivation, as shown, increases primarily Tf (4-fold) and k_b (2-fold), but not k_int or [Fe-V], as manifested by the large increase in Fe-binding capacity, Fe-binding rate constant, and the modest increase in Fe uptake, respectively. The 4-fold increase in Fe-binding capacity indicates induction of more iron-binding proteins at the plasma membrane. The increase in Fe-binding rate constant and the lower K_m for iron and for bicarbonate indicate intrinsic changes in the iron-binding sites. Conversely, the small increase in rate of internalization suggests that the number of Fe-binding units, [Fe-Tf], does not increase very much. Our kinetic results can be accommodated with the above kinetic model by inferring that, in Fe-deficient plasma membranes, small iron-binding units made of Fe-Tf are converted to larger iron-binding complexes with higher iron-binding capacity, without major changes in the number of Fe-binding units (or [Fe_n-Tf]_x [Fe_m-Tf]_y, where m n and x = y [approximately]). As mentioned above, we found recently that the increase in Fe binding in Fe-deficient cells is correlated with induction of three plasma membrane proteins, DTf (Schwarz et al., 2003a), DFox, and p130B, which associate with TTf (Paz et al., 2007). These results are consistent with the proposed kinetic model. The reasons why Dunaliella selected transferrins for iron binding are not clear. One reason may be that iron binding to transferrins is quite resistant to high salt. Another reason may be the high affinity and selectivity of transferrins for ferric ions, which would exclude competition by common divalent and trivalent metal ions that are often abundant in hypersaline and brackish water solutions.

The kinetic response to Fe deprivation differs very much from plants, other algae, yeast, and bacteria, where iron deprivation is usually associated with a dramatic increase in the rate of iron uptake.

However, such a mechanism is suitable for an organism that is exposed to large periodic changes in iron availability. The natural living habitats of Dunaliella are saline lakes and the open sea, which are often poor in bioavailable iron and obtain their iron supply from winter floods or dust carried by storms. The proposed mechanism of iron acquisition in D. salina offers a competitive advantage in comparison to carrier-mediated uptake mechanisms under such conditions because it enables efficient capture within a short time of large quantities of iron from diverse iron-binding ligands. Possible drawbacks of massive accumulation of proteins at the plasma membrane surface are the heavy energetic cost of protein synthesis, and it may interfere with other membrane functions because it occupies a large area of the plasma membrane.

The finding that D. salina internalizes iron into acidic vacuoles resembles iron uptake via the transferrin/transferrin receptor system in mammalian cells, but is unusual for plants that store iron primarily in the chloroplast bound to ferritin (Curie and Briat, 2003). Nevertheless, plant seeds store iron in acidic vacuoles (Lanquar et al., 2005) and it was recently reported that, in the presence of sufficient phosphate, Arabidopsis (Arabidopsis thaliana) stores iron as phosphate-iron complexes within the vacuole (Hirsch et al., 2006). Dunaliella, as well as other algae, store phosphate primarily as insoluble polyphosphates within acidic vacuoles (Pick and Weiss, 1991; Ruiz et al., 2001). Vacuolar polyphosphates may serve as an excellent sink for iron storage by providing a high-capacity iron-binding reservoir. Thus, acidic vacuoles may serve a central role in the regulation and storage of iron in D. salina. One manifestation of vacuolar regulation is the inhibition of iron binding and uptake as the vacuoles fill up. It should be realized, however, that the vacuoles are not the final station of iron in Dunaliella. We estimated from ⁵⁹Fe subcellular fractionation that, under steady-state growth conditions in Fe-sufficient medium, 70% to 80% of the cellular iron in D. salina is localized within the chloroplast, partly stored in ferritin, and partly incorporated into different proteins (U. Pick, unpublished data). Thus, similar to plants, in Dunaliella the chloroplast also seems to be the major consumer and storage site for iron and the acidic vacuoles are just an intermediary storage station. However, for an organism that takes up in a relatively short time enormous amounts of iron, vacuoles can serve as a safe buffering checkpoint that enables control of cytoplasmic levels of iron and avoidance of iron toxicity. Thus, Dunaliella has evolved a very exceptional mechanism for iron acquisition and homeostasis that may have special advantages for adaptation to hypersaline and iron-deficient environments.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Algal Strain and Growth Condition

Dunaliella salina, a green species, was obtained from the culture collection of Dr. W.H. Thomas (La Jolla, CA). All culture glassware was washed in acid and thoroughly rinsed with Mili-Q water. Cells were cultured in Fe-sufficient or in Fe-deficient medium as previously described (Fisher et al., 1997, 1998; Schwarz et al., 2003a). Standard growth medium contained 1 M NaCl, with or without FeCl₃/EDTA (1.5/6.0 µM) unless otherwise indicated.

Microscopic Techniques

Prussian Blue Staining of Iron
Ultrastructural localization of iron was made with acid ferrocyanide staining (Parmley et al., 1978). Iron-deficient cells were collected by centrifugation, washed once in 1 M NaCl, and suspended in iron uptake buffer containing growth medium without iron, 50 mM HEPES, pH 8.0, and 5 mM NaHCO₃. Cells were incubated with 10 µM Fe citrate for 30 min on ice (bound iron) or for 60 min at room temperature in the light under continuous shaking (internalized iron). Cells were fixed in 3.7% paraformaldehyde (p-formaldehyde) plus 1% glutaraldehyde and were next reacted for 30 min with fresh Perl's ferrocyanide solution, consisting of 0.5 g of potassium ferrocyanide in 49.5 mL of distilled water to which 0.5 mL of concentrated HCl (32%) was added. After extensive washing, fixed samples were rehydrated and embedded in Epon. Thin sections were examined in a Tecnai T12 (FEI) electron microscope operating at 120 kV and images were recorded using a MegaView III CCD camera (SIS GmbH).

Staining Acidic Vacuoles with LysoSensor Green
D. salina cells (2 x 10⁷ cells/mL in fresh growth medium, pH 8) were incubated for 10 min with 2 µM LysoSensor Green DND-189 (Molecular Probes) from a 1 mM stock solution in dimethylsulfoxide, washed twice, incubated for 10 min with 0.25% p-formaldehyde, and placed on a polylysine-coated glass slide. Cells were viewed and photographed in a laser-scanning confocal microscope (Fluoview FV500; Olympus) using an excitation wavelength of 458 nm and emission of 510 to 550 nm or of >660 nm to view LysoSensor Green and chlorophyll, respectively.

Immunolocalization
To immunolocalize acidic vacuoles, D. salina cells were labeled with antibodies against Chlamydomonas reinhardtii vacuolar H⁺-pyrophosphatase (V-H⁺-PPase), obtained from Dr. R. Decampo (University of Illinois, Urbana, IL). Cells were fixed for 1 h with freshly prepared 3.7% p-formaldehyde, permeabilized for 5 min with 1% NP-40, washed twice, incubated for 60 min with blocking medium containing 4% goat serum, 1% bovine serum albumin, and 0.05% Tween 20 in phosphate-buffered saline. The preparation was incubated for 12 h with rabbit anti V-H⁺-PPase serum (at 1:200 dilution) in blocking solution at 5°C, washed twice, incubated with fluorescein goat anti-rabbit polyclonal antibodies (Jackson ImmunoResearch Laboratories), washed twice, and viewed and photographed in a confocal microscope as above at excitation wavelength 488 nm and emission wavelengths 505 to 525 nm or >660 nm to view fluorescein and chlorophyll, respectively.

Iron Uptake Assay

Iron uptake in D. salina cells was measured as previously described (Fisher et al., 1998). Cells (5 x 10⁷ cells/mL) were incubated with ⁵⁹Fe citrate in the light for 1 h, the uptake was stopped by dilution into an acidified EDTA medium (pH 5.5), left for 15 to 30 min on ice, collected by centrifugation, and washed once again in the same medium. This treatment was found to remove all externally bound iron, but not internalized iron. Washed cells were counted in a -counter.

Iron-Binding Assay

Before the binding assay, cells were treated to unload prebound iron by preincubation for 1 h at 23°C in buffer containing growth medium without iron, 50 mM HEPES, pH 8.0, and 5 mM NaHCO₃ in the light. Iron binding to intact cells was measured essentially as previously described (Fisher et al., 1998). In brief, cells were incubated for 30 min at 4°C with ⁵⁹Fe citrate, washed in EDTA-free buffer (stop solution), and counted in a -counter. Under these conditions, >95% of the cell-associated iron was found to be externally bound to transferrins (bicarbonate dependent, EDTA sensitive). Plasma membrane vesicles were incubated for 1 h on ice with ⁵⁹Fe citrate in 50 mM Na-HEPES 8.0, 0.2 M KCl, and 5 mM NaHCO₃ and passed through semidry Sephadex G-50 columns to eliminate unbound ⁵⁹Fe as previously described (Schwarz et al., 2003b).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Model S1. Kinetic model of Fe binding and internalization.

	ACKNOWLEDGMENTS

 
We wish to thank Dr. Roberto Docampo from the University of Illinois for providing antibodies against C. reinhardtii V-H⁺-PPase.

Received April 10, 2007; accepted May 11, 2007; published May 18, 2007.

	FOOTNOTES

 
¹ This work was supported by the Israel Science Foundation and the Charles and Louise Gartner fund (grants to U.P.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Uri Pick (uri.pick{at}weizmann.ac.il).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.100644

^* Corresponding author; e-mail uri.pick{at}weizmann.ac.il; fax 972–8–9344118.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Allnutt FCT, Bonner WD (1987) Characterization of iron uptake from ferrioxamine B by Chloella vulgaris. Plant Physiol 85: 746–750[Abstract/Free Full Text]

Arbab AS, Frenkel V, Pandit SD, Anderson SA, Yocum GT, Bur M, Khuu HM, Read EJ, Frank JA (2006) Magnetic resonance imaging and confocal microscopy studies of magntically-labeled endothelial progenitor cells trafficking to sites of tumor angiogenesis. Stem Cells 24: 671–678[CrossRef][Web of Science][Medline]

Benderliev KM, Ivanova NI (1994) High-affinity siderophore-mediated iron transport system in the alga Scenedesmus incrassatulus. Planta 193: 163–166[Web of Science]

Boye M, van der Berg CMG (2000) Iron availability and the release of iron-complexing ligands in Emiliania huxleyi. Mar Chem 70: 277–287[CrossRef][Web of Science]

Curie C, Briat JF (2003) Iron transport and signaling in plants. Annu Rev Plant Biol 54: 183–206[CrossRef][Medline]

Eckhardt U, Buckout TJ (1998) Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to strategy I higher plants. J Exp Bot 49: 1219–1226[Abstract/Free Full Text]

Fisher M, Gokhman I, Pick U, Zamir A (1997) A structurally novel transferrin-like protein accumulates in the plasma membrane of the unicellular green alga Dunaliella salina grown in high salinities. J Biol Chem 272: 1565–1570[Abstract/Free Full Text]

Fisher M, Zamir A, Pick U (1998) Iron uptake by the halotolerant alga Dunaliella is mediated by a plasma membrane transferrin. J Biol Chem 273: 17553–17558[Abstract/Free Full Text]

Hell R, Stephan UW (2003) Iron uptake, trafficking and homeostasis in plants. Planta 216: 541–551[Web of Science][Medline]

Herbik A, Bölling C, Buckhout TJ (2002) The involvement of a multicopper oxidase in iron uptake by the green alga Chlamydomomonas reinhardtii. Plant Physiol 130: 2039–2048[Abstract/Free Full Text]

Hirsch J, Marin E, Floriani M, Chiarenza S, Richaud P, Nussaume L, Thibaud MC (2006) Phosphate deficiency promotes modification of iron distribution in Arabidopsis plants. Biochimie 88: 1767–1771[Medline]

Kim HJ, Kim HM, Kim JH, Ryu KS, Park SM, Jahng KY, Yang MS, Kim DH (2003) Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae. Appl Environ Microbiol 69: 1999–2005[Abstract/Free Full Text]

La Fontaine S, Quinn JM, Nakamoto SS, Page MD, Göhre V, Moseley JL, Kropat J, Merchant S (2002) Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii. Eukaryot Cell 1: 736–757[Abstract/Free Full Text]

Lanquar V, Lelievre F, Bolte S, Hames C, Alcon C, Neumann D, Vansuyt G, Curie C, Schroder A, Kramer U, et al (2005) Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination at low iron. EMBO J 24: 4041–4051[CrossRef][Web of Science][Medline]

Lynnes JA, Derzaph TLM, Weger HG (1998) Iron limitation results in induction of ferricyanide reductase and ferric chelate reductase activities in Chlamydomonas reinhardtii. Planta 204: 360–365[CrossRef][Web of Science]

Mori S (1999) Iron acquisition by plants. Curr Opin Plant Biol 2: 250–253[CrossRef][Web of Science][Medline]

Parmley RT, Spicer SS, Alvarez CJ (1978) Ultrastructural localization of nonheme cellular iron with ferrocyanide. J Histochem Cytochem 26: 729–741[Abstract]

Paz Y, Katz A, Pick U (2007) A multicopper ferroxidase involved in iron binding to transferrins in Dunaliella salina plasma membranes. J Biol Chem 282: 8658–8666[Abstract/Free Full Text]

Pick U (2004) The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta). Biometals 17: 79–86[CrossRef][Web of Science][Medline]

Pick U, Weiss M (1991) Polyphosphate hydrolysis within acidic vacuoles in response to amine-induced alkaline stress in the halotolerant algae Dunaliella salina. Plant Physiol 97: 1234–1240[Abstract/Free Full Text]

Pick U, Zeelon O, Weiss M (1991) Amine accumulation in acidic vacuoles protects the halotolerant alga Dunaliella salina against alkaline stress. Plant Physiol 97: 1226–1233[Abstract/Free Full Text]

Ruiz FA, Marchesini M, Seufferheld M, Govindjee, Docampo R (2001) The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase activity and are similar to acidocalcisomes. J Biol Chem 276: 46196–46203[Abstract/Free Full Text]

Schwarz M, Sal-Man N, Zamir A, Pick U (2003a) A transferrin-like protein that does not bind iron is induced by iron deficiency in the alga Dunaliella salina. Biochim Biophys Acta 1649: 190–200[Medline]

Schwarz M, Zamir A, Pick U (2003b) Iron-binding properties of TTf, a salt-induced transferrin from the alga Dunaliella salina. J Plant Nutr 26: 2081–2091[CrossRef][Web of Science]

Tallheden T, Nannmark U, Lorentzon M, Rakotoniraini O, Soussi B, Waagstein F, Jeppsson A, Sjogren-Jansson E, Lindhal A, Omerovic E (2006) In vivo MR imaging of magnetically-labeled human embryonic stem cells. Life Sci 79: 999–1006[CrossRef][Web of Science][Medline]

Vasconcelos M, Datta K, Oliva N, Khalekuzzaman M, Torrizo L, Krishnan S, Oliveira M, Goto F, Datta SK (2003) Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene. Plant Sci 164: 371–378[CrossRef][Web of Science]

Weiss M, Pick U (1991) Uptake of the fluorescent indicator atebrin into acidic vacuoles in the halotolerant alga Dunaliella salina. Planta 185: 494–501[Web of Science]

This Article Free via Open Access: OA OA Abstract Full Text (PDF) Supplemental Data OA All Versions of this Article: 144/3/1407    most recent pp.107.100644v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Paz, Y. Articles by Pick, U. Search for Related Content PubMed PubMed Citation Articles by Paz, Y. Articles by Pick, U. Agricola Articles by Paz, Y. Articles by Pick, U.