Atypical iron storage in marine brown algae: a multidisciplinary study of iron transport and storage in Ectocarpus siliculosus

Abstract

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood, with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins, while strategy II plants utilize high-affinity, iron-specific, binding compounds called phytosiderophores. In contrast, little is known about the corresponding systems in marine, plant-like lineages, particularly those of multicellular algae (seaweeds). Herein the first study of the iron uptake and storage mechanisms in the brown alga Ectocarpus siliculosus is reported. Genomic data suggest that Ectocarpus may use a strategy I approach. Short-term radio-iron uptake studies verified that iron is taken up by Ectocarpus in a time- and concentration-dependent manner consistent with an active transport process. Upon long-term exposure to ⁵⁷Fe, two metabolites have been identified using a combination of Mössbauer and X-ray absorption spectroscopies. These include an iron–sulphur cluster accounting for ~26% of the total intracellular iron pool and a second component with spectra typical of a polymeric (Fe³⁺O₆) system with parameters similar to the amorphous phosphorus-rich mineral core of bacterial and plant ferritins. This iron metabolite accounts for ~74% of the cellular iron pool and suggests that Ectocarpus contains a non-ferritin but mineral-based iron storage pool.

Introduction

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood, with two basic strategies for iron uptake being distinguished: strategy I plants, mainly dicotyledons, use a mechanism involving soil acidification and induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins (Moog and Bruggemann, 1994; Robinson et al., 1999); while strategy II plants (in particular monocotyledons/grasses) have evolved sophisticated systems, similar to those of bacteria and fungi, based on high-affinity, iron-specific, binding compounds called phytosiderophores (Romheld and Marschner, 1986).

In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages, particularly the multicellular macroalgae (seaweeds). This is important as the iron level in ocean waters is even lower than in most terrestrial environments due both to the low solubility of Fe(III) in oxic seawater and to the fact that a large fraction of the limited iron available is already tightly complexed (Bruland et al., 1991). Indeed, iron availability is now well known to limit primary productivity in certain oceanic regimes (Martin and Fitzwater, 1988). While there is some evidence that marine algae produce siderophore-like molecules (Trick et al., 1983; Naito et al., 2001), to date not a single extracellular metal chelator produced by eukaryotic algae has been characterized, and ambiguity remains about whether the siderophore-like molecules purported to be isolated from cultures of eukaryotic algae are actually produced by the algae themselves or by associated bacteria.

While efficient transport mechanisms for iron uptake are an essential element in all pro- and eukaryotic cells, its intracellular availability and storage have to be tightly regulated, not only to buffer supply and demand, but also to prevent cell damage from undesirable reactions of free radicals, formed catalytically by free Fe ions. Ferritin represents the most common form of iron storage in all domains of life. This water-soluble protein is composed of a tetraeicosameric shell built up of polypeptide subunits and a microcrystalline core of ferrihydrite within the protein cavity. A general structural model of ferritins has been derived from X-ray diffraction studies (Ford et al., 1984; Harrison et al., 1989; Lawson et al., 1991; Frolow et al., 1994; Trikha et al., 1994). Although the general topology of most ferritins is similar, a remarkable heterogeneity of the ferritin subunits is observed which is the basis of different classes of ferritins including various types of bacterial ferritins, namely haem-containing bacterioferritins (Bfrs), non-haem bacterial ferritins Ftn1 and Ftn2, ‘miniferritins’ (exhibiting a dodecahedral peptide assembly), and various animal and plant ‘maxiferritins’. Numerous functions have been attributed to these ferritins. One function is associated with ‘true’ iron storage. Under iron-rich growth conditions the metal is accumulated in order to provide an iron pool sufficiently high to prevent growth limitation effects in an iron-deficient environment. A second function is associated with the potentially harmful role iron can play in cell physiology by generating OH·^– and other oxygen radicals (Haber–Weiss–Fenton reaction cycle) (Matzanke, 1997). These oxygen radicals, and in particular OH·, may cause cellular oxidative damage and therefore participate in ageing processes and carcinogenesis (Halliwell and Gutteridge, 2007). A role for ferritin against oxidative stress has been shown in Arabidopsis (Ravet et al., 2009).

Brown algae (Phaeophyta) belong to a lineage that has been evolving independently of other major photosynthetic lineages, such as green plants (Chlorophyta) and red algae (Rhodophyta). Instead, they are classified within the Stramenopiles and Chromalveolates together with diatoms, golden-brown algae, and oomycetes (Baldauf, 2003). As a consequence of this singular evolutionary history, brown algae exhibit many unusual, and often unique, features. These features are adaptations to the marine coastal environments in which brown algae are usually the dominant organisms in terms of biomass, often forming extensive kelp forests. The key role of kelp forests, effectively constituting an interface between the ocean, the atmosphere, and land masses, in the biogeochemical cycle of halogens is well established (Küpper et al., 2008). However, the role of trace metals in brown algal-dominated ecosystems is poorly understood (as is brown algal trace metal metabolism), contrasting with the intense research interest which pelagic systems have received. This lack of knowledge is surprising in view of the fact that the industrial exploitation of brown algae is expanding partially due to interest in their use for production of alginate, fucans etc., but increasingly for their potential as biofuel where they have the advantage of high productivity without competing with terrestrial crops for farmland.

Ectocarpus siliculosus is a filamentous brown alga with a worldwide distribution along temperate coastlines, and is a nuisance as a ‘fouling’ organism on many man-made surfaces in the sea. It has some significant advantages as an experimental model and constitutes one of the best-studied seaweeds (Peters et al., 2004; Charrier et al., 2008). It can easily be cultivated in small volumes of seawater media both axenically and with associated bacteria, its entire, well-known life cycle can be completed within a few months in culture (Müller et al., 2008), and many molecular tools are available, including mutant collections, microarrays, and proteomics data. It has also recently become the first seaweed of which the entire genome has been sequenced and thus offers unprecedented opportunities for study (Cock et al., 2010).

While modern spectroscopic techniques such as Mössbauer spectroscopy and X-ray absorption spectroscopy (XAS) have played a major role in our understanding of iron uptake and storage in many terrestrial microorganisms (Winkler et al., 1994; Schünemann and Winkler, 2000; Matzanke et al., 2011), they have been little utilized in marine algal systems. Such spectroscopic techniques are powerful, non-invasive tools for the determination of both the in vivo redox, spin state, and coordination environment of iron, as well as for isolated biological (macro)molecules. Transmission Mössbauer spectroscopy (TMS) is valuable as it is specific for ⁵⁷Fe and thus no other transition metal obscures the experimental results. Due to the low natural abundance of the isotope (2%), ⁵⁷Fe enrichment is essential for almost any biological sample. This potential disadvantage, however, can be successfully exploited for iron uptake analyses since a sample prior to uptake typically exhibits ⁵⁷Fe in quantities below the detection limit of conventional Mössbauer spectroscopy. Thus only labelled newly acquired Fe is visible. XAS confirms and broadens the information obtained by Mössbauer. From XANES (X-ray absorption near edge structure), the average oxidation state of the metal centre and the metal ligand coordination geometry can be derived. Extended X-ray absorption fine structure (EXAFS) analysis provides metal–ligand bond distances at a high accuracy (±0.02 Å), metal–ligand type, and coordination number of the complex (albeit with lower accuracy, i.e. error bars of at least ±0.5). Herein the first ever study of the iron uptake and storage mechanisms in the brown alga E. siliculosus using such spectroscopic techniques in concert with more conventional radiolabelled uptake studies is reported.

Materials and methods

Ectocarpus siliculosus strain EcSil NZ KU 1–3♂ (CCAP 1310NZ1310-56) was obtained from the Culture Collection of Algae and Protozoa (CCAP) at the Scottish Association for Marine Science and grown axenically in modified Provasoli-enriched (Andersen, 2005) Scripps Pier seawater (SPSW) at 17 °C with a 12 h:12h light:dark photocycle. The iron content of SPSW was determined to be ~4nM and is thus defined as the concentration for iron-limited growth. Prior to all experiments, Ectocarpus was starved for a period of 5–10 d under iron-limited conditions. Iron-replete conditions were obtained by adding Fe-EDTA to SPSW at 30 µM.

Cell surface reductase activity

Cell surface reductase activity was determined as described by Kranzler et al. (2011) using C18 Sep-Pak columns (Waters) which absorb the ⁵⁵Fe(FZ)₃ complex formed by reduction of ⁵⁵FeEDTA in the presence of an excess of the Fe(II)-specific chelator ferrozine (FZ). After elution from the column with methanol, the ⁵⁵Fe(FZ)₃ was quantified by liquid scintillation counting as described below.

Iron uptake studies

⁵⁵FeCl₃ radionuclide was obtained from Perkin-Elmer and used to prepare the ⁵⁵FeEDTA solution used as an iron source. To remove any surface-adsorbed Fe(III) species, Ectocarpus samples were strained, and washed with 50ml of artificial seawater (ASW), followed by 10ml of a titanium (III) citrate/EDTA solution prepared as described by Hudson and Morel (1989). This was followed by a final wash with 50ml of ASW to remove all traces of Fe(II). Samples were then filtered onto 10 µm Millipore™ polycarbonate filters and washed with 50ml of ASW under a vacuum. Thorough washing with both the titanium (Ti) reagent and subsequently with ASW is essential to eliminate artefacts caused by surface binding. Filters containing Ectocarpus were placed in scintillation vials and 1ml of sodium hypochlorite was added to bleach the chlorophyll and reduce quenching effects. Vials were then heated in a 55 °C water bath for 1h and left to vent overnight at room temperature to allow chlorine evaporation. A 15ml aliquot of Hionic Fluor liquid scintillation fluid (Perkin-Elmer) was added to each of the vials which were incubated in the dark for at least 2h to eliminate any background chemiluminesence. The ⁵⁵Fe taken up was measured on a Beckman-Coulter LS 6500 scintillation counter using the tritium channel. Total iron uptake per mg wet weight Ectocarpus was calculated based on specific activity, measured count rates, scintillation counting efficiency, and biomass measurements. For inhibition studies, azide, carbonyl cyanide 3-chlorohydrazine (CCCP), ascorbate, FZ, and the ionophores gramicidin and valinomycin were added to separate iron-limited cultures 1h prior to inoculation with ⁵⁵FeEDTA.

Histochemistry

Ectocarpus siliculosus was grown under iron-replete conditions prior to fixation, dehydration, and embedding. Cells were fixated in a 0.1M phosphate buffer solution containing 2% (w/v) paraformaldehyde, 1% (w/v) glutaraldehyde, and 1% (w/v) caffeine for 2h. The fixed cells were then washed with 0.1M phosphate buffer and dehydrated in successive ethanol baths of 30, 50, 75, 85, 95, and 100% (three times). The cells were then embedded in 1:1 (v/v) ethanol/LR White resin (LWR; EMS, Hatfield, PA, USA) for 3h followed by 100% LWR overnight in gelatin capsules under vacuum. Sections of 3 µm were cut on a Leica EMUC6 microtome and deposited on glass slides. The Perls staining and diaminobenzidine (DAB) intensification procedure was performed as described by Meguro et al. (2007) and Roschzttardtz et al. (2009, 2010). Briefly, sections were incubated on glass slides with equal volumes of 4% (v/v) HCl and 4% potassium ferrocyanide (Perls staining solution) for 45min. After washing with distilled H₂O, sections were incubated in a methanol solution containing 0.01M NaN₃ and 0.3% (v/v) H₂O₂ for 1h and then washed with 0.1M phosphate buffer. DAB intensification was achieved by incubating sections in a 0.1M phosphate buffer solution containing 0.00025–0.005% (w/v) DAB (Sigma), 0.005% (v/v) H₂O₂, and 0.005% (w/v) CoCl₂ for 30min. The sections were then washed with H₂O before imaging with a Zeiss Axiovert 40 inverted microscope.

Transmission Mössbauer spectroscopy (TMS)

For TMS, Ectocarpus was grown for 33 d in modified Provasoli-enriched seawater containing 30 µM ⁵⁷FeEDTA. ⁵⁷Fe was obtained as the oxide from Isoflex (San Francisco, CA, USA) and converted to the chloride by repeated dissolution in concentrated HCl. The chloride was subsequently added to a concentrated solution of EDTA and the pH adjusted to 6.0. Algal samples were washed with the Ti citrate/EDTA reagent in order to remove adventitious iron from the algal surface, and the cells were harvested by vacuum-assisted filtration. Pellets were weighed, transferred into Delrin^® Mössbauer sample holders, frozen in liquid nitrogen, and kept at this temperature until measurement, except for overnight transport on dry ice. The Mössbauer spectra were recorded in the horizontal transmission geometry using a constant acceleration spectrometer operated in conjunction with a 512-channel analyser in the time-scale mode. The detector consisted of a proportional counter filled with argon–methane (9:1). The source was at room temperature and consisted of 1.4 GBq (⁵⁷Co) diffused in Rh foil (WissEl, Starnberg, Germany). The spectrometer was calibrated against α-iron at room temperature. For measurements at 77K, samples were placed in a continuous-flow cryostat (Oxford Instruments). For measurements at 4.3K and 2K, a helium bath cryostat (MD306, Oxford Instruments) was employed. Spectral data were transferred from the multichannel analyser to a PC for further analysis employing the public domain Vinda program on an Excel 2003^® platform. Isomer shift δ, quadrupole splitting ΔE_Q, B_hf, and percentage of the total absorption area were obtained by least-squares fits of Lorentzian lines to the experimental spectra. All values are rounded to the last given digit. The isomer shifts (δ), the quadrupole splitting (ΔE_Q), and the line width (Γ) are given in mm s^–1. The relative area is given in parts per hundreds.

EXAFS

Spectra were measured at Doris III, beamline A1 (DESY, Hamburg, Germany) using a fluorescence detector. Frozen samples were measured in a continuous flow cryostat (Oxford Instruments, Optistat) at 12K. The energy was calibrated in transmission mode against an iron foil while experimental spectra were recorded in the fluorescence mode. Algal samples were prepared as described above, and samples CC45 (Mössbauer) and CC47 (EXAFS) were prepared from the same batch. During sample handling, the sample temperature was kept below 195K.Thirty-nine energy scans from 6960eV to 8112eV were performed, resulting in 52 spectra. Spectra obtained during ring filling were skipped. Each scan was performed as follows: from 6960eV to 7085eV the energy step E was 5eV, from 7085eV to 7152eV ΔE was 0.5eV, and from 7152eV to 8112eV non-equidistant energy steps were used starting at ΔE=0.8eV. In the first two regions, a sampling time of 1 s was used. In the third region, an increasing sampling time was used corresponding to the non-equidistant energy steps starting at 1 s.

In each spectrum, the K-edge was localized by the zero crossing of the second derivative of the fluorescence signal and corrected against the iron foil K-edge. All spectra were summed up prior to correction. Afterwards, the sum was corrected as follows: the pre-edge range was defined relative to the determined K-edge from –150eV to –30eV and fitted with a line. The post-edge range for normalization was set relative to the K-edge from +150eV to +880eV and was fitted with a quadratic polynomial. The sum spectrum was corrected for its background, its pre-edge, and normalized to its post-edge. Forward Fourier transformation was performed for a k-range from 2 Å^–1 to 8.5 Å^–1 using a Hanning window function. The final overall fit of the two-component model (see below) uses scattering paths with distances <3.4 Å. The fits were performed in R-space using a Hanning type window from r=1.2 Å to 3.35 Å. For the least square fits in R-space, a k-weight of 2 was used. All corrections and fits were performed by the Athena/Artmis program package of Ravel (2005).

Results

Cell surface reductase activity

With the detection of a putative cell surface reductase in the Ectocarpus genome (Cock et al., 2010), experimental confirmation of external Fe(III) chelate reduction was sought. For these experiments, Ectocarpus cells previously grown under either iron-replete (30 µM) or limited (4nM) conditions (see the Materials and methods) were incubated in the presence of 30 µM ⁵⁵Fe FeEDTA and 100 µM of the Fe(II)-specific chelator FZ for 24h. Cells were then harvested and the ⁵⁵Fe(FZ)₃ complex formed in the supernatant assayed as described by Kranzler et al. (2011). As can be seen from Fig. 1, live, iron-limited, Ectocarpus cells were readily capable of reducing Fe(III) in the form of an EDTA complex at a rate of ~1ng Fe mg^–1 h^–1, that is commensurate with that of overall iron uptake (see below). Dead cells exhibited no activity, indicating that iron reduction was a specific metabolic process rather than a non-specific process driven by the presence of FZ. Under iron-replete conditions, ferric chelate reduction was reduced by almost half, suggesting that the reductase activity is inducible.

Fig. 1.

Fe(III) chelate reductase activity for (A) iron-replete (30 µM) and (B) iron-starved (4nM) cultures of Ectocarpus siliculosus grown as described in the Materials and methods. (C) Dead cells and (D) live cells minus FZ represent negative controls. Error bars represent ±1 SD from triplicate measurements.

Iron uptake

Iron ‘uptake’ without the Ti washing procedure described in the Materials and methods was always very high and time independent; observations that are consistent with strong, non-specific, cell surface binding. This non-specific binding was confirmed by TMS which showed a very strong signal with spectral parameters distinct from those much weaker signals seen following the Ti wash (see below). Careful and extensive washing of the cells with ASW following the Ti wash was also required to prevent the artefactual observation of residual surface-bound Fe(II) by TMS. However, with the appropriate care, ⁵⁵Fe from FeEDTA was seen to be taken up by iron-starved Ectocarpus in a time-dependent fashion. Uptake was relatively rapid (0.44ng mg^–1 h^–1) and approximately linear for ~24h, after which the uptake rate slowed and eventually ceased (Fig. 2). The uptake process appeared to be an active one as it was inhibited by a number of metabolic poisons or environmental effects. Pre-treatment of cells with azide, CCCP, ascorbate, gramicidin, or valinomycin resulted in 25–90% uptake inhibition (data not shown), whereas FZ had no effect. The uptake process was also saturable as determined by its concentration dependence (Fig. 3) the data of which could be fit well (R ²=0.9939) to a model with a V _max of 1.47±0.15ng mg^–1 h^–1, and a K _m of 1.5±0.5 µM.

Fig. 2.

Iron uptake from ⁵⁵FeEDTA as a function of time in Ectocarpus siliculosus cultures over 800h. Error bars represent ±1 SD from three separate experiments with replicate time points for each.

Fig. 3.

Concentration-dependent uptake of iron from ⁵⁵FeEDTA after 24h by Ectocarpus siliculosus. Error bars represent ±1 SD from duplicate experiments with replicate concentration points for each.

FeEDTA was used as an iron source as it is generally thought that the intact FeEDTA complex is not a biological substrate but rather it serves as an iron buffer maintaining a fixed concentration of free soluble Fe(III) known as Fe(III)’ at equilibrium while preventing the precipitation of insoluble Fe oxo-hydroxo polymeric species. To test the suitability of this hypothesis with Ectocarpus, the iron uptake rate at a fixed concentration of iron as a function of an increasing EDTA to Fe ratio was determined. Increasing the EDTA to Fe ratio should increase the concentration of FeEDTA and decrease the concentration of free Fe(III) at equilibrium. Thus, if FeEDTA itself was the biological substrate, the uptake rate should increase, while if free Fe(III) was the substrate, then uptake should decrease. Upon going from an EDTA/Fe ratio of 1.5:1 to 100:1, a 50-fold decrease in the uptake rate was observed, consistent with the idea that FeEDTA is serving simply as an iron buffer and the species actively involved in uptake is free Fe(III)’.

Histochemistry

Using the Perls–DAB staining protocol described by Roschzttardtz et al. (2009, 2010), it is possible to visualize the localization of iron at the subcellular level. The highest levels of iron consistently appeared as small dark granules clustered together inside the cell and not associated with the plastids, which in Ectocarpus typically adopt a spiral banded pattern (Fig. 4). Control cell slices not treated with Perls–DAB do not show such a staining pattern. While the exact nature of the structures that contain the majority of iron remains obscure, it is clear that large concentrations of iron appear to be stored inside Ectocarpus cells.

Fig. 4.

Micrographs (×63) of 3 µm sections of Ectocarpus cells embedded in LWR and stained by the Perls–DAB procedure as outlined in the Materials and methods. Upper panel: treatment at pH 7 with 0.0025% DAB. Lower panel: treatment at pH 1 with 0.005% DAB. The scale bars are 20 µm and the dark grains represent high concentrations of iron. (This figure is available in colour at JXB online.)

Mössbauer spectroscopy

After long-term incubation with ⁵⁷FeEDTA, TMS spectra exhibiting sufficient resonance absorption were obtained which display a single quadrupole doublet-like feature (Fig. 5). Since the algal filament was thoroughly washed with the Ti citrate/EDTA reagent, the presence of iron on the algal surfaces can be excluded and, therefore, the iron components observed by TMS are genuinely of intracellular origin. From this, it can be concluded that ⁵⁷Fe supplied as the EDTA complex in the medium is transported into, and metabolized inside, cells of Ectocarpus. However, despite its seeming simplicity, detailed analysis of the TMS spectrum showed the presence of two different and distinct iron species. The first corresponds to an (Fe₄S₄)²⁺ cluster. Biological [Fe₄S₄]ⁿ⁺ clusters are typically found in three cluster oxidation states: 1+, 2+, and 3+ (Schünemann and Winkler, 2000). All three types of [Fe₄S₄] cluster exhibit characteristic Mössbauer parameters which can be distinguished in most cases quite well. An iron–sulphur cluster in the +2 state displays at 4.3K isomer shifts in the range from 0.39mm s^–1 to 0.45mm s^–1 and quadrupole splittings from 0.98mm s^–1 to 1.22mm s^–1. Isomer shift and quadrupole splitting of the iron–sulphur species in the in situ Mössbauer spectrum of E. siliculosus are in the range found for such a cluster (26% of the absorption area, Table 1). This iron–sulphur cluster very probably represents a component of chloroplast and/or mitochondrial redox systems. The second iron compound detected by TMS displays a spectrum typical of a polymeric (Fe³⁺O₆) system which accounts for 74% of the absorption area (Table 1, Fig. 5). Polymeric biological (Fe³⁺O₆) systems found by in situ Mössbauer spectra very often represent the mineral cores of ferritins. The Mössbauer spectroscopic features of such systems are strongly temperature and size dependent, reflecting superparamagnetic relaxation of magnetic nanoparticles (Mørup, 2011). Bacterial ferritins (Bfr and Ftn) typically show superparamagnetic splitting (doublet–sextet transition) at temperatures below 4.3K, indicating an amorphous and frequently phosphate-rich crystal structure (Bauminger et al., 1980a , b ; Mann et al., 1987; Matzanke, 1997; Reindel et al., 2002; Boughamoura et al., 2008). The sextet lines show one-third of the resonance absorption compared with the doublet lines, and are, in addition, considerably broadened due to particle size distributions of the mineral. The Mössbauer spectrum of Ectocarpus at 1.8K shows less resonance absorption in the centre part compared with that seen at 4.3K (0.2 transmission intensity as compared with 0.45), indicating the partial disappearance of one doublet. The disappearance of the (Fe³⁺O₆) doublet can only be explained (since the Lamb–Mössbauer factor, f, at 1.8K cannot be smaller than that at 4.3K) by the formation of a broadened six-line pattern; the result of a doublet–sextet transition as found in bacterial ferritins. However, due to the low intensity of the (Fe³⁺O₆) doublet lines, the six-line pattern is at the detection limits of TMS (0.1% effect per line or even less). The residual absorption at 1.8K could be adequately fit with just the iron–sulphur species.

Fig. 5.

Mössbauer spectra of Ectocarpus siliculosus at 150K (upper), 4.3K (middle), and 1.8K (lower). The filled squares represent the experimental data. The subspectra obtained by least squares fits of Lorentzian lines are depicted by the light grey solid line (Fe₄S₄) and dark grey dotted line (polymeric FeO₆). The black solid line represents the overall fit. (This figure is available in colour at JXB online.)

Table 1.

Mössbauer fit parameters of isomer shift (δ), quadrupole splitting (ΔE _Q ), linewidth (Γ), and percentage of absorption area of E. siliculosus (CC45) at 150, 4.2, and 1.8KThe error in the last digit is 0.04mm s^–1.

	Parameter	In mm s–1 at 1.8K	In mm s–1 at 4.2K	In mm s–1 at 150K
[Fe3+O6Xm n]mn–9	δ	0.44	0.44	0.39
	ΔEQ	0.68	0.68	0.68
	Γ	0.66	0.66	0.66
	Area	50%	74%	74%
[Fe4-S4]2+	δ	0.43	0.43	0.40
	ΔEQ	1.13	1.13	1.13
	Γ	0.41	0.41	0.41
	Area	50%	26%	26%
SQRT		0.86	0.79	0.80

XAS

In addition to TMS, XAS was employed to probe the chemical nature of the internalized iron. Based on the above Mössbauer data, the presence of a superparamagnetically coupled polymeric Fe³⁺-oxo system and of an [Fe₄S₄]²⁺ cluster was indicated. Within this constraint, various models were tested. For the Fe³⁺-oxo species, the models tested included ferric (oxo, oxo-organic, oxo-phosphate), ferrihydrite, ferric phosphate, and FeO(OH). Models for these species were constructed and coordinates obtained after molecular mechanics energy minimization using the program Chem 3D. These coordinates were then used as input in the FEFF program to obtain the scattering paths. The best fits came from the oxo-phosphate model. However, the resulting fits were still unsatisfactory when only oxo-phosphate ligands were employed in the first ligand sphere. There was definitely an iron–sulphur contribution which was consistent with the Mössbauer spectra. Structural data for various iron–sulphur clusters were extracted from the appropriate data banks, and coordinates extracted after molecular mechanics energy minimization using the Program Chem3D. The resulting coordinates were again used for input in the FEFF program to obtain the scattering paths. Acceptable fits were only obtained for an [Fe₄S₄]²⁺ cluster.

The final fit of the EXAFS spectrum (Figs 6, 7) to the two-component model {i.e. polymeric Fe(III) oxo-phosphate and an [Fe₄S₄]²⁺ cluster} was very good where the second coordination shell of the Fe-oxo species contains 3.5±0.5 P and 1±0.5 Fe (Table 2). The Fe-oxo species comprises ~74% and the Fe–S species ~26% of the total iron. The average bond distances obtained from the EXAFS fit are summarized in Table 2. Attempts to add additional components such as an [Fe₂S₂]²⁺ (or additional Fe-oxo species) provided no statistically significant improvement to the fit. Overall, the EXAFS fit data are completely consistent with those obtained by TMS. In particular, they support both quantitatively and qualitatively the polymeric nature of the iron oxo species and the presence of an iron–sulphur protein as suggested by the Mössbauer spectra.

Fig. 6.

Extracted EXAFS spectrum of 52 merged spectra transformed in q-space. The filled squares represent experimental data, the black solid line represents the fit, and the light grey dotted line depicts the setting of the range. (This figure is available in colour at JXB online.)

Table 2.

Average distances and numbers of first and second shell ligands in Fe–O and Fe–S iron centres of sample CC47 obtained from the EXAFS fit as described in the textThe accuracy for ligand numbers n is ±0.5.

	Fe–O	Fe–(O)–P	Fe–(O)–Fe	Fe–S	Fe–(S)–Fe
Å	1.96	3.07	3.35	2.11	2.64
n	6	3.5	1	4	3

Elements in parentheses display bond angles indicating that the Fe–(X)–Y distance is shorter than the summation of individual bond lengths.

Discussion

Uptake

In bacteria there are a myriad of uptake systems and acquisition strategies designed to capture iron, many of which are often simultaneously operative in a single organism. These include uptake systems specific for siderophores, or other bound forms of iron (a similar system is also found in strategy II plants) as well as those based on ABC-type transporters capable of taking up ‘free’ Fe(III) and other transporters typically more or less specific for ferrous iron (Dassa and Bouige, 2001; Koester, 2001). Model eukaryotes typically adopt iron uptake schemes which involve reduction of Fe(III) to Fe(II) at some point, although there appear to be exceptions (Sutak et al., 2010). The first of these mechanisms is a reductive–oxidative pathway such as that found in yeast (Curie and Briat, 2003) and some green algae (La Fontaine et al., 2002), and the second is a cell surface reduction/divalent metal permease pathway such as that found in strategy I plants (Bauer and Bereczky, 2003; Morrissey and Guerinot, 2009; Weger et al., 2009; Sonier and Weger, 2010). Among the marine algae, the iron uptake systems of the diatoms have been the most thoroughly studied (Shaked et al., 2005; Kustka et al., 2007). Iron uptake in these related organisms has been described by the so-called Fe(II)s mechanism (Shaked et al., 2005) where reduction of free Fe(III)’ is the seminal step. This reductive step is followed either by direct uptake of the formed Fe(II) as in the pennate diatom Phaeodactylum tricornutum or by reoxidation of the Fe(II) by a multicopper oxidase and transport as Fe(III) as in the centric diatom Thalassiosira pseudonana (Kustka et al., 2007).

Genomic data suggest that Ectocarpus may, with some variation, use one or more of these approaches. In particular, Cock et al. (2010) have identified homologues of fro2, a proposed cell surface Fe(III) reductase. This Fe(III) reductase activity was also experimentally verified and its rate was commensurate with that of the overall iron uptake process. Additionally homologues to several divalent metal ABC transporters could be found (Bauer and Bereczky, 2003; Curie and Briat, 2003; Morrissey and Guerinot, 2009) as well as NRAMP, an M²⁺–H⁺ symporter with a preference for Fe(II) (Bauer and Bereczky, 2003; Curie and Briat, 2003; Morrissey and Guerinot, 2009), which would be consistent with the simple reductase/permease pathway. While no direct homologues to the multicopper oxidases (MCOs) Fox1 from Chlamydomonas or Fet3 of T. pseudonana could be found, two genes annotated as MCOs implicated in iron transport can be found in the Ectocarpus genome.

Short-term iron uptake studies verified that iron is taken up by Ectocarpus in a time-and concentration-dependent manner consistent with an active transport process. Derived kinetic parameters (Table 3) are qualitatively and quantitatively similar to those reported in the few available studies of other red, green, and brown algae (Manley, 1981; Matsunaga et al., 1991; Liu et al., 2000). While it is difficult to compare V _max rates due to the differing units employed in each of these studies as well as the different surface/volume ratios of the model organisms, the uptake rate for the slow growing Ectocarpus is similar that seen in Gracilaria and approximately an order of magnitude less than that of the faster growing Chlamydomonas, Laminaria, and Macrocystis (Manley, 1981; Matsunaga et al., 1991; Liu et al., 2000). However, the affinity constant (K _m) of ~1.5 µM is similar to the others which range from 0.1 to 3 µM. While open ocean iron concentrations are typically subnanomolar (Johnson et al., 1997), near-shore coastal areas can have iron concentrations orders of magnitude higher; that is, hundreds of nanomolar (Chase et al., 2002). Thus the affinity constants (based on total iron) seen for near-shore-dwelling macroalgae in the lower micromolar to high nanomolar range would appear to be reasonable. However, it should be noted that if free soluble Fe(III)’ rather than Fe(EDTA) itself is the actual substrate, as indicated by the constant Fe-variable EDTA concentration data presented earlier, then the true affinity constant is probably subnanomolar. However, the data are reported on the basis of total iron concentration in order to facilitate comparison with previous work (Manley, 1981; Matsunaga et al., 1991; Liu et al., 2000).

Table 3.

Transport parameters for iron uptake in marine algae

	Macrocystis	Gracilaria	Laminaria	Undaria	Ectocarpus
V max	1.6 pmol cm–2 h–1	0.26 pmol mg–1 h–1	2.7 pmol cm–2 h–1	6.4 pmol cm–2 h–1	0.25 pmol mg–1 h–1
K m	3.5 µM	0.6 µM	0.54 µM	6.4 µM	1.5 µM
Reference	Manley (1981)	Liu et al. (2000)	Matsunaga et al. (1991)	Matsunaga et al. (1991)	This work

Although an initial reductive process is clearly indicated both genomically and experimentally, whether the iron is taken up directly as Fe(II) or reoxidized and transported as Fe(III) (see above) remains unclear. While the lack of inhibition of iron uptake by FZ is surprising assuming a reductive process being operative, at the concentrations tested (50 µM) there is insufficient FZ to sequester completely all the Fe(II) formed via the reductase as a Fe(FZ)₃ complex. Since the rate of reduction appears to exceed that of actual uptake, there would remain a sufficient pool of uncomplexed Fe(II) to support continued uptake. It is also possible that the Fe(II) produced by the reductase is tightly coupled with an oxidase (see below) so that the ferrous ion is never in free equilibrium with the bulk solution and thus is unaffected by the presence of an external Fe(II) chelator. The strong inhibition by ascorbate, which at first glance also seems counterintuitive, suggests that a reductive/oxidative pathway may be the operative one as the ascorbate could be expected to inactivate a multicopper oxidase.

Unfortunately, attempts to determine the oxidation state of the initially transported iron by TMS were thwarted by the low sensitivity of the technique. Thus incubation times of the order of weeks in ⁵⁷Fe-enriched solutions are required to obtain useable spectra. This precluded following short-term iron uptake processes by this method. It was possible, however, to use TMS and XAS to provide evidence as to the fate of the transported iron after longer term exposure (3–4 weeks).

Storage

The first and most obvious conclusion from the long-term incubation studies is the lack of an observable ferrous iron pool in Ectocarpus. In previous in vivo Mössbauer studies of various bacterial, fungal, plant, and algal systems, significant amounts of intracellular high-spin ferrous iron octahedrally coordinated by oxygen ligands could be detected (Boenke and Matzanke, 1995; Semin et al., 2003; Matzanke et al., 2004, 2011; Kovacs et al., 2005, 2009). There is no evidence for such a species in Ectocarpus. Thus, despite the fact that the iron is probably transported across the cell membrane as Fe(II), it must be relatively rapidly reoxidized to Fe(III) and stored in that form. Of the two major metabolites which could be identified, one was an iron–sulphur protein which is likely to be a component of chloroplast and/or mitochondrial redox systems. However, a storage role for this species cannot be eliminated since in some archaeal systems polyferredoxins were detected, whose physiological function, while unclear, has been hypothesized to be to serve as electron sinks (Nölling et al., 1995; Wasserfallen et al., 1995). Nevertheless, the vast majority of organisms store iron in one or more of the various forms of the ubiquitous protein ferritin. However, genomic analysis reveals the presence of no ferritin or ferritin-like homologues in Ectocarpus (Cock et al., 2010). While this is unusual, it is not unprecedented. Thus while some diatoms such as P. tricornutum have ferritin genes, they have not been detected in others such as T. pseudonana (Marchetti et al., 2009).

In the absence of ferritins, two alternative or additional forms of iron storage have been identified in other organisms. The first, found in some fungi, is a siderophore-based storage system (Matzanke et al., 1987, 1988) clearly not present here. The second, which has been elucidated in yeast and several other eukaryotes including the halotolerant alga Dunaliella salina, is a vacuole based one (Martinoia et al., 2007; Paz et al., 2007). At present, there are few data in the literature about the chemical nature of vacuole sequestered iron stores. However, it seems likely that the iron would be stored in some sort of mineral phase. Indeed, it is reported that in Arabidopsis seeds some iron is located in vacuole globoids containing phytate which may bind ferric ions via phosphate groups (Lanquar et al., 2005) similar to what is proposed here. This notion is supported by the iron-specific histological staining which shows accumulation of high concentrations of granular-like iron stores inside Ectocarpus cells.

Since the spectroscopic parameters and relaxation properties (i.e. magnetic ordering temperatures) of condensed iron mineral phases are strongly dependent on particle sizes and their crystalline/amorphous structure, detailed temperature-dependent Mössbauer measurements can shed light on the nature of any iron stores. The observation that the second iron compound detected by TMS and XAS displays spectra typical of an (Fe³⁺O₆) system with parameters similar to the amorphous, phosphorus-rich mineral core of bacterial and plant ferritins suggests that Ectocarpus does indeed contain a mineral phase iron storage form. Whether this mineral phase is in the form of a ferritin-like protein which lacks significant homology to previously studied systems, or is sequestered in some sort of vacuole, remains to be determined. Work geared towards more fully characterizing this system is in progress.

Fig. 7.

EXAFS data of CC47 plotted in R-space. The graph was plotted with a k-weight of 1 and a phase correction. The k-range for the fast Fourier transformation was 2 Å^–1 to 8.5 Å^–1. The fit was performed with a model containing FeO₆ and Fe₄S₄ structures due to the limited R range up to r=3.35 Å as described in the text. Filled squares represent experimental data, the black solid line the fit, and the light grey dotted line the setting of the range.