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eXtra Botany COMMENTARY Iron acquisition and allocation in stramenopile algae John A. Raven1,2 1 Division of Plant Sciences, University of Dundee at the James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK 2 School of Plant biology, University of Western Australia, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia To whom correspondence should be addressed. Email: j.a.raven@ dundee.ac.uk Journal of Experimental Botany Vol. 64, No. 8, 2119–2127, 2013 doi:10.1093/jxb/ert121 Abstract The essential element iron has a low biological availability in the surface ocean where photosynthetic organisms live. Recent advances in our understanding of iron acquisition mechanisms in brown algae and diatoms (stramenopile algae) show the importance of the reduction of ferric to ferrous iron prior to, or during, transport in the uptake process. The uses of iron in photosynthetic stramenopiles resembles that in other oxygenic organisms, although (with the exception of the diatom Thalassiosira oceanica from an iron-deficient part of the ocean) they lack plastocyanin, instead using cytochrome c6, This same diatom further economizes genotypically on the use of iron in photosynthesis by decreasing the expression of photosystem I, cytochrome c6, and the cytochrome b6f complex per cell and per photosystem II relative to the coastal Thalassiosira pseudonana; similar changes occur phenotypically in response to iron deficiency in other diatoms such as Phaeodactylum tricornutum. In some diatoms grown under iron-limiting conditions, essentially all of the iron in the cells can be accounted for by the iron occurring in catalytic proteins. However, stramenopiles can store iron. Genomic studies show that pennate, but not centric, diatoms have the iron storage protein ferritin. While Mössbauer and X-ray analysis of 57Fe-labelled Ectocarpus siliculosus shows iron in an amorphous mineral phase resembling the core of ferritin, the genome shows no protein with significant sequence similarity to ferritin. Key words: allocation, Bacillariophyceae, deficiency, Ectocarpus, iron, Pelagophyceae, storage, transport. Introduction A recent paper by Böttger et al. (2013) reported very interesting findings on iron acquisition and allocation in the first brown alga to have its complete genome sequence assembled and annotated, Ectocarpus siliculosus. In commenting on these and related findings, I will emphasize evolutionary and phylogenetic approaches as well as ecological and palaeoecological, and especially the effects of iron limitation, extending the analysis of Böttger et al. (2013). The stramenopiles, also known as Heterokontophyta, Ochrophyta, or Ochrista, are a clade of eukaryotes containing fungus-like oomycetes such as the important plant pathogen Phytophthora, but are predominantly photosynthetic (van den Hoek et al., 1995; Graham et al., 2009; Brown and Sorhannus, 2010; Yang et al., 2012). The algal members include the unicellular or colonial (filamentous) diatoms, which are very significant primary producers in the plankton and benthos of marine and inland waters, on which there is a significant body of data on iron acquisition and metabolism, and the multicellular brown algae, which are major primary producers in the marine benthos, especially in temperate and polar oceans. Iron is an essential element for all organisms, being involved in large numbers of mainly redox catalysts, and has very significant roles in photosynthesis, respiration, reduction of oxidized nitrogen and sulfur compounds, and nitrogen fixation. In photosynthetic organisms, as much as half of the catalytically active iron can be involved in photosynthesis (Raven, 1988, 1990; Raven et al., 1999). These requirements for iron relate to the chemistry of the element (Williams and Rickaby, 2012) and also to the high availability in the anoxic Archean ocean when many components of the present-day processes evolved (Williams, 1981; Saito et al., 2003; Zerkle et al., 2006; Planavsky et al., 2010; Williams and Rickaby, 2012). The anoxic ocean with an excess of ferrous iron over sulfide (Crowe et al., 2008; Mikucki et al., 2009) gave way to the sulfidic ocean with an excess of sulfide over ferrous iron, resulting in the precipitation of much of the ferrous ions as ferrous sulfide. Such precipitation of ferrous sulfide by the mechanism described by Zerkle et al. (2006) occurred after the evolution and spread of oxygenic photosynthesis in the Global Oxygenation Event 2.3–2.4 Ga ago (Bekker et al., 2004; Blank and Sanchez-Baracaldo, 2010), with an oxygenated surface ocean and a sulfidic deeper ocean (Planavsky et al., 2010). After a brief ferruginous period during ‘Snowball Earth’ 750–635 Ma ago, increased atmospheric oxygenation resulted in the penetration of oxygenation deeper into the ocean (Planavsky et al., 2010; Ashkenazy et al., 2013). As a consequence, iron became even less available in the surface © The Author [2013]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For permissions, please email: [email protected] 2120 | Iron acquisition and allocation in stramenopile algae ocean, including the photic zone, yielding the essentially fully oxygenated version of the ocean found today (Williams, 1981; Saito et al., 2003; Zerkle et al., 2006; Planavsky et al., 2010; Williams and Rickaby, 2012; Ashkenazy et al., 2013). Attempts have been made to relate the higher content of iron found in green microalgae than in stramenopiles, which were derived by secondary endosymbiosis from a red alga, to seawater chemistry, at the time they became dominant in the fossil record in the Mesozoic from 251 million years onwards (Ho et al., 2003; Quigg et al., 2003, 2011; Falkowski et al., 2004). The stramenopiles arose well before the Mesozoic, with a sterane biomarker of the stramenopile class Pelagophyceae indicating an origin not later than the late Neoproterozoic almost 600 Ma ago, which is earlier than the date suggested by some molecular phylogenetic values (Raven, 2012). While the content of a trace element is not necessarily an indication of the requirement for the element, the phylogenetic differences in iron content, with higher values in the green lineage of eukaryotes than in the red lineage, are sustained in the latest analysis involving more species (29 rather than 14; Quigg et al., 2011). There seems to be no explanation for the phylogenetic difference in iron content between the green and red lines of microalgae in terms of the range of iron-containing catalysts and their stoichiometry found in these two groups. The chemical nature of iron meant that, with few exceptions (e.g. flavodoxin for ferredoxin, plastocyanin for cytochrome b6), no iron-free substitutes for the iron-containing catalysts have evolved (Raven et al., 1999), although natural selection has altered the stoichiometry of iron-containing components of photosynthesis, decreasing the iron cost of the photosynthetic apparatus for a given rate of inorganic carbon assimilation (Strzepek and Harrison, 2004). The decreased content of photosystem I and the cytochrome b6f complex in the open ocean diatom Thalassiosira oceanica relative to the coastal Thalassiosira weissflogii might be related a decreased ability to acclimate to fluctuations in photosynthetically active radiation (Strzepek and Harrison, 2004). The oxygenation of the atmosphere and first the upper layers and then the whole of most water bodies means that oxygenic phototrophs can have problems accessing iron from an environment dominated by ferric iron, which is insoluble unless it is in strong organic chelation, which brings its own problems for iron acquisition. These problems are particularly acute when iron availability is so low that iron limits (or co-limits) the growth rate, as in parts of the open ocean and also in some eutrophic coastal regions with high terrestrial inputs of available nitrogen, phosphorus, and organic ligands that can chelate iron (Suzuki et al., 1995; Cooke et al., 2004). The possibility of iron limitation could provide an explanation for the evolution of iron storage compounds, which sequester iron in a form that does not lead to the undesirable effects of free ferrous ions such as the Fenton reaction producing reactive oxygen species (Williams, 1982). Iron acquisition In considering iron acquisition by stramenopiles, most evidence is available for diatoms and brown algae, with some for the Pelagophyceae (Table 1). For diatoms, the general conclusion is that iron uptake resembles Strategy I of vascular plants, with reduction of the predominant ferric iron in the medium just before or during transport (Anderson and Morel, 1982; Kustka et al., 2007; for a general view of marine microbial iron acquisition, see Desai et al., 2012). Kustka et al. (2007) analysed the published genome of the centric diatom Thalassiosira pseudonana and identified genes related to iron acquisition. A plasmalemma-located NADPHoxidoreductase, which can convert external ferric iron to ferrous iron, is a component of a mechanism involving ferrous iron entry to the cytosol. The entry of ferrous iron could involve the product of genes for a divalent metal, including ferrous iron permease, which transfers ferrous iron into the cytosol by proton–ferrous iron symport. There is also a gene for plasmalemma-located ferroxidase, which converts the ferrous iron into ferric iron, which can then be acted on by an iron permease, which delivers ferric ions to the cytosol. These findings are consistent with the occurrence of two parallel mechanisms for iron entry to the cytosol. Whitney et al. (2011) examined a more restricted range of iron-acquisition genes in a coastal isolate of T. pseudonana and an oceanic isolate of T. weissflogii and found all the genes in both diatoms, although regulation as a function of iron availability differed between the two isolates. Kustka et al. (2007) also examined the pennate diatom Phaeodactylum tricornutum genome, and found only the ferric reductase, although Allen et al. (2008) found other sequences that could relate to iron influx at the plasmalemma (cf. BlabyHaas and Merchant, 2012; Morrissey and Bowler, 2012). The ferrireductase system in P. tricornutum is more strongly inducible than that of T. pseudonana when cells are transferred from high-iron to low-iron culture medium (Kustka et al., 2007; Allen et al., 2008; Sutak et al., 2013). Despite the varying interpretations of the evidence for genes encoding the proteins performing the transmembrane iron transport, P. tricornutum has an effective mechanism for transporting ferrous iron from the medium across the plasmalemma (Kustka et al., 2007). Analysis of the complete genome sequence of the pennate diatom Fragilariopsis cylindrus shows the presence of both: (i) a plasmalemma-located NADPH-oxidoreductase, which can convert external ferric iron to ferrous iron, which can then be transported by the ferrous iron transporter; and (ii) the presence of a plasmalemma-located ferroxidase, which conveys the ferrous iron into ferric iron, which can then be acted on by the iron permease, which delivers ferric ions to the cytosol (BlabyHaas and Merchant, 2012). These studies on P. triconutum and F. cylindrus clearly indicate that there are differences in iron transport at the plasmalemma among pennate diatoms. A novel finding by Sutak et al. (2013) is that the diatoms can take up ferric iron (without prior external reduction) as well as ferrous iron by a mechanism different from the other vascular plant iron-acquisition process (Strategy II) found only in grasses. This strategy in grasses, which involves secreted iron-binding organic molecules or siderophores, is not generally thought to occur in diatoms or any other eukaryotic alga but is found in cyanobacteria (Sutak et al., 2013). However, there is the possibility that domoic acid produced by many species of the diatom genus Pseudo-nitzschia Marchetti et al. (2009); Nuester et al. (2012); Böttger et al. (2013) ? Peers and Price (2006); Allen et al. (2008) Blaby-Haas and Merchant (2012); Lommer et al. (2012) Böttger et al. (2013) Cytochrome c6 rather than plastocyanin; Cu-Zn superoxide dismutase rather the Fe superoxide dismutase Nichols et al. (2001); Allen et al. (2008); Blaby-Haas and Merchant (2012); Lommer et al. (2012); Böttger et al. (2013) Ferric reductase; ferrous and ferric uptake systems Generally cytochrome c6, plastocyanin in Thalassiosira oceanica, which also has constitutive decreased expression of the Fe-rich photosystem I and cytochrome b6f complex Iron superoxide dismutase, but not Cu-Zn superoxide dismutase, in Thalassiosira pseudonana Not ferritin; associated with phosphate at low iron: phosphate ratios in vacuoles Cytochrome c6 rather than plastocyanin; Cu-Zn superoxide dismutase rather the Fe superoxide dismutase in Ectocarpus siliculosa Ferritin-like complex based on phosphate; iron–sulfur complex? Adaptive and acclimatory variations in iron allocation among enzymes and redox catalysts Iron storage Ferric reductase, ferrous and ferric uptake systems by functional assay; molecular genetic evidence for these systems strong for Fragilariopsis cylindrus, ambiguous in Phaeodactylum trivornutom Cytochrome c6 rather than plastocyanin (Phaeodactylum tricornutum), both cytochrome c6 and plastocyanin (Fragilariopsis cylindrus) Cu-Zn superoxide dismutase rather the Fe superoxide dismutase Decreased expression of photosystem I and cytochrome b6f complex relative to photosystem II under Fe-limiting conditions in Phaeodactylum tricornutum Ferritin Ferric reductase, ferrous and ferric uptake systems by functional assays and molecular genetic studies in Thalassiosira pseudonana Different opinions on the extent to which molecular genetic evidence supports the occurrence of ferric oxidase and ferrous and ferric transporters in Ectocarpus siliculosa Iron acquisition Bacillariophyceae (pennate) Bacillariophyceae (centric) Phaeophyceae Taxon Table 1. Iron acquisition, allocation among enzymes and redox catalysts, and storage in stramenopiles. Pelagophyceae References Iron acquisition and allocation in stramenopile algae | 2121 can function in extracellular chelation of iron and copper, with implications for the availability of these metals for Pseudo-nitzschia and also, perhaps, for co-occurring phytoplankton (Rue and Bruland, 2001; Maldonado et al., 2002; Wells et al., 2005; Trainer et al., 2012). However, iron does not appear to enter the cells as ferric iron ligated to domoic acid, as occurs for true siderophores. Instead, according to one model, the main effect of domoic acid is to increase the availability of the copper needed for the copper-containing plasmalemma ferrous iron oxidase, which provides ferric iron to a high-affinity ferric iron influx mechanism (Wells et al., 2005; Kustka et al., 2007; Trainer et al., 2012). While domoic acid production is increased when cells are grown with low iron availability, many other environmental factors also influence domoic acid production, and there is a lack of information on domoic acid production in Pseudo-nitzschia habitats with low iron availability (Ribalet et al., 2010; Trainer et al., 2012). Furthermore, domoic acid production is increased by iron (and copper) fertilization of regions of the ocean with initially sparse oceanic Pseudo-nitzschia populations (Trick et al., 2010; Trainer et al., 2012). At a higher (whole-cell) organisational level, Sunda and Huntsman (1995) examined iron-acquisition kinetics for six species of marine phytoplankton representing coastal and oceanic environments and four classes of algae, two of which were stramenopiles. The diatoms were coastal strains of the diatoms T. pseudonana and T. weissflogii and the oceanic T. oceanica, while the Pelagophyceae (a class closely related to the Bacillariophyceae: Brown and Sorhannus, 2010; Yang et al., 2012) were represented by the oceanic Pelagomonas calceolata. Expressing iron uptake on a surface-area basis as a function of total external iron concentration (see Fig. 7 of Sunda and Huntsman, 1995) for all six algae gives an approximately linear relationship. Sunda and Huntsman (1995) concluded, following Hudson and Morel (1993), that all six species are close to the limit on uptake imposed by the rate of diffusion of ligated iron and free iron from the bulk medium to the plasmalemma and of ligand exchange from bound iron in the medium to bound iron in the transporters. Sunda and Huntsman (1995) concluded that the main adaptive (genetic) response of phytoplankton to low iron availability is decreased size and hence, for a given shape, increasing the cell surface area per unit volume containing catalytically functional iron, and decreasing the catalytically functional iron per unit cell volume. Timmermans et al. (2005) investigated iron uptake in three species of picoplankton and showed that the affinity for total dissolved inorganic iron was somewhat higher in the pelagophycean Pelagomonas calceolata than in two other organisms, the cyanobacterium Synechococcus sp. and the prasinophyean Prasinomonas capsulatus (a species used by Sunda and Huntsman; 1995), so there is some room for variation within the relationship of Sunda and Huntsman (1995). Other data on iron uptake in the Pelagophyceae come from Nichols et al. (2001), who showed that the brown tide alga Aureococcus anophagefferens had a cell-surface ferric chelate reductase activity. Several phytoplanktonic stramenopiles undergo periodic (diel or longer) vertical migrations through the upper mixed layer of the ocean or epilimnion of lakes. These vertical 2122 | Iron acquisition and allocation in stramenopile algae movements can involve flagellar motion in stramenopiles such as the Raphidophyceae (Wada et al., 1985; Handy et al., 2005; Powers et al., 2012), and buoyancy regulation involving switching between sinking cells with high-density compounds (silica, chrysolaminarin) or floating cells with lower-density vacuolar solutions in large-celled marine diatoms (Boyd and Gradmann, 2002), relative to the suspending seawater. Such vertical movements are only possible when the surface waters are strongly stratified, with no vertical water movements at higher speeds than those attainable by the organisms, i.e. less than 1 mm s–1. The evolutionary rationale for these movements is usually said to lie in the opposite gradients of photosynthetically active radiation (highest near the surface) and of nutrients (highest near the bottom of the thermocline/ nutricline where eddy diffusion brings up nutrients regenerated in the deeper darker ocean waters). Diel migration general involves movement from the surface in the photoperiod to the deeper parts of the upper mixed layer at dusk, with the reverse migration at dawn (Wada et al., 1985; Handy et al., 2005). There is good evidence for upward movement in vertically migrating stramenopiles that are rich in nitrogen (obtained mainly as nitrate) and phosphorus (as phosphate) from near the thermocline/nutricline to more illuminated waters (Watanabe et al., 1983, 1988; Villareal et al., 1993; Villareal et al., 1996; Kimura et al., 1999; Villareal et al., 1999). ‘Nutricline’ as used above refers to nitrate (nitracline) and phosphate (phosphocline). What of iron? The deep chlorophyll maximum can correspond to a minimum concentration of dissolved iron, so the highest iron concentrations are in even deeper waters (Sedwick et al., 2005). Such iron minima could, at least in part, be related to the larger cellular iron quota (largely in thylakoid protein complexes, especially photosystem I and the cytochrome b6f complex) related to photoplankton growth at low irradiances (Raven, 1990; Sunda and Huntsman, 1997, 2011) and/or the currently unexplained large quota of certain trace metals in phytoplankton grown at low irradiances (Finkel et al., 2006). More generally, the ferricline can be well below the nitracline (Blain et al., 2008), so that vertical migration down to the nitracline would not take an alga to the ferricline. Despite this, there is some evidence consistent with a role of periodic vertical migration in iron acquisition at depth (Villareal et al., 1999; McKay et al., 2000). In E. siliculosus, the iron acquisition mechanism follows, from genomic data, Strategy I, i.e. involves external reduction of ferric iron with subsequent uptake of the resulting ferrous iron using the ferrous iron–proton symport mechanism (Böttger et al., 2013). Böttger et al. (2013) determined the Michaelis–Menten kinetic parameters (Kmand Vm) for iron uptake and compared them with literature values for four marine macroalgae, three of them stramenopiles. The E. siliculosus value for Km (1.5 µM) is in the range 0.54–6.4 µM for the other four seaweeds (Böttger et al., 2013). The fit to Michaelis–Menten kinetics suggests that diffusion boundary layers do not restrict iron uptake, despite the variable observed diffusion limitation of the uptake of other nutrients in macroalgae (e.g. MacFarlane and Raven, 1990). It is likely that uptake limitation by the diffusion boundary layer avoided for iron uptake by macroalgae is, at least in part, a function of diffusion not just of free ferric ions (on whose concentration the Km values are based) but also mainly of the diffusion of chelated ferric ions (in this case FeEDTA) (Böttger et al., 2013): boundary layer diffusion can be decreased if the equilibration of chelated and free ferric ions is rapid relative to the time taken to diffuse across the diffusion boundary layer and react with the reductase and transporter (Hudson and Morel, 1993). Intracellular allocation of iron Once iron has entered cells, it can be allocated to catalytic proteins (enzymes, redox agents) or to storage (assuming that all non-catalytic iron is stored in the sense of being capable of remobilization and use in the catalytic pool under sufficient iron limitation. As to the occurrence of iron-containing redox catalysts among stramenopiles (Table 1; Raven et al., 1999; Blaby-Haas and Merchant, 2012), all the stramenopiles whose genomes have been sequenced have the gene for the iron-containing cytochrome c6 as the catalyst of electron transfer from the cytochrome b6f complex to the P700 in photosystem I (BlabyHaas and Merchant, 2012). The pennate diatom F. cylindrus has the gene for the alternative electron carrier plastocyanin, which has no iron but contains copper, as well as the gene for cytochrome c6 (Blaby-Haas and Merchant, 2012). The centric diatom T. oceanica (Peers and Price, 2006) only contains plastocyanin, with undetectable levels of the alternative electron carrier cytochrome c6. Both of these diatoms occur in lowiron parts of the ocean. All of the stramenopiles examined, except T. pseudonana, have copper-zinc superoxide dismutase; T. pseudonana has iron-superoxide dismutase, as does E. siliculosus, while all stramenopiles have manganese superoxide dehydrogenase (Blaby-Haas and Merchant, 2012). Another adaptive (genetic) variation is the low expression of the ironrich photosystem I and cytochrome b6f complex relative to the iron-poor photosystem II in T. oceanica from iron-poor areas of the ocean (Strzepek and Harrison, 2004). The quantitative allocation of iron between the functional (catalytic) and storage pools, and within the catalytic pool, is not completely known for any stramenopile. Given that the total intracellular iron content is known, it is possible to determine the iron content of the major catalytic proteins in one of two ways. One is the direct way of measuring the contents of the individual proteins, such as the photosysytem I and photosystem II complexes, the cytochrome b6f complex, cytochrome c6 and ferredoxin of (and associated with) thylakoids, complexes 1, 2, 3, and 4 and cytochrome c of mitochondria, the reductases for oxidized nitrogen and sulfur sources, and catalase, peroxidise, and iron-superoxide dismutase. This approach has been applied to T. oceanica (from a part of the Pacific in which iron generally limits phytoplankton growth) and T. weissflogii (from coastal waters with greater iron availability), and other diatoms, by Strzepek and Price (2000), Strzepek and Harrison (2004), Peers and Price (2006), Allen et al. (2008) Iron acquisition and allocation in stramenopile algae | 2123 and Strzepek et al. (2011). It had previously been shown that T. oceanica contained a quarter of the iron found in coastal species of Thalaassiosira such as T. weissflogii (Sunda et al., 1991), and values are at least as low in diatoms from the Southern Ocean (Strzepek et al., 2011). The measurements of iron-containing catalytic components involve only the thylakoid components, and, as expected, the iron in these components in each cell is significantly less than the total iron per cell, and parallels, both genotypically and phenotypically, the iron content of the cell. However, the absence of a estimation of iron other than in or associated with the thylakoid membrane means that the there is an underestimation of the catalytic iron content, so the currently available data do not permit estimation of stored iron by subtracting the catalytic iron content from the total iron content. The other method involves estimating the catalytic iron requirement of cells from the cell growth rate, and the contributory rates of photosynthesis, respiration, and (with oxidized nitrogen and sulfur sources) the rates of reductive assimilation of nitrogen and sulphur, using the maximum iron-specific catalytic rates of the iron-containing catalytic proteins scaled for the requirement for their products in growth of a cell. The sum of the iron requirements computed in this way gives a minimum requirement of iron per unit biomass to give the observed growth rate (Raven, 1990; Raven et al., 1999; Cooke et al., 2004). The outputs of this method have been compared with data on the iron concentration in marine diatoms (Sunda et al., 1991; Maldonado and Price, 1996). While agreement has been reasonable between predictions and measurements, it is not possible to use this method to estimate the fraction of iron in the organism that is in a non-metabolic (storage) pool, i.e. is not in catalytic proteins. However, the method of Raven (1988, 1990) has been used to predict the iron cost of growth in the brown alga Hormosira banksii and to suggest, by comparison with the measured iron content, that iron availability is not limiting for growth (Cooke et al., 2004). While the outcome of either of these two methods of estimating the catalytic iron content of stramenopiles, subtracted from the observed iron content, should give an upper limit on the stored iron, neither method gives reliable results. A more direct estimate is needed. Böttger et al. (2013) used the combination of Mössbauer and X-ray absorption spectroscopy to partially characterize two multi-iron complexes in E. siliquosa. One of these, comprising about 74% of the iron signal, is a polymeric (Fe3+O6) complex resembling the amorphous phosphorus-containing core of ferritins from plants and bacteria. The core of Pisum sativum seed ferritin has 1800 Fe and 640 P per ferritin molecule, i.e. an iron:phosphorus ratio of 2.83 (Wade et al., 1993). However, there is no sequence in the E. siliculosus genome that would code for a gene that significantly resembles a ferritin. However, ferritin is known from stramenopiles: molecular genetic evidence shows that ferritin occurs in pennate, but not in centric, diatoms (Marchetti et al., 2009). The other signal is of an iron–sulfur cluster, which accounts for about 26% of the iron signal: it is not clear if this is catalytically active or represents storage. Böttger et al. (2013) did not give quantitative results (iron per g of dry matter) of the iron represented by the clusters, nor for values of total iron per g of dry matter. Accordingly, it is not possible to evaluate quantitatively the former suggestion in terms of the possible fraction of cellular iron in a single iron–sulfur protein complex, or even several complexes in terms of the known iron–sulfur complexes. How stramenopiles increase iron acquisition and decrease iron requirement in adapting or acclimating to low-iron environments There are several areas of the surface ocean where iron limits the growth of non-diazotrophic photoplankton: these are the north-east subarctic Pacific, the eastern equatorial Pacific, and the Southern Ocean (Martin et al., 1991). Low-iron environments in the ocean can be equated to high nutrient–low chlorophyll regions, i.e. those in which nitrate and phosphate concentrations are relatively high but chlorophyll concentration, as an indicator of phytoplankton biomass, is low (Martin et al., 1991). The hypotheses to account for incomplete use of nitrate and phosphate have included: (i) food web dynamics, which means a high impact of grazing and parasitism (e.g. by viruses), and (ii) deep mixing of the upper mixed layer, which causes light limitation of phytoplankton growth, but (iii) iron limitation is now accepted as a major cause of the high nutrient–low chlorophyll condition (Martin et al., 1991; Boyd, 2008; Smetacek et al., 2012). Iron limitation of growth can occur in the marine benthic environments, as mentioned above (Cooke et al., 2004). Dealing first with diatoms, the discussion above under general aspects of iron acquisition shows that all three diatoms and the pelagophycean investigated had iron uptake limited by physical (diffusion boundary layer) and chemical (exchange of iron between dissolved ligands and those in membrane transporters) means (Sunda and Huntsman, 1995, following Hudson and Morel, 1993). Accordingly, it would seem that changes to the kinetics of the transmembrane transporters would not significantly increase iron uptake from very low external iron concentrations. However, Strzepek et al. (2012) found differences in access to external ligated iron between Southern Ocean diatoms adapted to very low iron availability and coastal northern hemisphere diatoms adapted to much higher iron availability. Dealing next with intracellular iron allocation in diatoms, the observed genotypic responses of adaptation of the diatom T. oceanica to a low-iron environment is a decrease in the concentration of photosystem I and cytochrome b6f complex but not of photosystem II (Strzepek and Harrison, 2004), and the expression of plastocyanin rather than cytochrome c6, the plastocyanin gene presumably occurring as a result of horizontal gene transfer (Strzepek and Harrison, 2004, Peers and Price, 2006; Blaby-Haas and Merchant, 2012). While the photosynthetic capacity is not decreased relative to other species of Thalassiosira by these large changes in stoichiometry, T. oceanica has a much smaller capacity for acclimation to variations in the flux of photosynthetically active radiation 2124 | Iron acquisition and allocation in stramenopile algae (Strzepek and Harrison, 2004). Acclimatory changes in stoichiometry of thylakoid iron-containing protein complexes to iron limitation by P. tricornutum are qualitatively similar to the genotypic response in T. oceanica (Allen et al., 2008). Whitney et al. (2011) showed that, while a coastal isolate of T. pseudonana showed diel periodicity in the expression of the iron-containing ferredoxin and of the alternative, metal-free flavodoxin with no effect of iron availability, an oceanic isolate of T. weissflogii only expressed flavodoxin when iron was limiting growth. Whitney et al. (2011) also showed that the lack of response of flavodoxin expression in the T. pseudonana isolate was caused by the loss of the iron-responsive copy of the flavodoxin gene, retaining only the iron-unresponsive copy. Interactions between the supply of photosynthetically active radiation and of iron in controlling the growth rate of marine diatoms has been the subject of significant attention in predictions based on mechanistic models (Raven, 1988, 1990) and experiments (Sunda and Huntsman, 1997; Finkel et al., 2006; Sunda and Huntsman, 2011; Strzepek et al., 2012). Sunda and Huntsman (1997) found that more cellular iron is needed to support growth of a diatom under low light than at high light, as predicted by Raven (1988, 1990), although more complex results were found by Sunda and Huntsman (2011). One complication identified by Sunda and Huntsman (2011) is photolysis of iron chelates, which increases the availability of iron to the cells with increasing irradiances. Strzepek et al. (2012) used Southern Ocean diatoms, and found that, while iron use efficiency decreased at low light, this was solely a result of decreased growth rate with no increased intracellular iron concentration; the authors attributed this to acclimation to low light by increasing the size, rather than the number, of photosynthetic units, thus increasing the light-harvesting pigment content per unit of cellular iron, rather than an increase in the number of photosynthetic units. However, no data were obtained to test this hypothesis. These data on irradiance–iron interactions are relevant to the cause of high nutrient–low chlorophyll regions. One early suggestion for the high nutrient–low chlorophyll condition, especially in the Southern Ocean, was deep mixing, meaning a decreased mean incident photon flux density (Martin et al., 1991). These interactions of light and iron suggest that, even with the evidence from mesoscale iron fertilization experiments of stimulation of larger diatom growth by the addition of iron, light effects could be important in the iron response (Finkel et al., 2006). The low-iron-adapted F. cylindrus and Pseudo-nitzschia granii genomes code for an energy-transducing (protonpumping) rhodopsin, which provides an iron-independent means of photon energy conversion (Marchetti et al., 2012), although the photon use efficiency is lower than cyclic electron transport, as well as involving more protein per chromophore and a lower maximum rate of proton transport per unit protein, than cyclic electron flow in thylakoids (Raven, 2009). Furthermore, it is not clear whether any proton gradient produced by an energy-transducing rhodopsin can be used to phosphorylate ADP, as it does not seem to be clear that the rhodopsin is targeted to the thylakoid membrane (or the inner mitochondrial membrane) with an ATP synthase (Marchetti et al., 2012). Iron limitation of growth can occur in marine benthic environments (Cooke et al., 2004). Suzuki et al. (1995) showed that the growth of the two laminarian brown algae from a Japanese coastal site was stimulated by added iron. Furthermore, MacLachlan (1977) showed that iron limitation of the growth of embryos of the fucoid brown alga Fucus edentatus increased the growth of colourless hairs, as did nitrate and phosphate limitation, increasing the area available for nutrient uptake with a thinner diffusion boundary layer around the hairs than occurs around the bulk of the embryo. These results showed that growth of brown algae can be limited by iron supply in their natural environment and that they have developmental mechanisms that can increase iron uptake. Conclusions Much remains to be learned about how photosynthetic stramenopiles acquire iron, how they allocate intracellular iron among catalytically active iron proteins, and between such proteins and storage pools of iron, and also on how the algae genotypically or phenotypically respond to iron deficiency. However, progress is being made on both physicochemical limitations on iron uptake and on the distributions of the different plasmalemma ferric iron reductases and iron transporters among stramenopiles, although a number of questions remain, such as which transporters are involved in iron uptake in P. tricornutum. Progress has been made in determining intracellular iron allocation among photosynthetic catalysts in an open ocean and in a coastal diatom; more work of this type is needed, extending the studies to more species (including non-diatom stramenopiles) and also to non-photosynthetic catalysts. While characterization of the forms in which diatoms and brown algae store iron has been reported recently, quantitation is needed of the iron stores and hence of the allocation of intracellular iron between catalytic and storage pools in different organism and environments. Finally, while much progress has been made in understanding adaptation and acclimation to low-iron conditions in diatoms and, to a limited extent, brown algae, more detailed work is needed. Acknowledgements Discussions with John Beardall, Paul Falkowski, the late Michael Evans, Zoe Finkel, Mario Giordano, Beki Korb, Hans Lambers, and Antonietta Quigg have been very useful. Comments from an anonymous reviewer have been helpful. 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