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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
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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.
The University of Dundee is a registered Scottish charity, no.
SC015096.
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