21st Century Directions in Biology
Molecular Tools for Discovering
the Secrets of Diatoms
ANASTASIA SAADE AND CHRIS BOWLER
Diatoms are photosynthetic unicellular eukaryotes found in most aquatic environments. They are major players in global biogeochemical cycles, and
generate as much oxygen through photosynthesis as terrestrial rainforests do. Insights into their evolutionary origins have been revealed by the
whole-genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum. We now know that diatoms contain unusual
assortments of genes derived from different sources, including those acquired by horizontal gene transfer from bacteria. These genes confer novel
metabolic and signaling capacities that may underlie the extraordinary ecological success of diatoms on Earth today. The availability of a suite of
techniques that can be used to monitor and manipulate diatom genes is enhancing our knowledge of their novel characteristics. We highlight these
recent developments and illustrate how they are being used to understand different aspects of diatom biology. We also discuss the use of diatoms in
commercial applications, such as for nanotechnology and biofuel production.
Keywords: genomics, microarrays, nanotechnology, transgenic technology, biofuel
I
n the upper zone of the ocean and other water bodies, down
to depths where light can penetrate, one can typically find
an abundant group of eukaryotic algae known as diatoms.
These microscopic, unicellular organisms are characterized
by ornate, lacework-like, silicified shells and are distributed
all around the world. Diatoms are photosynthetic organisms
that can convert the energy from sunlight into chemical
energy in the form of ATP (adenosine triphosphate). This
chemical reaction confers on diatoms the ability to produce
their own nutrients (sugars), thus they have an autonomous
metabolism and are called photoautotrophs. Diatoms
absorb and fix large amounts of atmospheric carbon dioxide
(CO2) while capturing light and water to generate a major
fraction of the oxygen generated on Earth by photosynthesis. They are in fact believed to contribute between 20% and
25% of global primary production, equivalent to all terrestrial rainforests combined (Falkowski et al. 1998, Field et al.
1998, Smetacek 1999), and consequently play an essential
role in the well-being of our global ecosystem.
The scientist and artist Ernst Haeckel was one of the first
to observe and describe diatoms in the late 19th century
(Breidbach 2005). German biologist Robert Lauterborn
subsequently made exquisite microscopic descriptions of
subcellular events occurring during diatom cell division. A
century later, Jeremy Pickett-Heaps translated Lauterborn’s
observations from the original German and verified his discoveries using light and electron microscopy (Pickett-Heaps
et al. 1984, De Martino et al. 2009). Others began describing
diatoms’ habitats: Allen did research in the early 20th century
(Allen WE 1926), and experimental culturing became more
reliable with the finding that, in addition to light and macronutrients, certain micronutrients and vitamins were required
to cultivate them (Harvey 1939). Ecological and descriptive
studies continue to this day, with researchers now incorporating advanced techniques of molecular and cellular biology.
Consequently, knowledge of diatoms’ basic biology and their
potential for a range of commercial exploitations is now advancing rapidly.
Diatoms can be recognized in the microscope by their
highly ornamented silicified cell walls, known as frustules
(figure 1). How diatoms generate these beautiful structures
is largely unknown, although some insights are now being revealed (see below). The process is termed “biomineralization”
(defined as the formation of inorganic materials under biological control), and the species-specific patterns indicate
that it is genetically determined. Because marine organisms
use more than 6.7 gigatons of silicon per year (Tréguer et al.
1995), it is particularly important to understand silicon
uptake and deposition processes in diatoms.
Furthermore, diatoms are used as bioindicators of pollution and water quality. Because many heavy metals and
organic xenobiotics inhibit diatoms’ growth, other algae such
as cyanobacteria come to dominate (Berland et al. 1976). It
is therefore possible to determine water quality by analyzing
plankton diversity. Diatoms are also used as hydrographic
tracers because biogenic silica retains its primary oxygen
BioScience 59: 757–765. ISSN 0006-3568, electronic ISSN 1525-3244. © 2009 by American Institute of Biological Sciences. All rights reserved. Request
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21st Century Directions in Biology
Figure 1. Electron micrograph of the elaborate silicified
cell wall of a diatom (Thalassiosira oestrupii var. venrickae). The cell has a diameter of 9.5 microns. Image:
Courtesy of Diana Sarno (Service for Taxonomy and
Identification of Marine Phytoplankton, Stazione Zoologica Anton Dohrn, Naples, Italy).
isotopic composition after burial (Sancetta 1981). This property can be used to monitor past surface temperatures and
isotopic compositions of seawater (Shemesh et al. 1992).
Additionally, because the frustule can retain its structural
features over geological timescales, the diatom fossil record
is of high quality. These observations reveal that diatoms
have been major players in marine environments for at least
the past 90 million years (Kooistra et al. 2007).
Diatoms belong to the heterokont branch of the eukaryotes. This group lies within the hypothesized Chromalveolata
kingdom within which several major lineages, including
algae, can be found (Harper et al. 2005). These lineages were
originally defined using morphological and developmental
characters, and have subsequently been refined using molecular approaches—for example, sequence analysis of ribosomal
RNA genes and highly conserved proteins such as RuBisCo
(ribulose-1,5-bisphosphate carboxylase oxygenase) and elongation factor Tu (Baldauf et al. 1996, 2000). More recent,
larger-scale phylogenomics approaches based on multiple
sequence alignments are providing further insights into the
evolutionary relationships between diatoms (Baldauf 2003,
Li et al. 2006). Algal chloroplasts are believed to be derived
from photosynthetic prokaryotes that invaded or were engulfed by a eukaryotic cell and then became endosymbionts
more than 1.5 billion years ago (Gibbs 1981, Cavalier-Smith
1982, 1986). This event subsequently gave rise to the green and
red algal lineages. The chromalveolates are thought to have
derived from a second endosymbiotic event that occurred
around 1 billion years ago (Yoon et al. 2004), in which a red,
algal-like organism became associated a second time with a
heterotrophic eukaryote (figure 2). The most striking evidence
for this is the presence of four membranes surrounding the
758 BioScience • October 2009 / Vol. 59 No. 9
Figure 2. Schematic representation of the secondary
endosymbiotic process thought to have given rise to the
diatoms. An autotrophic red algal–like ancestor was endocytosed by a heterotrophic host cell. In the resulting
cell, gene transfer occurred between the endosymbiont
nucleus and the host nucleus, and probably also from the
plastid and mitochondrial genomes. The resulting diatom
cell contains the endosymbiont chloroplast, surrounded
by four membranes, the host nucleus, and the host mitochondria. New genes have also been acquired by horizontal gene transfer from bacteria. Nuclei are shown in blue.
Abbreviations: D, diatom; HGT, horizontal gene transfer;
m, mitochondria; pp, primary plastid; SE, secondary
endosymbiosis; sp, secondary plastid.
chloroplasts in many photosynthetic chromalveolates such as
the diatoms (Gibbs 1981). Diatoms are further divided into
two groups, the centrics and pennates, on the basis of their
radial and bilateral symmetry, respectively. Diatom fossils
representing centric species date from the Cretaceous, whereas
pennate diatoms appear to have arisen later, around 90 million years ago.
Studies of diatom biology have gone through a paradigm
shift following the recent incorporation of molecular and
cellular methods to dissect their biology. Most of these studies have been performed on two species, Thalassiosira pseudonana and Phaeodactylum tricornutum, now considered model
species for the centrics and pennates, respectively, because of
the availability of whole-genome sequences and molecular
tools to assess gene function (Armbrust et al. 2004, Poulsen
et al. 2007, Siaut et al. 2007, Bowler at al. 2008).
Diatom genome sequencing confirms
novel evolutionary histories
Both diatom genomes have been sequenced by the Joint
Genome Institute in California. The sequence from the centric diatom T. pseudonana was the first to be reported (Armbrust et al. 2004), and it was the first of any eukaryotic marine
phytoplankton species to be sequenced. The P. tricornutum
genome was subsequently completed (Bowler et al. 2008). Both
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21st Century Directions in Biology
species contain around 11,000 predicted genes in approximately 30 million base-pair (Mbp; 32 Mbp for T. pseudonana
and 27 Mbp for P. tricornutum) genomes. A careful functional
and phylogenetic annotation of these genes, facilitated by
the use of powerful computational approaches for predicting
functional domains and subcellular locations, has provided
new information to help understand the biology and evolutionary origins of diatoms. Whole-genome sequences
from a wider range of other algal species have also become
available, including a red alga, Cyanidioschyzon merolae
(Matsuzaki et al. 2004), and three green algae species: Chlamydomonas reinhardtii (Merchant et al. 2007), Ostreococcus tauri
(Derelle et al. 2006), and Ostreococcus lucimarinus (Palenik et
al. 2007).
In addition to whole-genome sequencing, expressed
sequence tags (ESTs) provide a cheaper and simpler way to
begin to acquire genomic data. The ESTs are generated from
mRNA extracted from cells of a species of interest, transformed
into complementary DNA (cDNA), and cloned into plasmids.
A small region of each cDNA can then be sequenced to generate a tag that can serve to identify what the gene encodes.
The ESTs from a range of unicellular algae have now been reported, including those from Fragilariopsis cylindrus, a diatom
found within the ice in polar regions, and Pseudo-nitzschia
multiseries, a bloom-forming diatom capable of synthesizing
the toxin domoic acid. Both of these genome sequences are
now being completed as well.
As noted above, before genome sequencing technologies
had been developed, a prevailing hypothesis was that diatoms
originated from a secondary endosymbiotic event between a
heterotrophic and an autotrophic eukaryote (figure 2). This
hypothesis is supported by genome analysis, which revealed
the presence of genes typical of both animal and plant classes
of eukaryotes, such as components encoding the urea cycle
and fatty acid oxidation, typical of animals, and genes encoding
photosynthesis, found in plants. The proposed red algal origin of the diatom chloroplast is also supported by genome
analysis (Oudot-Le Secq et al. 2007, Bowler et al. 2008). In
addition to providing strong support for these hypotheses,
examination of the predicted gene sets has also revealed the
presence of hundreds of genes that are likely to be derived
from horizontal gene transfer between bacteria and diatoms.
Diatom genomes therefore appear to be “melting pots” of
genes that have been derived from a variety of sources over
evolutionary time, and it has been hypothesized that this
unique cocktail of genes has conferred new metabolic and
regulatory capacities that have been key in establishing their
ecological finesse (Bowler et al. 2008).
Comparisons of gene repertoires between T. pseudonana
and P. tricornutum can also serve as a basis to explain the differences between centric and pennate diatoms. For example,
in contrast to centric diatoms, raphid pennate diatoms
possess a raphe, which permits them to move actively. They
are major biofoulers, they include toxic species, and they
generally respond most strongly to mesoscale iron fertilization (de Baar et al. 2005, Boyd et al. 2007, Kooistra et al.
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2007). They also have amoeboid isogametes in contrast to
motile sperm and oogamy in centric species. The availability
of these two genome sequences, combined with the tools described below, allow the molecular bases of these differences
to be explored and understood.
Analysis of gene expression in diatoms
Analysis of gene expression over time and in different conditions is a useful proxy for identifying the conditions in
which a gene product plays an important role. Gene-expression studies are most often performed by quantifying levels
of mRNA for the gene of interest. This is most commonly done
by first converting mRNA into cDNA, and then quantifying
the amount of expression with quantitative real-time polymerase chain reaction (qRT-PCR) using fluorescent dyes or
fluorescent probes. This technique can be carried out with
small amounts of mRNA, but it is crucial to have accurate reference genes to normalize expression levels. Siaut and colleagues (2007) identified several housekeeping genes in
diatoms whose expression remains relatively constant in different conditions, and in particular proposed the use of RPS,
a gene that encodes a 30S ribosomal protein subunit, and TBP,
a gene encoding the TATA-box binding protein, as rather
stable reference genes.
A more general approach is to use ESTs to identify whole
suites of genes expressed under particular conditions. When
performed on a large scale and without normalizing for
differences in mRNA levels of each gene, a global picture of
gene expression can be obtained. In P. tricornutum, ESTs
have been generated from cells grown in 16 different conditions, such as on different nitrogen sources, under iron
limitation, or in high CO2. Between 6000 and 12,000 ESTs are
available from each library, constituting a total of more than
130,000 ESTs. These sequences have been organized into a digital gene expression database that permits expression patterns
of individual genes to be examined, and also allows the facile
identification of genes displaying similar expression profiles
(Maheswari et al. 2009; www.biologie.ens.fr/diatomics/EST3).
Another approach to examining gene expression at the
whole-genome level is to use microarrays. Technologies are
now available to generate high-density arrays at a low cost, such
as those from Agilent (www.agilent.com) and Nimblegen
(www.nimblegen.com). A microarray typically contains
oligonucleotides representing each gene of interest, for example, for each annotated gene in a diatom genome. Compared with ESTs, an advantage of using microarrays is that
genes that are both up- and downregulated in a particular condition can be identified. On the other hand, ESTs can permit
the identification of bona fide expressed genes that were not
predicted by the in silico methods used for genome annotation. This limitation of typical gene-specific microarrays was
circumvented by Mock and colleagues (2008), who identified
additional unpredicted genes in T. pseudonana using tiled
arrays. Tiled arrays are a kind of microarray in which a whole
genome is represented by oligonucleotides, often on both
DNA strands. Using this method, they identified 3500 putaOctober 2009 / Vol. 59 No. 9 • BioScience 759
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tive new genes, some of which corresponded to noncoding and
antisense RNAs. Among those genes, 75 were identified by
gene-specific expression profiles as potentially involved in
silicon metabolism, and half of them encode proteins of
unknown function. Interestingly, these genes also provided
evidence of a link between silicon and iron metabolism
pathways.
The above-described methods for studying gene expression
can help infer a function of diatom-specific genes, and
several examples have now been reported in which these
methods have been used to explore specific aspects of diatom
biology, such as nutrient assimilation (Allen AE et al. 2008,
Mock et al. 2008), light responses (Siaut et al. 2007), and
gene expression during cell division (Gillard et al. 2008).
Such studies are especially important, given the unusual combinations of genes that have been found in diatoms, such that
empirical studies of gene expression in different conditions
are required to understand how they function together in a
coordinated manner.
Transgenic technology
It is also important to be able to manipulate the expression
of single genes and to assess the consequences of that modulation for the organism under study. Genetic transformation
technologies offer powerful ways of doing this by reverse
genetics. In diatoms, the most commonly used technique
for generating transgenic cells is based on helium-accelerated
bombardment of microparticles coated with the DNA that is
to be introduced. The methodology was initially reported for
Cyclotella cryptica (Roessler et al. 1994) and subsequently
for P. tricornutum (Apt et al. 1996), but has now been applied
to a range of other diatoms, including most recently T. pseudonana (Poulsen et al. 2007). Notwithstanding, the tools are most
highly developed for P. tricornutum—in particular, a series of
different transformation vectors made from the Gateway
cloning system from Invitrogen (www.invitrogen.com/gateway.
html; Siaut et al. 2007), which greatly facilitates the generation of chimeric gene constructs for a range of different applications, such as for the inactivation and overexpression of
genes, and for the localization of a gene product inside the cell
by fusing it to fluorescent reporters.
The green fluorescent protein (GFP) from the jellyfish
Aequorea victoria, the most versatile fluorescent tag currently
used in biology, was the subject of the project that won the
2008 Nobel prize for chemistry. Besides the wild-type green
version, a series of differently colored variants are now available, which can even be combined to label different proteins
in the same cell (figure 3). Such technologies complement the
more traditional protein localization approaches by subcellular fractionation and immunolocalization, in that they
permit localization to be visualized inside living cells. In
addition to GFP, other reporter proteins have also been used
in diatoms, such as luciferase and beta-glucuronidase, which
allow studies of gene expression in response to particular
conditions (Falciatore et al. 1999).
Using GFP as a reporter for protein localization, Kroth
(2007a, 2007b) studied the mechanisms of protein translocation and import into diatom chloroplasts, a fundamental
but little-understood process in diatoms. This is of further interest because diatom plastids are surrounded by four instead
of two membranes. These studies have shown that the outer
two membranes of diatom plastids appear to be derived from
Figure 3. Fluorescent image of a pair of transgenic Phaeodactylum tricornutum cells cotransformed
with a Sec4 protein-YFP fusion (green) and a Histone H4-CFP fusion (blue). The Sec4 protein
localizes to intracellular vesicles and to the plasma membrane, whereas H4 localizes to the
nucleus. Chlorophyll autofluorescence from the plastid is shown in red. A brightfield view of
two P. tricornutum cells (left) is shown for comparison. Images: Courtesy of Anton Montsant.
760 BioScience • October 2009 / Vol. 59 No. 9
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21st Century Directions in Biology
plasma membrane and endoplasmic reticulum, whereas the
inner two membranes resemble those found in green algae and
higher plants, as would be predicted if diatom plastids were
indeed acquired through secondary endosymbiosis (Gibbs
1981). Other studies have shown proteins localized in diatom
mitochondria and other subcellular organelles (Siaut et al.
2007). Furthermore, Vardi and colleagues (2008) recently
found that a protein associated with nitric oxide (NO) production localizes to diatom chloroplasts, in contrast to its plant
ortholog, which was found in mitochondria (Guo and Crawford 2005). Such fundamental differences were instrumental
in defining the role of the protein in diatoms (see below), thus
showing the utility of GFP-based reporters for protein localization studies.
Transgenes introduced in diatoms are under the control of
promoters, DNA sequences upstream of protein coding sequences that spatiotemporally regulate gene expression. When
a transgene is placed under the control of a chosen promoter,
it will usually display the expression pattern of the gene from
which the promoter was derived. Transgenes are most often
expressed from strong promoters such as FCP (fucoxanthin
chlorophyl protein) promoters (derived from genes encoding
light-harvesting fucoxanthin-chlorophyll a/c-binding proteins). Although these promoters are to some extent regulated
by light, they are generally considered to be rather constitutive. Transgenic technology also enables the modulation of
gene expression using inducible promoter systems, which
can be particularly useful when expression of a gene is lethal
for the cell. Poulsen and Kröger (2005) reported the first
method for inducible gene expression to study gene function
in diatoms, based on a nitrate reductase promoter that is
responsive to exogenous nitrate concentrations. With this
system, a transgene can in principle be switched on and off
simply by controlling the amount of nitrate in the growth
medium.
Overexpression of a gene of interest can help in understanding its function, particularly when its inactivation is
lethal. An overexpressed gene can cause a change in phenotype, thereby providing information that is useful for understanding its function. Overexpression of specific genes
has been reported several times in diatoms. A notable example is the overexpression of a gene encoding a glucose transporter to convert P. tricornutum cells from photoautotrophy
to heterotrophy (Zaslavskaia et al. 2001).
The inhibition of expression of a gene of interest has also
become a crucial method for elucidating gene function. The
method is most typically called gene silencing, and it consists
of generating small RNAs complementary to the target gene.
These small RNAs will bind to the transcribed product and
inhibit its translation into protein. The technique has recently been reported in diatoms, providing for the first time
a method of inactivating gene expression in these organisms
(De Riso et al. 2009).
In addition to the methods of reverse genetics described
above, forward genetics can discover genes responsible for a
defined phenotype. In forward genetics, cells are usually first
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mutagenized and then screened for interesting and unusual
phenotypes in the mutagenized population. This method
has proved to be extremely powerful for dissecting the basic
biology of a wide range of both unicellular and multicellular organisms (Candela and Hake 2008, Carradice and Lieschke
2008), although it has not yet been reported in diatoms
because of the difficulty of controlling their sexual cycle
(Chepurnov et al. 2008). This is important because diatom cells
are diploid, so any recessive mutation needs first to be fixed
on both copies by going through a round of meiosis, that is,
sex. To circumvent this shortcoming, a technique known as
activation tagging has been used in other organisms (Candela
and Hake 2008, Carradice and Lieschke 2008). The approach
is based on the random introduction into the genome of enhancers, DNA sequences that positively affect expression of
a gene when inserted close by. The use of such techniques to
generate dominant mutations in diatoms has not yet been reported, although it is likely to be a useful strategy for isolating mutants in forward genetic screens.
The benefits of using modern molecular
technologies to study diatoms
The use of molecular biology together with more classical
studies of diatom biology has led to a range of advances for
understanding their cell biology. The targeting of proteins
to the plastid (Gruber et al. 2007) and the dissection of
diatom cell-division mechanisms (Gillard et al. 2008) were
mentioned previously. The availability of diatom genome
sequences has also provided valuable starting points for
exploring their responses to key limiting nutrients such as
nitrogen, silicate, and iron (Allen AE et al. 2006). Iron
metabolism is of particular interest because diatoms tend to
dominate in mesoscale iron fertilization experiments (de
Baar et al. 2005, Boyd et al. 2007), suggesting that they are iron
limited under natural conditions. Iron responses have been
studied at the transcriptional level in both sequenced diatoms
(Allen AE et al. 2008, Bidle and Bender 2008, Mock et al. 2008).
While both diatoms possess classical ferric reductase enzymes, these enzymes are more numerous in P. tricornutum.
Furthermore, P. tricornutum possesses a number of gene
clusters—absent in the T. pseudonana genome—that are
highly expressed under iron limitation. Some of these genes,
prokaryotic in character, point to new iron acquisition systems
that have not yet been described in eukaryotic algae (Allen AE
et al. 2008). Also notable is the presence of the iron-storage
protein ferritin in P. tricornutum but not in T. pseudonana
(Marchetti et al. 2008). These differences between the sequenced centric and pennate diatoms may partly explain the
higher tolerance of P. tricornutum, and pennate diatoms in
general, to iron limitation (Kustka et al. 2002, de Baar et al.
2005, Boyd et al. 2007).
Carbon fixation in aquatic organisms can be enhanced by
CO2-concentrating mechanisms (CCMs) that increase the
availability of CO2 for the carbon-fixing RuBisCo enzyme.
These mechanisms are defined as biophysical, because of
the action of inorganic carbon uptake systems and carbonic
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anhydrases, and biochemical, which is based on the cycling
of CO2 through C4 intermediates. The mechanisms used by
diatoms remain controversial despite intensive research (Giordano et al. 2005). Phaeodactylum tricornutum has been proposed to have a greater capacity for biophysical CCM than
other diatoms (Kroth et al. 2008), and this is also supported
by the higher numbers, compared with T. pseudonana, of
bicarbonate transporters (seven and three, respectively) and
carbonic anhydrases (four and one) encoded in its genome.
Furthermore, one of the carbonic anhydrases is a beta-type,
plastid-localized enzyme that is absent in T. pseudonana
(Montsant et al. 2005). On the other hand, both diatoms
may use a C4-based biochemical CCM involving the cycling
of C4 intermediates between the inside of the plastid, the
periplastidic space, and the mitochondria (Kroth et al. 2008).
If confirmed, such a mechanism would be a highly novel
means of capturing CO2, and may also help the cells to dissipate excess light energy.
Illustrating the utility of molecular approaches to study
diatom responses of ecological relevance, recent studies reveal
the presence of complex inter- and intracellular signaling
mechanisms that regulate population proliferation and even
programmed cell death in response to environmental signals.
When zooplankton graze on diatom populations, the diatoms
release aldehydes such as decadienal that can reduce the reproductive capacity of the grazer population, potentially providing an antigrazing strategy. Increased aldehyde production
by diatoms can also occur as a general response to wounding
(Pohnert et al. 2004).Vardi and colleagues (2006) showed that
both P. tricornutum and T. weissflogii respond to treatment
with the aldehyde by producing NO, a phenomenon that is
most likely regulated by changes in intracellular calcium concentrations. At high concentrations, the aldehyde causes cell
death, whereas pretreatment with low concentrations can
prime cells to become immune. In a subsequent study, Vardi
and colleagues (2008) showed that expression of the PtNOA
gene (NO associated), which encodes a GTP-binding protein
belonging to the highly conserved YqeH subfamily, is increased in response to the aldehyde, and P. tricornutum cells
overexpressing PtNOA display increased NO production and
the appearance of several features symptomatic of stress. Adhesion of cells to surfaces was also compromised, implying the
importance of NO-regulated events for biofilm formation
(Thompson et al. 2008). These studies therefore identify a
diatom gene that appears to be central for regulating stress
sensitivity in diatoms.
Diatom silicification is one of the most distinctive features of diatoms (figure 1). The frustule (the cell wall) is
composed of two halves, a larger half and a smaller half called
the epivalve and the hypovalve, respectively. The frustule is
partly organic (proteins and polysaccharides) and partly
bioinorganic (hydrated silicon, SiO2·[H2O]n). This incredibly robust and highly ornamented structure has several proposed roles, including protection from grazers and parasites
through its mechanical resistance (Hamm et al. 2003, Pondaven et al. 2007), or as a proton-buffering agent (Milligan
762 BioScience • October 2009 / Vol. 59 No. 9
and Morel 2002). The frustule is synthesized during cell division within a membrane-bound organelle, termed the
silica deposition vesicle (SDV), which rapidly extends to
form a flat, large vesicle in which the new valve is synthesized.
When the new valve is complete, it is bulk exocytosed and
becomes the hypovalve of the new cell (Zurzolo and Bowler
2001).
Pioneering biochemical studies of frustule composition
have been performed by Kröger and colleagues in the pennate
diatom Cylindrotheca fusiformis, and have led to the identification of several components found only in diatoms (Kröger
et al. 1999, Kröger and Poulsen 2008). This work led to the
discovery of silaffins, novel peptides that may participate in
the basic biomineralization process within the SDV (Kröger
et al. 1999). Remarkably, these silaffins can promote the
formation of nanoscale silica spheres in vitro, and are the first
peptides shown to be able to do this. They are encoded by
modular genes whose gene product requires extensive posttranslational modifications such as the addition of phosphate and sugar groups during maturation from the precursor
protein to the mature peptides. Other major organic constituents of diatom biosilica are putrescine-derived, long-chain
polyamines, which, like the silaffins, can also induce rapid
silicic acid precipitation in vitro (Kröger et al. 2000). Different diatoms are likely to have different complements of
silaffins and polyamines that confer species-specific differences
to silica precipitation and thereby result in species-specific
nanopatterning, although they have so far been poorly characterized because of the difficulties of identifying the genes
involved on the basis of only homology. Furthermore, it
appears that the posttranslational modifications to these
peptides are in fact more important than their amino acid
sequence per se. Notwithstanding, silaffin genes have been
fused to GFP, and the fusion proteins are incorporated into
the silicified cell walls (Poulsen et al. 2007). Silaffin gene
expression is also upregulated significantly during valve
formation.
Other proteinaceous components of diatom cell walls
include frustulins and pleuralins (formerly called HEP
proteins; Kröger and Poulsen 2008), containing conserved
calcium-binding domains separated by hydroxyproline,
polyproline/hydroxyproline, or polyglycine-rich regions. Like
the silaffins, pleuralins are also tightly bound to silica and can
be removed from diatom cell walls only after the solubilization of silica with hydrogen fluoride. Pleuralins are encoded
by a small multigene family in C. fusiformis but have not
been found in T. pseudonana or P. tricornutum, and so they
are likely to represent a specific structural feature of this
diatom. Pleuralin-1 is not targeted to the SDV but is directly
secreted into the cleavage furrow that forms between the two
daughter cells (Kröger and Wetherbee 2000). Association
with the terminal girdle band of the hypotheca therefore
occurs in the extracellular space. It will be interesting to determine how many other wall-associated proteins avoid the
SDV during their secretion and incorporation into diatom
frustules.
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21st Century Directions in Biology
Frustulins are much more loosely associated with diatom
cell walls than are silaffins and pleuralins, and can be extracted with EDTA (ethylenediaminetetraacetic acid, a common chelating agent; Kröger and Poulsen 2008). They are
glycoproteins that can bind calcium because of the presence
of EF hands (helix-loop-helix structures in a family of calciumbinding proteins), and also contain characteristic acidic,
cysteine-rich domains. Although frustulins are most likely
conserved in all diatoms, including T. pseudonana and P. tricornutum, their function is not yet known. They are not
thought to be involved in silica deposition.
Biotechnology. The precision of the nanoscale pattern and
architecture of the frustule far exceeds the capabilities of
current materials and science engineers, suggesting that
understanding diatom cell-wall biosynthesis will one day be
exploitable in nanotechnological applications (Parkinson
and Gordon 1999, Lopez et al. 2005). Although the metabolic
pathways that drive cell-wall biosynthesis remain largely unexplored, they constitute a clear target for the discovery of
novel protein functions that are unlikely to be found in other
organisms and can be exploited in biotechnological applications. For example, diatom frustules can be incorporated
into membranes and used for the size-selective separation of
nanoparticles (Losic et al. 2006).
Biomimetic studies also seem promising: For example,
Vrieling and colleagues (2005) reported using water glass–
based and polyethylene oxide–based polymers to control the
synthesis of silica to generate ordered porous structures at the
nanometer and micrometer levels. The use of frustules to make
other functional materials by chemical conversion has also
been reported (Bao et al. 2007). These technologies open up
new opportunities to produce three-dimensional (3-D)
siliceous materials that have never before been engineered
(Kröger 2007, Kröger and Poulsen 2008). In addition, Gordon and Parkinson (2005) proposed another role for silica in
linear lithographic techniques that are used to engineer
microelectronics and that consist in replacement of silicon with
another atom while maintaining the 3-D structure of origin.
The genomic-enabled techniques described in this review
can be of great utility for understanding and ultimately exploiting the silicon nanaofabrication capacities of diatoms, and
some progress has already been made, as evidenced in the
previous section. Further progress will very likely require
additional high-quality biochemistry, as well as high-throughput, genetic-based screens, to identify diatom-specific genes
of currently unknown function.
The conversion of solar energy into chemical energy by
photosynthesis has become of great interest for the generation of renewable energy resources. Diatoms have a high
lipid content (up to 70% dry weight; Chisti 2007), and so they
have been proposed as a source of biofuels (Kroth 2007a).
Furthermore, the residual biomass is rich in protein and so
could be used as animal feed. Moreover, because diatoms do
not contain complex carbohydrate-based polymers such as
cellulose, ruminants’ digestion of diatom-derived biomass
www.biosciencemag.org
generates less methane and other potent greenhouse gases than
would be the case with other feed. Now that fossil fuels are
being depleted and becoming more expensive, these diatombased applications are particularly appealing. Compared with
plant-based sources of biofuels, diatoms and other algae are
much more efficient converters of solar energy and have a
much higher energy potential (Chisti 2008). Furthermore, they
do not compete with food production, they can be grown in
saltwater on marginal land, and they require less water inputs
(Lebeau and Robert 2003, Dismukes et al. 2008). Phaeodactylum tricornutum is an attractive target for proof of principle because it has a high lipid content (up to 30%), it can
be genetically manipulated, and it is already widely cultivated for commercial aquaculture. Genetic manipulation
can potentially be used to increase photosynthetic efficiency
to enable increased biomass yield, to enhance biomass growth
rate, to increase oil content in biomass, to improve temperature tolerance to reduce the expense of cooling, to eliminate
light saturation of photosynthesis, and to reduce photoinhibition and photooxidation. In addition, there is a need to identify new diatom strains with high oil content or to breed or
select for improved strains.
Conclusions
The recently available genome sequences from two diatoms
demonstrated the novel multilineage history of their gene
repertoires and revealed that their genomes encode an enormous metabolic and regulatory potential that perhaps underlies their ecological success. The noncanonical nature of their
genomes indicates that the functional exploration of diatomspecific genes is required to dissect their roles in diatom biology. Revealing the functions of these thousands of diatom
genes that do not have proxies in conventional model organisms is going to be a major challenge, and it is highly unlikely (because of the lack of financial resources and dedicated
personnel) that each can be experimentally assigned a function through reverse-genetics approaches such as gene knockouts. Nonetheless, new molecular techniques combined with
biochemical approaches provide an excellent starting point
for exploring novel aspects of diatom biology and for developing biotechnological applications. Additional computational approaches are likely to be required to help us predict
protein functions, and proteomics approaches can help to
associate specific proteins with specific cellular structures or
protein complexes.
As knowledge of diatom biology grows through laboratorybased experiments, additional technologies will need to be developed for exploring diatom biology in natural environments.
Metabolomics technologies could be of some help, in that they
can reveal the metabolic signatures of cells grown in specific
conditions, as was recently illustrated (Allen AE et al. 2008).
High-throughput genomics technologies that have yet to be
developed for diatoms would also be a major boost for rapidly
identifying mutations that result in major cellular perturbations (e.g., in the silicon nanofabrication process). In all such
scenarios, the available genome sequences clearly provide a
October 2009 / Vol. 59 No. 9 • BioScience 763
21st Century Directions in Biology
major advance in our knowledge and in our opportunities to
explore diatom biology. We eagerly await the forthcoming
sequences from the polar diatom Fragilariopsis cylindrus and
the toxin-producing Pseudo-nitzschia multiseries.
Acknowledgments
Funding for our work has been obtained from the European
Union (EU)–funded FP6 Diatomics project (LSHG-CT2004-512035), the EU-FP6 Marine Genomics Network of
Excellence (GOCE-CT-2004-505403), an ATIP (Actions Thématiques Initatives sur Programmes) Blanche grant from
the Centre National de la Recherche Scientifique, and the
Agence Nationale de la Recherche (France).
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Anastasia Saade ([email protected]) and Chris Bowler (cbowler@biologie.
ens.fr) are with the Department of Biology at the École Normale Supérieure
in Paris. Bowler is the director of research, and his laboratory studies signaling in higher plants and marine diatoms.
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