BioScience 2009 Saade 757 65

8/12/2019 BioScience 2009 Saade 757 65

1/9

In the upper zone of the ocean and other water bodies, downto depths where light can penetrate, one can typically findan abundant group of eukaryotic algae known as diatoms.

These microscopic, unicellular organisms are characterized

by ornate, lacework-like, silicified shells and are distributedall around the world.Diatoms are photosynthetic organisms

that can convert the energy from sunlight into chemical

energy in the form of ATP (adenosine triphosphate). Thischemical reaction confers on diatoms the ability to producetheir 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 photosynthe-

sis. They are in fact believed to contribute between 20% and

25% of global primary production, equivalent to all terres-

trial 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 Lauterbornsubsequently made exquisite microscopic descriptions of

subcellular events occurring during diatom cell division. A

century later, Jeremy Pickett-Heaps translated Lauterborns

observations from the original German and verified his dis-coveries 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 macro-

nutrients, certain micronutrients and vitamins were required

to cultivate them (Harvey 1939). Ecological and descriptivestudies continue to this day, with researchers now incorpor-

ating advanced techniques of molecular and cellular biology.

Consequently, knowledge of diatoms basic biology and theirpotential for a range of commercial exploitations is now ad-vancing 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 structuresis largely unknown,although some insights are now being re-

vealed (see below).The process is termedbiomineralization

(defined as the formation of inorganic materials under bio-

logical control), and the species-specific patterns indicatethat it is genetically determined. Because marine organisms

use more than 6.7 gigatons of silicon per year (Trguer et al.

1995), it is particularly important to understand silicon

uptake and deposition processes in diatoms.Furthermore, diatoms are used as bioindicators of pollu-

tion 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 analyzingplankton diversity. Diatoms are also used as hydrographic

tracers because biogenic silica retains its primary oxygen

BioScience59: 757765. ISSN 0006-3568, electronic ISSN 1525-3244. 2009 by American Institute of Biological Sciences. All rights reserved. Request

permission to photocopy or reproduce article content at the University of California Presss Rights and Permissions Web site at www.ucpressjournals.

com/reprintinfo.asp. doi:10.1525/bio.2009.59.9.7

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, andgenerate as much oxygen through photosynthesis as terrestrial rainforests do. Insights into their evolutionary origins have been revealed by thewhole-genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum. We now know that diatoms contain unusualassortments of genes derived from different sources, including those acquired by horizontal gene transfer from bacteria. These genes confer novelmetabolic and signaling capacities that may underlie the extraordinary ecological success of diatoms on Earth today. The availability of a suite oftechniques 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 incommercial applications, such as for nanotechnology and biofuel production.

Keywords: genomics, microarrays, nanotechnology, transgenic technology, biofuel

www.biosciencemag.org October 2009 / Vol. 59 No. 9 BioScience 757

21st Century Directions in Biology21st Century Directions in Biology


2/9

758 BioScience October 2009 / Vol. 59 No. 9 www.biosciencemag.org

21st Century Directions in Biology21st Century Directions in Biology

isotopic composition after burial (Sancetta 1981). This prop-erty 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 recordis 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 eukary-

otes. 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 developmentalcharacters, and have subsequently been refined using molec-

ular approachesfor example, sequence analysis of ribosomal

RNA genes and highly conserved proteins such as RuBisCo

(ribulose-1,5-bisphosphate carboxylase oxygenase) and elon-gation 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 derivedfrom photosynthetic prokaryotes that invaded or were en-

gulfed 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 riseto the green andred 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 aheterotrophic eukaryote(figure 2).The most striking evidence

for this is the presence of four membranes surrounding the

chloroplasts in many photosynthetic chromalveolates such asthe diatoms (Gibbs 1981). Diatoms are further divided intotwo groups, the centrics and pennates, on the basis of their

radial and bilateral symmetry, respectively. Diatom fossils

representing centric species datefrom the Cretaceous, whereas

pennate diatoms appear to have arisen later, around 90 mil-lion 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 stud-ies have been performed on two species, Thalassiosira pseudo-nanaandPhaeodactylumtricornutum, now considered modelspecies for the centrics and pennates,respectively, because of

the availability of whole-genome sequences and moleculartools to assess gene function (Armbrust et al. 2004, Poulsenet 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 cen-

tric diatomT. pseudonanawas the first to be reported (Arm-brust et al. 2004),and it was the first of any eukaryoticmarinephytoplankton species to be sequenced. TheP. tricornutumgenome was subsequently completed (Bowler et al.2008). Both

Figure 1. Electron micrograph of the elaborate silicified

cell wall of a diatom (Thalassiosira oestrupiivar.ven-

rickae). The cell has a diameter of 9.5 microns. Image:Courtesy of Diana Sarno (Service for Taxonomy and

Identification of Marine Phytoplankton, Stazione Zoo-

logica Anton Dohrn, Naples, Italy).

Figure 2. Schematic representation of the secondary

endosymbiotic process thought to have given rise to the

diatoms. An autotrophic red algallike ancestor was en-docytosed by a heterotrophic host cell. In the resulting

cell, gene transfer occurred between the endosymbiont

nucleus and the host nucleus, and probably also from theplastid and mitochondrial genomes. The resulting diatom

cell contains the endosymbiont chloroplast, surrounded

by four membranes, the host nucleus, and the host mito-

chondria. New genes have also been acquired by horizon-tal 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.


3/9

species contain around 11,000 predicted genes in approxi-mately 30 million base-pair (Mbp; 32 Mbp forT. pseudonanaand 27 Mbp for P. tricornutum) genomes.A careful functionaland phylogenetic annotation of these genes, facilitated by

the use of powerful computational approaches for predictingfunctional domains and subcellular locations, has provided

new information to help understand the biology and evo-lutionary origins of diatoms. Whole-genome sequences

from a wider range of other algal species have also becomeavailable, including a red alga, Cyanidioschyzon merolae(Matsuzaki et al.2004),and three green algae species:Chlamy-domonas reinhardtii(Merchantet al.2007),Ostreococcus tauri(Derelle et al.2006), andOstreococcus lucimarinus(Palenik etal. 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 frommRNAextracted from cellsof a species of interest, transformed

into complementary DNA (cDNA),and cloned into plasmids.

A small region of each cDNA can then be sequenced to gen-

erate a tag that can serve to identify what the gene encodes.The ESTs from a range of unicellular algae have now been re-

ported, including those from Fragilariopsis cylindrus, a diatomfound within the ice in polar regions, andPseudo-nitzschiamultiseries, a bloom-forming diatom capable of synthesizingthe 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 diatomsoriginated from a secondary endosymbiotic event between a

heterotrophic and an autotrophic eukaryote (figure 2). This

hypothesis is supported by genome analysis, which revealedthe presence of genes typical of both animal and plant classesof eukaryotes, such as components encoding the urea cycle

and fatty acid oxidation, typical of animals, andgenesencoding

photosynthesis, found in plants. The proposed red algal ori-

gin of the diatom chloroplast is also supported by genomeanalysis (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 derivedfrom 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 thisunique cocktail of genes has conferred new metabolic andregulatory capacities that have been key in establishing their

ecological finesse (Bowler et al. 2008).

Comparisons of gene repertoires betweenT. pseudonanaand P. tricornutum can also serveas a basis to explain the dif-ferences 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 theygenerally respond most strongly to mesoscale iron fertiliza-

tion (de Baar et al. 2005, Boyd et al. 2007, Kooistra et al.

2007). They also have amoeboid isogametes in contrast tomotile sperm and oogamy in centric species. The availability

of these two genome sequences, combined with the tools de-

scribed below, allow the molecular bases of these differences

to be explored and understood.

Analysis of gene expression in diatomsAnalysis of gene expression over time and in different con-

ditions is a useful proxy for identifying the conditions inwhich a gene product plays an important role. Gene-expres-

sion studies are most often performed by quantifying levels

of mRNAfor thegene of interest.This is most commonly done

by first converting mRNA into cDNA, and then quantifyingthe amount of expression with quantitative real-time poly-

merase 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 ref-erence genes to normalize expression levels. Siaut and col-

leagues (2007) identified several housekeeping genes in

diatoms whose expression remains relatively constant in dif-

ferent conditions,and in particular proposed the use ofRPS,a gene that encodes a 30S ribosomal protein subunit,andTBP,a gene encoding the TATA-box binding protein, as rather

stable reference genes.

A more general approach is to use ESTs to identify wholesuites 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, ESTshave been generated from cells grown in 16 different con-

ditions, such as on different nitrogen sources, under iron

limitation, or in high CO2. Between 6000 and 12,000ESTs areavailable from each library, constituting a total of more than130,000 ESTs.These sequences have been organizedinto a dig-

ital gene expression database that permits expressionpatterns

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-densityarrays at a low cost,suchas those from Agilent (www.agilent.com) and Nimblegen(www.nimblegen.com). A microarray typically containsoligonucleotides representing each gene of interest, for ex-

ample, for each annotated gene in a diatom genome. Com-pared with ESTs, an advantage of using microarrays is thatgenes that are both up- anddownregulated in a particular con-

dition can be identified.On the other hand, ESTs can permit

the identification of bona fide expressed genes that were not

predicted by thein silicomethods used for genome annota-tion. This limitation of typical gene-specific microarrays was

circumvented by Mock and colleagues (2008), who identified

additional unpredicted genes inT. pseudonanausing tiledarrays.Tiled arrays are a kind of microarray in which a wholegenome is represented by oligonucleotides, often on both

DNA strands. Using this method, they identified 3500 puta-


21st Century Directions in Biology


4/9

tive new genes, some of which corresponded to noncodingandantisense 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 providedevidence 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, andseveral 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), andgene expression during cell division (Gillard et al. 2008).

Such studies are especially important, given the unusual com-

binations of genes that have been found in diatoms, such that

empirical studies of gene expression in different conditionsare 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 mod-

ulationfor the organism under study. Genetictransformation

technologies offer powerful ways of doing this by reversegenetics. In diatoms, the most commonly used technique

for generating transgenic cells is based on helium-accelerated

bombardment of microparticles coated withthe DNA that is

to be introduced.The methodology was initially reported forCyclotella cryptica(Roessler et al. 1994) and subsequentlyfor P. tricornutum (Apt et al. 1996),but has now been applied

to a range of other diatoms,including most recentlyT. pseudo-nana(Poulsen et al.2007). Notwithstanding, thetools aremosthighly developed for P. tricornutumin particular, a series ofdifferent transformation vectors made from the Gateway

cloning system from Invitrogen (www.invitrogen.com/gateway.html; Siaut et al. 2007), which greatly facilitates the genera-

tion of chimeric gene constructs for a range of different ap-plications, such as for the inactivation and overexpression of

genes,and for the localization of a gene product inside thecellby fusing it to fluorescent reporters.

The green fluorescent protein (GFP) from the jellyfish

Aequorea victoria, the most versatile fluorescent tag currentlyused in biology, was the subject of the project that won the2008 Nobel prize for chemistry. Besides the wild-type green

version, a series of differently colored variants are nowavail-

able, which can even be combined to label different proteins

in thesame cell (figure 3). Such technologies complement themore traditional protein localization approaches by sub-

cellular fractionation and immunolocalization, in that they

permit localization to be visualized inside living cells. In

addition to GFP, other reporter proteins have also been usedin 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 translo-

cation and import into diatom chloroplasts, a fundamental

but little-understood process in diatoms. This is of further in-

terest because diatom plastids are surrounded by four insteadof 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 transgenicPhaeodactylum tricornutumcells cotransformed

with a Sec4 protein-YFP fusion (green) and a Histone H4-CFP fusion (blue). The Sec4 proteinlocalizes 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

twoP. tricornutumcells (left) is shown for comparison. Images: Courtesy of Anton Montsant.


5/9

plasma membrane and endoplasmic reticulum, whereas theinner twomembranes resemblethose 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 diatommitochondria and other subcellular organelles (Siaut et al.

2007). Furthermore, Vardi and colleagues (2008) recentlyfound that a protein associated with nitric oxide (NO) pro-

duction localizes to diatomchloroplasts, in contrast to its plantortholog,which was found in mitochondria (Guo and Craw-

ford 2005).Such fundamental differences were instrumental

in defining therole of the protein in diatoms (see below), thus

showing the utility of GFP-based reporters for protein local-ization studies.

Transgenes introduced in diatoms are under the control of

promoters, DNA sequences upstream of protein coding se-

quences that spatiotemporally regulate gene expression.Whena 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 (fucoxanthinchlorophylprotein) promoters (derived from genes encoding

light-harvesting fucoxanthin-chlorophylla/c-binding pro-teins). Althoughthese promoters are to some extent regulated

by light, they are generally considered to be rather constitu-tive. 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 Krger (2005) reported the firstmethod for inducible gene expression to study gene function

in diatoms, based on a nitrate reductase promoter that is

responsive to exogenous nitrate concentrations. With thissystem, a transgene can in principle be switched on and offsimply by controlling the amount of nitrate in the growth

medium.

Overexpression of a gene of interest can help in under-

standing its function, particularly when its inactivation islethal. An overexpressed gene can cause a change in pheno-

type, thereby providing information that is useful for un-

derstanding its function. Overexpression of specific genes

has been reported several times in diatoms.A notable exam-ple is the overexpression of a gene encoding a glucose trans-

porter to convertP. tricornutum cells from photoautotrophyto heterotrophy (Zaslavskaia et al. 2001).

The inhibition of expression of a gene of interest has alsobecome a crucial method for elucidating gene function. Themethod 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 re-cently 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 describedabove, forward genetics can discover genes responsible for a

defined phenotype.In forward genetics,cells are usually first

mutagenized and then screened for interesting and unusualphenotypes 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 multicellu-

lar organisms (Candela and Hake 2008, Carradice and Lieschke2008), although it has not yet been reported in diatoms

because of the difficulty of controlling their sexual cycle(Chepurnov et al. 2008). This is importantbecause diatom cells

are diploid, so any recessive mutation needs first to be fixedon 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 approachis based on the random introduction into the genome of en-

hancers, 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 notyet been re-ported, although it is likely to be a useful strategy for isolat-

ing 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 proteinsto 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 forexploring 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 todominate in mesoscale iron fertilization experiments (deBaar 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 bothsequenced diatoms

(AllenAE et al. 2008, Bidle and Bender 2008, Mock et al. 2008).While both diatoms possess classical ferric reductase en-

zymes, these enzymes are more numerous in P. tricornutum.Furthermore,P. tricornutum possesses a number of geneclustersabsent in theT. pseudonanagenomethat arehighly expressed under iron limitation. Some of these genes,

prokaryotic in character, point to newiron acquisition systems

that have not yet been described in eukaryotic algae (Allen AE

et al. 2008). Also notable is the presence of the iron-storageprotein ferritin inP. tricornutumbut not inT. pseudonana(Marchetti et al. 2008). These differences between the se-

quenced centric and pennate diatoms may partly explain the

higher tolerance ofP. tricornutum,and pennate diatoms ingeneral, 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 CO2for the carbon-fixing RuBisCo enzyme.

These mechanisms are defined as biophysical, because of

the action of inorganic carbon uptake systems and carbonic




6/9

anhydrases, and biochemical, which is based on the cyclingof CO

2through C4 intermediates. The mechanisms used by

diatoms remain controversialdespite intensive research(Gior-

dano et al. 2005). Phaeodactylum tricornutumhas been pro-posed to have a greater capacity for biophysical CCM thanother diatoms (Kroth et al. 2008), and this is also supported

by the higher numbers, compared withT. pseudonana, ofbicarbonate 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 cyclingof 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 dis-

sipate 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 signalingmechanisms that regulate population proliferation and even

programmed celldeath in response to environmental signals.

When zooplankton graze on diatom populations,the diatoms

release aldehydes such as decadienal that can reduce the re-productive capacity of the grazer population,potentially pro-

viding an antigrazing strategy. Increased aldehydeproduction

by diatoms can also occur as a general response to wounding

(Pohnert et al.2004).Vardiand colleagues (2006) showed thatbothP. tricornutumandT. weissflogiirespond to treatmentwith the aldehyde by producing NO, a phenomenon that is

most likely regulated by changes in intracellular calcium con-centrations. At high concentrations, the aldehyde causes celldeath, whereas pretreatment with low concentrations can

prime cells to become immune. In a subsequent study, Vardi

and colleagues (2008) showed that expression of the PtNOAgene (NO associated),whichencodes a GTP-binding proteinbelonging to the highly conserved YqeH subfamily, is in-

creased in response to the aldehyde, andP. tricornutumcellsoverexpressing PtNOA display increased NO production andthe appearance of several features symptomatic of stress. Ad-hesionof 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 stresssensitivity in diatoms.

Diatom silicification is one of the most distinctive fea-

tures 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 ispartly organic (proteins and polysaccharides) and partly

bioinorganic (hydrated silicon, SiO2[H

2O]n). This incredi-

bly robust and highly ornamented structure has several pro-

posed roles, including protection from grazers and parasitesthrough its mechanical resistance (Hamm et al. 2003, Pon-

daven et al. 2007), or as a proton-buffering agent (Milligan

and Morel 2002). The frustule is synthesized during cell di-vision 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 andbecomes the hypovalve of the new cell (Zurzolo and Bowler

2001).Pioneering biochemical studies of frustule composition

have been performed by Krger and colleagues in thepennatediatomCylindrotheca fusiformis,and have led to the identi-fication of several components found only in diatoms (Krger

et al. 1999, Krger and Poulsen 2008). This work led to the

discovery of silaffins, novel peptides that may participate inthe basic biomineralization process within the SDV (Krger

et al. 1999). Remarkably, these silaffins can promote the

formation of nanoscale silica spheres in vitro, and are the firstpeptides shown to be able to do this. They are encoded bymodular genes whose gene product requires extensive post-

translational modifications such as the addition of phos-

phate and sugar groupsduringmaturation from the precursor

protein to the mature peptides.Other major organic constitu-ents of diatom biosilica are putrescine-derived, long-chain

polyamines, which, like the silaffins, can also induce rapid

silicic acid precipitationin vitro(Krger et al. 2000). Differ-ent diatoms are likely to have different complements ofsilaffinsand polyamines thatconfer species-specific differences

to silica precipitation and thereby result in species-specific

nanopatterning, although they have so far been poorly char-

acterized because of the difficulties of identifying the genesinvolved on the basis of only homology. Furthermore, it

appears that the posttranslational modifications to these

peptides are in fact more important than their amino acidsequence per se. Notwithstanding, silaffin genes have beenfused 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; Krger 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 solubiliza-

tion of silica with hydrogen fluoride. Pleuralins are encodedby a small multigene family in C. fusiformisbut have notbeen found inT. pseudonanaorP. tricornutum,and so theyare 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 twodaughter cells (Krger and Wetherbee 2000). Association

with the terminal girdle band of the hypotheca therefore

occurs in the extracellular space. It will be interesting to de-

termine how many other wall-associated proteins avoid theSDV during their secretion and incorporation into diatom

frustules.




7/9

Frustulins are much more loosely associated with diatomcell walls than are silaffins and pleuralins, and can be ex-

tracted with EDTA (ethylenediaminetetraacetic acid, a com-

mon chelating agent; Krger and Poulsen 2008). They are

glycoproteins that can bind calcium because of the presenceof EF hands(helix-loop-helix structures in a family of calcium-

binding proteins), and also contain characteristic acidic,cysteine-rich domains. Although frustulins are most likely

conserved in all diatoms, including T. pseudonanaand P. tri-cornutum, their function is not yet known. They are notthought to be involved in silica deposition.

Biotechnology. The precision of the nanoscale pattern andarchitecture 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 (Parkinsonand Gordon 1999, Lopez et al.2005).Although the metabolic

pathways that drive cell-wall biosynthesis remain largely un-

explored, they constitute a clear target for the discovery of

novelprotein functions that are unlikely to be found in otherorganisms and can be exploited in biotechnological applica-

tions. 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 oxidebased polymers to control the

synthesis of silica to generate ordered porous structures at thenanometer and micrometer levels. The use of frustulesto make

other functional materials by chemical conversion has also

been reported (Bao et al. 2007). These technologies open upnew opportunities to produce three-dimensional (3-D)siliceous materials that have never before been engineered

(Krger 2007, Krger and Poulsen 2008). In addition, Gor-

donand Parkinson (2005) proposed another role for silica in

linear lithographic techniques that are used to engineermicroelectronics and thatconsist in replacementof siliconwith

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 ex-ploitingthe silicon nanaofabrication capacities of diatoms,and

some progress has already been made, as evidenced in the

previous section. Further progress will very likely require

additionalhigh-quality biochemistry, as well as high-through-put, genetic-based screens, to identify diatom-specific genesof currently unknown function.

The conversion of solar energy into chemical energy by

photosynthesis has become of great interest for the genera-

tion of renewable energy resources. Diatoms have a highlipid content (up to 70% dry weight; Chisti2007), 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 donot contain complex carbohydrate-based polymers such as

cellulose, ruminants digestion of diatom-derived biomass

generates less methane and other potent greenhouse gases thanwould be the case with other feed. Now that fossil fuels are

being depleted and becoming more expensive, these diatom-

based applications are particularly appealing.Compared with

plant-based sources of biofuels, diatoms and other algae aremuch more efficient converters of solar energy and have a

much higher energy potential (Chisti 2008). Furthermore,theydo 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).Phaeo-dactylumtricornutum is an attractive target for proof of prin-ciple because it has a high lipid content (up to 30%), it can

be genetically manipulated, and it is already widely culti-vated 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 temper-ature tolerance to reduce the expense of cooling, to eliminate

light saturation of photosynthesis,and to reduce photoinhi-

bitionand photooxidation.In addition,there is a need to iden-

tify new diatom strains with high oil content or to breed orselect for improved strains.

Conclusions

The recently available genome sequences from two diatomsdemonstrated the novel multilineage history of their gene

repertoires and revealed that their genomes encode an enor-

mous metabolic and regulatory potential thatperhaps under-

lies their ecological success.The noncanonical nature of theirgenomes indicates that the functional exploration of diatom-

specific genes is required to dissect their roles in diatom biol-

ogy. Revealing the functions of these thousands of diatomgenes that do not have proxies in conventional model or-ganisms is going to be a major challenge,and it is highly un-

likely (because of the lack of financial resources and dedicated

personnel) that each can be experimentally assigned a func-

tionthroughreverse-geneticsapproaches such as gene knock-outs. Nonetheless, new molecular techniques combined with

biochemical approaches provide an excellent starting point

for exploring novel aspects of diatom biology and for devel-

oping biotechnological applications. Additional computa-tional 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 knowledgeof diatombiologygrows through laboratory-

based experiments,additional technologies willneed to be de-

veloped for exploringdiatom biology in natural environments.

Metabolomics technologies could be of some help,in that they

can reveal the metabolic signatures of cells grown in specificconditions, 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 perturba-tions (e.g., in the silicon nanofabrication process). In all such

scenarios, the available genome sequences clearly provide a




8/9

major advance in our knowledge and in our opportunities to

explore diatom biology. We eagerly await the forthcoming

sequences from the polar diatomFragilariopsis cylindrusandthe toxin-producingPseudo-nitzschia multiseries.

Acknowledgments

Funding for our work has been obtained from theEuropeanUnion (EU)funded FP6 Diatomics project (LSHG-CT-

2004-512035), the EU-FP6 Marine Genomics Network of

Excellence (GOCE-CT-2004-505403), an ATIP (Actions Th-

matiques Initatives sur Programmes) Blanche grant from

the Centre National de la Recherche Scientifique, and the

Agence Nationale de la Recherche (France).

References citedAllen AE, Vardi A, Bowler C. 2006. An ecological and evolutionary context

for integrated nitrogen metabolism and related signaling pathways in

marine diatoms. Current Opinion in Plant Biology 9: 264273.

Allen AE,LaRoche J,Maheswari U, LommerM, Schauer N,LopezPJ,Finazzi

G, Fernie AR,BowlerC. 2008. Whole-cell responseof thepennatediatomPhaeodactylum tricornutum to ironlimitation.Proceedingsof the NationalAcademy of Sciences 105: 1043810443.

Allen WE.1926.Remarks on surfacedistributionof marine plankton diatoms

in the East Pacific. Science 63: 9697.

Apt KE,Kroth-Pancic PG, Grossman AR. 1996. Stable nuclear transforma-

tion of the diatom Phaeodactylum tricornutum. Molecular Genetics andGenomics 252: 572579.

Armbrust EV, et al. 2004. The genome of the diatom Thalassiosira pseudo-nana: Ecology, evolution, and metabolism. Science 306: 7986.

Baldauf SL. 2003. The deep roots of eukaryotes. Science 300: 17031706.

Baldauf SL,Palmer JD,Doolittle WF. 1996.The rootof the universaltree and

the origin of eukaryotes based on elongation factor phylogeny. Pro-

ceedings of the National Academy of Sciences 93: 77497754.

Baldauf SL, Roger AJ, Wenk-Siefert I, Doolittle WF. 2000. A kingdom-level

phylogeny of eukaryotes based on combined protein data. Science 290:972977.

Bao Z, et al. 2007. Chemical reduction of three-dimensional silica micro-

assemblies into microporous silicon replicas. Nature 446: 172175.

Berland BR, Kapkov VI, Maestrini SY, Arlhac DP. 1976. Toxic effect of

four heavy metals on the growth of unicellular marine algae. Comptes

rendus hebdomadaires des sances de lAcadmie des Sciences D 282:

633636.

Bidle KD, Bender SJ. 2008. Iron starvation and culture age activate meta-

caspasesand programmedcell death in themarinediatom Thalassiosirapseudonana.Eukaryotic Cell 7: 223236.

Bowler C, et al.2008. ThePhaeodactylumgenome reveals the evolutionaryhistory of diatom genomes. Nature 456: 239244.

Boyd PW, et al. 2007. Mesoscale iron enrichment experiments 19932005:

Synthesis and future directions. Science 315: 612617.

Breidbach O. 2005. Art Forms from the Ocean: The Radiolarian Prints ofErnst Haeckel. Prestel.

Candela H, Hake S. 2008. The art and design of genetic screens: Maize.

Nature Reviews Genetics 9: 192203.

Carradice D, Lieschke GJ. 2008. Zebrafish in hematology: Sushi or science?

Blood 111: 33313342.

Cavalier-Smith T. 1982. The evolutionary origin and phylogeny of eukary-

ote flagella. Symposia of the Society for Experimental Biology 35:

465493.

. 1986. The kingdoms of organisms. Nature 324: 416417.

Chepurnov VA, Mann DG, Von Dassow P,Vanormelingen P, Gillard J, Inz

D, Sabbe K, Vyverman W. 2008. In search of new tractable diatoms for

experimental biology. Bioessays 30: 692702.

Chisti Y. 2007. Biodiesel from microalgae. Biotechnology Advances 25:

294306.

. 2008. Biodiesel from microalgae beats bioethanol. Trends in

Biotechnology 26: 126131.

de Baar HJW, et al. 2005. Synthesis of iron fertilization experiments: From

theironage in the ageof enlightenment.Journal of GeophysicalResearch

Oceans 110: C09S16. doi: 10.1029/2004JC002601

De MartinoA, AmatoA, Bowler C.2009.Mitosis in diatoms:Rediscovering

an old model for cell division. Bioessays 31: 874884.

De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A. 2009.

Gene silencing in the marine diatom Phaeodactylum tr icornutum.Nucleic Acids Research 37: e96. doi:10.1093/nar/gkp448

Derelle E, et al. 2006. Genome analysis of the smallest free-living eukaryote

Ostreococcus tauriunveils many unique features. Proceedings of theNational Academy of Sciences 103: 1164711652.

Dismukes GC, Carrieri D, Bennette N, Ananyev GM, Posewitz MC. 2008.

Aquatic phototrophs: Efficient alternatives to land-based crops for

biofuels. Current Opinions in Biotechnology 19: 235240.

FalciatoreA, Casotti R,Leblanc C,AbresciaC, Bowler C.1999.Transformation

of nonselectable reporter genes in marine diatoms.Marine Biotechnol-

ogy 1: 239251.

Falkowski PG, Barber RT, Smetacek V. 1998. Biogeochemical controls and

feedbacks on ocean primary production. Science 281: 200207.

FieldCB,BehrenfeldMJ, Randerson JT, FalkowskiP.1998.Primary production

of the biosphere:Integratingterrestrial and oceanic components. Science

281: 237240.

Gibbs SP. 1981. The chloroplastsof somealgal groups mayhave evolved from

endosymbiotic eukaryotic algae. Annals of the New York Academy of

Sciences 361: 193208.

Gillard J, et al. 2008. Physiological and transcriptomic evidence for a close

coupling between chloroplast ontogeny and cell cycle progression in

the pennate diatom Seminavis robusta. PlantPhysiology 148:13941411.Giordano M, BeardallJ, Raven JA.2005.CO

2concentrating mechanisms in

algae: Mechanisms, environmental modulation, and evolution.Annual

Review of Plant Biology 56: 99131.

GordonR, Parkinson J.2005.Potential roles fordiatomists in nanotechnol-

ogy. Journal of Nanoscience and Nanotechnology 5: 3540.

Gruber A, Vugrinec S, Hempel F, Gould SB, Maier UG, Kroth PG. 2007.

Protein targeting into complex diatom plastids: Functional characteri-

sation of a specific targetingmotif. Plant Molecular Biology 64: 519530.GuoFQ, Crawford NM.2005.Arabidopsisnitric oxide synthase1 is targetedtomitochondria and protects against oxidativedamageand dark-induced

senescence. Plant Cell 17: 34363450.

Hamm CE, Merkel R, Springer O, Jurkojc P, Maier C,Prechtel K, Smetacek

V. 2003. Architecture and material properties of diatom shells provide

effective mechanical protection. Nature 421: 841843.

Harper JT, Waanders E, Keeling PJ. 2005. On the monophyly of chromal-

veolates using a six-protein phylogeny of eukaryotes. International

Journal of Systematic and Evolutionary Microbiology 55: 487496.

Harvey PH. 1939. Hereditary variation in plant nutrition. Genetics 24:

437461.

Kooistra WHCF, Gersonde R, Medlin LK, Mann DG. 2007. Pages 207249

in Falkowski PG, Knoll AH, eds. Evolution of Primary Producers in the

Sea. Academic Press.

Krger N. 2007. Prescribing diatom morphology: Toward genetic engi-neering of biological nanomaterials. Current Opinion in Chemical

Biology 11: 662669.

Krger N, Poulsen N. 2008. Diatomsfrom cell wall biogenesis to nano-

technology. Annual Review of Genetics 42: 83107.

Krger N,WetherbeeR. 2000.Pleuralins are involved in thecadifferentiation

in the diatomCylindrotheca fusiformis.Protist 151: 263273.Krger N,Deutzmann R,Sumper M.1999.Polycationic peptidesfrom dia-

tom biosilica that direct silica nanosphere formation. Science 286:

11291132.

Krger N, Deutzmann R, Bergsdorf C, Sumper M. 2000. Species-specific

polyamines from diatoms control silica morphology. Proceedings of

the National Academy of Sciences 97: 1413314138.

Kroth PG. 2007a. Molecular biology and the biotechnological potential of

diatoms. Advances in Experimental Medicine and Biology 616: 2333.




9/9

. 2007b.Genetic transformation: A tool to study protein targeting in

diatoms. Methods in Molecular Biology 390: 257268.

Kroth PG, et al. 2008.A model for carbohydrate metabolism in the diatom

Phaeodactylum tricornutumdeduced from comparative whole genomeanalysis.PLoS One 3: e1426.

KustkaA, Carpenter EJ,Saudo-Wilhelmy SA.2002.Iron and marinenitrogen

fixation: Progress and future directions. Research in Microbiology 153:

255262.

Lebeau T, Robert JM.2003.Diatom cultivation and biotechnologicallyrele-

vant products, pt. 1: Cultivation at variousscales.Applied Microbiology

and Biotechnology 60: 612623.

Li S, Nosenko T, Hackett JD, Bhattacharya D. 2006. Phylogenomic analysis

identifies red algalgenes of endosymbiotic originin the chromalveolates.

Molecular Biology and Evolution 23: 663674.

Lopez PJ, Descls J,AllenAE, Bowler C.2005.Prospects in diatom research.

Current Opinion in Biotechnology 16: 180186.

Losic D, Rosengarten G, Mitchell JG, Voelcker NH.2006. Pore architecture

of diatom frustules: Potential nanostructured membranes for molecu-

lar and particle separations. Journal of Nanoscience and Nanotechnol-

ogy 6: 982989.

Maheswari U,MockT,ArmbrustEV, Bowler C.2009.Update of theDiatom

EST Database: A new tool for digital transcriptomics. Nucleic Acids

Research 37: D1001D1005.

MatsuzakiM, et al.2004.Genome sequence of theultrasmall unicellular red

algaCyanidioschyzon merolae10D. Nature 428 : 653657.Marchetti A, Parker MS, Moccia LP, Lin EO,Arrieta AL, Ribalet F, Murphy

ME, Maldonado MT, Armbrust EV. 2008. Ferritin is used for iron

storagein bloom-forming marine pennatediatoms. Nature 457: 467470.

Merchant SS,et al.2007.The Chlamydomonasgenome reveals the evolutionof key animal and plant functions. Science 318: 245250.

Milligan AJ, Morel FM. 2002. A proton buffering role for silica in diatoms.

Science 297: 18481850.

Mock T, et al. 2008. Whole-genome expression profiling of the marine

diatom Thalassiosira pseudonana identifies genes involved in siliconbioprocesses. Proceedings of the National Academy of Sciences 105:

15791584.

Montsant A, Jabbari K, Maheswari U, Bowler C. 2005. Comparative ge-

nomics of the pennate diatom Phaeodactylum tricornutum. Plant

Physiology 137: 500513.Oudot-Le Secq MP, Grimwood J,Shapiro H,ArmbrustEV, Bowler C,Green

BR.2007. Chloroplast genomes of thediatomsPhaeodactylum tricornutumand Thalassiosira pseudonana: Comparison withother plastid genomesof the red lineage. Molecular Genetics and Genomics 277: 427439.

Palenik B,et al.2007.The tiny eukaryote Ostreococcusprovides genomic in-sights intothe paraox of plankton speciation.Proceedings of theNational

Academy of Sciences 104: 77057710.

Parkinson J, Gordon R. 1999. Beyond micromachining: The potential of

diatoms. Trends in Biotechnology 17: 190196.

Pickett-Heaps J, SchmidA-MM, Tippit DH. 1984. Cell division in diatoms:

A translation of part of Robert Lauterborns treatise of 1896 with some

modern confirmatory observations. Protoplasma 120: 132154.

Pohnert G, Adolph S, Wichard T. 2004. Short synthesis of labeled and un-

labeled 6Z, 9Z, 12Z, 15-hexadecatetraenoic acid as metabolic probes

for biosynthetic studies on diatoms.Chemistry and Physicsof Lipids 131:

159166.

PondavenP, Gallinari M,Chollet S,Bucciarelli E,Sarthou G, Schultes S,Jean

F. 2007. Grazing-induced changes in cell wall silicification in a marine

diatom. Protist 158: 2128.

Poulsen N, Krger N. 2005. A new molecular tool for transgenic diatoms:

Control of mRNA and protein biosynthesis by an inducible promoter-

terminator cassette. FEBS Journal 272: 34133423.

Poulsen N, Berne C, Spain J, Krger N. 2007. Silica immobilization of an

enzyme through genetic engineeringof the diatom Thalassiosirapseudo-nana.Angewandte Chemie International Edition in English 46: 18431846.

Roessler PG,BleibaumJL, ThompsonGA, Ohlrogge JB.1994. Characteristics

of thegene that encodes acetyl-CoA carboxylasein thediatom Cyclotellacryptica.Annals of the New York Academy of Sciences 721: 250256.

SancettaC. 1981. Diatoms as hydrographictracers: Example fromBering Sea

sediments. Science 211: 279281.

Shemesh A, Charles CD, Fairbanks RG. 1992. Oxygen isotopes in biogenic

silica: Global changes in ocean temperature and isotopic composition.

Science 256: 14341436.

Siaut M,Heijde M,MangognaM,MontsantA, CoeselS,AllenA, Manfredonia

A, FalciatoreA, Bowler C.2007. Molecular toolbox for studyingdiatom

biology inPhaeodactylum tricornutum.Gene 406: 2335.Smetacek V. 1999. Diatoms and the ocean carbon cycle.Protist 150: 2532.

Thompson SEM, Taylor AR, Brownlee C, Callow ME, Callow JA.2008. The

role of nitric oxide in diatom adhesion in relation to substratum prop-

erties. Journal of Phycology 44: 967976.

Trguer P, Nelson DM, Van Bennekom AJ, Demaster DJ, Leynaert A,

Quguiner B. 1995. The silica balance in the world ocean: A reestimate.

Science 268: 375379.

Vardi A,Formiggini F, Casotti R,De Martino A,Ribalet F, Miralto A,Bowler

C. 2006. A stress surveillance system based on calcium and nitric oxide

in marine diatoms.PLoS Biology 4: e60.

VardiA, BidleKD, Kwityn C, HirshDJ, Thompson SM,Callow JA,Falkowski

P, Bowler C. 2008. A diatom gene regulating nitric-oxide signaling and

susceptibilityto diatom-derived aldehydes. CurrentBiology 18:895899.

Vrieling EG,Sun Q, Beelen TP, HazelaarS, GieskesWW,van Santen RA,Som-

merdijk NA. 2005.Controlled silica synthesis inspired by diatomsilicon

biomineralization. Journal of Nanoscience and Nanotechnology 5:6878.

Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D. 2004. A molec-

ular timeline for the origin of photosynthetic eukaryotes. Molecular

Biology and Evolution 21: 809818.

Zaslavskaia LA, Lippmeier JC, Shih C, Ehrhardt D, Grossman AR, Apt KE.

2001. Trophic conversion of an obligate photoautotrophic organism

through metabolic engineering. Science 292: 20732075.

Zurzolo C, Bowler C. 2001. Exploring bioinorganic pattern formation in

diatoms.A story of polarized trafficking.Plant Physiology 127:13391345.

Anastasia Saade ([email protected])andChrisBowler ([email protected]) are with the Department of Biology at the cole Normale Suprieurein Paris. Bowler is the director of research, and his laboratory studies signal-ing in higher plants and marine diatoms.



Documents

BioScience 2009 Saade 757 65