Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms.

Evolution and Functional Diversification of Fructose
Bisphosphate Aldolase Genes in Photosynthetic Marine
Diatoms
Andrew E. Allen,1,2 Ahmed Moustafa,�,2 Anton Montsant,1,3 Angelika Eckert,4 Peter G. Kroth,4 and
Chris Bowler*,1,3
1Environmental and Evolutionary Genomics Section, Institut de Biologie de l’Ecole Normale Supe´reure, CNRS UMR8186 INSERM
U1024, Ecole Normale Supe´rieure, Paris, France
2Microbial and Environmental Genomics, J. Craig Venter Institute, San Diego, California
3Laboratory of Cell Signaling , Stazione Zoologica Anton Dohrn, Naples, Italy
4Plant Ecophysiology, Fachbereich Biologie, Universita¨t Konstanz, Konstanz, Germany
�Present address: Department of Biology and Biotechnology Graduate Program, American University in Cairo, New Cairo, Egypt
*Corresponding author: E-mail: cbowler@biologie.ens.fr.
Associate editor: Charles Delwiche
Abstract
Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse
phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily
monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong
evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of
gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire.
As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases
(FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other
chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression,
and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very
little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears
to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates
from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal
endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and
chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated
with b-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating
mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient
limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically
localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the
stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.
Key words: carbon concentrating mechanism (CCM), carbon metabolism, carbonic anhydrase, diatom, fructose
bisphosphate aldolase, pyrenoid.
Introduction
It is believed that the diatom lineage emerged less than 300
Ma. Considering the diversity of extant diatom species,
comparable to that of angiosperms (Round et al. 1990), di-
atoms have displayed a remarkable diversification rate and
plasticity for adaptation to new environments. Mecha-
nisms for assimilation and recycling of major nutrients,
such as carbon (C), nitrogen (N), phosphorus (P), or iron
(Fe) likely present notable differences from those known in
green algae and plants and may provide insights about the
ability of diatoms to thrive and diversify in aquatic environ-
ments (Wilhelm et al. 2006; Armbrust 2009; Bowler et al.
2010). For example, diatoms possess a urea cycle, previously
known only in metazoans, through which organic nitrogen
may be recycled (Armbrust et al. 2004; Allen et al. 2006,
2011), and they may utilize siderophore-based iron uptake
like in cyanobacteria (Allen et al. 2008).
Diatoms are responsible for up to 40% of annual primary
productivity in the ocean (Field et al. 1998; Granum et al.
2005). Diatom RuBisCO (ribulose 1,5-bisphosphate carboxyl-
ase/oxygenase) enzymes, however, have half saturation con-
stants for CO2 of 30–60 lM (Badger et al. 1998), whereas
seawater typically contains around 10 lM CO2, which implies
that marine diatoms would be CO2-limited in the absence of
a carbon concentrating mechanism (CCM) (Riebesell et al.
© The Author(s) 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
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Mol. Biol. Evol. 29(1):367–379. 2012 doi:10.1093/molbev/msr223 Advance Access publication September 8, 2011 367
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1993) (Hopkinson et al. 2011; Reinfelder 2011). The relatively
high affinity of diatom cells for CO2, coupled with the obser-
vation that diatom populations commonly reach bloom den-
sities, indicates that diatoms possess efficient CCMs (Badger
et al. 1998; Roberts et al. 2007a, 2007b; Bowler et al. 2010;
Hopkinson et al. 2011; Reinfelder 2011).
In recent years, the CCMs, through which diatoms en-
sure the supply of CO2 to RuBisCO, have attracted in-
creasing attention (Roberts et al. 2007b). Green algae
typically utilize bicarbonate transporters in combination
with plastid, cytosol, and cell surface carbonic anhydrases
(CAs), which catalyze conversion between CO2 and bicar-
bonate, as a mechanism of promoting CO2 supply to Ru-
BisCO, much like the biophysical CCMs that have been
reported in cyanobacteria (Kaplan and Reinhold 1999).
Diatom genome sequences unambiguously indicate the
presence of different HCO3- transport systems (Kroth
et al. 2008). In Phaeodactylum tricornutum, at least one
such transporter has an N-terminal plastid-targeting pre-
cursor (Allen et al. 2008; Kroth et al. 2008). Phaeodactylum
tricorutum is also known to contain a plastid-targeted
b-carbonic anhydrase (b-CA) (Montsant et al. 2005; Kitao
et al. 2008; Tachibana et al. 2011).
In diatoms, a putative C4-like mechanism has also been
proposed to explain their efficient C fixation under CO2 and
Zn limitation (Reinfelder et al. 2000, 2004; Reinfelder 2011).
However, conflicting experimental data for support of C4
photosynthesis in diatoms has been reported (Roberts
et al. 2007a; McGinn and Morel 2008), and genomic data
does not fully clarify the presence and localization of the en-
zymes that may drive this mechanism (Kroth et al. 2008;
Parker et al. 2008; Bowler et al. 2010). It is possible that in-
dividual diatom species may rely to a different extent on
both biophysical and biochemical CCMs in order to opti-
mally regulate efficient inorganic carbon acquisition.
Potential links between diatom CCMs and other aspects
of C metabolism such as the direction of C flow between C3
and C6 sugar pools and sustained regeneration of the C5
RuBisCO substrate ribulose 1,5-bisphosphate (Ru 1,5-BP)
have not been addressed. b-CA and putatively plastidic
fructose 1,6-bisphosphate aldolase (FBA) are downregu-
lated and upregulated, respectively, in iron-limited P. tricor-
nutum cells (Allen et al. 2008), which are known to display
compromised photosystem reaction centers, reduced pho-
tosynthetic electron transfer rates, decreased reductant pro-
duction, and an inability to efficiently process absorbed
photons (Behrenfeld et al. 1996, 2006; Milligan and Harrison
2000). This has prompted speculation that plastidic b-CA
and FBA are part of a coordinated effort to regulate carbon
flux within the plastid during periods of energy limitation
(Allen et al. 2008). FBA genes have been noted for their pe-
culiar phylogenetic distribution and highlighted in several
comparative genomic studies (Kroth et al. 2005, 2008;
Montsant et al. 2005) and due to important roles in balanc-
ing C6 and C3 sugar pools in the Calvin–Benson cycle and
glycolysis, as likely key regulators for the flow of small organic
C molecules and CO2 in the plastid and cytosol. There are
two evolutionarily unrelated FBAs, termed class I and class II,
with a very complex phylogenetic distribution. These two
types do not share sequence similarity with each other
and their catalytic mechanism is different; unlike class I FBAs,
class II FBAs are dependent on divalent cations and, there-
fore, constitute a case of convergent functional evolution
(Marsh and Lebherz 1992).
Class I and class II FBAs catalyze the interconversion be-
tween C3/C4 molecules and C6/C7 molecules (Flechner
et al. 1999). Therefore, FBAs can be involved both in carbon
fixation and in glycolysis. In Calvin–Benson cycle reactions,
the C5 molecule ribulose 1,5-bisphosphate (Ru 1,5-BP) is
carboxylated and split into C3 molecules that are reduced
to glyceraldehyde 3-phosphate (G3P). The formation of the
hexose sugars glucose and fructose as well as regeneration
of Ru 1,5-BP requires the action of FBA, which catalyzes the
formation of the C6 compound fructose 1,6-bisphosphate
from two C3 G3P molecules and dihydroxyacetone phos-
phate (DHAP). On the other hand, in glycolytic reactions
FBAs split C6 molecules into C3 molecules, which are trans-
formed into pyruvate. Pyruvate can then be transformed to
acetyl coenzyme A and oxidized to CO2 through the Krebs
cycle in the mitochondria or enter biosynthetic pathways
such as fatty acid or amino acid biosynthesis.
In animals, only class I FBAs are known, whereas fungi ap-
pear to rely solely on FBA II (Jacobshagen and Schnarrenberger
1990; Marsh and Lebherz 1992; Pelzer-reith et al. 1993) (table
1). Red algae and glaucocystophytes have been reported to
have class II FBAs in their cytosol and class I FBA in their
plastids, although the sampling of these eukaryotic lineages
has been limited to date (Anita 1967; Gross et al. 1994, 1999).
The two classes of FBA have been detected in Eubacteria
(including Cyanobacteria), although these organisms most
typically utilize class II FBAs (Sanchez et al. 2002). Although
FBA I activity was detected in the diatom P. tricornutum in
an early study (Anita 1967), secondary endosymbiotic algae,
commonly referred to as Chromalveolata (Cavalier-Smith
2000), such as diatoms, dinoflagellates, haptophytes, and
cryptophytes, which are believed to have inherited their plas-
tids from red algae in a unifying single event (Yoon et al.
2002), are generally thought to utilize class II FBAs both
in cytosol and in plastids (Anita 1967; Rogers and Keeling
2004). The universal occurrence of class II type A FBA in chro-
malveolate plastids coupled with an apparent lack of red
alga-like plastid-targeted FBA I was previously interpreted
as evidence for a chromalveolate-specific gene replacement
that supports the single origin of chromalveolate plastids
(Patron et al. 2004). More recently, statistical analyses of
phylogenomic data have been used to argue for falsification
of the single origin of Chromalveolata in favor of more
complex evolutionary scenarios such as serial acquisition
of secondary plastids by distantly related hosts (Baurain
et al. 2010). Whether chromalveolate plastids evolved from
a single endosymbiotic event or from serial transfer be-
tween diverse hosts, genomic data for key outgroups indi-
cates an unambiguously common line of descent for
Chromalveolata plastids (Janouskovec et al. 2010).
Although a single gene family such as Fba will not resolve
the conflicts surrounding the origin of chromalveolate plastids,
Allen et al. · doi:10.1093/molbev/msr223 MBE
368

information related to the phylogenetic distribution and
function of different FBA types will help to clarify the evo-
lutionary and physiological significance of different FBAs
within and among different algal groups. Preliminary com-
parative analyses of the distribution of FBA family genes in
diatoms based on the genome sequence of the centric
diatom Thalassiosira pseudonana and expressed sequence
tag (EST) data from P. tricornutum revealed several surpris-
ing features (Montsant et al. 2005). On the one hand, dia-
toms appeared to be the only eukaryotes to possess
a typically bacterial class I FBA, termed FBA4 (Kroth et al.
2005), for which no targeting sequence was detected (i.e.,
likely a cytosolic enzyme). Additionally, the pennate dia-
tom P. tricornutum appeared to have a plastid-localized
class I FBA, FBAC5 (Kroth et al. 2005; Montsant et al.
2005), which are usually found in the green and red algal
lineages rather than in chromalveolate algae (Patron et al.
2004). This phylogenetic puzzle poses functional questions
when considering that both T. pseudonana and P. tricornu-
tum also have the class II plastid and cytosolic FBAs ex-
pected in chromist algae (Patron et al. 2004), denoted
FBAC1, FBAC2, and FBA3 in P. tricornutum (Kroth et al.
2005). We have therefore characterized each P. tricornutum
FBA phylogenetically, using genome sequence or EST data
now available for each of the major lineages of eukaryotic
photosynthetic algae. To generate further information
about functionality, we have also examined their subcel-
lular localization and evaluated gene expression under
different conditions. We specifically evaluated FBA tran-
scriptional responses to Fe limitation because previous
studies based primarily on EST sequencing suggested that
certain FBA genes are responsive to Fe availability (Allen
et al. 2006; Maheswari et al. 2010). Iron availability is also
known to heavily influence diatom physiology and me-
tabolism (Kustka et al. 2007; Allen et al. 2008) and distri-
bution (Behrenfeld et al. 2006).
Materials and Methods
Phaeodactylum tricornutum Cultures
Axenic cultures of P. tricornutum Bohlin clone CCMP2560
(Provasoli-Guillard National Center for Culture of Marine Phy-
toplankton, Bigelow) were grown in f/2 medium made with
0.2 lm filtered and autoclaved seawater supplemented with
f/2 vitamins and inorganic nutrients (Guillard 1975). Cell cul-
tures were grown to midexponential phase at a temperature
of 18 �C and a 16:8 light (150 lE/m/sec):dark regime. Sterility
was monitored by inoculation into peptone-enriched media
to detect bacterial growth. For iron limitation experiments, Fe
was precomplexed with EDTA (1:1.1 mol:mol) and added
(5 nM Fe) to Fe-free media. Cells were cultured in semicon-
tinuous mode until growth rates stabilized. Cultures were di-
luted with fresh medium at a rate of approximately 0.1–0.2
per day depending on growth rate and were maintained in
a state of exponential growth. Fe#, the sum of all unchelated
species, calculated according to equations of Sunda and
Huntsman (2003) was (Fe# 5 0.0024 FeT) under our condi-
tions. Therefore, the Fe-limiting condition had an Fe# level of
12.0 pmol�liters�1 Fe# and the Fe replete condition had an
Fe# value of 2.6 nmol�liters�1 Fe#.
The availability of free Fe ions in the culture media was
effectively buffered to maintain consistent Fe# levels over
the course of the experiments. Growth rates were moni-
tored by cell counting via microscopy before and after di-
lution. Generally, the growth rate of cells in iron-limited
cultures was around 60% of the maximum growth rate
in iron-replete cultures. Iron limitation was confirmed by
following the recovery of high growth rate following addi-
tion of iron to a small volume of the culture.
Cloning of P. tricornutum Genes
The five Fba genes and a plastid-localized b-CA (CA1) de-
scribed earlier (Tanaka et al. 2005) were cloned into Gateway
Table 1. Distribution of FBA Genes in Eukaryotes.
Eukaryotic Lineage Eukaryotic Crown Group Plastid FBA Cytosol FBA References
Plants Viridiplantae I I, IIB Jacobshagen and Schnarrenberger
(1990); Pelzer-Reith et al. (1993)
Red algae Viridiplantae I Ia, II Anita (1967); Kroth et al. (2005); This work
Glaucophytes Viridiplantae IIB Ia, II Gross et al. (1994); This work
Prasinophytes Viridiplantae Ia, IIa Ia, (1 Ib)a This work
Oomycetes Heterokonts — II Kroth et al. (2005)
Diatomsc Heterokonts (Ipy)a, II, (IIpy)a II, Ia, (1 Ib) Patron et al. (2004); Kroth et al.
(2005); Montsant et al. (2005)
Haptophyta Chromalveolatesd (Ipy)a, IIa, (IIpy)a Ia, IIa This work
Cryptophytes Chromalveolatesd I, (IIpy)a Ia Patron et al. (2004); This work
Apicomplexa Alveolates — I Rogers and Keeling (2004)
Dinoflagellates Alveolates (Ipy), (IIpy)a Ia, IIa Kroth et al. (2005); This work
Chlorarachniophyte Rhizaria I I, IIa Rogers and Keeling (2004)
Animals Opisthokonts — I Marsh and Lebherz (1992)
Fungi Opisthokonts — II Marsh and Lebherz (1992)
NOTE.—The two known types of FBAs can participate in glycolytic reactions in the cytosol and in Calvin–Benson cycle reactions in the plastid. The two types are also widespread
between Archaea and Eubacteria. py indicates plastidic FBAII or FBAI localized to the pyrenoid. Pyrenoid localization of FBA has only been confirmed in P. tricornutum.
a Denotes first report in this work.
b This cytosolic type-I FBA is a divergent, bacterial-derived variant dissimilar to type-I FBAs known in other eukaryotes.
c Phaeodactylum tricornutum has three putative plastid FBAs (the respective genes termed FBAC1, FBAC2, and FBAC5) and two cytosolic FBAs (FBA3 and FBA4). FBA4 and
FBAC5 are type-I FBAs.
d Haptophya and Cryptophyta are closely related to Heterokonts and together with diatoms and brown algae (stramenopiles), and dinoflagellates (alveolates) are known as
chromalveolates.
Diversification of FBAs in Marine Diatoms · doi:10.1093/molbev/msr223 MBE
369

diatom expression vectors (Siaut et al. 2007). The primers
and cDNA templates used for polymerase chain reaction
(PCR) amplification are indicated in supplementary table
S1 (Supplementary Material online). Correctly sized ampli-
cons were cloned into Gateway pENTR/D-TOPO vectors (In-
vitrogen) and sequenced. All FBA pENTR clones were then
recombined into a diatom adapted pDEST_5# enhanced yel-
low fluorescent protein (EYFP) through LR recombination
reactions (LR clonase mix, Invitrogen), to generate pEXPR_
gene_EYFP vectors driving expression of the genes of interest
with an EYFP tag at their C-termini (Siaut et al. 2007). FBA4,
the bacteria-like class I FBA, was cloned both with and with-
out a stop codon and recombined into pDEST vectors to
generate N- and C-terminal EYFP fusions. The b-CA1 was
used as a plastid marker for colocalization with FBA proteins.
This gene was PCR amplified from the plasmid pre138-
mptca1-enhanced green fluorescent protein (kindly pro-
vided by Prof Yusuke Matsuda, Kwansei-Gakuin University,
Hyogo, Japan), cloned into a pENTR vector, and recombined
into C-terminal cyan fluorescent protein (CFP) fusion pDEST
vectors to generate pEXPR_PtCA1_CFP.
Transformation of Wild-Type P. tricornutum Cells
The pEXPR plasmids (3 lg) described above were each mixed
with pAF6 plasmid (3 lg), which drives expression of the
phleomycin-resistance ShBle gene and precipitated onto
tungsten microparticles (M-17, Biorad) of 1.1 lm in diameter.
The constructs were transfected into P. tricornutum wild-type
cells by helium-accelerated microparticle bombardment us-
ing a BioRad PDS 1000 device following standard procedures
described previously (Falciatore et al. 1999). Transformants
were selected on solid medium (f/2 with agarose) containing
100 lg/ml phleomycin during 2 weeks, and individual colo-
nies were then transferred to liquid medium for screening for
EYFP signal. EYFP positive transformants were maintained in
liquid media in the presence of the phleomycin analog xeocin
(50 lg/ml); which is lethal for wild-type cells. The EYFP pos-
itive transformants have not lost their YFP signal over a period
of 3–5 years, and the cultures have maintained resistance in-
dicating long-term stability of transformed constructs.
Epifluorescence Microscopy
Transformant clones were screened for fluorescence 2 days
after transfer to antibiotic-containing liquid medium. A
Zeiss Axioskop equipped with an HBO 50W UV lamp
was used, and the filter 93/XF104-2 (OMEGA), with exci-
tation at 500 nm and emission at 545 nm, was used to de-
tect YFP signal. The filter 93/XF102-2 (excitation 560 nm,
emission 595 nm, OMEGA) was also used to acquire chlo-
rophyll autofluorescence images.
Confocal Microscopy
EYFP-positive P. tricornutum clones detected by epifluores-
cence microscopy were observed using a Leica TCS SP2
confocal laser scanning microscope using an HCX PL
APO 100x/1.40-0.70 oil objective. EYFP and chlorophyll
were excited at 514 nm, CFP was excited at 458 nm,
and the resulting fluorescence was filtered using a beam
splitter (DD458/514). Fluorescence was detected using
a photomultiplier tube with a bandwidth of 525–571
nm for EYFP, 465–551 nm for CFP, and 620–710 nm for
chlorophyll autofluorescence. Images were captured with
the Leica confocal software and processed with the ImageJ
1.34 software (Wayne Rasband, NIH).
Transmission Electron Microscopy
Three hundred microliters of an FBAC5-EYFP clone batch cul-
ture were grown to late exponential phase and harvested by
gentle centrifugation. Cells were fixed in 2% glutaraldehyde
and embedded in LRWhite medium grade resin (London
Resin Company). Ultrathin sections were treated with rabbit
anti-GFP antiserum (AbCam) followed by goat anti-rabbit
conjugated to electron-dense 10-nm gold particles as de-
scribed previously (Lichtle´ and McKay 1992). Sections were
observed with a Jeol CX2 electron microscope at 80 kV.
Quantitative RT-PCR
Cells were cultured in duplicate under Fe replete and deplete
conditions, and mRNA was extracted from the four pellets
(TRIzol, Invitrogen). RNA concentration was measured by
means of an ND-1000 Spectrophotometer (Nanodrop), and
SuperScript III reverse polymerase (Invitrogen) was used to
convert 100-ng total RNA into cDNA by reverse transcriptase-
polymerase chain reaction (RT-PCR). The Fba gene tran-
scripts were quantified by qPCR along with 18S rRNA as
an internal control, in triplicate, with each reaction (25
ll) containing 1 ll of the cDNA preparation, 200 nM of for-
ward and reverse primers (supplementary table S2, Supple-
mentary Material online), and 2� SYBR Green I PCR Master
Mix (Applied Biosystems, CA). Reactions were run in an Op-
ticon Chromo4 MJ Research Thermal Cycler (Bio-Rad,
Hercules, CA). The cycling conditions comprised 10-min
polymerase activation at 95 �C and 40 cycles at 95 �C for
15 s and 60 �C for 60 s. The reaction was ended with
a 5-min elongation step at 72 �C. Amplicon dissociation
(melting) curves were recorded after cycle 40 by heating from
60 to 95 �C with a ramp speed of 0.5 �C every second. The
Fba threshold cycles (CT) were normalized to three endog-
enous control gene 18S rRNA, TATA binding protein, and H4
(Siaut et al. 2007). Fold difference between Fe replenished
and Fe starved cultures measured by means of the 2�DD
CT method (Livak and Schmittgen 2001), where DDCT
5 (CT, target � CT, 18S )5 nM � (CT, target � CT, 18S )1 lM.
FBA Activity Assays
Protein concentrations of cellular extracts were determined
by Bradford assay (BioRad, Hercules) according to protocols
of the manufacturer. Enzyme activity was measured in an
optimized coupled test based on the protocol by Gross
et al. (1999). The standard reaction assay contained 100
mM Tricine, pH 7.5; 10 mM MgCl2; 1 U/ml triosephosphate
isomerase (TIM); 1 U/ml glycerol-3-phosphate dehydro-
genase; NADH (0.2 mM); cellular extract (varying vol-
umes). The decreasing concentration of NADH was
monitored spectrophotometrically at k 5 366 nm. The
reaction was started at 25 �C by the addition of 2 mM
Allen et al. · doi:10.1093/molbev/msr223 MBE
370

fructose-1,6-bisphosphate. Cellular extracts were preincu-
bated in the presence of bivalent ions (10 mM either MgCl2,
MnCl2, ZnCl2) or 10 mM EDTA on ice for 1 h. Class II FBAs
are dependent on divalent metal ions and are, therefore, in-
hibited by EDTA (Perham 1990).
Phylogenetic Inference
For each of the diatom FBA genes, the predicted protein
model was searched for homologs in a comprehensive ge-
nomic database, which we assembled from the nonredun-
dant GenBank CDS database (nr), JGI microbial genomes,
and EST libraries (NCBI; ESTdb) for lineages that do not
have complete genome data (e.g., red algae and dinoflagel-
lates). Using the iTree phylogenomic pipeline (Moustafa
et al. 2010), the homologs for the queries were consolidated
at each of the taxonomic ranks (i.e., species, genus, class,
order, phylum, and kingdom) to maximize taxon coverage,
while maintaining a practical number of taxa. Each query
and its homologs were aligned using MAFFT (Katoh et al.
2002; Armbrust et al. 2004; Moustafa et al. 2010) with up to
1,000 refinement iterations. Multiple sequence alignments
were manually examined and refined to ensure that key
taxa were not missing and to remove ambiguous sites from
the alignments. Next, molecular phylogenies were inferred
using PhyML (Guindon et al. 2009) with a maximum likeli-
hood and LG amino acid substitution model (Le and Gascuel
2008) and discrete gamma distribution (Yang 1994).
Branch confidence values were estimated using the ap-
proximate likelihood-ratio test (Anisimova and Gascuel
2006) implementation in PhyML. Only support values
greater than 50% were indicated on the graphical repre-
sentation of the phylogenetic trees. Accession numbers
for all sequences analyzed are given in supplementary
table S3 (Supplementary Material online).
Results and Discussion
Intracellular Localization and Phylogeny of P.
tricornutum FBAs
Plastidic FBAs
Observation of multiple positive transformants of C-
terminally tagged FBAC1, FBAC2, and FBAC5 confirmed that
all three of these proteins are localized in the plastid. The
FBAC1-EYFP fusion appears as small spots (usually only
one or two) inside the plastid, in a very discrete and precise
fashion (fig. 1A), with virtually no EYFP background observed
elsewhere in the plastid or the cytosol. Although, phyloge-
netically, FBAC2 seems to be a duplication of FBAC1 (Kroth
et al. 2005), its localization was not like FBAC1 but rather
appeared to be diffuse throughout the plastid (fig. 1B).
On the other hand, the class I plastid FBA, FBAC5, typical
of higher plants and algae with primary plastids, formed
well-defined spots similar to FBAC1-YFP (fig. 1C).
To further investigate the subplastidal localization of
FBAC1 and FBAC5, P. tricornutum b-type CA, PtCA1, which
was recently reported to localize to the pyrenoid (Tachibana
et al. 2011) was cloned and tagged with a C-terminal CFP
fusion, and cotransformed with YFP-tagged FBAC1 and
FBAC5. The CA1-CFP signal indeed appeared as punctate
spots within the plastid, which colocalized with both
FBAC1 and FBAC5 (fig. 2A and B). It therefore appears
that all three proteins localize to the same intraplastidial
compartment.
Immunolocalization of EYFP in transformants expressing
FBAC5-EYFP showed clearly that the protein localizes to
the pyrenoid (fig. 3). We can conclude that PtCA1, FBAC1,
and FBAC5 all localize to pyrenoids within diatom plastids.
Pyrenoids are electron-dense bodies known to contain
a protein matrix that consists mainly of RuBisCO (Griffiths
1970). Although some classes of microalgae, including cer-
tain dinoflagellates, have been reported to activate a CCM
in the absence of an observable pyrenoid (Morita et al.
1998; Ratti et al. 2007), the pyrenoid is generally considered
to be an important factor for CCM function when present;
however, precise function remains unclear (Giordano et al.
2005). Notably, enlargement of pyrenoids has been ob-
served under CO2 limitation (Izumo et al. 2007), and model
simulations of mass spectrometric measurements of cellu-
lar carbon fluxes in P. tricornutum wild-type and b-CA over
expression cells suggested that the pyrenoid is the likely site
of CO2 elevation to concentrations required to saturate
FIG. 1. Localization of plastid FBA-EYFP fusion proteins. (A) FBAC1-
EYFP, (B) FBAC2-EYFP, (C) FBAC5-EYFP. On the left light microscopy
image, chlorophyll autofluorescence is shown in red and YFP
fluorescence in green. Bars, 2 lm.
Diversification of FBAs in Marine Diatoms · doi:10.1093/molbev/msr223 MBE
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