chimeric plastid proteome in the florida “red tide...
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and Evolution. All rights reserved. For permissions, please e-mail: [email protected] The Author 2006. Published by Oxford University Press on behalf of the Society for Molecular Biology
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Research Article:
Chimeric Plastid Proteome in the Florida “Red Tide” Dinoflagellate
Karenia brevis
Tetyana Nosenko, Kristy L. Lidie1, Frances M. Van Dolah1, Erika Lindquist2, Jan-Fang Cheng3, US
Department of Energy - Joint Genome Institute4, and Debashish Bhattacharya*
University of Iowa, Department of Biological Sciences and the Roy J. Carver Center for Comparative
Genomics, 446 Biology Building, Iowa City, Iowa 52242, USA
1Marine Biotoxins Program, NOAA National Ocean Service, Center for Coastal Environmental Health
and Biomolecular Research, Charleston, South Carolina
2DOE Joint Genome Institute and Lawrence Berkeley National Laboratory, 2800 Mitchell Drive, Walnut
Creek, CA 94598, USA
3Genomics Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 84-171,
Berkeley, CA 94720, USA
4Corporate author, see Supplementary Material
*Corresponding author: University of Iowa, Department of Biological Sciences and the Roy J. Carver
Center for Comparative Genomics, 446 Biology Building, Iowa City, Iowa 52242-1324, USA,
Telephone: (319) 335-1977, Fax: (319) 335-1069, Email: [email protected]
Key words: endosymbiosis, endosymbiotic gene transfer, Karenia brevis, proteome, red tide
Running head: Karenia plastid proteome
MBE Advance Access published July 28, 2006
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Abstract
Current understanding of the plastid proteome comes almost exclusively from studies of plants and red
algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single
cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine
environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that
contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is
known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic
“Florida red tide” dinoflagelate Karenia brevis that has undergone a tertiary plastid endosymbiosis.
Comparative analyses identified 30 nuclear encoded plastid-targeted proteins in this chromalveolate that
originated via endosymbiotic or horizontal gene transfer from multiple different sources. We identify a
fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of
mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the “red”
and “green” clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to
engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent
endosymbioses and horizontal gene transfers. One of these genes is an electron transfer protein
(plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions
of iron depletion.
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Introduction
Oxygenic photosynthesis, the ability to harvest solar energy to fix carbon dioxide and produce organic
material evolved in cyanobacteria 2.5-3 billion years ago (Schopf 1993; Hedges et al. 2001). The advent
of photosynthesis was arguably the most important event in the evolution of life. It changed the redox
balance permitting the development and further metabolic and structural diversification of eukaryotic life
forms (Blankenship 2001). Eukaryotes acquired the ability to photosynthesize about 1.5 billion years ago
(Hedges et al. 2004; Yoon et al. 2004; Hackett et al. 2006) through the capture and retention of a
cyanobacterium in the “host” cytoplasm. This primary endosymbiosis gave rise to the plastid (e.g.,
chloroplast) in the common ancestor of three extant primary lineages: green algae and land plants
(Viridiplantae), red algae (Rhodophyta), and Glaucophyta (Bhattacharya and Medlin 1995; Cavalier-
Smith 2004), together known as the Plantae and more recently, the Archaeplastida (Adl et al. 2005).
Thereafter, seven other groups of eukaryotes gained their plastid through secondary endosymbiosis,
whereby a non-photosynthetic protist engulfed an existing alga and retained its plastid (Bhattacharya,
Yoon, and Hackett 2004). Separate secondary endosymbioses with green algae are believed to have given
rise to the plastid in euglenoids and chlorarachniophytes. Red algae contributed the plastid to a variety of
protist lineages putatively united in the supergroup Chromalveolata that includes alveolates
(apicomplexans, ciliates, and dinoflagellates) and chromists (cryptophytes, haptophytes, and
stramenopiles) (Cavalier-Smith 1999). The number of red algal secondary endosymbiotic events that gave
rise to the chromalveolate plastid is under debate (Bhattacharya, Yoon, and Hackett 2004) although recent
data suggest a single origin (Harper and Keeling 2004; Yoon et al. 2005; Li et al. 2006; Weber, Linka,
and Bhattacharya 2006). The ancestral red algal plastid was however not maintained in all
chromalveolates with its loss in ciliates, its diminution (apicoplast) to a remnant genome in the obligate
parasites, apicomplexans, and its replacement with a plastid from a different alga on at least five separate
occasions in dinoflagellates (Saldarriaga 2001; Ishida and Green 2002; Bhattacharya, Yoon, and Hackett
2004; Hackett et al. 2006). In Lepidodinium viride the broadly distributed peridinin containing
dinoflagellate plastid of red algal origin was replaced by one of green-algal origin. In other species the
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ancestral plastid was replaced with one from a cryptophyte, stramenopile, or haptophyte through tertiary
endosymbiosis (i.e., capture of a plastid of secondary endosymbiotic origin (Hackett et al. 2004a; Yoon et
al. 2005).
The dinoflagellates that acquired their plastid from a haptophyte alga are known as fucoxanthin
dinoflagellates because of the presence of this light harvesting protein in the endosymbiont. Given this
complex series of events, and the potential for large-scale transfer of endosymbiont genes from all of its
genomes to the host nucleus (endosymbiotic gene transfer, EGT) (Martin et al. 2002), the complement of
plastid proteins (proteome) in fucoxanthin dinoflagellates could quite literally be composed of a
menagerie of proteins of red and haptophyte (and potentially other) origins. Much of what we know about
plastid proteomes comes from analysis of the green and red lineages that have a significantly simpler
evolutionary history with a single cyanobacterial endosymbiosis and no known cases of phagotrophy.
Dinoflagellates on the other hand have undergone plastid replacements and are important grazers in the
marine environment. The capacity to engulf different algae and bacteria as prey provides the opportunity
to accumulate foreign genes in the dinoflagellate nucleus. These horizontally transferred genes could
potentially add to or replace genes involved in plastid function (Archibald et al. 2003; Hackett et al.
2004b; Li et al. 2006).
To determine the origins of the plastid proteome in a tertiary endosymbiosis, we reconstructed the
evolutionary history of nuclear encoded plastid targeted proteins in the fucoxanthin “Florida red tide”
dinoflagellate Karenia brevis. The plastid-targeted proteins were identified in two extensive EST libraries
from this species. The phylogenetic approach allowed us to reconstruct the chain of endosymbiotic gene
transfer events that resulted in the complex plastid proteome in K. brevis. We also demonstrate a
significant contribution of green algal-derived genes to the chromalveolate plastid proteome. Our data
underline the great plasticity of the plastid biochemical machinery and provide insights into the molecular
mechanisms of plastid establishment.
Materials and Methods
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Generating the K. brevis ESTs
Two cDNA libraries were generated from clonal K. brevis Wilson cells cultured under 25 ± 1°C on a
16:8-hour light-dark cycle. The Dark library was constructed using log phase cells grown on seawater
enriched with f/2 medium and during the dark phase of the diel cycle. The cDNA clones were sequenced
from the 5’ end. A total of 7,001 ESTs from the Dark library are available from the NCBI expressed
sequence tag database (ESTdb and at www.marinegenomics.org), which comprise 5,280 non-redundant
gene clusters. For complete details about Dark library generation, sequencing, and processing see Lidie et
al. (2005). The non-normalized Stress library was constructed using K. brevis Wilson cells grown under
five different culture conditions: 1) nitrate-depletion, 2) phosphate-depletion, 3) in log phase under replete
conditions, harvested during the light phase 4) in the presence of oxidative metals, 5) undergoing heat
stress. Total RNA from each treatment was purified with Trireagent (Molecular Research Center,
Cincinnati, OH) and pooled to construct a cDNA library in the Lambda Zap II vector system (Strategene,
La Jolla, CA) as previously described. A total of 22,272 ESTs from the Stress library and an additional
9,984 ESTs from the Dark library were sequenced from both 5’- and 3’-termini. This work was done by
JGI.
The EST data were processed using Trace2dbest V2.1 (Parkinson and Blaxter 2004). Low
complexity regions of the DNA sequences were masked using lower case and RepeatMasker V3.15
(http://www.repeatmasker.org/). Clustering and assembly of the EST data was done with TGICL
(http://www.tigr.org/tdb/tgi/software/) (Pertea et al. 2003) and resulted in 9,784 non-redundant gene
clusters.
Identification of Plastid Proteins and Phylogenetic Analysis
Plastid-targeting proteins were identified using sequence similarity search (BLAST) against the GenBank
non-redundant database (nr). The primary structure of identified sequences has been analyzed to confirm
nuclear localization of the gene (presence of poly-A tail, 3’ untranslated region [UTR] and plastid
localization of its product [i.e., presence of a 5’ plastid targeting leader peptide]). The 5’ leader peptides
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were analyzed using the following protein prediction programs: SignalP
(http://www.cbs.dtu.dk/services/SignalP/), TargetP (plant version)
(http://www.cbs.dtu.dk/services/TargetP), PSORT (http://www.psort.org/), and PATS
(http://gecco.org.chemie.uni-frankfurt.de/pats/pats-index.php). Protein precursors in chromalveolates are
considered to be plastid targeted if they contain an N-terminal extension composed of a signal peptide
(SignalP probability ≥ 0.7) and transit peptide. Any amino acid sequence separating the signal peptide and
mature protein is considered a transit peptide. The length of the N-terminal extension predicted by the
computer programs was verified using the protein alignments of the K. brevis sequence with its homologs
from other taxa. Identified K. brevis sequences encoding plastid-targeted proteins have been deposited in
GenBank under accession numbers DQ531572 – DQ531601 that are listed in Table 1.
Protein alignments were constructed using homologous sequences identified using BLAST
searches (e- value cut-off < 10-10) against the GenBank nr and dbEST databases and public protist
databases including C. merolae (Matsuzaki et al. 2004) (http://merolae.biol.s.u-tokyo.ac.jp/), Galdieria
sulphuraria (Weber et al. 2004) (Michigan State University Galdieria Database
http://genomics.msu.edu/galdieria/sequence_data.html), Porphyra yezoensis (Asamizu et al. 2003),
(http://www.kazusa.or.jp/en/plant/porphyra/EST), Chlamydomonas reinhardtii (http://genome.jgi-
psf.org/Chlre3/Chlre3.info.html), Phaeodactylum tricornutum (Scala et al. 2002)
(http://avesthagen.sznbowler.com/), Thalassiosira pseudonana (Armbrust et al. 2004), and unpublished
Cyanophora paradoxa data (ongoing project in our lab). In addition, search by gene/protein name was
used to identify homologous sequences from the Protist EST Program database
(http://amoebidia.bcm.umontreal.ca/public/pepdb/welcome.php).
Amino acid sequences for each protein were aligned using ClustalW and manually refined under BioEdit
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The 6-protein concatenated protein alignment was
assembled using BioEdit. For each data set, a phylogeny was reconstructed under maximum likelihood
(ML) using the PHYML V2.4.3 computer program (Guindon and Gascuel 2003; Guindon et al. 2005)
using the WAG + Γ + I evolutionary model and tree optimization. The alpha values for the gamma
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distribution were calculated using eight rate categories. To assess the stability of monophyletic groups in
the ML trees, we calculated PHYML bootstrap (100 replicates) support values (Felsenstein 1985). In
addition, we calculated bootstrap values (500 replications) using the neighbor joining (NJ) method with
JTT+ Γ distance matrices (PHYLIPV3.63, http://evolution.genetics.washington.edu/phylip.html). The NJ
analysis was done with randomized taxon addition. We calculated Bayesian posterior probabilities for
nodes in the ML tree using MrBayes V3.0b4 (Huelsenbeck and Ronquist 2001) and the WAG + Γ model.
The Metropolis-coupled Markov chain Monte Carlo from a random starting tree was run for 1,000,000
generations with trees sampled each 1,000 cycles. The initial 20,000 cycles (200 trees) were discarded as
the ‘‘burn in.’’ A consensus tree was made with the remaining 800 phylogenies to determine the posterior
probabilities at the different nodes. Manual inspection of the trees produce by the MCMCMC tree search
suggested convergence by 200,000 generations.
Results
We identified sequences encoding 30 different plastid-targeted proteins from two EST libraries of K.
brevis (see Table 1). The evolutionary origins of 22 of these genes have been resolved. The sequences
encoding K. brevis light harvesting proteins were counted as one protein and were omitted from the
phylogenetic analysis due to the presence of multiple paralogs in different photosynthetic lineages. Our
phylogenetic analyses of the remaining genes show that most of the K. brevis plastid-targeted proteins are
shared by different lineages of chromalveolates. Thirteen of them were derived by the chromalveolate
ancestor from its red algal endosymbiont, six proteins originate from green algae, and three plastid-
targeted proteins have arisen through the duplication of genes encoding cytosolic proteins in the ancestral
chromalveolate (Table 1). After the replacement of the peridinin-containing plastid with the haptophyte-
derived plastid, 12 of these genes were acquired by fucoxanthin dinoflagellates from their tertiary
haptophyte endosymbiont and one gene was derived from the genome of the dinoflagellate ancestor.
Origins of the remaining nine proteins in the tertiary plastid proteome were unresolved. Here we classify
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proteins by their origin in the secondary plastid (i.e., red algal-derived) proteome that is typical for most
chromalveolates.
Red Algal Contribution to the K. Brevis Proteome through Secondary and Tertiary
Endosymbioses
The 13 proteins acquired by chromalveolates from their secondary red algal endosymbiont trace their
origins to the primary endosymbiosis: i.e., 11 “cyanobacterial” proteins and two proteins of pro-
mitochondrial origin recruited for a plastid function before the Archaeplastida divergence. We resolved
unambiguously the phylogeny of six of the “cyanobacterial” proteins: ATP synthase gamma subunit
(encoded by atpC), oxygen-evolving-enhancer 1 protein (psbO), cytochrome b6 (petC), photosystem II
12kD extrinsic protein (psbU), phosphoglycerate mutase (PGAM), and beta-ketoacyl-ACP reductase
(fabG) (see Fig. S1 in the Supplementary Material). These genes were transferred from a cyanobacterium
to a red alga (primary endosymbiosis), from the red alga to the chromalveolate ancestor (secondary
endosymbiosis), and finally from a haptophyte alga to fucoxanthin dinoflagellates (tertiary
endosymbiosis). To robustly position K. brevis within a tree of plastid-targeted nuclear proteins, we
generated a 6-protein alignment using these proteins. The resulting tree strongly supports the monophyly
of the K. brevis and haptophyte sequences and the expected monophyly of red algae and chromalveolates
(bootstrap proportions, maximum likelihood, BPml = 100%; neighbor joining, BPnj = 100%; Bayesian
posterior probability, BPP = 1.0 for both nodes; see Fig. 1). The red algal-derived plastid clade includes
representatives of both groups of chromalveolates, the chromists represented by haptophytes and
stramenopiles and the alveolates represented by peridinin dinoflagellates. This tree of nuclear-encoded
proteins provides significant support for the origin of all chromalveolates from a single common ancestor
that all initially shared a red algal secondary endosymbiont (Bhattacharya, Yoon, and Hackett 2004; Li et
al. 2006; Weber, Linka, and Bhattacharya 2006).
The phylogeny of translation elongation factor G (EF-G encoded by fusA) is similar to the tree
shown in Fig. 1, but is a host contribution to the primary plastid proteome. EF-G catalyzes the
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translocation step of protein synthesis in prokaryotic ribosomes including plastids and mitochondria
(Breitenberger and Spremulli 1980). Analysis of organellar EF-Gs in photosynthetic algae using
immunological techniques revealed that the two proteins have distinct structures (Breitenberger and
Spremulli 1980). Surprisingly, plastid EF-G, unlike the mitochondrial protein, has a significant structural
similarity to Escherichia coli EF-G. This is believed to be a case of convergent evolution (Breitenberger
and Spremulli 1980). Our analysis provides a phylogenetic perspective on this issue. The phylogenetic
analyses support the origin of plastid fusA from alpha proteobacteria (BPml = 99%, BPnj = 100, BPP =
1.0; Fig. 2). It is believed that a Rickettsia-like proteobacterium gave rise to the mitochondrion in
eukaryotes (Gray 1998). The inclusion of mitochondrial-targeted sequences into the fusA tree shows that
they form a separate clade that is closely related to delta-proteobacteria, spirochaetes, and planctomycetes
(BPml = 100%, BPnj = 100%, BPP = 1.0). Our interpretation of these results is that a fusA sequence
derived by HGT from a non-photosynthetic bacterium was recruited for protein translation in
mitochondria and later replaced the original mitochondrial fusA. This recruitment occurred prior to the
divergence of plants, animals, and fungi (see Fig. 2). Thereafter, the original pro-mitochondrial fusA
retained in the host nuclear genome replaced its homolog from the cyanobacterial endosymbiont and gave
rise to the plastid fusA in the common ancestor of Archaeplastida. Chromalveolates acquired the gene
encoding plastid EF-G from the red-algal secondary endosymbiont. In turn, fucoxanthin dinoflagellates
gained this gene from the haptophyte tertiary endosymbiont (BPml = 94%, BPnj = 95%, BPP = 1.0).
The evolutionary origin in K. brevis of plastid fructose-1,6-bisphosphatase (FBPase) that is a core
enzyme of the Calvin cycle and glycolysis, parallels fusA. Plastid FBPase arose through the duplication of
the primary host cytosolic FBPase that occurred early in eukaryotic evolution. Similarity of the cytosolic
FBPase of eukaryotes to its proteobacterial homolog supports the pro-mitochondrial origin of this enzyme
(Martin and Schnarrenberger 1997). Here we show that plastid FBPase of chromalveolates was acquired
from the red algal secondary endosymbiont (BPml = 68%, BPnj = 69, BPP = 1.0; Fig. 3). The origin of
FBPase in tertiary endosymbiosis remains however unresolved.
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Green Algal Contribution to the Tertiary Plastid Proteome
Six K. brevis plastid-targeted proteins originated in chromalveolates from green algae. These proteins
represent components of the primary plastid proteome: four of them are of a cyanobacterial origin and
two are of host origin (Table 1, Fig. 4). These “green genes” will be dealt with separately.
Glutamate 1-semialdehyde 2,1-aminomutase (GSA-AT) is an enzyme of the class-III
pyridoxal-phosphate-dependent aminotransferase family that is involved in chlorophyll and heme
biosynthesis in the plastid stroma in plants and algae (Kannangara and Gough 1978). Phylogenetic
analysis shows this protein to have a cyanobacterial provenance (BPml = 86 %, BPP = 1.0) and has
originated in chromalveolates from a green algal lateral transfer (BPml = 64%, BPnj = 73%, BPP = 1.0,
Fig. 4A). Chromists and alveolates form a monophyletic group (BPml = 81 %, BPnj = 100%, BPP = 1.0)
suggesting that acquisition of the green gene encoding GSA-AT by chromalveolates predates the
divergence of these two lineages. The position of K. brevis relative to haptophytes and peridinin
dinoflagellates is however unresolved.
Plastid ferredoxin NADP(H) reductase (FNR encoded by petH). FNRs are flavoprotenoids that
catalyze the reversible electron transfer between NADP(H) and electron carrier proteins ferredoxin or
flavodoxin (Arakaki, Ceccarelli, and Carrillo 1997). Plants contain two tissue-specific types of plastidic
FNRs. One of these is present only in plastids from photosynthetic leaf tissue and the second isoform is
present in plastids from non-photosynthetic organs such as root, fruits, and petals (Ceccarelli et al. 2004).
The two isoforms have arisen through duplication of a cyanobacterial gene that occurred early in the
evolution of the Archaeplastida. After the divergence of the three primary photosynthetic lineages, the
ancestral gene was apparently lost in the red algae and the glaucophytes, whereas the green algae lost the
“leaf” type of petH. We find that chromalveolates have only one isoform of petH (Fig. 4B). The
phylogenetic analysis strongly supports (BPml = 100%, BPnj = 89%, BPP = 1.0) the monophyly of
chromists and the “green” clade of the “root” (green-algal) type. PetH in alveolates, apicomplexan, and
peridinin dinoflagellates belong to the “root” type. However these sequences are highly divergent and we
are unable to resolve the position of alveolates in the petH tree. The substitution of the red-algal gene with
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one of green-algal origin presumably occurred in the common ancestor of chromalveolates (Fig. 4B). The
petH tree topology supports the monophyly of fucoxanthin dinoflagellates and haptophytes implying that
fucoxanthin dinoflagellates acquired their “green” petH from the tertiary endosymbiont.
Plastocyanin (Pc encoded by petE) is a small (ca. 100 –150 aa) thylakoid lumen copper-binding
protein that is an electron carrier between Photosystems II and I (Gross 1993). Plastocyanin is believed to
be unique to plants, green algae, and some cyanobacteria (Ho and Krogmann 1984; Gross 1993). In red
algae and in chromalveolates that contain a red algal-derived plastid, electron transport from cytochrome
b6/f to Photosystem I is achieved via the iron binding analog of plastocyanin, cytochrome C6 (CytC6)
(Sandmann et al. 1983; Price et al. 1991). The complete genome sequences of Cyanidioschyzon merolae,
Galdieria sulfuraria, and Thalassiosira pseudonana confirm the absence of petE encoding plastocyanin
from the genomes of red algae and stramenopiles. Both, petE and CytC6 have a cyanobacterial origin.
Many cyanobacteria and green algae still retain both genes. In these organisms, CytC6 is the functional
backup of plastocyanin that is activated under copper deficiency (Wood 1978; Ho, Krogmann 1984).
We identified 18 clones encoding the petE sequence in the K. brevis EST libraries; i.e., two
clones from the “dark” library and 16 clones from the “stress” library (see Materials and Methods). These
ESTs are encoded by five closely related genes that have arisen through recent gene duplications. BLAST
searches against NCBI identified petE sequences from K. micrum (ABA55542.1). In addition, we found
two petE encoding ESTs in the haptophyte alga Emiliania huxleyi (CX773129 and CX773088). These
two sequences have identical 3’UTRs indicating that they are derived from a single locus. The K. brevis
and E. huxleyi petE sequences are polyadenylated, encode a complete petE ORF, have a stop codon in the
position conserved among all photosynthetic eukaryotes, and a 3’UTR. The E. huxleyi petE sequences do
not include an N-terminal extension, whereas K. micrum petE encodes an N-terminal extension that has a
structure typical for dinoflagellate plastid targeting signals; i.e., it contains a cleavable 19 aa signal
peptide that directs the protein precursor to the endoplasmic reticulum (ER) followed by a plastid
targeting transit peptide (Fig S2A). Interestingly, the N-terminal extension of all K. brevis petE sequences
differ from the canonical dinoflagellate plastid-targeting peptides (Fig S2B). The N-terminal 48 aa form a
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highly hydrophobic, putatively uncleaved signal peptide (P = 0.979 according to SignalP) followed by a
92 aa sequence. The C-terminal thylakoid-spanning motif of the K. brevis plastocyanin leader peptide
shares significant similarity with the corresponding sequence in K. micrum plastocyanin. According to
PSORT (http://www.psort.org/) and TMpred (http://www.ch.embnet.org/software/TMPRED_form.html),
a 48 aa segment can be classified as a type II signal anchor peptide that orients the protein in the N’-
cytoplasm/C’-ER lumen topology in the ER membrane. We propose that plastocyanin targeting to the
plastid involves some alternative mechanism in K. brevis. Study of the prion protein, a brain glycoprotein
involved in various neurodegenerative diseases, provides an example of an alternative protein trafficking
mechanism (Lopez et al. 1990). The prion protein precursor contains an uncleaved N-terminal segment
that functions as a membrane-anchor motif in cell-free systems. However in vivo prion protein precursors
are translocated across the ER membrane with the assistance of cytosolic factors and trafficked to the cell
surface.
The distribution of petE suggests that haptophytes and fucoxanthin dinoflagellates acquired this
gene either from a green alga or a cyanobacterium. Phylogenetic analysis of the protein provides strong
support (BPml = 89%, BPP = 1.0) for the divergence of haptophyte and dinoflagellate petE within the
green clade (Fig. 4C). Based on this tree topology, we suggest that the haptophyte ancestor acquired petE
from a green alga. Thereafter fucoxanthin dinoflagellates captured this gene from the haptophyte tertiary
endosymbiont. CytC6 has not been found in K. brevis, but it was identified in the EST library of K.
micrum (PEPdb accession number KME00008061;
http://amoebidia.bcm.umontreal.ca/public/pepdb/welcome.php), in different species of haptophytes
including E. huxleyi (GenBank accession number CX774985), and in peridinin dinoflagellates
(Alexandrium tamarense, Heterocapsa triquetra, Lingulodinium polyedrum, and Amphidinium carterae).
Thus, fucoxanthin dinoflagellates, like green algae, possess both electron carrier proteins. The results of a
recent microarray analysis show that K. brevis petE is expressed under both normal and iron-limited
culture conditions (KL Lidie and F Van Dolah unpublished data). PetE transcript accumulation is
apparently not affected by a change in iron concentration in the medium. Based on these observations, we
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hypothesize that in fucoxanthin dinoflagellates, like in green algae, copper-binding plastocyanin is used to
transfer electrons from the cytochrome b6/f to Photosystem I dominant over CytC6. The expression
pattern of CytC6 in fucoxanthin dinoflagellates remains to be determined.
Gamma-tocopherol O-methyltransferase (γ-Tmt) is the final enzyme of the γ-tocopherol (vitamin
E) synthesis pathway that is found only in photosynthetic organisms (Sattler et al. 2003). It is believed
that tocopherol protects membrane fatty acids from oxidative degradation by scavenging reactive oxygen
species produced by photosynthesis (Fryer 1993). In plants and algae, tocopherol biosynthesis occurs on
the plastid membrane. Until now, proteins involved in tocopherol biosynthesis have been reported only in
plants, green and red algae, and cyanobacteria. We found an EST encoding the entire γ-Tmt coding region
in K. brevis. Partial γ-Tmt sequences have been identified in the EST libraries of K. micrum and a
haptophyte alga Isochrysis galbana (PEPdb accession numbers KME00005944 and ISE00003655,
respectively). The bootstrap (BPml = 87%, BPnj = 91%) and Bayesian analyses (BPP = 0.99) provide
support for the monophyly of green algae, plants, haptophytes, and fucoxanthin dinoflagellates suggesting
the same scenario for the origin of γ-Tmt in these genomes as described above for plastocyanin (Fig. 4D).
Soluble inorganic pyrophosphatases (sPPases) are ubiquitous enzymes that are responsible for the
removal of inorganic pyrophosphate produced by a variety of vital biosynthetic reactions (Perez-
Castineira et al. 2001). Eukaryotic cells contain several isoforms of sPPase located in the cytosol and
mitochondrion of animals and fungi, in the plastid and mitochondrion of photosynthetic eukaryotes, and
only in the mitochondrion of heterotrophic protists. All of these isoforms, with the exception of plant and
green algal mitochondrial sPPase, arose though the duplication of the eukaryotic gene, which replaced the
bacterial homologs of the proteobacterial and cyanobacterial endosymbionts. Analysis of the plastid
sPPase distribution shows this protein to be present in both photosynthetic plastid-bearing
chromalveolates as well as non-photosynthetic apicoplast-bearing apicomplexans. We however excluded
the highly divergent apicoplast targeted proteins from the analysis (results not shown). The sPPase
phylogenetic tree strongly supports the monophyly of chromalveolates and the origin of this gene from a
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green alga (BPml = 96%, BPP = 0.96; Fig. 4E). Similar to GSA-AT and petH, “green” sPPase originated
in chromalveolates prior to the split of chromists and alveolates. The strongly supported (BPml = 100%
BPnj = 100, BPP = 1.0) position of K. brevis within the haptophyte clade indicates that this gene
originated in fucoxanthin dinoflagellates from the tertiary endosymbiont.
Serine protease IV (sppA) is a thylakoid-bound stroma-exposed protease involved in the light-
induced plastid protein degradation in plants (Lensch, Herrmann, and Sokolenko 2001). Phylogenetic
analyses show that sppA encoding plastid-targeted proteins from all photosynthetic taxa form a
monophyletic group (BPml = 100%, BPnj = 100 %, BPP = 1.0; Fig. 4F) suggesting a common ancestry.
The plastid-targeted sppA genes presumably have arisen in Archaeplastida through the duplication of
mitochondrial sppA (BPml = 95%, BPnj = 94%, BPP = 1.0). However, an unresolved phylogeny within
the mitochondrial/eubacterial clade leaves open the possibility for an origin of plastid sppA through HGT
from a non-cyanobacterium. The nested position of the K. brevis plastid sppA inside of the green
algal/plant clade (BPml = 93%, BPnj = 99%, BPP = 1.0) suggests a green algal origin of the gene.
Absence of plastid sppA homologs from other chromalveolate taxa does not allow us to infer the time of
entry of this “green” gene into the chromalveolate lineage.
The Secondary Host Contribution to the Tertiary Plastid Proteome
In chromalveolates, plastid targeted fructose-1,6-bisphosphate aldolase (FAB II) and glyceraldehyde-3-
phospahte dehydrogenase (GAPDH) arose through the duplication of the secondary host cytosolic
isoforms that replaced homologs of plastid-targeted proteins of the secondary endosymbiont (Harper and
Keeling 2003; Patron, Rogers, and Keeling 2004). The results of our phylogenetic analysis of K. brevis
and K. micrum FAB II support the haptophyte origin of this gene in fucoxanthin dinoflagellates that is
consistent with previously reported FAB II phylogenies (Patron, Waller, and Keeling 2006).
The cytosolic GAPDH that is the precursor of chromalveolate plastidic GAPDH belongs to the
most common form of these enzymes, NADH dependent GAPDH (NAD-GAPDH) that is involved in
glycolytic and gluconeogenetic pathways in all organisms (Forthergill-Gilmore and Michels 1993). In
15
plants, plastid NAD-GAPDH, which originated through the duplication of the gene encoding a plant
cytosolic NAD-GAPDH, is involved in glycolysis that takes place in non-photosynthetic plastids and in
the dark in chloroplasts (Plaxton 1996). Photosynthetic CO2 assimilation in plant, green algal, and red
algal plastids is catalyzed by the cyanobacterial-derived NADPH dependent GAPDH (Martin et al. 1993).
In chromalveolate plastids that lack the NADPH dependent GAPDH, NAD-GAPDH presumably is
involved in both the Calvin cycle and glycolysis.
Several NAD-GAPDH phylogenies including fucoxanthin dinoflagellates have been previously
published (Takishita, Ishida, and Maruyama 2004; Yoon et al. 2005). These data show the dinoflagellate
nuclear genome to encode two cytosolic forms. One of them (gapC3) has been derived either from
spirochaetes or from euglenoids by horizontal gene transfer, whereas the second (gapC2) is a normal
vertically inherited isoform of NAD-GAPDH. Duplication of the gapC2 gave rise to the gene encoding
the plastid-targeting protein. The plastid-targeted NAD-GAPDH found in fucoxanthin dinoflagellates has
been derived from the haptophyte tertiary endosymbiont. Analyses of the K. brevis EST libraries allowed
us to identify four distinct isoforms of GAPDH: two cytosolic (gapC2 and gapC3) and two plastid-
targeted (gapC1-pd and gapC1-fd) isoforms (Fig. S3). One of the plastid isoforms is similar to the
described previously gapC1-fd (Yoon et al. 2005) and clusters with plastid-targeted sequences of the
haptophyte algae, whereas another isoform, gapC1-pd, is positioned within the clade that includes plastid-
targeted proteins from peridinin dinoflagellates (Fig. 5). These results demonstrate for the first time that
K. brevis retains both tertiary endosymbiont haptophyte-derived and secondary endosymbiont red algal-
derived forms of NAD-GAPDH.
Discussion
Plastid Establishment and Endosymbiotic Gene Transfer
The evolution of the fucoxanthin dinoflagellate plastid proteome was shaped by primary, secondary, and
tertiary endosymbioses (Yoon et al. 2005). Study of the green and red algal plastids that resulted from
primary endosymbiosis have until now received the greatest attention. Using these models, it was
16
demonstrated that establishment of the primary plastid proteome involved the integration of the molecular
machineries of both the endosymbiont and the host (Martin et al. 2002; Richly and Leister 2004). This
process was facilitated by large-scale gene movement from the cyanobacterial endosymbiont to the host
nuclear genome, with the subsequent re-targeting of these gene products to the plastid (Martin et al.
1998). Using present estimates, ca. 900 – 1200 plastid-targeted proteins encoded by the Arabidopsis
thaliana nuclear genome and 676 proteins in the thermoacidiphilic red alga Cyanidioschyzon merolae are
of cyanobacterial origin (Reumann, Inoue, and Keegstra 2005; Sato et al. 2005). For Arabidopsis, these
numbers comprise up to 50% of its nuclear-encoded plastid-targeted proteins. The remaining proteins
(around 1200) are derived from the host nuclear or pro-mitochondrial genomes, or from various sources
via HGT. Phagocytosis is considered to be the major mechanism in free living eukaryotes for the
acquisition of new genes via HGT (Doolittle 1998). The inability of virtually all red and green algae to
carry out phagocytosis has most certainly limited the influx of new genetic material through HGT into
these lineages. However, it is likely that HGT played an important role in the early formation of the
Archaeplastida primary plastid proteome (Martin and Schnarrenberger 1997) because phagotrophy was an
ancestral character in this group that most likely allowed the capture of the primary plastid.
Following primary endosymbiosis red and green plastids were acquired by other protist lineages
through secondary endosymbiosis (Bhattacharya, Yoon, and Hackett 2004). In secondary endosymbiosis,
most of the genes encoding plastid-targeted proteins were transferred from the endosymbiont nucleus to
the new host nucleus and reused for plastid function and regulation. The results of our phylogenetic
analysis of the plastid-targeted proteins confirm this prediction with 13 out of 30 proteins in K. brevis
being derived by chromalveolates from a red alga through secondary endosymbiosis (Table 1). This
number includes 11 proteins of cyanobacterial origin and two proteins (fusA and FBPase) derived from
the pro-mitochondrial genome of the primary host (Figs. 6A, B).
Despite the large-scale influx of endosymbiont genes into the host nuclear genome, the secondary
plastid proteome should not be considered simply as a copy of that in the primary plastid. Phylogenetic
analyses show significant changes in the proteome of the red algal-derived plastid that occurred in the
17
chromalveolates during the establishment of secondary endosymbiosis. These changes can be classified
into two major types. The first involves the substitution of cyanobacterial genes with host paralogs. Two
examples are GAPDH and FBA (Harper and Keeling 2003; Patron, Rogers, and Keeling 2004). Both of
these substitutions occurred prior to the divergence of the chromalveolate lineages (Fig. 6B). The second
type of change in the red algal-derived plastid proteome is the substitution of genes derived from the red
algal endosymbiont with green algal or bacterial homologs. This category also includes the acquisition of
“foreign” genes, homologs of which are absent from red algae. There are two possible, but not mutually
exclusive explanations for the occurrence of “foreign” genes in chromalveolates, 1) continuous HGT over
chromalveolate evolution, or 2) an ancient green algal endosymbiosis potentially preceding the red algal-
derived plastid in the chromalveolate ancestor. The latter idea was proposed by Funes et al. (2002) as an
explanation for the occurrence of the green algal derived mitochondrial protein COXII in apicomplexans.
The red algal secondary endosymbiosis that gave rise to the chromalveolate plastid occurred about
1.3 billion years ago (Yoon et al. 2004). However, unlike Archaeplastida, many extant photosynthetic
chromalveolates still retain the ability for phagocytosis (Kugrens and Lee 1990; Kawachi et al. 1991;
Wilcox and Wedemayer 1991; Jones, Leadbeater, and Green 1994) that makes possible the influx of new
genes via HGT. If continuous HGT is a major mechanism for chromalveolate plastid proteome evolution,
we would expect to see the substitutions of red algal-derived genes occurring at different time points in
the chromalveolate tree. Three examples of such substitutions have been reported so far. One of them is
plastid protein delta-aminolevulinic acid dehydrogenase (hemB) that has a green origin in dinoflagellates
and a red origin in stramenopiles (Hackett et al. 2004b). A second example is the substitution of the
normal plastid encoded RuBisCo with its proteobacterial homolog that is nuclear encoded in
dinoflagellates (Palmer 1996). And finally, a plastid-targeted protein encoded by fused genes for the
shikimate biosynthetic enzyme AroB and an O-methyltransferase (OMT) derived by HGT from a
cyanobacterium has been found in dinoflagellates (Waller, Slamovits, and Keeling 2006). These examples
provide evidence for the importance of intra- and interdomain HGT in chromalveolate plastid evolution.
Other reported cases of plastid proteins of non-red algal origin in chromalveolates include two “green”
18
genes, chlorophyll A synthase (chlG, Li et al. 2006) and phosphoribulokinase (PRK, Li et al. 2006;
Petersen et al. 2006) that are shared by different chromalveolate lineages. Red algal derived homologs of
these genes have not yet been found in chromalveolates.
Analysis of the K. brevis EST libraries identified six proteins of green algal origin: GSA-AT,
petH, and sPPase that are shared by chromists and alveolates, petE and γ-Tmt that are shared by
haptophytes and fucoxanthin dinoflagellates, and sppA, homologs of which have not been found in other
lineages of chromalveolates (Fig. 6B, C). Phylogenetic analyses of petE and γ-Tmt show that tertiary
EGT from the haptophyte endosymbiont is the most plausible explanation for the presence of these genes
in fucoxanthin dinoflagellates (Figs. 6C, D). There are two possible scenarios for the occurrence of these
genes in haptophytes. Under the first scenario, haptophytes gained petE and γ-Tmt through an
independent HGT. The observation that many extant species of photosynthetic haptophytes still retain the
ability for phagotrophy (Kawachi et al. 1991; Jones, Leadbeater, and Green 1994) makes plausible this
scenario. Under the second scenario, these genes entered the chromalveolate nuclear genome prior to the
divergence of the constituent lineages and have subsequently been lost from stramenopiles and alveolates
after the divergence of the cryptophyte-haptophyte branch (for the chromalveolate phylogeny see Hackett
et al. 2006). Limited molecular data from cryptophyte algae preclude us from reaching a decisive
conclusion on the time of petE and γ-Tmt recruitment by chromalveolates. Phylogenies of GSA-AT,
petH, sPPase, PRK, and chlG however suggest that a major influx of green genes occurred early in the
evolution of chromalveolates prior to the divergence of the chromists and alveolates.
It remains a daunting task to distinguish between the scenarios that the “green” genes are derived
by EGT from a single endosymbiont prior to the establishment of the “red” plastid or alternatively via
HGT from multiple green algal donors after this event. One possible approach to resolving this issue is to
compare the phylogenetic composition of foreign genes acquired by Chromalveolata before and after the
split of its constituent lineages. The present data show that the list of organisms that donated genes to the
chromalveolate lineages after their divergence from each other includes green algae, proteobacteria, and
19
cyanobacteria (see above). However all foreign genes acquired by chromalveolates before their split are
derived from a single donor lineage, the green algae. One reasonable explanation for these data is the
presence of a green algal endosymbiont in the chromalveolate ancestor prior to the establishment of the
red algal-derived plastid. Unfortunately, the red algal endosymbiosis likely resulted in the substitution of
most green algal nuclear genes of plastid function with red algal homologs, effectively erasing the major
evidence for the previous endosymbiosis.
The results of our study show that secondary endosymbiosis brought together components of highly
diverged red and green algal photosynthetic machineries. Examples are psbU, that is unique in red algae
and green algal plastocyanin, structural homologs of which are absent from the red algal lineage. A
significant flexibility of the light-harvesting complex (LHC) in plastids has been previously demonstrated
(Grabowski, Cunningham, and Gantt 2001), showing that LHC proteins from a red alga, Porphyridium
cruentum, could functionally bind pigments that had evolved separately in different evolutionary lineages
(e.g., plants, stramenopiles, dinoflagellates). The successful incorporation of green algal-derived plastid
proteins into the proteome of the red plastid demonstrated by our study is consistent with these results
(Grabowski, Cunningham, and Gantt 2001). These data support the significant biochemical plasticity of
key components of plastid energy metabolism, such as the electron transport chain and the pyrophosphate
utilization machinery.
Tertiary endosymbiosis introduced an additional level of complexity to the process of plastid
regulation. In this case, the genomes of both the endosymbiont and the host contain genes of plastid
function. The discovery of nuclear-encoded plastid-targeted PSBO in fucoxanthin dinoflagellates that
originated from the haptophyte tertiary endosymbiont led Ishida and Green (Ishida and Green 2002) to
propose the replacement of ancestral red algal derived genes in these photosynthetic taxa with the
haptophyte endosymbiont homologs. Until recently, this was the leading hypothesis for the origin of the
tertiary plastid proteome. Patron, Waller, and Keeling (2006) however reported several plastid-targeted
proteins of the ancestral peridinin-dinoflagellate-type that were retained in the nuclear genome of the
fucoxanthin dinoflagellate K. micrum. These proteins include thylakoid-bound ascorbate peroxidase and
20
phosphoribulokinase. Based on their involvement in processes unrelated to photosynthesis, Patron,
Waller, and Keeling (2006) suggested that the fucoxanthin dinoflagellate ancestor contained a non-
photosynthetic (apicoplast-like) plastid that required a reduced set of genes to express its function. Thus,
genes of the haptophyte tertiary endosymbiont may not have replaced, but rather reestablished the
photosynthetic machinery with genes that were lost prior to this endosymbiotic event. Analysis of the K.
brevis EST data turned up 13 genes encoding plastid-targeted proteins of haptophyte origin: atpC, psbO,
petC, psbU, PGAM, fabG, petH, petE, γ-Tmt, fusA, sPPase, FBA II, and gapC1-fd (Fig. 6C).
In addition to haptophyte-derived GAPDH encoded by gapC1- fd, we found the ancestral red algal
derived gapC1-pd sequence in K. brevis. This the first report of both ancestral and tertiary endosymbiont-
derived genes encoding homologous plastid-targeted proteins in fucoxanthin dinoflagellates. This
suggests that fucoxanthin dinoflagellates likely retain two functional sets of other, yet undetected nuclear
genes encoding plastid-targeted proteins. However, the clear dominance of expressed genes of haptophyte
provenance suggests that most ancestral genes encoding plastid-targeted proteins have been lost or
replaced after the establishment of the tertiary endosymbiont. This situation may mirror the more ancient
substitution of green algal genes with genes derived from the red algal endosymbiont postulated above.
Serial Endosymbiosis and HGT as Mechanisms for Adaptation in Chromalveolates
Chromalveolate algae are the most abundant group of eukaryotic marine phytoplankton. It is however
unclear why these taxa have risen to pre-eminence. Grzebyk et al. (2003) posited as explanation the
presence of a larger set of genes involved in photosynthesis and energy transduction in chromalveolate
plastids than in reds and greens (plastid portability hypothesis) and structural traits of the chromalveolate
host cell such as an armored cell wall as potential explanations.
We propose that the ability to recruit a new genetic material through serial endosymbiosis and
HGT is a key chromalveolate trait that provides the ability for rapid adaptation to changing environmental
conditions, thereby increasing their evolutionary fitness. For example, Karenia brevis is remarkably
tolerant to low iron conditions (10-9M) that will not support the growth of diatoms (A Neeley, G DiTullio,
21
FM Van Dolah unpublished data). Green algal-derived petE is an example of a gene that is absent in red
algae and provides a novel mechanism in fucoxanthin dinoflagellates that may contribute to this trait.
Like green algae and some cyanobacteria, haptophytes and fucoxanthin dinoflagellates possess two types
of functionally homologous electron carrier proteins, copper-binding plastocyanin and iron-binding
CytC6. In fucoxanthin dinoflagellates, like in green algae and cyanobacteria, plastocyanin is the main
protein carrying electrons between the cytochrome b6/f complex and Photosystem I. Based on our results
we suggest that the acquisition of plastocyanin is an adaptation of fucoxanthin dinoflagellates to iron-
limitation in its natural habitat. Another known adaptation to iron deficiency in many algae and
cyanobacteria is a physiological switch from the iron–sulfur protein ferredoxin to the non-iron protein
flavodoxin in the electron transport chain (Erdner et al. 1999; Geiss et al. 2001). Analysis of the K. brevis
EST data show that both proteins, ferredoxin and flavodoxin, are present in this fucoxanthin
dinoflagellate. Consistent with its role in facilitating photosynthetic electron transport under low iron
conditions, flavodoxin transcript levels increase, whereas those of ferredoxin decrease under iron
limitation (KL Lidie and FM Van Dolah unpublished data), and flavodoxin protein expression is induced
under iron limitation, whereas only ferredoxin is present in nutrient replete conditions (A Neeley, G
DiTullio, and FM Van Dolah unpublished data). Thus, the two types of plastid electron carrier proteins,
red algal-derived flavodoxin (Fig. 6B, Table 1) and green algal-derived plastocyanin, likely provide
fucoxanthin dinoflagellates with the ability to thrive under conditions of iron deficiency in their natural
habitats.
22
Supplementary Material
Figures S1-S3 and the list of contributors in the JGI Production Sequencing Group are available at
Molecular Biology and Evolution online.
23
Acknowledgements
The K.brevis EST data were produced under the auspices of the US Department of Energy's Office of
Science, Biological and Environmental Research Program and the University of California, Lawrence
Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National
Laboratory under contract No. DE-AC03-76SF00098 and Los Alamos National Laboratory under
contract No. W-7405-ENG-36. This was Community Sequencing Program project #200407. D. B. and T.
N. were supported by grants from the National Science Foundation and the National Aeronautics and
Space Administration awarded to D. B. (EF 04-31117, NNG04GM17G). T. N. was partially supported by
an Avis E. Cone Fellowship from the University of Iowa. F. M. V. D. and K. L. L. acknowledge support
from the NOAA HAB Initiative.
24
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32
Fig. 1. - A 6-protein plastid phylogeny using genes implicated in tertiary EGT in K. brevis. This ML tree
was inferred from the combined plastid-targeted protein sequences of atpC, psbO, petC, psbU, PGAM,
and fabG. The numbers above and below the branches are the results of ML and NJ bootstrap analyses,
respectively. Only bootstrap values ≥ 60% are shown. The thick branches indicate ≥ 0.95 posterior
probability from Bayesian inference. Branch lengths are proportional to the number of substitutions per
site (see scale bars). Chr, chromista; Alv, alveolates; Ra, red algae; Gr, green algae/plants; CB,
cyanobacteria. Numbers in bold indicate bootstrap supports for the monophyly of chromalveolates and
red algae and K. brevis and haptophytes.
Fig. 2. - ML tree of plastid- and mitochondrial-targeted translation elongation factor G (fusA). The
numbers above and below the branches are the results of ML and NJ bootstrap analyses, respectively.
Only bootstrap values ≥ 60% are shown. The thick branches indicate ≥ 0.95 posterior probability from
Bayesian inference. Branch lengths are proportional to the number of substitutions per site (see scale
bars). The lineage designations are as in Fig. 1. Numbers in bold indicate bootstrap support for the
monophyly of plastid and alpha-proteobacterial clades, and K. brevis and haptophytes.
Fig.3. - ML tree of plastid targeted and cytosolic fructose-1, 6-bisphosphatase (FBPase) sequences. The
numbers above and below the branches are the results of ML and NJ bootstrap analyses, respectively.
Only bootstrap values ≥ 60% are shown. The thick branches indicate ≥ 0.95 posterior probability from
Bayesian inference. Branch lengths are proportional to the number of substitutions per site (see scale
bars). The lineage designations are as in Fig. 1. Numbers in bold indicate bootstrap support for the
monophyly of plastid and cytosolic isoforms of FBPase, and chromalveolates and red algae.
Fig. 4. - Proteins of green algal origin in the K. brevis tertiary plastid proteome. (A) ML tree of GSA-AT.
(B) ML tree of petH. (C) ML tree of petE. (D) ML tree of γ-Tmt. (E) ML tree of sPPase. (F) ML tree of
33
sppA. The numbers above and below the branches are the results of ML and NJ bootstrap analyses,
respectively. Only bootstrap values ≥ 60% are shown. Dashes represent bootstrap values bellow 60%. The
thick branches indicate ≥ 0.95 posterior probability from Bayesian inference. Branch lengths are
proportional to the number of substitutions per site (see scale bars). The lineage designations are as in Fig.
except that Gl is glaucophytes. Numbers in bold indicate bootstrap support for the monophyly of
chromalveolates and green algae/plants and fucoxanthin dinoflagellates K. brevis and K. micrum and
haptophytes.
Fig. 5. - ML tree of chromalveolate plastid targeted NAD-GAPDH. The numbers above and below the
branches are the results of ML and NJ bootstrap analyses, respectively. Only bootstrap values ≥ 60% are
shown. The thick branches indicate ≥ 0.95 posterior probability from Bayesian inference. Branch lengths
are proportional to the number of substitutions per site (see scale bars). Chr, chromista; Alv, alveolates.
Numbers in bold indicate bootstrap support for the monophyly of gapC1-pd isoform of K. brevis and
peridinin dinoflagellates and gapC1-fd isoform of fucoxanthin dinoflagellates and haptophytes. This tree
represents a portion of the complete NAD-GAPDH tree provided in supporting materials (Fig. S3).
Fig. 6. - Origin of plastid-targeted proteins in K. brevis. Fucoxanthin dinoflagellate plastid proteome
evolution through primary, secondary, and tertiary endosymbioses is shown from top to bottom. The
sources of K. brevis genes encoding plastid targeted proteins are denoted using different colors in three
sections of the figure corresponding to primary, secondary, and tertiary endosymbioses. (a) Genes derived
from the primary host through vertically (black font/arrow) and cyanobacteria through EGT (blue
font/arrow). (b) Genes derived from the secondary host vertically (black font/arrow), red algal
endosymbiont through EGT (red font/arrow), and green algae through HGT (green font/arrow). (c) Genes
derived by fucoxanthin dinoflagellates from the peridinin dinoflagellate host vertically (black font/arrow)
and haptophyte tertiary endosymbiont through EGT (purple font/arrow). The question mark indicates
34
uncertainty about the time for the gene transfer. * - numbers of cyanobacteria- and host-derived genes are
indicated for Arabidopsis plastid proteome (Reumann, Inoue, and Keegstra 2005).
36
Table 1. Plastid targeted proteins in the fucoxanthin dinoflagellate Karenia brevis.
Gene source in1’ 2’ 3’
# Protein name Gene/Protein
abbreviation
Accessionnumber
Leaderpeptide
plastid proteome
Electron transport and carbohydrate metabolism1 Glyceraldehyde-3-phosphate
dehydrogenase isoform C1-fdgapC1-fd/
GAPDHp-fdDQ531601* st Cyt _ H
2 Glyceraldehyde-3-phosphatedehydrogenase isoform C1-pd
gapC1-pd/GAPDHp-pd
DQ531575 st Cyt _ PD
3 Cytochrome b6-f complex iron-sulfur subunit
petC/ RISP DQ531586 t CB _ RA _ H
4 Ferredoxin petF/ Fd DQ531588* st CB _ nr _ nr5 Ferredoxin NADPH reductase petH/ FNR DQ531589 st CB _ GA _ H6 Flavodoxin isiB /Flv DQ531584* st CB _ RA _ nr7 Oxygen-enhancer 1 protein psbO DQ531590 st CB _ RA _ H8 Photosystem II 12kD extrinsic
proteinpsbU DQ531591* st CB _ RA _ H
9 Fructose-1,6-bisphosphatealdolase class II
fbaC1/FBA-II
DQ531583 st Cyt _ H
10 Adenylate kinase ADK DQ531597 - CB _ RA _ nr11 Soluble inorganic
pyrophosphatase sPPase DQ531593* st Euk _ GA _ H
12 ATP synthase gamma subunit atpC DQ531581 st CB _ RA _ H13 Plastocyanin petE/ Pc DQ531587* at CB _ GA _ H14 Phosphoglycerate mutase PGAM DQ531576 st CB _ RA _ H15 Fructose-1,6 bisphosphatase fbp/ FBPase DQ531600 st Mt_Cyt _ RA _ nr16 Putative ribulose-1,5
bisphosphate carboxylaseRBCMT DQ531595 st nr
17 LH chlorophyll binding proteins not analyzed
37
Translation18 Translation elongation factor G fusA/ EF-G DQ531572 - Mt _ RA _ H19 Translation elongation factor Ts EF-Ts DQ531577 st CB _ nr _ nr Transcription and DNA synthesis20 DNA gyrase subunit A gyrA DQ531573 - CB _ RA _ nr Fatty acid synthesis21 Beta-ketoacyl-ACP reductase fabG/ BKR DQ531582 st CB _ RA _ H Protein import and processing22 Clp protease clpC DQ531574 - nr23 Clp protease clpN DQ531578 st CB _ nr _ nr24 DegP serine-type peptidase degP DQ531596 t CB _ RA _ nr25 Periplasmic serine protease IV sppA DQ531592 st CB _ GA _ nr26 M48-like peptidase - DQ531585 st nr Plastid biogenesis27 RNA helicase VDL VDL DQ531579 st nr Chlorophyll biosynthesis28 Glutamate 1-semialdehyde 2,1-
aminomutaseGSA-AT DQ531598 - CB _ GA _ nr
Terpenoid biosynthesis29 Gamma-tocopherol O-
methyltransferase_-Tmt DQ531594 st CB _ GA _ nr
Unknown function30 Conserved hypothetical protein
CO065457- DQ531580 st CB _ RA _ nr
* The sequence represents one member of a closely related family of proteins in K. brevis; s - signal peptide (> 0.9 SignalP
probability); a - signal anchor peptide (> 0.9 SignalP probability); t - transit peptide; 1’ – Archaeplastida; 2’ – Chromalveolata; 3’-
fucoxanthin dinoflagellates. CB - cyanobacteria; RA - red algae; GA - green algae; H - haptophytes; PD - peridinin dinoflagellates; Mt
38
- gene of mitochondrial or pro-mitochondrial origin; Cyt - host gene encoding cytosolic protein; Euk – host gene of eukaryotic origin
(if the original gene copy was lost from the host genome); nr- protein phylogeny was not resolved.
Emiliania huxleyiIsochrysis galbana
Prymnesium parvumKarenia brevis - Alv
Thalassiosira pseudonanaPhaeodactylum tricornutum
Heterocapsa triquetraAlexandrium tamarense
Amphidinium carteraeGaldieria sulphurariaCyanidioschyzon merolae
EudicotArabidopsis thaliana
Oryza sativaChlamydomonas reinhardtii
Nostoc sp. PCC7120Crocosphaera watsonii WH8501
Synechococcus elongatus PCC6301Gloeobacter violaceus PCC7421
10095
100
100
97100
70
100
100
100100
100
10096
62
100-
100
100
-
100
100
100100
10087 95
73
100
-
Chr
Chr
Alv
Ra
Gr
Cb
0.1 substitutions/site
Prymnesium parvumEmiliania huxleyi
Karenia brevis Thalassiosira pseudonanaFragilariopsis cylindrusCyanidioschyzon merolae
Porphyra yezoensisArabidopsis thaliana Glycine maxOryza sativa
Chlamydomonas reinhardtiiRhodobacter sphaeroidesOceanicola batsensis HTCC2597
Mesorhizobium loti MAFF303099Brucella melitensis biovar Abortus 2308
Rhodospirillum rubrum ATCC 11170Magnetospirillum magneticum AMB-1
marine alpha proteobacterium HOT2C01Rickettsia akari str. Hartford
Mus musculusRattus norvegicus
Homo sapiensCryptococcus neoformans
Neurospora crassaAspergillus nidulans
Kluyveromyces lactisArabidopsis thalianaOryza sativa
Galdieria sulphurariaCyanidioschyzon merolae
Dictyostelium discoideumBorrelia garinii PBi
Treponema pallidum subsp. pallidum str. NicholsSyntrophobacter fumaroxidans MPOB
Desulfotalea psychrophila LSv54Leptospira interrogans serovar Lai str. 56601
Blastopirellula marina DSM 3645Rhodopirellula baltica SH 1
Synechococcus elongatus PCC6301Nostoc sp. PCC7120
Gloeobacter violaceus PCC7421
6794
99
93 10099
100
99
100
100
69
91100
100100
98100
8791
100
100
6497
100
74100
Alpha-proteobacteria
Spirochaetesand Delta-proteobacteria
Planctomycetes
10078
100
100
96100
9495
100
96
100 100100
67
100
100
100
9799
100
95
6894 69
97
100
95
mitochondria
Cb
Gr
Ra
Chr
Chr- Alv
0.1 substitutions/site
-
-
plastid
Arabidopsis thalianaBrassica oleraceaSolanum tuberosum
Triticum aestivumOryza sativa
Spinacia oleraceaScherffelia dubiaChlamydomonas reinhardtiiIsochrysis galbana
Prymnesium parvumLingulodinium
Karenia brevisGaldieria sulphuraria 1
Galdieria sulphuraria 2Cyanidioschyzon merolae
Cyanidioschyzon merolaeGaldieria sulphuraria
Thalassiosira pseudonanaAmphidinium carterae
Phytophthora infestansCaenorhabditis elegans
Ustilago maydisCryptococcus neoformansSolanum tuberosum
Spinacia oleraceaArabidopsis thalianaBrassica oleracea
Oryza sativa
0.1 substitutions/site
919860
62100
99
100
9972
6879
98
65
100
9289
100
100 10067
97100
63
93
98 100
6967
10082
100
100
100 100
-
-
Gr
Alv
Ra
cytosol
plastid
Chr
0.1 substitutions/site
Prymnesium parvum - ChrKarenia brevisEmiliania huxleyi
Thalassiosira pseudonanaAmphidinium carterae
Lingulodinium polyedrumOryza sativaArabidopsis thaliana
Chlamydomonas reinhardtiiCyanidioschyzon merolae
Galdieria sulphurariaCyanophora paradoxa
Dictyostelium discoideumKluyveromyces lactis
Debaryomyces hanseniiSchizosaccharomyces pombe
Ustilago maydisCryptococcus neoformans
Porphyra yezoensisGaldieria sulphuraria
Cyanidioschyzon merolaeDrosophila melanogaster
Caenorhabditis elegansHomo sapiens
Rattus norvegicusGallus gallus
Danio rerio
79100
94
9697
100
94
95
100
94
98
10066
98
99
-
-
-
71
-
100100
-
100-
63
100 67
100
8699
mitochondrialand cytosolicsPPase
- GlRa
Gr
Alv
- AlvChr
-
0.1 substitutions/site
Amphidinium carteraeKarlodinium micrumKarenia brevis
Alexandrium tamarenseThalassiosira pseudonana
Phaeodactylum tricornutumIsochrysis galbana
Emiliania huxleyiPavlova lutheri
Arabidopsis thalianaBrassica oleraceaNicotiana tabacumLycopersicon esculentum
Hordeum vulgareOryza sativa
Polytomella sp.Chlamydomonas reinhardtii
Cyanidioschyzon merolaeGaldieria sulphuraria
Crocosphaera watsonii WH8501Nostoc sp. PCC7120
Synechococcus elongatus PCC6301Gloeobacter violaceus PCC7421
9281
64 100
10087
100
100
98
8480
86
99-
100
73
100
100
100
98100
82-
-
99
-83
-
Gr
Alv
Chr
Ra
Cb
A
E
0.1 substitutions/site
Isochrysis galbanaPrymnesium parvum
Emiliania huxleyiKarlodinium micrumKarenia brevis
Thalassiosira pseudonana 1Thalassiosira pseudonana 2
Phaeodactylum tricornutumChlamydomonas reinhardtiiVolvox carteriOryza sativaZea mays
Nicotiana tabacumPisum sativumArabidopsis thalianaCeratopteris richardii
Toxoplasma gondiiHeterocapsa triquetra
Amphidinium carteraeOryza sativa
Arabidopsis thalianaPisum sativum
Nicotiana tabacumCyanidioschyzon merolae
Glaucocystis nostochinearumCyanophora paradoxaGaldieria sulphuraria - Ra
Nostoc sp. PCC7120Synechococcus elongatus PCC6301Crocosphaera watsonii WH8501
Gloeobacter violaceus PCC7421
10067
10086
100
94
100 99
97100
93
60
10068
98
9699
90 Cb
- Ra Gl
Alv
Alv
Gr
Gr"leaf" isoform
Chr
Chr
B100
100
100100100
100 86
-
89
100
-
-
-
100
6999
-
100
9299
74
Solanum tuberosumPisum sativum
Nicotiana tabacumArabidopsis thaliana
Zea maysOryza sativaHordeum vulgarePhyscomitrella patens
Emiliania huxleyiKarlodinium micrum
Karenia brevisPediastrum duplex
Pediastrum boryanumChlorella vulgaris
Chlamydomonas reinhardtiiScherffelia dubia
Scenedesmus obliquusTrichodesmium erythraeum IMS101
Gloeobacter violaceus PCC7421Nostoc punctiforme PCC73102
Phormidium laminosumSynechococcus sp. WH 8102
Prochlorococcus marinus CCMP1375Crocosphaera watsonii WH8501
Synechocystis sp. PCC 6803
676389
89
7294
9499
90
100
91
10096
96
-
-
Gr
Gr
- ChrAlv
Cb
--
0.1 substitutions/site
C Arabidopsis thalianaBrassica oleracea
Helianthus annuusPicea sitchensis
Pinus taedaTriticum aestivumOryza sativa
Physcomitrella patensChlamydomonas reinhardtii
Isochrysis galbanaKarlodinium micrum
Karenia brevisCyanidioschyzon merolae
Galdieria sulphuraria Trichodesmium erythraeum IMS101
Anabaena variabilis ATCC 29413Crocosphaera watsonii WH8501Synechocystis sp. PCC 6803
10073
10093
88 10069
87100
98
100
D 100
66
10080
99
93
9193
99
1000.1 substitutions/site
- ChrAlv
Ra
Cb
"root"isoform
Gr
Vitis viniferaNicotiana tabacumArabidopsis thaliana
Sorghum bicolorOryza sativa
Hordeum vulgareChlamydomonas reinhardtii
Karenia brevisCyanidioschyzon merolae
Galdieria sulphuraria Solibacter usitatus Ellin6076
Chlorobium tepidum TLSThermobifida fusca YX
Magnetospirillum MS-1Mycobacterium leprae
Chlamydomonas reinhardtiiDictyostelium discoideuml
Galdieria sulphurariaOceanicaulis alexandrii HTCC2633Cyanidioschyzon merolae
Thalassiosira pseudonanaIdiomarina baltica OS145
Shewanella sp. MR-7Porphyromonas gingivalis W83
Cytophaga hutchinsoniiNostoc punctiforme PCC73102Crocosphaera watsonii WH8501
Trichodesmium erythraeum IMS101Cb
Ra
Gr
mitochondrialand EubacterialsppA
- Alv
0.1 substitutions/site
F 75
9494
99
93
100
80
7187
7376
95
7370
100
91100
70
-
-99
-
65-
72
100
84-
6877
91
62
100-64
10079
99
GSA-AT petH
petE G-Tmt
sPPase sppA
Symbiodinium muscatineiAmphidinium carterae
Gymnodinium simplexKarenia brevis
Lingulodinium polyedrumHeterocapsa triquetra
Fucus distichusOchromonas danica
Mallomonas rasilisGuillardia theta
Pyrenomonas salinaPhaeodactylum tricornutum
Odontella sinensisKarenia mikimotoiKarenia brevis
Karlodinium micrumPrymnesium parvum
Isochrysis galbanaPavlova lutheri
Toxoplasma gondii
9087
7687
6091
100 97
97
100
100100
100100
93
100
--
--
-
92
97
100
96
100
100
9599
9785
AlvgapC1-pd
Alv gapC1-fd
Chr
Chr
- Alv
0.1 substitutions/site
Cytosolic gapC2
TERTIARY ENDOSYMBIOSIS
PRIMARY ENDOSYMBIOSIS
Peridinin Dinoflagellates
Fucoxanthin Dinoflagellates
Haptophytes
ENDOSYMBIOTIC / HORIZONTAL GENE TRANSFER
FBPasefusAsppAsPPase(~1200 genes*)
Large-scaleEGT fromCyanobacterium(~900 genes*)
PGAMpsbOpsbUC0065457VDLGSA-ATpetH sPPase
gapC1-pd
petHgapC1-fdPGAMpsbOpsbUsPPaseTmt
Rhodophytes
petETmt
SECONDARY ENDOSYMBIOSIS
gapC1FBAII
Origin of Plastid Targeted Proteins in Fucoxanthin Dinoflagellates
Archaeplastida
ChromalveolatesatpCdegPfabGFBPaseFlvfusAgyrApetC
atpCfabGFBAIIfusApetCpetE
?
HOST GENES
A
B
C
sppA?