Proposal of a Twin Aarginine Translocator System-Mediated
Constraint against Loss of ATP Synthase Genes from
Nonphotosynthetic Plastid Genomes
Ryoma Kamikawa,*,1,2 Goro Tanifuji,3 Sohta A. Ishikawa,4 Ken-Ichiro Ishii,1 Yusei Matsuno,2
Naoko T. Onodera,5 Ken-Ichiro Ishida,3 Tetsuo Hashimoto,3,6 Hideaki Miyashita,1,2
Shigeki Mayama,7 and Yuji Inagaki3,6
1
Graduate School of Global Environmental Studies, Kyoto University, Kyoto, Japan
Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Japan
3
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
4
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
5
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada
6
Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
7
Department of Biology, Tokyo Gakugei University, Tokyo, Japan
*Corresponding author: E-mail: [email protected].
Associate editor: Aoife McLysaght
2
Abstract
Letter
Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of
these organelles. Yet the functional roles of some retained genes—such as those coding for ATP synthase—remain
mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic
diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between
thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins
into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related
nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel
loss of photosynthesis in multiple independent lineages of algae/plants.
Key words: apochlorotic diatoms, nonphotosynthetic plastid genome, genome reduction, ATP synthase complex, twin
arginine translocator.
Plastids are organelles that carry out photosynthesis in addition to many other biosynthetic pathways. Plastids have been
spread by separate, multiple endosymbioses into phylogenetically diverse eukaryotes (Archibald 2009; Gray 2010; Keeling
2010; Kamikawa, Tanifuji, et al. 2015). Secondary losses of
photosynthetic ability are also often observed in separated
branches in the tree of eukaryotes (such as the malaria parasite Plasmodium falciparum; McFadden et al. 1996; Wilson
et al. 1996), and loss of photosynthesis has been found to be
coupled with plastid genome reduction in both size and gene
content to varying degrees (de Koning and Keeling 2006;
Delannoy et al. 2011; Molina et al. 2014; Smith and Lee 2014).
Many genes for photosynthesis are still retained as probable functional genes or as pseudogenes in reduced plastid
genomes of the parasitic liverwort, the mycoheterotrophic
monocot, the broomrapes, and the mycoheterotrophic orchids (Wickett et al. 2008; Logacheva et al. 2011, 2014; Barrett
and Davis 2012; Wicke et al. 2013; Barrett et al. 2014). On the
other hand, the nonphotosynthetic plastids in Cryptomonas
paramecium (cryptophyte alga), Euglena longa (euglenophyte
alga), and Prototheca wickerhamii (opportunistic parasite
green alga) contain plastid genomes that lack nearly all
photosynthetic genes and many biosynthetic genes (Gockel
and Hachtel 2000; Knauf and Hachtel 2002; Donaher et al.
2009). Nevertheless, the three nonphotosynthetic plastid genomes mentioned above still possess genes for ribulose-1,5bisphosphate carboxylase/oxygenase (RuBisCo) and/or the
ATP synthase complex (Gockel and Hachtel 2000; Knauf
and Hachtel 2002; Donaher et al. 2009). The presence of
genes for RuBisCo in nonphotosynthetic plastids is explained
by their involvement in lipid biosynthesis via conversion of
hexose to pyruvate, resulting in improvement of carbon efficiency during the formation of acetyl-CoA and lipids
(Schwender et al. 2004; Krause 2008). In contrast, the reasons
for the retention of ATP synthase genes in nonphotosynthetic
plastid genomes have, until now, been a mystery (Wickett
et al. 2008; Wicke et al. 2013; Barrett et al. 2014). Here, we
report analyses of the complete plastid genome and the transcriptome data of the apochlorotic diatom alga Nitzschia sp.
NIES-3581 which has four-membrane-bound plastids residing
reduced thylakoids (Kamikawa, Yubuki, et al. 2015). Based on
these analyses and considering other nonphotosynthetic plastid genomes, we propose a new hypothesis for the retention
of ATP synthase genes in nonphotosynthetic plastids.
ß The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
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Mol. Biol. Evol. 32(10):2598–2604 doi:10.1093/molbev/msv134 Advance Access publication June 5, 2015
ATP Synthase Genes in Nonphotosynthetic Plastid Genomes . doi:10.1093/molbev/msv134
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A
B
FIG. 1. Physical map of the Nitzschia sp. NIES-3581 plastid genome and plastid-encoded protein phylogeny of diatoms. (A) Protein-coding and
ribosomal RNA-coding regions are shown by closed boxes, whereas transfer RNAs (tRNAs) are depicted by lines. Genes shown on the outside of
the circle are transcribed clockwise. Large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions are labeled. (B) ML tree was inferred
from the 58-protein data set. The 58-protein data set was analyzed by the ML method with the LG + G + F model (Le and Gascuel 2008) using RAxML
ver.7.2.8 (Stamatakis 2006). Values at nodes represent maximum likelihood bootstrap values (MLBPs). MLBPs <50% are omitted from the figure.
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Kamikawa et al. . doi:10.1093/molbev/msv134
Table 1. Plastid Genomes in Nonphotosynthetic and Photosynthetic
Diatoms.
Sizes (kb)
Noncoding (%)
A + T (%)
Proteinsa
RNAsa
Translation
Transcription
Photosynthesis
Others
tRNAs
rRNA
Other
Nitzschia sp.
NIES-3581
68
5
77
41
4
7
10
Photosynthetic
Speciesb
116–166
12–39
68–71
45
4
51
22
26
3
—
27
3
2
NOTE.—rRNA, ribosomal RNA; tRNA, transfer RNA; —, not detected.
a
Genes conserved in all the photosynthetic diatom plastid genomes used in this
analysis, and duplicated genes were counted only once.
b
This analysis considered Phaeodactylum tricornatum, Fistulifera sp. JPCC DA0580,
Ulnaria acus, Kryptoperidinium foliaceum (dinotom), Durinskia baltica (dinotom),
Odontella sinensis, Thalassiosira pseudonana, T. oceanica, Leptocylindrus danicus,
Coscinodiscus radiatus, Eunotia naegelii, Lithodesmium undulatum, Asterionellopsis
glacialis, Asterionella formosa, Cylindrotheca closterium, and Didymosphenia geminata.
Presence or absence of each gene is shown in supplementary table S1,
Supplementary Material online. Data of photosynthetic diatoms are those from
Ruck et al. (2014).
The circularly mapped complete plastid genome of
Nitschia sp. NIES-3581 was found to be the smallest in size
and gene content in diatoms (fig. 1A, table 1, and supplementary table S1, Supplementary Material online; Ruck et al.
2014). Reflecting the high AT content (table 1), the apochlorotic diatom plastid genome displayed a greater abundance of [dA]/[dT] homopolymers than those of
photosynthetic diatoms (supplementary fig. S1,
Supplementary Material online), and more than 95% of the
detected [dA]/[dT] homopolymers accumulated within
coding regions (supplementary fig. S1, Supplementary
Material online). As a result, the evolutionary rate of proteins
encoded on the plastid genomes was apparently accelerated
as reflected in the maximum-likelihood (ML) phylogeny with
the 58 plastid-encoded protein data set in which Nitzschia sp.
was an enormously long branch like other nonphotosynthetic
plastid genomes (Vernon et al. 2001; Donaher et al. 2009; fig.
1B). Nitzschia sp. NIES-3581 was also a long branch in individual analyses of the 58 plastid-encoded proteins (supplementary fig. S2, Supplementary Material online). The
difference in the evolutionary rate between Nitzschia sp.
NIES-3581 and other (photosynthetic) diatoms was confirmed by the likelihood ratio test based on the ML tree
(supplementary table S2, Supplementary Material online
and supplementary document, Supplementary Material
online).
The Nitzschia sp. NIES-3581 plastid genome lacks all the
gene sets for the photosystem I, photosystem II, cytochrome
b6/f complexes, photosystem repair (i.e., ftsH; de Vries et al.
2013), carbon fixation through the Calvin cycle, and the chlorophyll a biosynthesis pathway (supplementary table S1,
Supplementary Material online). None of these genes were
detected even from our transcriptome data (data not shown),
2600
strongly suggesting complete loss of them but not gene transfer to the nucleus (Martin et al. 1998; Timmis et al. 2004),
although all these genes are found in the photosynthetic
diatom plastid genomes determined to date (Ruck et al.
2014). The absence of these genes in the Nitzschia sp.
NIES-3581 plastid confirms that this apochloritic species is
genuinely nonphotosynthetic.
In spite of the reduced plastid genome structure of the
nonphotosynthetic diatom, several dozens of protein-coding
genes are still present. Of the 58 functionally assignable proteins encoded in the plastid genome, most (e.g., ribosomal
proteins and a molecular chaperone) are involved in protein
expression and quality control. The genome also has genes
encoding Sec membrane translocators and the Sec-independent twin arginine translocator (Tat). The remaining nine
other genes code for proteins relevant to two distinct biosynthesis pathways: iron–sulfur cluster assembly and ATP
synthase. Retention of genes on photosynthetic plastid genomes has been well justified in the “colocation for redox
regulation (CoRR)” hypothesis (Allen 2003), whereby regulation of redox control was argued to be a very important
selective force for gene retention. However, as the plastid of
Nitzschia sp. is no longer photosynthetic and appears to have
few (if any) redox-related functions, gene retention is unlikely
to be explained by CoRR in this case. As the genes above
mentioned are all members of protein complexes or assembly
of those complexes, retention of some of them on the plastid
genome may instead come from purifying selection to maintaining “control by epistasy of synthesis” (Choquet and Vallon
2000; Wostrikoff et al. 2004). That is, the regulation of assembly of protein complexes requires one or more subunits to be
organelle encoded.
In photosynthetic diatoms, the plastid ATP synthase complex is constituted by nine subunits, eight of which are carried
on the plastid genomes, and it is thought that the ATP
synthase complex localizes to thylakoid membranes. Of the
eight plastid-encoded subunit genes, we found seven genes,
namely atpA, atpB, atpD, atpF, atpG, atpH, and atpI, in the
plastid genome of Nitzschia sp. NIES-3581. The gene atpE was
undetected in the Nitzschia sp. plastid genome—this gene
encodes an endogenous regulator (inhibitor) of the ATP hydrolase activity of ATP synthase (e-subunit; Richter et al. 1984;
Richter 2004). Expression of the above ATP synthase genes on
the RNA level was confirmed by detection of corresponding
sequences in the transcriptome data (sequences deposited in
DNA Data Bank of Japan: accession nos LC052663–
LC052665).
Because of the absence of most of the photosynthesisrelated genes, retention of the ATP synthase genes was unexpected. To gain further insights into the functionality of the
ATP synthase complex, we searched for nuclear-encoded
plastid ATP synthase transcripts in the transcriptome data.
We successfully detected atpC transcripts encoding the ATP
synthase g subunit, the only nuclear-encoded subunit of ATP
synthase complex (supplementary table S3, Supplementary
Material online). The deduced amino acid sequence of the
atpC transcript contains the N-terminal bipartite plastid-targeting signal comprised of the signal peptide region followed
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ATP Synthase Genes in Nonphotosynthetic Plastid Genomes . doi:10.1093/molbev/msv134
A
Cytosol
AtpD
DNA
TatC
Sec61
SELMA
Omp85
AtpI
AtpF
ADP
NTT1
AtpH
AtpG
H+
TIC
?
Hcf106/Tha4
TAT
TPP
?
ATP
ATP
NTT2
H+
ATP synthase
AtpC
AtpB
AtpA
Cytosol
(d)NTP
Stroma
Transcription
& Translation
H+
H+
Protein
H+
H+
o
N tos
itz y
sc nth
hi et
a ic
R
sp d
ho
. N iat
C do
IE om
ry m
S- s
pt on
35
om a
81
on s s
a
Li
a
l
i
n
s na
pa
O den
ra
ro b
e
m
ba r
ec
nc g i a
iu
he p
m
h
i
sp l i p
p. p e
ns
is
Thylakoid membrane
Ph
B
PSI
*
PSII
*
Cytochrome b6/f complex
Chlorophyll synthesis
*
Carbon fixation
*
ATP synthase complex
Tat system
Diatoms
Cryptophytes
Orobancheae
FIG. 2. A model of the ATP synthase function in the nonphotosynthetic diatom plastid and distribution of particular plastid functions in nonphotosynthetic plastids. (A) A model of localization of ATP synthase complex and Tat system, protein import, and ATP import in the nonphotosynthetic
plastid of Nitzschia sp. NIES-3851 is depicted. In this model, ATP synthase genes, atpA, atpB, atpD, atpF, atpH, and atpI, and the core Tat gene, tatC, are
expressed in the stroma. Cytosolically expressed nuclear-encoded atpC encoding g-subunit of plastid ATP synthase complex, and Tat-related genes,
hcf106/tha4, and thylakoid processing peptidase gene, are imported into the nonphotosynthetic plastid through Sec61 complex for the outermost,
Symbiont-specific ERAD-like machinery (SELMA) for the second outermost, and TIC for the innermost membrane. The mechanism of protein
translocation through the second innermost membrane is currently unknown as the homolog of Omp85, a component for protein import of the
second innermost membrane, has not yet been identified in Nitzschia sp. NIES-3581. The ATP synthase complex localizes on thylakoid membrane and
functions for generating proton gradient between stroma and thylakoid lumen by hydrolysis of ATP imported from cytosol through ATP/ADP
translocators NTT-1 and NTT-2. Any systems by which ATP/ADP are imported through second outermost and second innermost membranes are
unknown in diatoms (depicted by question mark; Ast et al. 2009). Utilizing the proton gradient, the Tat also localizing on thylakoid membrane facilitates
protein translocation into thylakoid lumen although any proteins localizing in thylakoid lumen of the nonphotosynthetic plastid remain unknown. The
protein import model is reconstructed on the basis of Ast et al. (2009) and Hempel et al. (2014). (B) Presence or absence of genes for photosystem I
(PSI), photosystem II (PSII), cytochrome b6/f complex, chlorophyll synthesis, carbon fixation, ATP synthase complex, and Tat system are shown for three
distantly related lineages, diatoms, cryptophytes, and the broomrape Orobancheae. Nonphotosynthetic species and closely related photosynthetic
species are shown on right and left in each lineage. Genes present on plastid genomes are depicted as green squares, whereas genes absent from plastid
genomes are depicted as open squares. Genes absent from plastid genomes but present in nuclear genomes are depicted as green triangles in open
squares. Presence of pseudogenes on plastid genomes are shown as asterisks in open squares. The data here are derived from Donaher et al. (2009),
Wickett et al. (2011), Wicke et al. (2013), Ruck et al. (2014), and this study.
by transit peptide-like regions (Apt et al. 2002; Gruber et al.
2007; supplementary fig. S3 and table S3, Supplementary
Material online). Most importantly, the two regions are separated by “ASAFAP” motif-like sequence (supplementary fig.
S3 and table S3, Supplementary Material online), which is the
typical diatom plastid-targeting signal (Gruber et al. 2007). If
this product is imported into the nonphotosynthetic plastids,
protein translocons for plastids must be retained. We also
detected transcripts for proteins relevant to the protein
import system of diatom plastids in the transcriptome data
(supplementary table S3, Supplementary Material online).
Given the typical plastid targeting signal found in the atpC
transcripts and the plastid protein import machine, it appears
that nuclear-encoded plastid ATPase g subunit is imported
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Kamikawa et al. . doi:10.1093/molbev/msv134
into the nonphotosynthetic plastids. Furthermore, detection
of the ATP synthase subunits encoded in the transcriptome
suggests that the ATP synthase complex is functional or has
been functional until very recently. Like plastid ATP synthase
complexes in other plastids, the ATP synthase complex is
likely to be localized on the thylakoid membrane in fourmembrane-bound plastids of Nitzschia sp. NIES-3581 that
we previously observed by the transmission electron microscopic analysis (fig. 2A; Kamikawa, Yubuki, et al. 2015), although the precise localization of ATP synthase is to be
investigated by biochemical experiments in future.
It is possible that these ATP synthase complexes might be
retained for ATP synthesis using a proton gradient generated
through an as yet unknown, photosynthesis-independent
mechanism. Here we suggest an alternative function: The
plastid ATP synthase complex genes of Nitzschia sp. NIES3581 and these other nonphotosynthetic plastids might
function in ATP hydrolysis. ATP hydrolysis is known to be
performed even by an ATP synthase complex lacking e subunit (Richter et al. 1984; Richter 2004), a gene that we did not
detect (and maybe absent) in the nonphotosynthetic diatom
plastid genome (see above). A previous study suggested that
the ATP synthase complex in root plastids of various land
plants performs proton pumping into the thylakoid lumen,
driven by ATP hydrolysis, in order to maintain a sufficient
proton gradient for some processes (Kohzuma et al. 2012). In
plastids, three mechanically distinct systems, termed Tat,
Albino3, and Sec, are involved in translocation of proteins
into the thylakoid lumen (Gould 2008). Most importantly,
the Tat system depends on a proton gradient (Mori and
Cline 2001; Robinson et al. 2001; Jarvis and Robinson 2004),
and even nonphotosynthetic plastids need to discharge protons from the stromal compartment to the thylakoid lumen
to facilitate protein translocation. As mentioned above, we
identified a gene for Tat in the plastid genome of Nitzschia sp.
NIES-3581. Furthermore, in addition to tatC, we have detected
Tat-related nucleus-encoded genes for plastid Hcf106/tha4
and thylakoid processing peptidase with bipartite plastid-targeting signals (supplementary fig. S3 and table S3,
Supplementary Material online), suggesting that Nitzschia
sp. NIES3581 also has potential to import these components
known to be required for a functional Tat system into the
nonphotosynthetic plastids (fig. 2A and supplementary table
S3, Supplementary Material online; Jarvis and Robinson 2004).
Thus, we propose that following loss of photosynthesis, the
ATP synthase complex in the nonphotosynthetic diatom
plastids has functioned to hydrolyze ATP to maintain a
proton gradient between the thylakoid lumen and stroma,
required for the Tat-dependent protein translocation system
(fig. 2A). As this proposed function (fig. 2A) requires ATP, we
queried our transcriptome data and found transcripts encoding ATP/ADP translocators with N-terminal plastid-targeting
signals. These are homologous to plastid ATP/ADP transporters, NTT-1 and NTT-2, whose functions for ATP import into
plastids have been clarified with the model photosynthetic
diatom Phaeodactylum tricornatum (Ast et al. 2009; supplementary fig. S3 and table S3, Supplementary Material online),
and we suggest that they are involved in importing ATP from
2602
cytosol into the stroma (fig. 2A). This hypothesis straightforwardly explains why the ATP synthase genes are still found in
the nonphotosynthetic plastid genome of Nitzschia even after
the drastic loss of all the other genes for photosynthesis.
Nonphotosynthetic plastid genomes of several parasitic
plants and a unicellular alga have been also found to retain
ATP synthase genes (Donaher et al. 2009; Wicke et al. 2013). It
is notable that the plastid genome of the nonphotosynthetic
cryptophyte C. paramecium, phylogenetically distantly related
to diatoms, also carries genes for TatC, the core protein of the
Tat system, as well as genes for an ATP synthase complex
(Donaher et al. 2009; fig. 2B). Moreover, we found nucleusencoded plastid-type tatC homolog sequences in transcriptome data of Orobanche aegyptiaca, the nonphotosynthetic
broomrape species (e.g., ID: OrAe3GB1_13393; Parasitic Plant
Genome Project [http://ppgp.huck.psu.edu/blast.php, last
accessed May 13, 2015]; Westwood et al. 2012; fig. 2B).
Although, to the best of our knowledge, the plastid
genome sequence of O. aegyptiaca is not available, a nucleus-encoded sequence for the g subunit of plastid ATP
synthase is present in the transcriptome data (e.g., ID:
OrAe3GB1_8525; Parasitic Plant Genome Project [http://
ppgp.huck.psu.edu/blast.php, last accessed May 13, 2015];
Westwood et al. 2012; fig. 2B), strongly suggesting the presence of ATP synthase genes in the plastid genome of O.
aegyptiaca, as reported for its close relatives O. crenata and
O. gracilis (Wicke et al. 2013). Accordingly, we suggest that the
Tat system also functions (or has worked) in these nonphotosynthetic plastids, and could again be the main reason for
the retention of ATP synthase genes in these nonphotosynthetic plastid genomes. If so, this requirement for the maintenance of a proton gradient by ATP synthase genes has been
important in the early phase of plastid genome reduction in
these various independent nonphotosynthetic “algal”/plant
lineages including both parasitic and free-living species.
Materials and Methods
Detailed procedures for this manuscript were described in the
supplementary document, Supplementary Material online.
Supplementary Material
Supplementary document, figures S1 and S2, and tables
S1–S4 are available at Molecular Biology and Evolution
online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
The authors appreciate the useful comments of Professors
Michael W. Gray and Andrew J. Roger (Dalhousie
University). They also thank Dr Daniel Moog (Dalhousie
University) for his technical advice regarding in silico prediction of plastid localization. This work was supported by a
grant from the Institute for Fermentation, Osaka, Japan
(awarded to R.K.), and by grants from the Japanese Society
for Promotion of Sciences (JSPS; nos 15H05606 and 15K14591
awarded to R.K.). S.A.I., T.H., and Y.I. were supported by a
grant from JSPS (nos 24007, 23247038, and 23117006,
respectively).
ATP Synthase Genes in Nonphotosynthetic Plastid Genomes . doi:10.1093/molbev/msv134
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