Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis.
ABSTRACT The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.
Article: An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences.[show abstract] [hide abstract]
ABSTRACT: It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.Molecular Biology and Evolution 11/1995; 12(6):1166-73. · 5.55 Impact Factor
Article: Evidence for a common origin of chloroplasts with light-harvesting complexes of different pigmentation
Nature 09/1995; 376(6540):473-4. · 36.28 Impact Factor
Phylogeny of Galactolipid Synthase Homologs Together with their
Enzymatic Analyses Revealed a Possible Origin and Divergence Time
for Photosynthetic Membrane Biogenesis
YUICHI Yuzawa1, HIDENORI Nishihara1, TSUYOSHI Haraguchi1, SHINJI Masuda2, MIE Shimojima2,
ATSUSHI Shimoyama1, HIDEYA Yuasa1, NORIHIRO Okada1, and HIROYUKI Ohta2,*
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B-65 Nagatsuta-cho, Midori-
ku, Yokohama 226-8501, Japan1and Center for Biological Resources and Informatics, Tokyo Institute of Technology,
4259 B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan2
*To whom correspondence should be addressed. Tel. þ81 45-924-5736. Fax. þ81 45-924-5823.
Edited by Satoshi Tabata
(Received 26 September 2011; accepted 29 November 2011)
The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably
similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts
are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, al-
though MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic ana-
lysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi
have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses
for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the
MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant
MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis
and photosystems have been already functionally associated at least at the time of Chloroflexi divergence.
As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence
time of type A and B MGDs. Our analysis indicated that these genes diverged ∼323 million years ago,
when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photo-
synthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolution-
ary model that has increased our understanding of photosynthesis evolution.
Key words: monogalactosyldiacylglycerol; MGDG synthase; galactolipid; Roseiflexus castenholzii;
It is generally accepted that chloroplasts evolved
from a photosynthetic prokaryote that entered into
an endosymbiotic relationship with a non-photosyn-
thetic host cell.1It is also accepted that this photosyn-
thetic prokaryote was an ancestor of the phylum
Cyanobacteria.2It is plausible, therefore, that most
of the genes encoding chloroplast-localized proteins,
including components of the photosynthetic machin-
ery, are derived from this ancestral cyanobacterium.3
Cyanobacteria and chloroplasts of higher plants
share common features in many biological contexts,
including photosynthesis and cell division.4,5In add-
ition, the lipid compositions of biological membranes
high degrees of similarity between cyanobacteria
and chloroplasts.6Thylakoid membranes within a
in particular) show
#The Author 2011. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
DNA RESEARCH 19, 91–102, (2012)
Advance Access Publication on 30 December 2011
chloroplast are ?80% galactolipids, whereas the bio-
logical membranes of prokaryotic and eukaryotic
Monogalactosyldiacylglycerol (MGDG) is the most
abundant lipid in thylakoid membranes of both
cyanobacteria and higher plants. Thus, it is likely
that genes encoding enzymes of the galactolipid syn-
thetic pathways were acquired in higher plants
through the endosymbiotic event.
MGDG is essential for oxygenic phototrophs. Crystal
structure analyses of cyanobacteria have revealed that
MGDG binds to photosystem complexes, suggesting
that MGDG is an important component of the
photosynthetic machinery.7,8Furthermore, in MGDG-
deficient Arabidopsis thaliana, photosynthetic activity
is completely absent, and development is arrested at
the early seedling stage.9Thus, MGDG is involved in
both photosynthesis and chloroplast development.
The pathways for MGDG synthesis in cyanobacteria
and higher plants, however, are quite different.10
Higher plants utilize uridine diphosphate galactose
(UDP-Gal) and diacylglycerol (DAG) to synthesize
MGDG. In contrast, cyanobacteria must first synthe-
glucose (UDP-Glc) and DAG, which is subsequently
epimerized to produce MGDG. In addition, sequence
comparison indicates that MGDG synthase (MGD)
genes belong to the glycosyltransferase 28 (GT28)
family, whereas MGlcDG synthase (MGlcD) genes
belongto the family
Carbohydrate-Active Enzymes database (www.cazy.
org)].11–13In Viridiplantae (green algae and land
plants), therefore, the precise origin of the pathway
for MGDG synthesis is not clear.
As MGDG is essential for photosynthesis, under-
standing the evolution of this pathway could poten-
phototroph evolution in general. MGDG is found in
some anoxygenic phototrophs as well as oxygenic
phototrophs.14Chloroflexi is an anoxygenic photo-
trophic group of bacteria whose divergence preceded
that of Cyanobacteria.15–17The MGD homolog of
Chloroflexus aurantiacus, a member of the phylum
Chloroflexi,18belongs to the GT28 family and one of
the encoded proteins has an MGDG producing activ-
However, the phylogenetic
between these prokaryotic MGD homologs and
higher plant MGDs has not been well established.
Here, we performed comprehensive phylogenetic
analyses on MGD homologs from both prokaryotic
and eukaryotic organisms and revealed that eukaryot-
ic MGDs including Viridiplantae, Rhodophyta, and
have a single ancestor. Moreover, we found that
some of Chloroflexi MGD homologs are highly asso-
ciated with the eukaryotic MGDs. Analyses of the
sugar specificity and anomeric configuration of the
sugar head groups for three MGD homologs in
Chloroflexi, Roseiflexus castenholzii,20revealed that
only one of the MGD homologs in R. castenholzii exhi-
bits a genuine MGD activity, suggesting a possible
origin of higher plant MGDs.
To understand plastid evolution, it is also important
to characterize MGD gene duplication. In A. thaliana,
there are two types of MGD enzymes, type A
(MGD1) and type B (MGD2 and MGD3).21Type A
MGDs reside in membranes of the inner envelope
and, under normal circumstances, generate most
MGDGs found in the chloroplast. In contrast, type B
MGDs are found in membranes of the outer envelope
and are involved in membrane remodeling under
phosphate-starved conditions.22MGDG synthesized
in this context is converted into digalactosyl-DAG,
which is used to generate cell membranes when phos-
pholipids are not available. As type B MGDs are only
found in angiosperms, this type of membrane lipid re-
modeling is an important characteristic of angios-
interestingly, the expression of type B MGD are low
in leaves but high in floral organs.27Thus, it is possible
that the emergence of type B MGDs was an important
Spermatophyta (seed plant) evolution. Two types of
MGDs are essential for plants to survive under low-
phosphate conditions, and yet a detailed analysis of
MGD gene divergence has not been reported. In this
study, we present detailed data concerning MGD
gene divergence. Our data provide a new insight into
the plastid evolution based on the biogenesis of
2.Materials and methods
2.1. Phylogenetic analyses
To obtain MGD gene sequences, blastp and tblastn
searches were performed against the Protein (nr),
GenBank, and EST databases at the National Center
for Biotechnology Information website (www.ncbi.
nlm.nih.gov/), using Arabidopsis MGD amino acid
sequences as queries. In addition, a blastp search
was conducted against
Department of Energy Joint Genome Institute (DOE-
JGI) website (www.jgi.doe.gov/). The resulting data
are listed in Supplementary Table S1.
MGD amino acid sequences were aligned using
MACSIMS28and MUSCLE,29followed by a partial
manual correction. To refine these data for phylogen-
etic analysis, the 50- and 30-terminal regions of MGD
gene and regions where .15% of the species had a
sequence gap were eliminated from the alignment.
The refined data were then subjected to phylogenetic
92Evolution of Plastid Membrane Biogenesis [Vol. 19,
analyses with Bayesian inference and maximum likeli-
hood (ML). MrBayes ver 3.1.230was used for Bayesian
inference under the WAG þ G4model. Two runs with
four chains of Markov chain Monte Carlo (MCMC)
iterations were performed for 4 000 000 generations.
Trees were sampled every 100 generations, and the
first 10 000 trees (1 000 000 generations) were dis-
carded as burn-in. Treefinder ver. October 200831
was used for ML analyses under the WAG-F þ G8
model. Approximate bootstrap supports (LR-ELW;
expected likelihood weights by the local rearrange-
RAxML ver. 7.0.432was also used for ML analyses
under the WAG-F þ G4model with 1000 bootstrap
To calculate the divergence time of type A and type
B MGDs, the nucleotide sequences of land plant MGDs
(the first and second codon positions) were subjected
to Bayesian and ML phylogenetic analyses. Sequence
data are listed in Supplementary Table S2. MrBayes
ver 3.1.2 was used for Bayesian-tree inference with
1 000 000 generations under the GTR þ G8 model.
Trees were sampled every 100 generations, and the
first 2500 trees (250 000 generations) were dis-
carded as burn-in. The ML tree was estimated using
RAxML ver. 7.0.4 under the GTR þ G4 model with
1000 bootstrap replications. In both phylogenetic
analyses, different model parameters were estimated
between the first and the second codon position
data. Divergence times were estimated based on the
first and second codon sequences under a relaxed
clock model using the MCMCTREE program in the
PAML4 package.33The model parameters were separ-
ately estimated between the first and the second
codon sequences under the HKY þ G5 model. The
number of MCMC samples and burn-in were set to
10 000 and 2500, respectively. The following con-
straints were used for time calibrations (similar to
Yoon et al.34): (i) divergence of vascular plants from
other land plants ,432 million years ago (MYA)
(Kenrick and Crane35provided the origin of land
plants), (ii) divergence of seed plants from other
vascular plants .355 MYA (Gillespie et al.36provided
the oldest known seeds), (iii) 290–320 MYA for
the Angiospermae–Gymnospermae split, and (iv)
90–130MYA for the
with 10 000 replicates.
2.2.Expression of MGD homologs
The genes of three MGD homologs from R. casten-
holzii were amplified by polymerase chain reaction
and cloned into the pET28a vector, respectively.
Vectors were transformed into BL21 (DE3) competent
Escherichia coli. Transformed cells were grown in
Luria–Bertani medium at 378C until they reached
an OD600 reading of 0.7. Protein expression was
induced using 1 mM isopropyl b-D-thiogalactopyra-
noside for 3 h [we designated three genes as MGD
of R. castenholzii (RcMGD), MGlcD of R. castenholzii
(RcMGlcD), and diglucosyl-DAG (DGlcDG) synthase
of R. castenholzii (RcDGlcD) according to their func-
2.3. Assay for glycolipid synthesis
described by Awai et al.13Radiolabeled lipids were
separated by one-dimensional thin-layer chromatog-
raphy (TLC) in chloroform–hexane–isopropanol–
tetrahydrofuran–water (50:100:80:1:2, by volume)
(40:0.4:50:10, by volume) and detected by Image
Sunnyvale, CA, USA, or BAS2000, FUJIX).
activity wasassayed as
2.4.Isolation of glycolipids
Cultures of E. coli expressing RcMGD, RcMGlcD, or
RcDGlcD were centrifuged at 3500g for 10 min at
48C. Pellets were homogenized in 5 ml of 50 mM
TES-KOH buffer (pH 7.0) and 125 ml of 1 M MgCl2.
The enzyme solutions were sonicated and mixed
with 115 ml of 30 mM UDP-Gal or UDP-Glc. These
mixtures were incubated at 378C for 3 h and total
lipids were extracted as described previously.38Total
lipids were then purified by column chromatography
(InterSep SI, GL Sciences). Elution solvents included
chloroform, acetone–isopropanol (9:1, by volume),
and methanol. Glycolipids were collected in the
one-dimensional TLC as above or using hexane–
by volume). Finally, glycolipids were eluted using
chloroform–methanol (2:1, by volume).
Roseiflexus castenholzii provided from Prof. Keizo
Shimada was grown as described by Yamada et al.39
Roseiflexus total lipid was also extracted as described
above.38This lipid was developed by two-dimensional
TLC using chloroform–methanol–7 N ammonium
hydroxide (120:80:8, by volume) in the first dimen-
sion and chloroform–methanol–acetic acid–water
(170:20:15:3, by volume) in the second.
2.5.Acetylation of glycolipids
The glycolipids isolated by one-dimensional TLC
were evaporated and then eluted using 0.5 ml of pyri-
dine and 0.25 ml of acetic acid anhydride. These mix-
Subsequently, 5 ml of toluene was added to these
mixtures and evaporated. This step was repeated
three times. Finally, acetylated lipids were dissolved
No. 1]Y. Yuzawa et al. 93
in chloroform and used for nuclear magnetic reson-
2.6.Structural analysis of glycolipids by NMR
Acetylated lipids were dissolved in CDCl3. These
structures were analyzed by
(Bruker AV-600), 400 MHz (Varian 400-MR), or
270 MHz (JEOL JNM-EX-270) for protons. Internal
tetramethylsilane [s 0 parts per million (ppm)] in
CDCl3 was used as the standard. Chemical shifts
were expressed in ppm with reference to the standard.
The multiplicity of signals was abbreviated as follows:
s, singlet; d, doublet; dd, doublet of doublets; t, triplet;
and m, multiplet. The protons of the sugar head
groups were denoted as H-1, H-10, H-2, H-20, H-3,
H-30, H-4, H-40, H-5, H-50, H-6a, H-60a, H-6b, and H-
60b. The protons of the glycerol backbone were
denoted as H-100a, H-100b, H-200, H-300a, and H-300b. In
MGDG synthesized by RcMGD,
CDCl3) d 5.39–5.38 (d, 1H, J30,40 ¼ 3.3 Hz, H-4),
5.36–5.34 (m, 1.7H, fatty acid), 5.20–5.17 (m, 2H,
H-2 and H-200), 5.01 (dd, 1H, J20,30 ¼ 10.3 Hz, J30,40 ¼
3.3 Hz, H-3), 4.48 (d, 1H, J10,20 ¼ 7.9 Hz, H-1), 4.31
(dd,1H,J1a,1b¼ 12.0 Hz,
4.18–4.08 (m, 3.5H, H-6a, H-6b, and H-100b), 3.96
(dd, 1H, J3a,3b¼ 11.0 Hz, J2,3a¼ 5.0 Hz, H-300a), 3.90
(t, 1H, J50,60a¼ J50,60b¼ 6.7 Hz, H-5), and 3.77–3.66
(m, 1.7H, J3a,3b¼ 11.0 Hz, J2,3b¼ 5.7 Hz, H-300b). In
(600 MHz, CDCl3) d 5.35–5.34 (m, 1H, fatty acid),
5.20 (t, 1H, J20,30 ¼ J30,40 ¼ 9.5 Hz, H-3), 5.21–5.18
(m, 1H, H-200), 5.08 (t, 1H, J30,40 ¼ J40,50 ¼ 9.5 Hz, H-
5), 4.97 (dd, 1H, J10,20 ¼ 7.9 Hz, J20,30 ¼ 9.5 Hz, H-2),
4.52 (d, 1H, J10,20 ¼ 7.9 Hz, H-1), 4.30 (dd, 1H,
J60a,60b¼ 12.0 Hz, J50,60 ¼ 3.4 Hz, H-6a), 4.26 (dd, 1H,
J1a,1b¼ 12.4 Hz, J1a,2¼ 4.7 Hz, H-100a), 4.14–4.11
(m, 2H, H-6b and H-100b), 3.95 (dd, 1H, J3a,3b¼
10.9 Hz, J2,3a¼ 4.9 Hz, H-300a), and 3.69–3.66 (m,
3H, H-5, H-300b, and fatty acid). In DGlcDG synthe-
sized by RcDGlcD,
5.36–5.33 (m, 4H, fatty acid), 5.21–5.16 (m, 3H,
H-200, H-4, and H-40), 5.07 (t, 1H, J200,300 ¼ J300,400 ¼
10.0 Hz, H-30), 4.98 (dd, 1H, J10.20 ¼ 7.9 Hz, J20,30 ¼
9.5 Hz, H-2), 4.92 (dd, 1H, J100,200 ¼ 9.7 Hz, J200,300 ¼
7.9 Hz, H-20), 4.89 (t, 1H, J20,30 ¼ J30,40 ¼ 9.5 Hz, H-3),
4.57 (d, 1H, J10,20 ¼ 7.9 Hz, H-1), 4.47 (d, 1H, J100,200 ¼
7.9 Hz, H-10), 4.31–4.26 (m, 2H, H-100a and H-60a),
4.14–4.09 (m, 2H, H-100b and H-60b), 3.97 (dd, 1H,
J60a,60b¼ 10.8 Hz, J50,60 ¼ 5.1 Hz, H-6a), 3.87 (dd, 1H,
J3a,3b¼ 10.8 Hz, J2,3a¼ 2.0 Hz, H-300a), and 3.73–
lated from R. castenholzii,1H-NMR (270 MHz, CDCl3) d
5.38 (d, 1H, J30,40 ¼ 3.3 Hz, H-4), 5.20 (dd, 1H, J10,20 ¼
7.9 Hz, J20,30 ¼ 10.3 Hz, H-2), 5.01 (dd, 1H, J20,30 ¼
10.5 Hz, J30,40 ¼ 3.3 Hz, H-3), 5.00–4.93 (m, 1H,
1H-NMR at 600 MHz
1H-NMR (600 MHz,
J1a,2¼ 3.4 Hz,H-100a),
1H-NMR (400 MHz, CDCl3) d
H-200), 4.47 (d, 1H, J10,20 ¼ 7.9 Hz, H-1), 4.20–4.05
(m, 2H, H-6a and H-6b), 3.88 (dd, 2H, J ¼ 11.9,
6.0 Hz, H-5 and H-100a), 3.60 (dd, 1H, J1a,1b¼ 11.1 Hz,
J1b,2¼ 4.1 Hz, H-100b), and 3.15–3.10 (m, 1.7H, H-300a
3.Results and discussion
3.1. Evolutionary relationship of MGD homologs
To comprehensively understand the relationships
among MGD homologs, we compared amino acid
sequences of 56 MGD homologs from prokaryotes
and phototrophic eukaryotes. The homologs similarly
located were excluded from the phylogenetic tree. A
completelist of these
Supplementary Table S1. Our phylogenetic analyses
(heterokonts), which were clearly supported by both
(Treefinder and RAxML; node A in Fig. 1). Our data
provide compelling evidence that a single origin of
MGD in a common ancestor of these groups and the
pathway for MGDG synthesis is monophyletic among
Viridiplantae, Rhodophyta, and Heterokontophyta. As
formed a single group, suggesting that the common
ancestor of Heterokontophyta acquired MGDs (node
B in Fig. 1). This grouping was strongly supported
Rhodophyta and Heterokontophyta formed a single
group (node C in Fig. 1). Recent phylogenetic analyses
of plastid proteins have revealed an extremely close
relationship between heterokontophytes and rhodo-
ginated from endosymbiosis with Rhodophyta.40,41
MGDs of heterokonts were likely acquired from
Rhodophyta through this endosymbiotic event.
Gloeobacter violaceus is one of the most ancient
cyanobacterial lineages, and the Gloeobacter MGD
homolog is a likely candidate for the origin of
plastid MGDs. Our phylogenetic analysis of MGDs,
however, indicated that this gene formed a clade
with different MGD homologs in green non-sulfur
bacteria (C. aurantiacus and Chloroflexus aggregans42)
(node D in Fig. 1). Since chloroplasts may be derived
from ancient types of cyanobacteria, it was expected
that the eukaryote MGDs may be directly evolved
from a cyanobacterial MGD origin that might be
acquired by the primary endosymbiotic event, al-
though it might be lost in most cyanobacterial
species. However, here, the grouping between the
Gloeobacter and Chloroflexi MGD homologs was ro-
bustly supported by MrBayes, Treefinder, and RAxML
(node D in Fig. 1). This result strongly suggests that
94 Evolution of Plastid Membrane Biogenesis[Vol. 19,
the G. violaceus MGD homolog is not the origin of
MGDs in Viridiplantae and that horizontal gene trans-
fer may have occurred much more recently between
Gloeobacter and Chloroflexi.
Chloroflexi strains, Chloroflexus and Roseiflexus, were
located closest to the clade of chloroplasts (node E
in Fig. 1). Chloroflexus au. 1, an MGD of C. aurantiacus,
belongs to the GT28 family and the encoded protein
has an MGDG synthetic activity.19In addition, R. cas-
tenholzii has three MGD homologs that we have
named RcMGD, RcMGlcD, and RcDGlcD according
Figure 1. Phylogenetic relationship of MGD homologs. (A) An accepted model of chloroplast evolution.2, 22, 53Each red point indicates an
endosymbiotic event. (B) A phylogenetic tree of all MGD homologs from plastids and bacteria (based on amino acid sequences). To
obtain MGD sequences, blastp and tblastn searches were performed using Arabidopsis MGD amino acid sequences as queries. Above
each branch is shown: 1) the posterior probability estimated with Bayesian inference, 2) LR-ELW edge supports from Treefinder
analysis, and 3) the bootstrap probability from RAxML analysis (in that order). Viridiplantae, Rhodophyta, Heterokontophyta,
Chloroflexi, and Cyanobacteria are indicated by green, light magenta, yellow, light green, and light blue, respectively. MURG synthase
genes were used as the outgroup. The original phylogenetic trees from the 3 separate analyses (Bayesian, Treefinder, and RAxML)
are shown in supplementary figure 1–3. Arrowheads indicate (A) the acquisition of MGD in plastids, (B) the acquisition of MGD
homologs in Heterokontophyta, (C) the grouping of red algal plastids, (D) the horizontal gene transfer between Gloeobacter and
Chloroflexus, and (E) the close relationship between plastids and Chloroflexi.
No. 1] Y. Yuzawa et al.95
to their functions determined (see below). These
genes also belong to the GT28 family, and RcMGD
au. 1. Although MGD homologs from Chloroflexi and
chloroplasts were closely related phylogenetically, in-
formation regarding substrate specificity was only
available for Chloroflexus au. 1. We have therefore
examined in more detail the enzymatic properties of
Chloroflexi MGD homologs.
3.2.Glycolipid synthetic activities of R. castenholzii
To characterize the glycolipid synthetic activities of
the three R. castenholzii MGD homologs, we expressed
these recombinant proteins in E. coli. When radiola-
beled UDP-Gal was used as a substrate, RcMGD was
capable of synthesizing a radiolabeled lipid (Fig. 2).
We used UDP-Gal as a substrate because the proposed
pathway for MGDG synthesis of higher plants utilizes
(CsMGD) was used as a positive control for MGDG
synthesis in this experiment. In contrast, RcMGD
extract did not contain a radiolabeled product, when
UDP-Glc was provided as a substrate, suggesting that
lipid synthesis via RcMGD was UDP-Gal-dependent.
On the otherhand, the
RcMGlcD synthesized MGlcDG when UDP-Glc was
provided as a substrate. The MGlcD of Synechocystis
sp. PCC 6803 (Sll1377) was used as a positive
control for MGlcDG synthesis in this experiment.
Using TLC, lipids from two samples (RcMGD/UDP-
Gal and RcMGlcD/UDP-Glc) were purified and sub-
jected to1H-NMR analysis to reveal the sugar head
groups of these lipids (Fig. 3). In Figs 3A and B, the
doublet peak indicated by the black arrow at 4.5–
4.6 ppm indicates H1 of the hexose moiety of the
lipid. These peaks demonstrated that the sugar head
group was bound to a glycerol backbone via a b-
anomeric configuration at the C3 position, an
plants.14Consequently, the clade in Fig. 1, which
includes Chloroflexus and Roseiflexus, represents green
non-sulfur bacterial MGDs. The present results indi-
cate that MGD genes occur at least at the time of
Chloroflexi divergence (an ancient bacterial lineage).
Botte ´ et al.43has independently performed phylogen-
etic analysis of MGDs. Although they indicated similar
results for phylogeny of MGDs, they have not included
prokaryotic MGD homologs except for Chloroflexi in
Figure 2. Analysis of sugar transferase activity. Radiolabeled UDP-
Gal (þGal) or UDP-Glc (þGlc) were used as substrates. The
resulting lipids were separated by TLC (chloroform-hexane-
tetrahydrofuran-isopropanol-water, 50:100:1:80:2 by volume)
and visualizedby autoradiography.
(pET24b and pQE-32) were used as controls for MGDG and
Figure 3. Analysis of glycolipids by
synthesized by RcMGD and RcMGlcD was analyzed by1H-NMR
at 600 MHz. (A) MGDG synthesized by RcMGD expression and
(B) MGlcDG synthesized by RcMGlcD expression were isolated
from E. coli. H-1 doublet peaks (arrows) indicate that these
glycolipids formed b-anomeric configurations to the glycerol
1H-NMR. Each glycolipid
96Evolution of Plastid Membrane Biogenesis[Vol. 19,
their phylogenetic analysis, and thus, the phylogenetic
relationship between eukaryotic and prokaryotic
MGD homologs remains uncertain. Our detailed ana-
lyses suggest that this ancestral type of MGD genes is
the origin of higher plant MGDs.
RcDGlcD synthesized a glycolipid that consisted of a
number of sugars, using UDP-Glc as a substrate
(Fig. 2). The crude lipid extract from E. coli overexpres-
sing RcDGlcD contained much phospholipids derived
from E. coli. To remove phospholipids and concentrate
glycolipids, the extract was purified by silica column
chromatography. The separated fractions were further
developed by TLC and stained using the anthrone
reagent (Fig. 4A). The lipid mobility was associated
with DGlcDG.19 1H-NMR analysis identified this lipid
as DGlcDG. The analysis showed two doublet peaks
peaks revealed that this lipid contained two glucoses
and these glucoses were bound to each other via a b-
coses was also bound to a glycerol backbone via a b-
anomeric configuration. MGD gene homologs share a
high degree of sequence similarity, yet sugar-donor
specificity and the number of molecules involved in
the reaction are clearly diverse. Detailed comparisons
of MGD homolog sequences could potentially reveal
novel protein domains that determine sugar-donor
3.3.Lipid analysis of R. castenholzii
Subsequently, to define whether R. castenholzii pos-
sesses MGDG, MGlcDG, and DGlcDG or not, we ana-
lyzed the lipid composition of R. castenholzii. Total
lipid was extracted and developed by two-dimension-
al TLC, and three glycolipids were detected by the
anthrone reagent (Fig. 5A). We compared the mobil-
ity on two-dimensional TLC between A. thaliana and
R. castenholzii (Supplementary Fig. S4). The mobility
Arabidopsis MGDG. Using
that this glycolipid was a galactolipid and formed a
b-anomeric configuration to the glycerol backbone
(Fig. 5B). From these results, we concluded that this
glycolipid is MGDG. Although the peak pattern
derived from the glycerol backbone was slightly differ-
ent from MGDG synthesized by RcMGD in E. coli, it is
probably due to the difference in fatty acid compos-
ition of these MGDGs (Supplementary Table S3).
This result strongly supports that RcMGD actually
functions in vivo to synthesize MGDG.
As indicated above, we also detected two other gly-
colipids on two-dimensional TLC. One of them was
assumed to be sulfoquinovosyl-DAG (SQDG) from
the mobility on TLC. In fact, there is a homolog of
Arabidopsis SQDG synthase 1 in Roseiflexus and the
presence of SQDG in C. aurantiacus was reported in
a previous paper.44On the other hand, in another
one (UL4, unknown lipid 4, in Fig. 5A), the corre-
sponding lipid was not existent in A. thaliana. This gly-
colipid was likely DGlcDG. However,1H-NMR analysis
revealed that this lipid was not DGlcDG, although it
consisted of at least two sugar head groups (data
not shown). We could not detect MGlcDG in total
lipid fraction from Roseiflexus. In Cyanobacteria,
MGlcDG is a minor lipid, less than 1% of total
lipid.10Similarly, MGlcDG may not be abundant in
Roseiflexus. Therefore, these results suggest that
RcDGlcD mainly incorporates glucose to a major
monoglycolipid MGDG, not MGlcDG in vivo.
wasvery similar to
1H-NMR, we confirmed
Figure 4. Analysis of glycolipids synthesized by RcDGlcD. Glycolipids
synthesized by RcDGlcD were analyzed by TLC (A) and1H-NMR
at 400 MHz (B). (A) RcDGlcD was incubated with UDP-Glc and
DAG (þsub), or without substrate (2sub). Synthesized lipids
were purified by column chromatography. Fractions eluted
either with chloroform (Chl), acetone-isopropanol (9:1, by
volume, Ace-Iso), or methanol (Met) were developed using
volume). Glycolipids were detected by the anthrone reagent
(MGDG, DGDG, and the lipid indicated by an arrow). (B)1H-
NMR lipid analysis resulted in doublet H-1 peaks (arrows),
indicating b-anomeric configurations.
No. 1]Y. Yuzawa et al.97
To confirm the glucose transfer activity of RcDGlcD,
we used MGDG and MGlcDG as sugar acceptors and
compared the diglycolipid producing activity (Fig. 6).
Obviously, RcDGlcD could transfer glucose to MGDG,
although DGlcDG is still produced by it’s processive
activity. When MGlcDG was used as a substrate, the
band corresponding to DGlcDG was increased, con-
firming that RcDGlcD could also transfer glucose to
MGlcDG. However, UL4 was not DGlcDG and MGDG
is one of the most abundant lipids in Roseiflexus
presume that the main sugar acceptor of RcDGlcD in
vivo is MGDG and UL4 is glycosylgalactosyl-DAG.
However, since we only utilized UDP-Gal and UDP-
Glc as in vitro substrates (Fig. 2), its true substrate is
still uncertain, and further experiments are needed
to determine the structure of UL4.
Thylakoid membranes or membranous structures
of anoxygenic photosynthetic bacteria involve com-
plexes composed of proteins, pigments, and lipids. In
particular, these types of membranes are character-
ized by a unique and conserved lipid composition.
Genes related to the photosynthetic membrane bio-
genesis, therefore, have also played an important
role in phototroph evolution. Recently, we reported
that a distinct type of MGD belonging to the GT1
family was found in the green sulfur bacterium
Chlorobacterium tepidum and proved to be involved
in chlorosome biogenesis.45This work also demon-
strated that MGDG is commonly important in both
anoxygenic and oxygenic photosynthetic organisms,
Figure 6. Analysis of sugar transferase activity of RcDGlcD.
Radiolabeled UDP-Gal (þGal) or UDP-Glc (þGlc) were used as
substrates. Additionally, MGDG or MGlcDG eluted in EtOH
were added as acceptors. The resulting lipids were separated
40:0.4:50:10, by volume) and visualized by autoradiography.
Figure 5. Analysis of MGDG isolated from R. castenholzii. (A)
Description of 2-dimensional TLC in R. castenholzii. The TLC
analysis was performed with chloroform-methanol-
ammonium hydroxide (120:80:8, by volume) in the first
dimension and chloroform-methanol-acetic
(170:20:15:3, by volume) in the second. The lipids were
visualized by spraying with primulin reagent and viewing under
ultraviolet light. The glycolipids were visualized by anthrone
reagent and The phospholipids were visualized by Dittmer’s
syldiacylglycerol; PI, phosphatidylinositol; UL, unknown lipid.
(B) MGDG isolated from R. castenholzii was analyzed by
NMRat 270 MHz.The
configuration to the glycerol backbone.
98Evolution of Plastid Membrane Biogenesis[Vol. 19,
although green sulfur and non-sulfur bacteria have in-
dependently acquired different types of MGDG syn-
thetic machineries. Given the conservation of MGD
Chloroflexi, a functional association between the
photosystem and the membrane galactolipid MGDG
was established before endosymbiosis event, leading
to the establishment of highly organized photosyn-
Our results suggested that the photosynthetic eu-
karyote has acquired MGD gene from ancestral
Cyanobacteria hasa different
pathway. These pathways were subsequently con-
served during the course of evolution and have
played important roles in chloroplast biology. A
recent large-scale phylogenetic analysis also revealed
that a considerable amount of non-cyanobacterial
genomic material has contributed to the establish-
ment of the plastid before the split of red and green
algae.46Eukaryotic MGD genes may be acquired
through this kind of large-scale gene transfer from an-
presence ofMGDG in
3.4.The divergence point of type A and type B MGDs
Determining the time at which the two types of
MGDs (type A and type B) diverged is important to
understand the evolution of photosynthetic mem-
branes in land plants. Using both Bayesian inference
and ML methods, we performed phylogenetic ana-
lyses on MGD gene sequences of land plants, exclud-
ing third codon positions (Supplementary Figs S5
and S6). Results from these analyses generally
agreed with accepted phylogenetic relationships of
land plants. For example, Angiospermae were clearly
divided into Monocotyledoneae (monocots) and
Dicotyledoneae (dicots) in both types of MGDs. The
Bayesian inference and ML methods (Supplementary
Figs S5 and S6). These results indicate that MGDs
have been conserved throughout the course of land
plant evolution. Importantly, one full-length MGD
gene sequence of Picea glauca was available among
Gymnospermae, and both Bayesian and ML analyses
placed the Picea sequence close to the type B MGD
clade. Although a Gymnospermae type A MGD has
not yet been identified, this result suggests that the
type A/B divergence may have preceded that of
Angiospermae and Gymnospermae.
Based on the phylogenetic tree, we estimated an
MGD type A/B divergence time using four calibration
points (similar to Yoon et al.34). Our analysis indicated
that MGDs likely diverged ?323 MYA, with 298–
357 MYA representing the 95% confidence interval
(Fig. 7). This divergence time corresponds to the
support from both
propose, therefore, that the acquisition of two types
of MGDs occurred in the common ancestor of
Our data indicate that MGD gene duplication and
subsequent functional differentiation occurred in
Spermatophyta (Fig. 8). Jiao et al.47showed that a
whole-genome duplication (WGD) event occurred in
the common ancestor of all seed plants. This WGD
allowed for major adaptive changes in these organ-
isms and contributed to the dominance of seed
plants. Interestingly, type B MGD expression is ele-
plants.27During the Carboniferous period, spore-pro-
ducing plants were dominant,48whereas this domin-
ance shifted to seed plants shortly thereafter. The
Carboniferous and Permian periods were also charac-
terized by dramatic changes to the global climate, pri-
marily drying. Expression of type B MGDs is elevated
during phosphate deprivation, a response that is regu-
lated by both auxin and cytokinin.49These regulatory
mechanisms may have worked in response to these
climate changes. More extensive research concerning
the MGDs of Gymnospermae is required for a thor-
ough understanding of MGD gene evolution. It is
Figure 7. The divergence time of type A and type B MGDs. (A) A
Divergence time estimation among MGDs of land plants based
on the nucleotide dataset of the 1st and 2nd codon positions.
Horizontal bars (blue and orange) indicate 95% credible
intervals of the divergence time estimates. The 5 nodes used
as time constraints are indicated with blue bars. The original
ML and Bayesian trees used for the divergence time estimation
are shown in supplementary figure 4 and 5. Arrowhead
indicates the divergence of type A and type B MGDs. DE,
Devonian; CA, Carboniferous; PE, Permian; TR, Triassic; JU,
Jurassic; CR, Cretaceous; CE, Cenozoic.
No. 1]Y. Yuzawa et al. 99
important to determine whether there is a species of
Gymnospermae that has two types of MGDs. The evo-
lution of Gymnospermae, however, has not been ad-
equately mapped, in part because a complete
genome sequence is not available. Determining the
Gymnospermae may prove critical if we are to under-
stand the divergence of land plants.
A andB MGDs in
Based on phylogenetic and enzymatic analyses, we
proposed the evolutionary model of MGDs (Fig. 8).
Although no cyanobacteria analyzed possessed the
ancestral type of higher plant MGDs, we identified
the possible MGD ancestor from Chloroflexi. The
results suggest that higher plant MGD originated
from this type of Chloroflexi MGD. Eukaryotic MGD
may be acquired from ancient Chloroflexi via horizon-
tal gene transfer in parallel with the major endosym-
biotic event and contributed to the early plastid
evolution. After the acquirement, MGD duplication
and functional differentiation of two types of MGD,
types A and B, occurred along with land plant evolu-
tion. This functional differentiation probably had im-
portant meanings in further prosperity of seed
plants. It is universally accepted that an ancestral
cyanobacterium is the origin of the plastid organelle;
however, plastid evolution is still heavily debated.
These disagreements can be attributed to a very
complex process that involved both horizontal and
intracellular gene transfer during the course of pro-
karyotic and eukaryotic evolution.50,51To generate a
more accurate picture of photosynthesis evolution,
we believe that focused comprehensive analyses of
specific physiological processes are just as important
as more global gene sequence comparisons. In par-
ticular, galactolipids are highly enriched in photosyn-
thetic membranes, and deciphering the evolution of
galactolipid synthetic pathways provides critical infor-
mation toward understanding the early evolution of
Dr Robert P. Olinski for help in constructing an
aligned data set of MGDG synthases, and we also
thank Prof.Keizo Shimada
We would liketothank
for help withthe
Supplementary Data: Supplementary Data are
available at www.dnaresearch.oxfordjournals.org.
This work was supported by the Global Center of
Excellence Program, from the Earth to ‘Earths’, at the
Tokyo Institute of Technology and The University of
endosymbiosis and the origin of plastids, J. Phycol., 37,
2. Nelissen, B., Van de Peer, Y., Wilmotte, A. and De
Wachter, R. 1995, An early origin of plastids within
the cyanobacterial divergence is suggested by evolution-
ary trees based on complete 16S rRNA sequences, Mol.
Biol. Evol., 12, 1166–73.
3. Martin, W., Rujan, T., Richly, E., et al. 2002, Evolutionary
analysis of Arabidopsis, cyanobacterial, and chloroplast
genomes reveals plastid phylogeny and thousands of
cyanobacterial genes in the nucleus, Proc. Natl Acad.
Sci. USA, 99, 12246–51.
4. Wolfe, G.R., Cunningham, F.X., Durnford, D., Green, B.R.
and Gantt, E. 1994, Evidence for a common origin of
chloroplasts with light-harvesting complexes of different
pigmentation, Nature, 367, 566–8.
5. Osteryoung, K.W. and Vierling, E. 1995, Conserved cell
and organelle division, Nature, 376, 473–4.
6. Joyard, J., Marechal, E., Miege, C., Block, M.A., Dorne, A.J.
and Douce, R. 1998, Structure, distribution and biosyn-
thesis of glycerolipids from higher plant chloroplasts. In:
Photosynthesis: Structure, Function and Genetics, Kluwer
Academic Publishers: Dordrecht, pp. 21–52.
G.I. 2001, Primaryandsecondary
Murata, N.,eds.Lipid in
Figure 8. Proposed model for MGD evolution. The requirement of
MGDG in photosynthetic processes was established in ancient
times. ForMGDs, duplication
occurred in Spermatophyta. Black dashed arrows indicate
horizontal gene transfer. Large, shaded black arrows indicate
100Evolution of Plastid Membrane Biogenesis [Vol. 19,
7. Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W.
and Krauss, N. 2001, Three-dimensional structure of
Nature, 411, 909–17.
8. Loll, B., Kern, J., Saenger, W., Zouni, A. and Biesiadka, J.
2005, Towards complete cofactor arrangement in the
3.0 A˚ resolution structure of photosystem II, Nature,
9. Kobayashi, K., Kondo, M., Fukuda, H., Nishimura, M. and
Ohta, H. 2007, Galactolipid synthesis in chloroplast
inner envelope is essential for proper thylakoid biogen-
esis, photosynthesis, and embryogenesis, Proc. Natl
Acad. Sci. USA, 104, 17216–21.
10. Sato, N. and Murata, N. 1982, Lipid biosynthesis in the
blue-green alga, Anabaena variabilis I. Lipid classes,
Biochim. Biophys. Acta, 710, 271–8.
11. Shimojima, M., Ohta, H., Iwamatsu, A., Masuda, T.,
Shioi, Y. and Takamiya, K. 1997, Cloning of the
gene for monogalactosyldiacylglycerol synthase and its
evolutionary origin, Proc. Natl Acad. Sci. USA, 94,
12. Do ¨rmann, P. and Benning, C. 2002, Galactolipids rule in
seed plants, Trends Plant. Sci., 7, 112–8.
13. Awai, K., Kakimoto, T., Awai, C., et al. 2006, Comparative
genomic analysis revealed a gene for monoglucosyldia-
cylglycerol synthase, an enzyme for photosynthetic
membrane lipid synthesis in cyanobacteria, Plant
Physiol., 141, 1120–7.
14. Ho ¨lzl, G. and Do ¨rmann, P. 2007, Structure and function
of glycoglycerolipids in plants and bacteria, Prog. Lipid
Res., 46, 225–43.
15. Woese, C.R. 1987, Bacterial evolution, Microbiol. Rev.,
16. Gupta,R.S., Mukhtar,
aurantiacus, cyanobacteria, Chlorobium tepidum and
proteobacteria): implications regarding the origin of
photosynthesis, Mol. Microbiol., 32, 893–906.
17. Xiong, J., Fischer, W.M., Inoue, K., Nakahara, M. and
Bauer, C.E. 2000, Molecular evidence for the early evo-
lution of photosynthesis, Science, 289, 1724–30.
18. Pierson, B.K. and Castenholz, R.W. 1974, A phototrophic
Microbiol., 100, 5–24.
19. Ho ¨lzl, G., Za ¨hringer, U., Warnecke, D. and Heinz, E.
2005, Glycoengineering of cyanobacterial thylakoid
membranes for future studies on the role of glycolipids
in photosynthesis, Plant Cell Physiol., 46, 1766–78.
20. Hanada, S., Takaichi, S., Matsuura, K. and Nakamura, K.
2002, Roseiflexus castenholzii gen. nov., sp. nov., a
thermophilic, filamentous, photosynthetic bacterium
that lacks chlorosomes, Int. J. Syst. Evol. Microbiol., 52,
21. Awai, K., Mare ´chal, E., Block, M.A., et al. 2001, Two types
of MGDG synthase genes, found widely in both 16:3
and 18:3 plants, differentially mediate galactolipid syn-
theses in photosynthetic and nonphotosynthetic tissues
in Arabidopsis thaliana, Proc. Natl Acad. Sci. USA, 98,
Iat 2.5 A˚resolution,
of hot springs,
22. Kobayashi, K., Awai, K., Nakamura, M., Nagatani, A.,
Masuda, T. and Ohta, H. 2009, Type-B monogalactosyl-
diacylglycerol synthases are involved in phosphate star-
vation-induced lipid remodeling, and are crucial for
low-phosphate adaptation, Plant J., 57, 322–31.
23. Andersson, M.X., Stridh, M.H., Larsson, K.E., Liljenberg, C.
and Sandelius, A.S. 2003, Phosphate-deficient oat
replaces a major portion of the plasma membrane
phospholipids with the galactolipid digalactosyldiacyl-
glycerol, FEBS Lett., 537, 128–32.
24. Gaude, N., Tippmann, H., Flemetakis, E., Katinakis, P.,
Udvardi, M. and Do ¨rmann, P. 2004, The galactolipid
digalactosyldiacylglycerol accumulates in the peribac-
soybean and Lotus, J. Biol. Chem., 279, 34624–30.
25. Jouhet, J., Mare ´chal, E., Baldan, B., Bligny, R., Joyard, J. and
Block, M.A. 2004, Phosphate deprivation induces trans-
fer of DGDG galactolipid from chloroplast to mitochon-
dria, J. Cell Biol., 167, 863–74.
26. Russo, M.A., Quartacci, M.F., Izzo, R., Belligno, A. and
Navari-Izzo, F. 2007, Long- and short-term phosphate
deprivation in bean roots: plasma membrane lipid
alterations and transient stimulation of phospholipases,
Phytochemistry, 68, 1564–71.
27. Kobayashi, K., Awai, K., Takamiya, K. and Ohta, H. 2004,
Arabidopsis type B monogalactosyldiacylglycerol syn-
thase genes are expressed during pollen tube growth
and induced by phosphate starvation, Plant Physiol.,
28. Thompson, J.D., Muller, A., Waterhouse, A., et al. 2006,
MACSIMS: multiple alignment of complete sequences
information management system, BMC Bioinformatics,
29. Edgar, R.C. 2004, MUSCLE: multiple sequence align-
Nucleic. Acids Res., 32, 1792–7.
30. Ronquist, F. and Huelsenbeck, J.P. 2003, MrBayes 3:
Bayesian phylogenetic inference under mixed models,
Bioinformatics, 19, 1572–4.
31. Jobb, G., von Haeseler, A. and Strimmer, K. 2004,
TREEFINDER: a powerful graphical analysis environment
for molecular phylogenetics, BMC Evol. Biol., 4, 18.
32. Stamatakis, A. 2006, RAxML-VI-HPC: maximum likeli-
hood-based phylogenetic analyses with thousands of
taxa and mixed models, Bioinformatics, 22, 2688–90.
33. Yang, Z. 2007, PAML 4: phylogenetic analysis by
maximum likelihood, Mol. Biol. Evol., 24, 1586–91.
34. Yoon, H.S., Hackett, J.D., Ciniglia, C., Pinto, G. and
Bhattacharya, D. 2004, A molecular timeline for the
origin of photosynthetic eukaryotes, Mol. Biol. Evol.,
35. Kenrick, P. and Crane, P.R. 1997, The origin and early
evolution of plants on land, Nature, 389, 33–9.
36. Gillespie, W.H., Rothwell, G.W. and Scheckler, S.E. 1981,
The earliest seeds, Nature, 293, 462–4.
37. Crane, P.R., Friis, E.M. and Pedersen, K.R. 1995, The
origin and early diversification of angiosperms, Nature,
38. Bligh, E.G. and Dyer, W.J. 1959, A rapid method of total
lipid extraction and purification, Can. J. Biochem.
Physiol., 37, 911–7.
No. 1] Y. Yuzawa et al.101
39. Yamada, M., Zhang, H., Hanada, S., Nagashima, K.V.,
Shimada, K. and Matsuura, K. 2005, Structural and
spectroscopic properties of a reaction center complex
from the chlorosome-lacking filamentous anoxygenic-
J. Bacteriol., 187, 1702–9.
40. Yoon, H.S., Hackett, J.D., Pinto, G. and Bhattacharya, D.
2002, The single, ancient origin of chromist plastids,
Proc. Natl Acad. Sci. USA, 99, 15507–12.
41. Harper, J.T. and Keeling, P.J. 2003, Nucleus-encoded,
plastid-targeted glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) indicates a single origin for chromal-
veolate plastids, Mol. Biol. Evol., 20, 1730–5.
42. Hanada, S., Hiraishi, A., Shimada, K. and Matsuura, K.
1995, Chloroflexus aggregans sp. nov., a filamentous
phototrophic bacterium which forms dense cell aggre-
Bacteriol., 45, 676–81.
43. Botte ´, C.Y., Yamaryo-Botte ´, Y., Janouskovec, J., et al.
2011, Identification of
Chromera velia, a photosynthetic relative of malaria
parasites, J. Biol. Chem., 286, 29893–903.
44. Knudsen, E., Jantzen, E., Bryn, K., Ormerod, J. and
Sireva ˚g, R. 1982, Quantitative and structural character-
ization of lipids in Chlorobium and Chloroflexus, Arch.
Microbiol., 132, 149–54.
45. Masuda, S., Harada, J., Yokono, M., et al. 2011, A mono-
galactosyldiacylglycerol synthase found in the green
sulfur bacterium Chlorobaculum tepidum reveals import-
ant roles for galactolipids in photosynthesis, Plant Cell,
46. Suzuki, K. and Miyagishima, S.Y. 2010, Eukaryotic and
eubacterial contributions to the establishment of
plant-like galactolipids in
plastid proteome estimated by large-scale phylogenetic
analyses, Mol. Biol. Evol., 27, 581–90.
47. Jiao, Y., Wickett, N.J., Ayyampalayam, S., et al. 2011,
Ancestral polyploidy in seed plants and angiosperms,
Nature, 473, 97–100.
48. DiMichele, W.A., Pfefferkorn, H.W. and Gastaldom, R.A.
2001, Response oflate carboniferous and early
Permian plant communities to climate change, Annu.
Rev. Earth Planet Sci., 29, 461–87.
49. Kobayashi, K., Masuda, T., Takamiya, K. and Ohta, H.
2006, Membrane lipid alteration during phosphate
starvation is regulated by phosphate signaling and
auxin/cytokinin cross-talk, Plant J., 47, 238–48.
50. Raymond, J., Zhaxybayeva, O., Gogarten, J.P., Gerdes, S.Y.
and Blankenship, R.E. 2002, Whole-genome analysis
of photosynthetic prokaryotes, Science, 298, 1616–20.
51. Keeling, P.J. and Palmer, J.D. 2008, Horizontal gene
transfer in eukaryotic evolution, Nat. Rev. Genet., 9,
52. Turner, S., Pryer, K.M., Miao, V.P. and Palmer, J.D.
1999, Investigating deep phylogenetic relationships
among cyanobacteria and plastids by small subunit
rRNA sequence analysis, J. Eukaryot. Microbiol., 46,
53. Blankenship, R.E. 2010, Early evolution of photosyn-
thesis, Plant Physiol., 154, 434–8.
54. Pryer, K.M., Schneider, H., Smith, A.R., et al. 2001,
Horsetails and ferns are a monophyletic group and
the closest living relatives to seed plants, Nature, 409,
55. Palmer, J.D., Soltis, D.E. and Chase, M.W. 2004, The plant
tree of life: an overview and some points of view,
Am. J. Bot., 91, 1437–45.
102Evolution of Plastid Membrane Biogenesis[Vol. 19,