Opposite Chilarity of a-Carotene in Unusual Cyanobacteria
with Unique Chlorophylls, Acaryochloris and Prochlorococcus
Shinichi Takaichi1,*, Mari Mochimaru2, Hiroko Uchida3, Akio Murakami3, Euichi Hirose4,
Takashi Maoka5, Tohru Tsuchiya6 and Mamoru Mimuro6
Department of Biology, Nippon Medical School, Kawasaki, 211-0063 Japan
Department of Natural Sciences, Komazawa University, Setagaya, Tokyo, 154-8525 Japan
3
Kobe University for Research Center for Inland Seas, Awaji, 656-2401 Japan
4
Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan
5
Research Institute for Production Development, Kyoto, 606-0805 Japan
6
Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, 606-8501 Japan
*Corresponding author: E-mail, [email protected]; Fax, +44 733 3584.
(Received May 17, 2012; Accepted September 4, 2012)
2
Among all photosynthetic and non-photosynthetic prokaryotes, only cyanobacterial species belonging to the genera
Acaryochloris and Prochlorococcus have been reported to
synthesize a-carotene. We reviewed the carotenoids, including their chirality, in unusual cyanobacteria containing
diverse Chls. Predominantly Chl d-containing Acaryochloris
(two strains) and divinyl-Chl a and divinyl-Chl b-containing
Prochlorococcus (three strains) contained b-carotene
and zeaxanthin as well as a-carotene, whereas Chl b-containing Prochlorothrix (one strain) and Prochloron (three isolates) contained only b-carotene and zeaxanthin but no
a-carotene as in other cyanobacteria. Thus, the capability
to synthesize a-carotene seemed to have been acquired only
by Acaryochloris and Prochlorococcus. In addition, we unexpectedly found that a-carotene in both cyanobacteria had
the opposite chirality at C-60 : (60 S)-chirality in Acaryochloris
and normal (60 R)-chirality in Prochlorococcus, as reported in
some green algae and land plants. The results represent the
first evidence for the natural occurrence and biosynthesis of
(60 S)-a-carotene. All the zeaxanthins in these species were
of the usual (3R,30 R)-chirality. Therefore, based on the identification of the carotenoids and genome sequence data, we
propose a biosynthetic pathway for the carotenoids, particularly a-carotene, including the participating genes and
enzymes.
Keywords: Acaryochloris a-Carotene Carotenoid Chirality Prochlorococcus.
Abbreviations: CD, circular dichroism; DV, divinyl; MV,
monovinyl; NMR, nuclear magnetic resonance.
Introduction
More than 750 distinct carotenoids are distributed in nature
(Britton et al. 2004). They are produced by all photosynthetic
Regular Paper
1
organisms including plants, algae, cyanobacteria and anoxygenic photosynthetic bacteria, and by some non-photosynthetic bacteria, archaebacteria and fungi. Photosynthetic
organisms synthesize several specific molecular species of carotenoids, which serve as light-harvesting pigments (Mimuro
and Akimoto 2003, Akimoto and Mimuro 2005) and as photoprotective agents under excess light as scavengers of reactive
oxygen species or quenchers of singlet and triplet Chl (Bonente
et al. 2011, Jahns and Holzwarth 2012). All carotenoids of
photosynthetic organisms are derived from acyclic C40 lycopene, but only some purple bacteria synthesize carotenoids
from neurosporene, and heliobacteria synthesize C30 diaponeurosporene (Takaichi 2009). All oxygenic photosynthetic
organisms including cyanobacteria generally synthesize
b-carotene and/or its derivatives such as zeaxanthin, violaxanthin, neoxanthin, fucoxanthin and peridinin as major components. In contrast, a-carotene and its derivatives such as
e-carotene, lutein, loroxanthin and siphonaxanthin possessing
at least one e-end group are restricted to a few phyla of eukaryotic photosynthetic organisms: Rhodophyta, Cryptophyta,
Chlorarachniophyta, Chlorophyta and land plants (Britton et al.
2004, Takaichi 2011). Furthermore, only (60 R)-chirality has been
reported for a-carotene and its derivatives examined in eukaryotes (Britton et al. 2004).
Most ordinary cyanobacteria with Chl a and phycobiliproteins (allophycocyanin and phycocyanin, and phycoerythrin in
some cases) contain b-carotene and its derivatives such as
zeaxanthin, nostoxanthin and echinenone as major carotenoid
components, and also contain unique carotenoid glycosides
such as myxol fucoside and ketomyxol chinovoside (Takaichi
and Mochimaru 2007). However, unusual cyanobacterial species containing unique Chls, belonging to the two genera
Acaryochloris and Prochlorococcus, have been reported to accumulate a-carotene in abundance (Goericke and Repeta 1992,
Miyashita et al. 1997, Strickforth et al. 2003).
Plant Cell Physiol. 53(11): 1881–1888 (2012) doi:10.1093/pcp/pcs126, available online at www.pcp.oxfordjournals.org
! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 53(11): 1881–1888 (2012) doi:10.1093/pcp/pcs126 ! The Author 2012.
1881
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Acaryochloris marina was accidentally isolated from didemnid ascidians living in a tropical coral lagoon and reported as an
unusual Chl d-containing cyanobacterium (Miyashita et al.
1997). Chl d accumulated in them as the major Chl pigment
(92–97% of total Chls) along with Chl a in small amounts. The
major carotenoids were a-carotene and zeaxanthin, but the
presence of b-carotene was not described (Miyashita et al.
1997). Another strain of Chl d-containing cyanobacterium,
Acaryochloris sp. strain Awaji, was found as an epiphytic microorganism on a macrophytic red alga in temperate water on the
Japanese coast (Murakami et al. 2004). Its Chl composition was
very similar to that of A. marina. Acaryochloris marina is the
only species taxonomically described in this genus.
Prochlorococcus marinus is distributed in the oligotrophic
open ocean in tropical and subtropical regions at depths of
50–200 m as a major primary producer (Goeriche and Repeta
1992). Prochlorococcus exceptionally contains divinyl (DV)-type
Chls (DV-Chl a and DV-Chl b) instead of the monovinyl (MV)type Chls commonly found in almost all cyanobacteria, algae
and land plants. The DV-Chl b/DV-Chl a ratios in Prochlorococcus vary considerably, depending on the strains and light conditions (Mimuro et al. 2011). Prochlorococcus marinus is
the only species taxonomically described in this genus, and
>30 strains have been isolated to date. In all strains, most
components (genes) of phycobiliproteins have been lost,
except for subunits of phycoerythrin (Hess et al. 1996,
Wiethaus et al. 2010).
Prochlorothrix and Prochloron commonly contain Chl b as a
second minor Chl and have completely lost phycobiliproteins
(Herbstová et al. 2010, Donia et al. 2011). Prochlorothrix is free
living and planktonic in fresh and brackish water of temperate
regions. Prochloron is obligatorily symbiotic in didemnid ascidians inhabiting tropical and subtropical coral reefs, and has not
been cultured to date. Prochlorothrix hollandica and Prochloron
didemni are the only species taxonomically described in their
genera.
These four genera of unusual cyanobacteria with unique
Chls, Acaryochloris, Prochlorococcus, Prochloron and Prochlorothrix, are evolved independently of each other, as indicated by
molecular phylogenetic analyses (Criscuolol and Gribaldo 2011,
Schirrmeister et al. 2011). In this work, we reviewed the identification of the carotenoids and their chirality in these species.
a-Carotene of Acaryochloris showed the unprecedented (60 S)chirality, whereas Prochlorococcus showed the usual (60 R)-chirality reported for other oxygenic photosynthetic organisms. We
also propose a biosynthetic pathway for the carotenoids including the corresponding genes and enzymes.
Results
Pigment composition in Chl d-containing
Acaryochloris marina
Fig. 1 shows an elution profile for the organic solvent-soluble
pigments extracted from A. marina strain MBIC 11017
1882
Fig. 1 HPLC elution profile of pigments extracted from Acaryochloris
marina strain MBIC 11017 using the mBondapak C18 column.
Absorbance at 440 nm is shown.
separated by HPLC equipped with a mBondapak C18 column
with the eluent methanol. Based on their absorption spectra,
the pigments in peaks 2 and 3 at approximately 5.5 and 7.3 min
were identified as Chls d and a, respectively, whereas the peaks
at 2–3 min were neither Chls nor carotenoids.
Peak 4 at approximately 13.8 min (Fig. 1) was further separated on a Novapak C18 column with the eluent acetonitrile/
methanol/tetrahydrofuran (58 : 35 : 7, by vol.), and two carotene peaks were detected (Supplementary Fig. S1). The
major carotene, with absorption maxima at 269, 338, 427
(shoulder), 449 and 476 nm, was eluted earlier, and the spectral
fine structure showed a %III/II of 58 (Supplementary Fig. S2,
solid line); this is the ratio of the peak heights of the longest and
the middle wavelength absorption bands from the trough between the two peaks (Takaichi and Shimada 1992). The maxima
of the minor carotene were 277, 349, 432 (shoulder), 455 and
480 nm, with a %III/II of 23 (Supplementary Fig. S2, dashed
line). These absorption spectra were compatible with those of
a-carotene and b-carotene, respectively (Takaichi 2000). After
purification, both had the same relative molecular mass of 536.
The proton nuclear magnetic resonance (1H-NMR) spectrum of
the major carotene (Supplementary Table S1) was compatible
with that of a-carotene (Englert 1995). The circular dichroism
(CD) spectrum of the major carotene (Fig. 2, solid line) in diethyl ether/2-pentane/ethanol (5 : 5 : 2, by vol.) was the mirror
image of that of standard (60 R)-a-carotene (Buchecker and
Noack 1995). Accordingly, the major carotene was identified
as the enantiomeric (60 S)-a-carotene, and the minor one as
b-carotene, whose IUPAC-IUBMB semi-systematic names are
(60 S)-b,e-carotene and b,b-carotene, respectively (Fig. 3). Note
that both the absorption and NMR spectra were identical
for (60 S)-a- and (60 R)-a-carotene (Supplementary Fig. S2,
Table S1).
In the HPLC eluent methanol, the absorption maxima of
xanthophyll in peak 1 at approximately 4.4 min (Fig. 1) were
276, 429 (shoulder), 450 and 478 nm, and the %III/II was 33,
Plant Cell Physiol. 53(11): 1881–1888 (2012) doi:10.1093/pcp/pcs126 ! The Author 2012.
a-Carotene in Acaryochloris and Prochlorococcus
absorption and CD spectra of a-carotene were compatible
with those of (60 S)-a-carotene from A. marina (Supplementary
Fig. S2, solid line; Fig. 2, solid line, respectively). It had the
same relative molecular mass of 536. The major carotenoids
were (60 S)-a-carotene and zeaxanthin. The composition was
almost the same as that of A. marina (Table 1).
Pigment composition of the DV-Chl a- and
DV-Chl b-containing Prochlorococcus marinus
Fig. 2 The CD spectra of (60 S)-a-carotene from Acaryochloris marina
strain MBIC 11017 (solid line) and (60 R)-a-carotene from Prochlorococcus marinus strain CCMP 1986 (dashed line) in diethyl ether/
2-pentane/ethanol (5 : 5 : 2, by vol.).
Fig. 3 Structures of (60 S)-a-carotene and (60 R)-a-carotene.
values compatible with those of zeaxanthin (Takaichi and
Shimada 1992). It had a relative molecular mass of 568, and
its CD spectrum was compatible with that of the standard
(3R,30 R)-zeaxanthin (Buchecker and Noack 1995, Iwai et al.
2008). The carotenoid was accordingly identified as (3R,30 R)zeaxanthin, (3R,30 R)-b,b-carotene-3,30 -diol.
In enriched fractions from purification of the major carotenoids, b-cryptoxanthin (elution at approximately 7.7 min,
absorption spectra compatible with zeaxanthin and relative
molecular mass of 552), and zeinoxanthin (elution at approximately 7.6 min, and absorption spectra compatible with
a-carotene) were found in HPLC analyses with the
mBondapak C18 column. The carotenoid composition (mol%
of total carotenoids) was (60 S)-a-carotene (53%), zeinoxanthin
(<1%), b-carotene (3%), b-cryptoxanthin (1%) and (3R,30 R)zeaxanthin (43%) (Table 1). The relative compositions were
somewhat variable; in other batches, the composition of
b-carotene was approximately 5%, but a-carotene and zeaxanthin were still major components.
The HPLC elution profiles with the mBondapak C18 column
for the pigments extracted from Acaryochloris sp. strain Awaji
(Supplementary Fig. S3A) (Murakami et al. 2004) were comparable with those from A. marina (Fig. 1). Its major pigments
were Chls a and d, and a-carotene and zeaxanthin. The
The HPLC elution profiles with the mBondapak C18 column for
the pigments extracted from P. marinus strain CCMP 1986
showed several pigment peaks (Supplementary Fig. S3B).
DV-Chls a and b were identified based on their absorption
spectra (Chisholm et al. 1992).
The carotene peak was further analyzed with the Novapak
C18 column, resulting in two peaks. The absorption maxima of
the major pigment were 269, 336, 426 (shoulder), 448 and
476 nm, and the %III/II was 53. Major and minor carotenes
were similarly identified as a-carotene and b-carotene based
on compatible absorption spectra and specific retention times
on HPLC as compared with those from A. marina (Supplementary Fig. S2, solid and dashed lines, respectively). The major
carotene had a relative molecular mass of 536, and its CD spectrum was compatible with that of normal (60 R)-a-carotene,
(60 R)-b,e-carotene (Fig. 2, dashed line; Fig. 3) (Buchecker and
Noack 1995).
The xanthophyll peak had an absorption spectrum compatible with that of zeaxanthin from A. marina. It had the relative
molecular mass of 568, and its CD spectrum was compatible
with that of (3R,30 R)-zeaxanthin from A. marina. Carotenoids
were (60 R)-a-carotene (8%), b-carotene (<1%) and (3R,30 R)zeaxanthin (92%) (Table 1).
The HPLC elution profiles with the mBondapak C18 column
for the pigments extracted from P. marinus strains CCMP 1375T
and CCMP 2773 were compatible with those from P. marinus
strain CCMP 1986 (Supplementary Fig. S3B). Their major pigments were DV-Chls a and b, and a-carotene and zeaxanthin.
a-Carotene from both strains gave absorption and CD spectra
compatible with those of (60 R)-a-carotene from P. marinus
strain CCMP 1986 (Fig. 2, dashed line), and had the same relative molecular mass of 536. The carotenoid composition in
strain CCMP 1375T was (60 R)-a-carotene (22%) and zeaxanthin
(78%), and that in strain CCMP 2773 was (6R,60 R)-e-carotene
(1%), (60 R)-a-carotene (49%) and (3R,30 R)-zeaxanthin (50%)
(Table 1).
Pigment composition in the MV-Chl a- and
MV-Chl b-containing cyanobacteria
The pigments in Prochlorothrix hollandica strain PCC 9006T and
Prochloron sp. isolated separately from ascidian colonies (Trididemnum cyclops, Trididemnum miniatum and Lissoclinum
timorense) were separated by HPLC on the mBondapak C18
column, and the carotenes of these species were further separated on the Novapak C18 column. Only b-carotene was detected in the carotene fractions, and a-carotene was absent.
Plant Cell Physiol. 53(11): 1881–1888 (2012) doi:10.1093/pcp/pcs126 ! The Author 2012.
1883
S. Takaichi et al.
Table 1 Carotenoid compositions (mol% of total carotenoids) in Acaryochloris, Prochlorococcus and related species
Carotenoid
a-Carotene
e-Carotene
Zeinoxanthin
b-Carotene
b-Cryptoxanthin
Zeaxanthin
a
b
c
d
e
Chl type
Chl d and minor Chl a
DV-Chl a and DV-Chl b
Acaryochloris
marina strain
MBIC 11017
Prochlorococcus
marinus strain
CCMP 1986
53b
Acaryochloris
sp. strain
Awaji
38b
8c
22c
Chl a and minor Chl b
Prochlorococcus
marinus strain
CCMP 2773
Prochlorothrix
hollandica strain
PCC 9006T
Prochloron
sp.a
49c
0
0
d
0
0
0
0
0
<1
<1
<1
<1
<1
0
0
3
1
<1
<1
<1
32
1
0
42–51
1
0
0
0
0
0
2–4
43e
61
92e
78
50e
68
45–56
Cells were isolated separately from the three species of didemnid ascidians.
(60 S)-a-carotene.
(60 R)-a-carotene.
(6R,60 R)-e-carotene.
(3R,30 R)-zeaxanthin.
The carotenoid composition in P. hollandica was b-carotene
(32%) and zeaxanthin (68%), and that in Prochloron sp. isolated
from three different host ascidians was b-carotene (42–51%),
b-cryptoxanthin (2–4%) and zeaxanthin (45–56%) (Table 1).
Discussion
The major pigments of A. marina strain MBIC 11017 have been
reported to be Chls a and d, and a-carotene and zeaxanthin
(Miyashita et al. 1997). Those of P. marinus strain CCMP 1375T
have been reported to be DV-Chls a and b, and a-carotene and
zeaxanthin (Goericke and Repeta 1992). In addition to these
major pigments, we detected b-carotene, b-cryptoxanthin and
zeinoxanthin in Acaryochloris (two strains), and b-carotene,
e-carotene and zeinoxanthin in Prochlorococcus (three strains)
(Table 1, Fig. 4). These minor components were disregarded
in the previous reports, but they are essential information to
understand the carotenogenesis pathway in unusual
cyanobacteria.
We analyzed the chirality of a-carotene and zeaxanthin, and
found A. marina strain MBIC 11017 and Acaryochloris sp. strain
Awaji to contain the unusual chirality of (60 S)-a-carotene,
whereas P. marinus strains CCMP 1986, CCMP 1375T and
CCMP 2773 had the normal (60 R)-chirality (Fig. 3) reported
for green algae and land plants. They also had the usual
(3R,30 R)-zeaxanthin. Among all photosynthetic and nonphotosynthetic prokaryotes, a-carotene and its derivatives are
found in only two genera of cyanobacteria (Britton et al. 2004,
Takaichi and Mochimaru 2007). Furthermore, this is the first
finding of the natural occurrence of (60 S)-a-carotene (Britton
et al. 2004).
In A. marina strain MBIC 11017, the identification of the
carotenoids in this study and the acquisition of the full genome
sequence (Swingley et al. 2008) have facilitated the elucidation
of the biosynthetic pathways of carotenoids (Fig. 4) and the
corresponding enzymes and genes (Table 2). Some carotenogenesis genes have been suggested by nucleotide sequence
1884
Prochlorococcus
marinus strain
CCMP 1375T
homologies (Swingley et al. 2008). Geranylgeranyl diphosphate
synthase (CrtE) and phytoene synthase (CrtB) produce phytoene, and then phytoene desaturase (CrtP), z-carotene desaturase (CrtQ) and cis-carotene isomerase (CrtH) produce
lycopene as in other cyanobacteria (Takaichi and Mochimaru
2007). Lycopene is cyclized to b-carotene through g-carotene
by lycopene b-cyclase, whose functional genes have not yet
been revealed, although candidates were suggested (Swingley
et al. 2008). Then b-carotene is hydroxylated to zeaxanthin
through b-cryptoxanthin by b-carotene hydroxylase (CrtR).
For the biosynthesis of a-carotene, one end group of lycopene
is cyclized by lycopene e-cyclase (CrtL-e) to d-carotene, and
then another end group is cyclized by lycopene b-cyclase
(CrtL-b) to a-carotene. The b-end group of a-carotene can
be hydroxylated by CrtR to form zeinoxanthin. Since CrtL-b
(PMM1064) and CrtL-e (PMM0633) of P. marinus strain
CCMP 1986 have similarity as described below, AM1_6075 of
A. marina strain MBIC 11017 has homologous to both lycopene
cyclases (Table 2). We are investigating the functions of CrtL-b
and CrtL-e including this gene.
In P. marinus strain MED4 (CCMP 1986), two homologous
lycopene cyclases, CrtL-b and CrtL-e, have been functionally
confirmed (Table 2) (Stickforth et al. 2003). CrtL-b
(PMM1064) has only lycopene b-cyclase activity to form
b-carotene from lycopene, and has sequence similarity to
CrtL of Synechococcus elongatus strain PCC 7942 (Cunningham
et al. 1994). On the other hand, CrtL-e (PMM0633) is a
bifunctional enzyme, lycopene b-cyclase and lycopene
e-cyclase, and lycopene can be converted to b-carotene,
a-carotene, e-carotene and d-carotene in Escherichia coli,
which produces lycopene (Stickforth et al. 2003). We also reconfirmed that PMM0633 from P. marinus strain CCMP 1986
was used to obtain b-carotene, a-carotene, g-carotene,
e-carotene and d-carotene from lycopene in E. coli. Since the
similarity between the two lycopene cyclases of P. marinus
strain CCMP 1986 is 47%, crtL-e presumably arose by duplication of crtL-b.
Plant Cell Physiol. 53(11): 1881–1888 (2012) doi:10.1093/pcp/pcs126 ! The Author 2012.
a-Carotene in Acaryochloris and Prochlorococcus
Fig. 4 Proposed biosynthetic pathways of carotenoids and their enzymes in Acaryochloris. In the case of Prochlorococcus, (60 R)-a-carotene is the
alternative product of (60 S)-a-carotene.
Table 2 Presumed enzymes and the open reading frame (ORF) number of Acaryochloris marina and the three strains of Prochlorococcus
marinus used
Gene
Enzyme
Query sequence for BLASTPa
ORF number and identity (%)/e-value
Acaryochloris
marina MBIC
11017
Prochlorococcus
marinus CCMP
1986
Prochlorococcus
marinus CCMP
1375T
Prochlorococcus
marinus CCMP
2773
PMM1070, 57/e-88
Pro1129, 59/3e-92
PMT1109, 63/2e-98
PMT2003, 57/2e-97
crtE
Geranylgeranyl
diphosphate synthase
Thermosynechococcus elongatus CrtEb
AM1_4854, 74/e-124
crtB
Phytoene synthase
Synechocystis sp. PCC 6803 CrtBc
AM1_4825, 66/e-119
PMM0143, 54/2e-94
Pro0166, 59/2e-99
crtP
Phytoene desaturase
Synechocystis sp. PCC 6803 CrtPd
AM1_4826, 70/0.0
PMM0144, 71/0.0
Pro0167, 71/0.0
PMT2004, 72/0.0
crtQ
z-Carotene desaturase
Synechocystis sp. PCC 6803 CrtQe
AM1_3692, 73/0.0
PMM0115, 60/e-165
Pro0136, 64/e-179
PMT1968, 64/e-174
crtH
cis-Carotene isomerase
Synechocystis sp. PCC 6803 CrtHf
AM1_1491, 72/0.0
PMM1155, 60/0.0
Pro0584, 65/0.0
PMT1051, 66/0.0
crtL-b
Lycopene b-cyclase
Prochlorococcus marinus
CCMP 1986 CrtL-bg
AM1_6075, 45/e-97
PMM1064j, query
Pro1136, 56/e-130
PMT1123, 53/e-125
crtL-e
Lycopene e-cyclase
Prochlorococcus marinus
CCMP 1986 CrtL-eh
AM1_6075, 47/e-107
PMM0633j, query
Pro0790, 59/e-149
PMT1773, 60/e-149
crtR
b-Carotene hydroxylase
Synechocystis sp. PCC 6803 CrtRi
AM1_3637, 64/e-115
PMM0236k, 53/6e-89
Pro0266, 55/e-100
PMT1816, 57/e-100
Database searches were carrid out with the BLASTP.
a
Enzymes, whose functions have already been confirmed, were chosen for query sequences.
Accesssion numbers are as follows: b tll0020 (Cyanobase) (Ohto et al. 1999); c slr1255 (Cyanobase) (Martı́nez-Férez et al. 1994); d slr1254 (Cyanobase) (Martı́nez-Férez
and Vioque 1992); e slr0940 (Cyanobase) (Breitenbach et al. 1998); f sll0033 (Cyanobase) (Masamoto et al. 2001); g PMM1064 and h PMM0633 (Stickforth et al. 2003)
and i sll1468 (Cyanobase) (Masamoto et al. 1998, Lagarde and Vermass 1999).
j
Functions of these genes have been confirmed (Stickforth et al. 2003).
k
‘crtR pseudo’ NC_005072 (NCBI).
The three strains of P. marinus examined have homologous
carotenogenesis genes (Table 2): crtE, crtB, crtP, crtQ, crtH,
crtL-b and crtL-e. The two strains CCMP 1375T and CCMP
2773 also carry the homologous gene crtR. Strain CCMP 1986
also contained zeaxanthin in this study, and accordingly is expected to carry CrtR. Klassen (2010) has reported that crtR is
homologous to sequence PMM0236 in strain CCMP 1986,
whereas it is indicated as ‘crtR pseudo’ in the NCBI database
(NC_005072). Indeed, PMM0236 has high homology, 75% and
66%, to Pro0266 and PMT1816, respectively (see Table 2).
The opposite (60 S)-chirality of a-carotene and its derivatives
has to date not been known in nature (Britton et al. 2004).
Enzymatic reactions to produce the e-end group from
the c-end group of lycopene cannot dictate the (60 R)- or
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1885
S. Takaichi et al.
(60 S)-chirality, since only the direction of H+ elimination can
differ (Britton et al. 1977). For the e-end group formation of
a-carotene, H+ attaches to C-20 of the c-end group of lycopene,
and a single bond between C-10 and C-60 is formed as intermediate (Britton et al. 1977), which has hydrogen at C-60 and,
therefore, (60 R)- and (60 S)-chirality are possibly present. Then,
H+ is eliminated from C-40 to produce a double bond between
C-40 and C-50 , and (60 R)- and (60 S)-e-end groups are formed
(see Fig. 3). Consequently, enzymatic reactions to produce the
e-end group from the c-end group cannot dictate the (60 R)- or
(60 S)-chirality. Note that when H+ is eliminated from C-6, the
b-end group is formed. However, the binding site(s) of carotenoid(s) on pigment–protein complexes may restrict one
enantiomer by steric hindrance. Identification of lycopene
e-cyclase and further investigations of enzymatic mechanisms
are needed, and isolation of the pigment–protein complexes
and investigations of their binding sites are also required.
Two examples of opposite chirality in carotenoids have been
reported (Britton et al. 2004). Standard (3S,30 S)-astaxanthin is
synthesized in the alphaproteobacteria Paracoccus and
Brevundimonas, and the green alga Haematococcus by a combination of two enzymes, b-carotene hydroxylase (CrtZ/CrtR)
and b-carotene ketolase (CrtW/BKT) (Misawa 2011). In contrast, a red yeast, Xanthophyllomyces, produces (3R,30 R)-astaxanthin using one bifunctional enzyme, astaxanthin synthase
(ASY/CrtS), which has both b-carotene hydroxylase and
b-carotene ketolase activities (Ojima et al. 2006). Concerning
the 20 -hydroxy-g-carotene derivative of 30 ,40 -didehydro-10 ,20 dihydro-b,c-carotene-10 ,20 -diol, the (20 R)-chirality found in
the fungi Plectania and Cryptococcus is named plectaniaxanthin, whereas the (20 S)-chirality found in Gram-positive
bacteria Mycobacterium and Gordonia (Takaichi et al. 2008) is
named phleixanthophyll (Britton et al. 2004). Myxol and myxol
glycosides, which have an additional 3-hydroxyl group, found in
some cyanobacteria (Takaichi and Mochimaru 2007) and a
flavobacterium Robiginitalea (Shindo et al. 2007) display (20 S)chirality (Rønneberg et al. 1985, Britton et al. 2004). Their synthetic enzymes are unknown at present.
We reconfirmed the presence of a-carotene in Acaryochloris
and Prochlorococcus, and unusual Chl d and DV-Chls a and b,
respectively, are also present only in these genera (Chisholm
et al. 1992, Miyashita et al. 1997, Murakami et al. 2004).
Whether the production of a-carotene is related to the occurrence of the unusual Chls is unknown. A further search for
a-carotene among the cyanobacteria is needed. The major pigments of P. hollandica, in contrast to these two genera, have
been reported to be MV-Chls a and b, and b-carotene and
zeaxanthin (Burger-Wiersma et al. 1986), and those of
Prochloron sp. from ascidian colonies are also MV-Chls a and
b, and b-carotene and zeaxanthin (Foss et al. 1987). We carefully reviewed that the MV-Chl b-containing cyanobacteria of
Prochlorothrix and Prochloron did not contain a-carotene.
Among eukaryotic phototrophs, MV-Chl b is present in the
land plants, Charophyceae, Prasinohyceae, Chlorophyceae,
Ulvophyceae, Euglenophyceae and Chlorarachniophyceae, and
1886
these organisms contain normal a-carotene and/or its derivatives. On the other hand, Cryptophyceae (MV-Chl a and Chl c)
and the macrophytic type of Rhodophyceae (MV-Chl a) contained a-carotene and/or its derivatives, but not Chl b (Takaichi
2011). Thus, the distributions of a-carotene and its derivatives,
and MV- and DV-Chl b are different between cyanobacteria and
eukaryotic phototrophs. This observation is an interesting
point in the evolution and adaptation of photosynthetic
pigments.
Materials and Methods
Cyanobacterial strains and culture conditions
Acaryochloris marina strain MBIC 11017 (NBRC 102967) was
grown at 25 C under light (10 mmol photons m–2 s–1) conditions, as described previously (Tsuchiya et al. 2012). Acaryochloris sp. stain Awaji (Murakami et al. 2004) was grown
at 20 C under white fluorescent light (20 mmol photons
m–2 s–1) using ASN III medium as previously reported (Akimoto
et al. 2006).
Prochlorococcus marinus strains CCMP 1375T (SS120T),
CCMP 1986 (MED4) and CCMP 2773 (MIT 9313) were cultured
autotrophically in Pro99 medium as reported (Mimuro et al.
2011). The light intensity of the fluorescent light was adjusted
depending on the strains: 40 mmol photons m2 s1 were
used for the high-light-adapted strain CCMP 1986, but only
4 mmol photons m2 s1 for the low-light-adapted strains
CCMP 1375T and CCMP 2773. A light–dark regime was applied
(12 h light:12 h dark).
Prochlorothrix hollandica strain PCC 9006T was grown in BG
11 medium at 20 C under a white fluorescence lamp (20 mmol
photons m–2 s–1). Uncultivable Prochloron sp. was obtained
from Prochloron-harboring didemnid ascidian colonies,
T. cyclops, T. miniatum and L. timorense, which were collected
by snorkeling at Bise, Okinawajima Island (Ryukyu Archipelago,
Japan) on October 10, 2011. Intact symbiotic cells of Prochloron
were extruded separately from colonies of each host ascidian
following cutting with a steel razor blade (Hirose et al. 2009).
All cyanobacterial cells were collected by centrifugation
(3,000–15,000 r.p.m., 10–30 min) and stored at –85 C until
use for pigment analyses.
HPLC analysis
The HPLC system for total pigments was equipped with a
mBondapak C18 column (8100 mm, RCM type; Waters)
eluted with methanol (1.8 ml min–1) (Takaichi and Shimada
1992). The HPLC system for carotenes was equipped with a
Novapak C18 column (8100 mm, RCM type; Waters) eluted
with acetonitrile/methanol/tetrahydrofuran (58 : 35 : 7, by vol.,
2.0 ml min–1) (Takaichi 2000).
Extraction and purification of pigments
The carotenoids were extracted, isolated and purified as
follows. Pigments were extracted with acetone/methanol
Plant Cell Physiol. 53(11): 1881–1888 (2012) doi:10.1093/pcp/pcs126 ! The Author 2012.
a-Carotene in Acaryochloris and Prochlorococcus
(7 : 2, v/v) using an ultra sonicator, centrifuged, and the solvent
was evaporated. The pigments were loaded on a column of
DEAE-Toyopearl 650 M (Tosoh), and the carotenoids were
eluted with n-hexane/acetone (1 : 1, v/v), but Chls and polar
lipids remained on the column. They were then purified by
silica gel thin-layer chromatography (TLC; Merck) developed
with petroleum ether/hexane (7 : 3, v/v), and two yellow
bands were collected. Finally, the non-polar carotene band
was purified and separated into two carotenes using the
Novapak C18 HPLC column, and the polar xanthophyll band
was purified using the mBondapak C18 HPLC column described
above.
Spectroscopic analysis
We measured the absorption spectra of the pigments with an
MCPD-3600 photodiode array detector (Otsuka Electronics)
attached to the HPLC apparatus (Takaichi and Shimada
1992). The CD spectra of the purified carotenoids were
measured with a J-820 spectropolarimeter (JASCO) in diethyl
ether/2-pentane/ethanol (5 : 5 : 2, by vol.) at room temperature.
The relative molecular masses of the carotenoids were measured using an FD-MS; M-2500 double-focusing gas chromatograph–mass spectrometer equipped with a field-desorption
apparatus (Hitachi, Japan). The 1H-NMR (500 MHz) spectra
including NOESY and qCOSY of the carotene in CDCl3 at
room temperature were measured with a UNITY INOVA-500
system (Varian).
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Japan Society for the
Promotion of Science [No. 24570115 to S.T., No. 23370013 to
A.M. and E.H., No. 22370017 to T.T. and M.M., and No.
24658080 to T.T.].
Acknowledgments
The authors wish to thank Professors M. Tanaka and T. Sanji,
Tokyo Institute of Technology, for measuring CD spectra.
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