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INTRODUCTION |
Photosynthetic organisms capture light energy by their
light-harvesting systems that consist of core and peripheral antenna complexes (1). Core antenna complexes of oxygen-evolving photosynthetic organisms are highly conserved and have chlorophyll a as a
pigment, whereas peripheral antenna complexes, especially for
photosystem II (PSII),1 have
various pigments such as chlorophyll b, chlorophyll
c, phycobilins, fucoxanthin, and peridinin depending on the
group of photosynthetic organisms (2). Cyanobacteria and red algae have
phycobilins that harvest light energy in a wavelength region between
500 and 650 nm. Chlorophytes and prochlorophytes contain chlorophyll
b, which captures light energy at around 470 and 650 nm.
These antenna pigments are acquired during the evolution of
photosystems. These new antenna systems are thought to play an
important role in the adaptation to various light conditions or in
competition with other organisms because they capture light energy that
had not been harvested by pre-existing pigments. Thus, the new pigments have been a driving force in the divergence of photosynthetic organisms. However a new pigment(s) would not have its specific binding
sites in pre-existing pigmented proteins.
All pigments bind to their specific proteins to form pigment-protein
complexes. Studies of the crystal structure of pigment-protein complexes at the atomic level indicate that the arrangement and molecular species of pigments are strictly determined in the complexes (3, 4), and this strict determination is thought to be important for
efficient energy transfer among pigments. This idea is supported by the
results of studies using the light-harvesting complex II (LHCII) of
higher plants. The chlorophyll b content of LHCII in higher
plants is highly conserved (between 45 and 50%) (5). LHCII proteins of
chlorophyll b-less mutants of higher plants are unstable in
thylakoid membranes (6-8) and do not accumulate without chlorophyll
b, probably because of the breakdown of apoproteins. In vitro reconstitution experiments have shown that folding
of LHCII required both chlorophyll a and chlorophyll
b (9). These are thought to be the mechanisms by which
chlorophyll a/b ratios were conserved in LHCII.
In contrast to LHCII of higher plants, some studies suggest that the
chlorophyll b content of LHCII of green algae are variable.
The chlorophyll a/b ratio of LHCII of Dunaliella tertiolecta varies according to the light
intensity, and the content of chlorophyll b in LHCII
regulates the effective absorption cross-section of PSII (10),
indicating that the flexibility of proteins for pigments plays an
important role in adaptation to light environments. It has also been
reported that LHCII proteins are stable in thylakoid membranes of a
chlorophyll b-less mutant of Chlamydomonas
reinhardtii (11). Moreover, the antenna size of PSI in the mutant
is similar to that in the wild type (11), suggesting that chlorophyll
a molecules bind to LHC at chlorophyll b binding sites.
Many studies have indicated that the protein and pigment compositions
of core antenna complexes are highly conserved among oxygen-evolving
photosynthetic organisms (1). PSI and PSII core complexes purified by
nondenatured polyacrylamide gel electrophoresis (PAGE) from green
plants had very little or no chlorophyll b (12). These
observations led to the conclusion that core antenna complexes of
chlorophytes have chlorophyll a and do not bind chlorophyll b despite the presence of chlorophyll b.
The ability of the proteins to bind pigments has been studied by
biochemical, physiological, and biophysical methods. However, we
considered that the introduction of a new pigment into cells by a
molecular genetics method would be a useful means of
investigating the distribution of a new pigment among light-harvesting
complexes to understand their pigment-binding activity. We therefore
introduced the chlorophyll b synthesis gene,
i.e. chlorophyll a oxygenase (CAO)
(13), into a cyanobacterium that does not synthesize chlorophyll b. This is the first report on the introduction of a new
pigment into a photosynthetic organism. Chlorophyll b was
synthesized in transformant cyanobacteria cells and incorporated into
the P700-chlorophyll a-protein complex (CP1). The
chlorophyll a-protein was then functionally transformed to
the chlorophyll a/b protein. It was found that
CP1 of C. reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b. We propose herein
a hypothesis for the evolution of light-harvesting systems on the basis
of flexibility of antenna proteins.
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EXPERIMENTAL PROCEDURES |
Introduction of the CAO Gene into the Synechocystis sp. PCC6803
Genome--
We predicted the cleavage site after the 50th amino acid
residue of Arabidopsis CAO and therefore used a cDNA
clone encoding the coding region starting with the 51st residue
(lysine) (14). The CAO cDNA with a strong promoter of
psbAII was introduced within the open reading frame
slr2031 at the HpaI site in a glucose-tolerant strain of Synechocystis sp. PCC 6803 together with a
chloramphenicol-resistant cartridge (cb1-3). The control mutant
was generated with DNA lacking the CAO gene but having the
psbAII promoter and a kanamycin-resistant cartridge. It is
very likely that the insertion of CAO had no effect on the
cells, because slr2031 is not functional in this strain due
to spontaneous deletion of a 154-base pair segment including a
putative initiation codon for translation (15).
Culture Conditions--
Synechocystis cells were
grown at 22 °C in BG11 medium (16) under continuous illumination (30 micro-einsteins/m2/s) with 20 µg/ml of kanamycin
(control) or 20 µg/ml of chloramphenicol (cb1-3). Cell density was
monitored using a Shimadzu UV-160A to measure the absorbance of the
culture medium at 677 nm.
Preparation of Thylakoid Membranes from Synechocystis sp. PCC6803
and C. reinhardtii--
Cells of Synechocystis sp. PCC6803
or C. reinhardtii were harvested by centrifugation,
suspended in 50 mM HEPES (pH 8.0), and broken with a
Vibrogen with glass beads at 0 °C. After removal of the beads by
centrifugation, homogenates were centrifuged at 10,000 × g for 20 min. The green pellets were suspended in 50 mM HEPES (pH 8.0) containing 5 mM EDTA and
centrifuged at 10,000 × g for 20 min. The pellets were
resuspended in 50 mM HEPES (pH 8.0) and sonicated. After
centrifugation at 2,500 × g for 5 min, the
supernatants were used as thylakoid membranes.
Optical Measurements--
Fluorescence spectra of cells were
measured using a Hitachi F4500 spectrofluorometer. For the excitation
spectra at 77 K, a Dewar bottle and a home-made holder were used
(17).
Separation of Chlorophyll-Protein
Complexes--
Thylakoid membranes from
Synechocystis sp. PCC6803 (0.5 mg chlorophyll/ml) were
dissolved in 0.5% SDS and electrophoresed with 8% polyacrylamide disc
gels at 4 °C in the dark for 1 h (12).
Isolation of the P700-Chlorophyll a-Protein Complex from
C. reinhardtii--
Thylakoid membranes prepared from wild type (c-9)
and a chlorophyll b-less mutant (cbs3) of C. reinhardtii (13) were solubilized with 2% SDS at room
temperature. Solubilized membranes were loaded on a 12% polyacrylamide
gel and electrophoresed for 14 h in the dark. Green bands
corresponding to CP1 were cut and immersed in solubilizing buffer
containing 2% SDS. After heat treatment at 90 °C for 2 min, gels
were loaded on a 12% polyacrylamide gel, electrophoresed, and stained
with silver.
Northern Blot Analysis--
Total RNA was extracted from
Synechocystis cells. Two µg of total RNA was
electrophoresed on a 1% agarose/formaldehyde gel. The RNA was blotted
onto a Hybond-N nylon membrane and hybridized with a probe for
CAO.
Isolation of Chlorophyll from the Green Bands--
The green
bands on the gel were cut and homogenized with 0.1% SDS. Chlorophyll
was extracted from the gel slices with 0.1% SDS. After centrifugation
of the homogenate, 100% acetone was added to the green supernatant to
a final concentration of 80% and centrifuged.
Chlorophyll Determination--
Chlorophyll was extracted with
80% acetone and subjected to high-performance liquid chromatography
(HPLC). The chlorophyll was quantified from the chromatographic peak
areas after calibration of the chromatographic response with known
quantities of the relevant pigments (18).
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RESULTS |
Expression of the CAO Gene in Synechocystis--
Enzymatic studies
have shown that CAO catalyzes out two-step oxygenation and
converts chlorophyllide a to chlorophyllide b by
itself (14), suggesting that photosynthetic organisms can synthesize
chlorophyll b when they acquire the CAO gene. We
introduced Arabidopsis CAO cDNA into the
genome of Synechocystis sp. PCC6803 (cb1-3) under the
control of a PsbAII promoter. Northern blot analysis showed
that the introduced CAO gene was expressed in a cb1-3
transformant (Fig. 1). We then analyzed
the pigment composition by HPLC. Besides chlorophyll a, we
observed a new peak (X) (Fig. 2) for
which the absorption maximum was located at 650 nm in acetone. The
results of coinjection experiments on HPLC (data not shown) and
fluorescence spectra (Fig. 3) showed that
peak X corresponded to chlorophyll b. The chlorophyll
b content varied with the growth phase. During the early
phase, chlorophyll b was actively synthesized, and the
chlorophyll b content was in the range of 7.0 to 10.6% (Fig. 4). However, the chlorophyll
b content decreased as the culture period was prolonged. A
possible explanation for this decrease is that chlorophyll b
synthesis was halted by the nature of the promoter used for expression
of CAO or that chlorophyll b was cleared out
because of a greater turnover of chlorophyll-binding proteins that
contain chlorophyll b. At present, we have no direct evidence, and further studies are required. There was no significant difference between cell growth in the control group and that in the
cb1-3 transformant group during the whole growth phase (Fig. 4),
indicating that chlorophyll b did not induce photodamage and was not toxic for this species.

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Fig. 1.
Northern blot analysis of CAO
mRNA. The coding region of the CAO was fused
with the promoter for psbAII and introduced into the
Synechocystis sp. PCC6803 genome together with a
chloramphenicol-resistant cartridge (cb1-3). The control mutant
(control) was generated with DNA lacking the CAO
but having the psbAII promoter and a kanamycin-resistant
cartridge. Total RNA from Synechocystis cells was hybridized
with a probe for CAO.
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Fig. 2.
HPLC elution profiles of chlorophyll.
Chlorophylls extracted with 80% acetone from C. reinhardtii, a control, and the cb1-3 transformant were separated
by reversed-phase HPLC. The peak X shows a newly synthesized
pigment.
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Fig. 3.
Fluorescence spectra of chlorophylls.
Fractions of HPLC corresponding to chlorophyll b from
Chlamydomonas and the peak X from the cb1-3
transformant were collected, and their fluorescence spectra were
measured in methanol by excitation at 470 nm.
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Fig. 4.
Chlorophyll b content
varied with the growth phase. Cells grown under continuous light
were harvested at various times. Chlorophyll was extracted and
determined by HPLC. Cell densities were monitored by measuring
absorbance at 677 nm of culture.
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Function of Chlorophyll b in PSI--
The localization of
chlorophyll b in pigment-protein complexes was investigated.
Thylakoid membranes were prepared from Synechocystis cells,
and chlorophyll-protein complexes were resolved by green gel (15) (Fig.
5). It was found that 57.5 and 34.9% of
chlorophyll a was confined to the P700-chlorophyll
a-protein complex (CP1) and the chlorophyll
a-protein complexes of PSII (CP43/47), respectively (Table
I). Less than 7% of chlorophyll
a was of a free form, indicating the reliability of
separation. Most of the chlorophyll b (76.5%), on the other
hand, was associated with CP1 with a small amount (15.0%) in CP43/47
and free chlorophyll (8.5%). The chlorophyll b content of
CP1 to total chlorophyll was 8%, higher than that of CP43/47 (2.7%)
and of free chlorophyll (6.8%), indicating that chlorophyll
b was associated preferentially with CP1. Considering that
CP1 has 100 chlorophyll molecules/P700, 8 chlorophyll b
molecules were bound to CP1 in the cb1-3 transformant. On the other
hand, CP43/47 bound at most 1 chlorophyll b molecule on the
basis of 40 chlorophyll a molecules in CP43/47.

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Fig. 5.
Separation of chlorophyll-protein
complexes. Thylakoid membranes were isolated from
Synechocystis cells, and chlorophyll-protein complexes were
separated by nondenaturing SDS-PAGE (12).
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Table I
Distribution of chlorophyll among chlorophyll-protein complexes
After nondenatured SDS-PAGE, the green bands of CP1, CP43/47, and free
pigments were excised, and chlorophyll was extracted.
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Next, we carried out spectral analysis to determine whether chlorophyll
b in Synechocystis cells could function as
photosynthetic pigment. Upon excitation of chlorophyll a at
440 nm, a typical PSII fluorescence was observed at 685 nm both at room
temperature and at 77 K, and an additional two fluorescence components
were detected at 695 and 722 nm, as in the case of the control cells (data not shown). Upon excitation of chlorophyll b at 465 nm, no fluorescence from chlorophyll b was observed on
intact cells either at room temperature or at 77 K (data not shown),
indicating that free chlorophyll b did not exist in cells.
This finding is consistent with the results that most of the
chlorophyll b was bound to some chlorophyll proteins as
shown by the green gel. When we measured the excitation spectra of the
PSI fluorescence (725 nm) at 77 K (Fig. 6,
top), a significant
difference was observed at around 470 nm between the control and the
cb1-3 transformant; the difference spectrum corresponded to the
chlorophyll b Soret band. These results, together with those
of the green gel, clearly showed that chlorophyll b was
incorporated into CP1 and transferred light energy to PSI chlorophyll
a. On the other hand, the excitation spectrum of PSII
chlorophyll a fluorescence at 685 nm showed that the
contribution of chlorophyll b to sensitization of
chlorophyll a was very small (Fig. 6, bottom),
consistent with the low chlorophyll b content of CP43/47
purified by the green gels. These results indicate that the
P700-chlorophyll a-protein complex was functionally transformed to a P700-chlorophyll a/b-protein
complex when this cyanobacterium acquired chlorophyll b.

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Fig. 6.
Excitation spectra of individual
photosystems at liquid nitrogen temperatures.
The excitation spectra of chlorophyll of intact cells were measured by
monitoring fluorescence at 685 nm (PSII) and 725 nm
(PSI). The difference spectrum of PSI excitation spectra
between the control and the cb1-3 transformant exhibited a peak at 465 nm, indicating an energy transfer from chlorophyll b to
chlorophyll a in PSI.
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Existence of Chlorophyll b in the PSI Core Complex in C. reinhardtii--
Because the amino acid sequences of CP1 apoproteins
in cyanobacteria and green plants are very similar, binding of
chlorophyll b to CP1 in Synechocystis cells would
indicate the possibility that CP1 of green plants also binds
chlorophyll b. We therefore re-examined whether CP1 of green
plants binds chlorophyll b. Thylakoid membranes were
prepared from Chlamydomonas cells and subjected to PAGE of
the Laemmli system (19) without heat treatment. This PAGE system
was so harsh that all of the chlorophyll was released from CP43/47, and
a large amount of chlorophyll was also released from LHCII. However,
CP1 still bound a considerable amount of chlorophyll (Fig.
7a). A CP1-LHCI complex and
CP1 were resolved by the commonly used green gel (20), but only the
latter was found with the Laemmli system. We then investigated the
protein composition of CP1; CP1 prepared by the green gel was heated in the presence of SDS and subjected to PAGE. The results showed that CP1
consisted only of CP1 apoproteins (Fig. 7b), indicating that
LHCI was released completely from CP1 in the green gel. However, the
HPLC analysis showed that the chlorophyll b content of
purified CP1 was 4.4%, indicating that CP1 of green algae bound
chlorophyll b. Namely, the core complex of PSI in green
algae was not a P700-chlorophyll a-protein complex but a
P700-chlorophyll a/b-protein complex in situ.

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Fig. 7.
P700-chlorophyll
a-protein complexes of C. reinhardtii. a, protein composition of CP1
of Chlamydomonas. Left, thylakoid membranes were
prepared from wild-type (c-9; WT) and a chlorophyll
b-less mutant (cbs3; CBS-3) of
Chlamydomonas (13), solubilized under 2% SDS at room
temperature, and subjected to green gel as described under
"Experimental Procedures." Right, protein composition of
CP1. The green bands corresponding to CP1 were cut and
immersed in solubilizing buffer containing 2% SDS. After heat
treatment at 90 °C for 2 min, gels were loaded on a 12%
polyacrylamide gel, electrophoresed, and stained with silver.
b, HPLC elution profile of chlorophyll associated with
CP1.
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DISCUSSION |
In a previous paper, we described a possible reaction mechanism of
CAO based on the results of in vitro experiments
with CAO gene products (14). CAO catalyzed
two-step oxygenation reactions and converted chlorophyllide
a to chlorophyllide b without any other enzymes.
The results of those in vitro experiments are supported by
the results of present experiments showing that cyanobacteria accumulated chlorophyll b by acquiring only the
CAO gene. An in vitro study (14) also showed that
reduced ferredoxin was required for the oxygenation reactions by
CAO. The cb1-3 transformant cells probably utilized
endogenous ferredoxin for chlorophyll b synthesis.
Chlorophyll b synthesized in the cb1-3 transformant cells
was incorporated preferentially into CP1 apoproteins, and chlorophyll b transferred light energy to chlorophyll a in
PSI. Fluorescence from free chlorophyll b was not observed
on the transformant cells. These results indicate that chlorophyll
b was incorporated into the chlorophyll a binding
sites of CP1 instead of chlorophyll a, because it is
generally observed that nonspecifically bound chlorophyll fluoresces
even in a low yield (21). The notion of specific binding of chlorophyll
b was also supported by the observation that chlorophyll
b was found only in CP1. CP43/47 and free chlorophyll bands
on the green gels and that of other colorless proteins never bound
chlorophyll b. The chlorophyll b content of 8%
in CP1 indicated that at least eight chlorophyll binding sites could be
replaced with chlorophyll b. These results are consistent
with recent reports that some chlorophyll binding sites in LHC are
replaceable by either chlorophyll a or chlorophyll b and that the chlorophyll b content of LHCII
could change (10, 17).
Native SDS-PAGE is a powerful tool for isolating chlorophyll-protein
complexes without contamination. This method has been used in many
studies to determine the chlorophyll contents of chlorophyll-protein
complexes. Most of these studies showed that CP1 has no chlorophyll
b (12), although some studies suggested the presence of
chlorophyll b in CP1 (22). Ikegami and Ke (23), using the
ether extraction method, reported the existence of chlorophyll b in PSI reaction center particles in which the
chlorophyll/P700 ratio was 13. These discrepant results concerning the
chlorophyll a/b ratio of CP1 may be due in part
to the method used for chlorophyll determination, because high
chlorophyll a/b ratios cannot be determined spectroscopically. Although CP1 is believed to be a chlorophyll a-protein complex, there have been no results providing
clear evidence of this idea. In the present study, we found by
HPLC that CP1, which was completely free from LHCI, bound a
considerable amount of chlorophyll b. Our results suggested
that CP1 is not a chlorophyll-a protein complex but a
chlorophyll a/b-protein complex in C. reinhardtii. Our observation was also supported by the results of
experiments showing that cyanobacterial CP1 apoproteins, which have
amino acid sequence homology to that of green plants, bound chlorophyll
b. Further studies are needed to determine whether core
antenna complexes of oxygen-evolving photosynthetic organisms bind
exclusively chlorophyll a.
On the basis of the above results, we propose the progression of an
antenna system in which a new pigment is acquired without the presence
of a corresponding new protein. Our results demonstrated that the PSI
core complex in a prokaryote has the capacity to incorporate
chlorophyll b flexibly to its functional sites and that the
complex in green algae indeed binds chlorophyll b. These findings led us to hypothesize as to how chlorophyll b, a
new pigment, is incorporated into an antenna system of a prototype of
cyanobacteria. When photosynthetic organisms acquired a CAO gene during an early evolutionary phase (24), chlorophyll b began to be synthesized and bound to the core antenna of PSI through its flexibility. Chlorophyll b in CP1 immediately began to
function as a photosynthetic pigment, and the organisms became able to use light energy at around 470 and 650 nm, which would be
favorable for competition for light energy capturing. It would
also have been important for the organisms that a new pigment did not
induce photodamage. Our experiments reproduced this process.
Cyanobacteria do not contain chlorophyll b, and it is
therefore probable that they lost the CAO gene.
Prochlorophytes bind chlorophyll b to prochlorophyte
chlorophyll b-binding protein (25) by acquiring a new
protein. In the evolutionary progression to eukaryotes, CP1 in green
plants has retained an ancestral character to bind chlorophyll
b by keeping a CAO gene in the common ancestor.
On the other hand, chlorophyll b in peripheral antenna
systems has changed its locations from prochlorophyte chlorophyll
b-binding protein to LHC, which appears after an
endosymbiotic event by the duplication of high
light-inducible protein (26).