Department of Biological Sciences, Brock University, St.
Catharines, Ontario, Canada L2S 3A1
The light state transition regulates the distribution of
absorbed excitation energy between the two photosystems (PSs) of photosynthesis under varying environmental conditions and/or metabolic demands. In cyanobacteria, there is evidence for the redistribution of
energy absorbed by both chlorophyll (Chl) and by phycobilin pigments,
and proposed mechanisms differ in the relative involvement of the two
pigment types. We assayed changes in the distribution of excitation
energy with 77K fluorescence emission spectroscopy determined
for excitation of Chl and phycobilin pigments, in both wild-type and
state transition-impaired mutant strains of
Synechococcus sp. PCC 7002 and
Synechocystis sp. PCC 6803. Action spectra for the
redistribution of both Chl and phycobilin pigments were very similar in
both wild-type cyanobacteria. Both state transition-impaired mutants
showed no redistribution of phycobilin-absorbed excitation energy, but
retained changes in Chl-absorbed excitation. Action spectra for the
Chl-absorbed changes in excitation in the two mutants were similar to
each other and to those observed in the two wild types. Our data show
that the redistribution of excitation energy absorbed by Chl is
independent of the redistribution of excitation energy absorbed by
phycobilin pigments and that both changes are triggered by the same
environmental light conditions. We present a model for the state
transition in cyanobacteria based on the x-ray structures of PSII, PSI,
and allophycocyanin consistent with these results.
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INTRODUCTION |
The effective absorption of sunlight
by antenna pigments is the critical first step in photosynthesis. All
oxygenic photosynthetic organisms share a common core antenna pigment
complement of about 40 chlorophyll (Chl) a in PSII and about
100 Chl a in PSI (Rögner et al., 1990
). Photosynthetic
organisms do not, however, limit their photon capturing ability to this
level, but rather use some form of additional peripheral antenna
pigments to increase the effective "absorption cross section" of
one or both PSs. Higher plants and algae have evolved diverse
mechanisms to increase their ability to absorb sunlight. In
cyanobacteria, the soluble phycobiliproteins are organized into
phycobilisomes (PBSs), which are primarily associated excitonically
with PSII in a manner analogous to the family of intrinsic thylakoid
membrane Chl a/b-containing light-harvesting complex polypeptides (LHCII), which serve the same function in higher
plants (Glazer, 1984; Zilinskas and Greenwald, 1986).
Both cyanobacteria and higher plants can regulate the efficiency of
excitation energy transfer to the two PSs. The light state transition
appears designed to adjust the relative activities of PSII and PSI in
response to a dynamic environment or to changing metabolic demands (Yu
et al., 1993). The mechanism in higher plants involves a reversible
association of LHCII with PSII and PSI triggered by the redox state of
intersystem electron transport carriers and driven by the reversible
phosphorylation of LHCII (for review, see Allen, 1992; Wollman, 2001).
There is no consensus for the mechanism of the state transition in
phycobilisome-containing cyanobacteria (for review, see Van Thor et
al., 1998; Mullineaux, 1999).
The state transition in cyanobacteria is triggered in the same way as
that in higher plants. State 1 is achieved by oxidation of intersystem
electron carriers (usually by "excess" excitation of PSI).
Reduction of intersystem electron carriers, most likely plastoquinone
(Mullineaux and Allen, 1990), either by "excess" excitation of PSII
or by a dark respiratory pathway (Mullineaux and Allen, 1986), triggers
the conversion to state 2. State 2 is characterized by a decrease in
PSII variable fluorescence, a decrease in the PSII absorbance cross
section, and an increase in the PSI absorbance cross section as
compared with state 1 (Mullineaux, 1992). It is clear that excitation
energy absorbed by the PBS has a lower probability of reaching
PSII and a higher probability of reaching PSI in state 2 than in state
1. How this change is effected and the role of Chl a in the
state transition mechanism have been controversial points (Salehian and
Bruce, 1992; Mullineaux, 1994).
Early proposals for mechanisms for the state transition were based on
either the idea of a mobile PBS (Allen and Holmes, 1986), which changes
its association with PSII and PSI, or a "spillover" of energy from
PSII Chl a to PSI Chl a (Biggins and Bruce, 1989; Bruce et al., 1989; Rouag and Dominy, 1994). Recent work supporting the
idea of a mobile PBS comes from fluorescence recovery after photobleaching (FRAP) measurements that clearly but surprisingly indicate that PBSs are much more mobile than PSII (Mullineaux et al.,
1997). Mutants of the apcD gene, which codes for the
-subunit of the allophycocyanin (APC) B core subunit
(AP-B), have been shown to be impaired in
state transitions and appear to be stuck in state 1 (Zhao et al.,
1992). This has been interpreted to support the idea of a mobile PBS
mechanism and that the apcD gene product is the site of
energy transfer from the PBS to PSI (Ashby and Mullineaux, 1999). The
mobile PBS model is not sufficient to fully explain the state
transition, however, because changes in the relative contribution of
Chl a-absorbed excitation energy to PSII and PSI are also
observed. This result is not predicted by the mobile PBS model. The
observation of changes in the distribution of Chl
a-absorbed excitation has supported the "spillover"
model for the state transition, originally proposed by Murata (1969). The spillover model suggests that changes in the rate constant for
excitation energy transfer between PSII Chl a and PSI Chl a are responsible for the observed changes in the
distribution of both PBS- and Chl a-absorbed energy between
the two PSs. This mechanism depends on a strong coupling between the
PBS and PSII Chl a and a variable coupling between PSII Chl
a and PSI Chl a. The spillover model predicts
that the state transition-induced change in the relative contribution
of PBS to PSII would have to be equal to or less than the relative
change in the contribution of Chl a to PSII. A number of
reports show, however, that the relative changes in the distribution of
Chl a-absorbed energy are somewhat smaller than the relative
changes in the distribution of PBS-absorbed energy (Mullineaux and
Holzwarth, 1990; Salehian and Bruce, 1992).
Freeze fracture electron microscopy has shown that large-scale
organization changes of ectoplasmic face particles (containing PSII) in the thylakoid membrane accompany the state transition in
cyanobacteria (Olive et al., 1986). The ectoplasmic face particles exhibit a nonrandom alignment into long rows in state 1 and a more
random distribution in state 2. Linear dichroism studies have also
shown that the state transition in cyanobacteria is associated with
changes in the orientation of APC and Chl a (Bruce and
Biggins, 1985; Brimble and Bruce, 1989; Homer-Dixon et al., 1994).
Clearly, the state transition mechanism in cyanobacteria is more
complex than either the simple mobile PBS or spillover mechanism, and
somehow involves changes in both PBS and Chl a. A structural model for the state transition in cyanobacteria based primarily on data
from electron microscopy suggests that changes in both dimerization of
PSII and trimerization of PSI are involved, as well as differential
association of the PBS with PSII and PSI (Bald et al., 1996). It
appears likely that a number of changes are involved in the state
transition mechanism that affect both the relative association of PBS
with PSII and PSI and also the probability of excitation energy
transfer between PSII and PSI Chl a. Excellent recent work
has not simplified the situation. For example, it was reported that
a genetically engineered strain of Synechocystis sp. PCC
6803, in which thylakoid membranes were more rigid because of the
absence of di- and tri-unsaturated fatty acids, were unable to do
state transitions at temperatures below the lipid phase transition
temperature (El Bissati et al., 2000). Thus, membrane fluidity plays an
important role in cyanobacterial state transitions, supporting the idea
of some kind of involvement of mobile PSII and PSI in the mechanism,
even though the FRAP data indicate that the PBS are more mobile than
PSII (Mullineaux et al., 1997). Reversible changes in the association
of isolated PSII and PSI induced by changing detergent concentration
have been shown to be associated with state transition-like changes in
77K emission spectra, which again suggest a role for changes in
the association of PSII and PSI (Federman et al., 2000).
An insertional inactivation mutant in Synechocystis sp. PCC
6803 (
sll1926 or rpaC
)
was unable to perform state transitions and to grow more slowly than
the respective wild type under light-limiting conditions (Emlyn-Jones
et al., 1999). The deleted gene product, designated RpaC (regulator of
PBS association C), bears no sequence similarity to any known
photosynthesis-associated polypeptide, and no recognizable sequence
motifs. Interestingly, the mutant lacked the characteristic differences
in 77K fluorescence emission spectra indicative of a state transition
for excitation of the PBS, but did appear to retain some state
transition-like changes when emission spectra were collected for
excitation of Chl a (Emlyn-Jones et al., 1999). That work
suggested the possibility of differing origins for the fluorescence
changes indicative of state transitions in cyanobacteria for excitation
of Chl a and PBS.
Are the redistributions of Chl a- and PBS-absorbed
excitation energy with PSII and PSI associated with the state
transition independent of each other? If the PBS and Chl antenna do act
independently, are their light-induced changes in distribution
triggered by the same environmental light conditions?
To address these questions, we used two different species of
cyanobacteria. The Synechocystis sp. PCC 6803 wild type and
rpaC strain described above were
compared with Synechococcus sp. PCC 7002 wild type and a
mutant with impaired PBS function,
apcD. The apcD mutant
lacks the AP-B subunit of the APC core, which
has been suggested to facilitate the transfer of absorbed excitation
energy from the PBS to PSI (Maxson et al., 1989; Ashby and Mullineaux,
1999). The apcD mutant has also been reported to be unable
to perform state transitions (Zhao et al., 1992).
Our work confirms the observation that the
rpaC strain of
Synechocystis PCC 6803 exhibits state transition-associated
Chl fluorescence yield changes upon excitation of Chl but not upon excitation of PBS and shows for the first time, to our knowledge, that
this is also the case for the apcD
strain of Synechococcus sp. PCC 7002. In addition, action
spectra for the fluorescence yield changes in both species clearly show that the redistribution of both Chl and phycobilin antennae, although separable, are driven by the same environmental light conditions and,
thus, share the same triggering mechanism. We also present a new
structural model for the light state transition in cyanobacteria that
is consistent with independent pathways for the redistribution of PBS-
and Chl-absorbed excitation. Our model is based on the x-ray structures
of PSII, PSI, and APC and is supported by recent kinetic modeling
studies of excitation energy transfer in PSII (Vasil'ev et al.,
2001).
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RESULTS |
Room Temperature Fluorescence
Figure 1 displays the steady-state
room temperature variable fluorescence kinetics of
Synechococcus sp. PCC 7002 wild type and apcD
mutant and Synechocystis sp. PCC 6803 wild type and the rpaC mutant determined with a PAM fluorometer. In both
wild-type cyanobacteria, illumination with blue light induces an
increase in maximal level of variable fluorescence
(Fm) indicative of a transition to state 1. The approximately 20% increases in Fm
displayed by both of the wild-type cyanobacteria are absent in the room temperature fluorescence traces of their respective mutants.
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Figure 1.
Room temperature pulse-amplitude-modulated (PAM)
fluorescence kinetic traces for Synechococcus sp. PCC 7002 wild-type and apcD cells and
Synechocystis sp. PCC 6803 wild-type and
rpaC cells. Cells were dark adapted
to state 2 and the arrow indicates the application of blue light in an
attempt to drive the cells to state 1. See "Materials and Methods"
for details.
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77K Fluorescence Emission Spectra
77K fluorescence emission spectra of Synechococcus sp.
PCC 7002 wild type and apcD mutant and of
Synechocystis sp. PCC 6803 wild type and rpaC
mutant are shown in Figure 2. In the wild
type of both species, increases in PSII fluorescence emission (685- and
695-nm peaks) relative to PSI fluorescence emission (715-nm peak in
Synechococcus and 725-nm peak in Synechocystis)
are associated with the transition to state 1 and observed for
excitation of both the PBS at 580 nm (lower) and Chl a at
435 nm (upper). In contrast, no significant changes in the shape of the
emission spectra are observed in the mutant cells of either species for excitation of the PBS, although a significant increase in the PSII
emission relative to the PSI emission is seen in emission spectra of
the mutant cells when excited at 435 nm. In both species, the wild-type
cells exhibit state transition-like changes in the relative emission
yields of PSII relative to PSI for excitation of either PBS or Chl
antenna, whereas the mutant cells show changes only for excitation of
Chl.
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Figure 2.
77K fluorescence emission spectra of
Synechococcus sp. PCC 7002 wild-type and
apcD cells and
Synechocystis sp. PCC 6803 wild-type and
rpaC cells. Spectra were collected
for excitation of Chl a at 435 nm (top) and for excitation
of phycobilin pigments at 580 nm (bottom). All cells were
pre-illuminated before freezing in liquid nitrogen with either 420 nm
of light in an attempt to drive a transition to state 1 (solid lines)
or 560 nm of light in an attempt to drive a transition to state 2 (dotted line). See "Materials and Methods" for details.
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Action Spectra
To investigate the environmental light conditions that trigger the
state transition-associated changes in ratio of PSII emission relative to PSI emission
(F695/F715 in
Synechococcus;
F695/F725 in
Synechocystis) observed in the 77K fluorescence emission
spectra, we determined action spectra for the ratio as a function of
wavelength of the pre-illumination light used to drive the state
transition. As described in "Materials and Methods," cells were
pre-illuminated at room temperature at the wavelengths used to
construct the action spectra, quickly frozen in liquid nitrogen, and
then assayed for 77K fluorescence emission.
In Figure 3 action spectra for the
Synechococcus sp. PCC 7002 wild type and apcD
mutant are shown. The
F695/F715 ratio is used as
an indicator of the "state" attained by any particular pre-illumination wavelength. State 1 is characterized by a high F695/F715 ratio and state 2 is characterized by a low
F695/F715 ratio. For each
sample, the F695/F715 ratio
was determined for excitation of the PBS (580 nm) and Chl a
(435 nm). This allowed the construction of two action spectra, one for
pre-illumination-induced changes in distribution of light absorbed by
the PBS and one for pre-illumination-induced changes in distribution of
light absorbed by Chl a. In each action spectrum, the dashed
horizontal lines show values of
F695/F715 characteristic of
state 1 (blue light driven) and state 2 (dark adapted or orange light
driven). In wild-type cells, the action spectra for the
F695/F715 ratio for excitation of PBS and for excitation of Chl a are very
similar to each other. They show that pre-illumination with blue and
red light, which is preferentially absorbed by Chl a, drives
the cells to state 1 and that pre-illumination with green or orange
light, where light is preferentially absorbed by the PBS, drives the cells to state 2 regardless of whether changes in the distribution of
PBS- or Chl a-absorbed light are measured. In contrast, the apcD cells show no
pre-illumination-induced changes in
F695/F715 when fluorescence
emission was assayed with PBS excitation, but do show an action
spectrum of smaller magnitude but very similar shape to the spectra of
the wild type when assayed with Chl a excitation.
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Figure 3.
Action spectra for the light state transition in
Synechococcus sp. PCC 7002 wild-type and
apcD cells. The relative amplitude
of PSII fluorescence emission divided by PSI emission
(FPSII/FPSI) at
77K was plotted versus the pre-illumination wavelength used to drive
the state transition at room temperature. Action spectra in the top two
panels were generated from analysis of emission spectra excited with
light-absorbed by Chl a, whereas the action spectra in the
bottom two panels were generated from an analysis of emission spectra
from the same samples excited with light absorbed by phycobilin
pigments. See "Materials and Methods" for details. For each
pre-illumination wavelength, the mean of three independent measures is
plotted, and the error bars show the SD.
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Figure 4 shows action spectra for the
F695/F725 ratio as a
function of pre-illumination wavelength of the wild-type and
rpaC cells of
Synechocystis sp. PCC 6803. This set of action spectra show
the same pattern observed in Figure 3 for the action spectra of
wild-type and apcD cells of
Synechococcus sp. PCC 7002. All action spectra, with the
exception of that from the rpaC
cells under PBS excitation, show clear evidence of a state transition. These action spectra have very similar shapes to each other and to the
action spectra in Figure 3 for Synechococcus sp. PCC 7002. The F695/F725 ratio is high
with blue and red pre-illumination (Chl a-absorbed light,
preferential excitation of PSI) and low with green or orange
pre-illumination (PBS-absorbed light, preferential excitation of PSII).
In the action spectrum of the rpaC
cells, determined for PBS excitation, the characteristic peaks and
troughs indicative of a state transition are absent.
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Figure 4.
Action spectra for the light state transition in
Synechocystis sp. PCC 6803 wild-type and
rpaC cells. The relative amplitude
of PSII fluorescence emission divided by PSI emission
(FPSII/FPSI) at
77K was plotted versus the pre-illumination wavelength used to drive
the state transition at room temperature. Action spectra in the top two
panels were generated from analysis of emission spectra excited with
light-absorbed by Chl a, whereas the action spectra in the
bottom two panels were generated from an analysis of emission spectra
from the same samples excited with light absorbed by phycobilin
pigments. See "Materials and Methods" for details. For each
pre-illumination wavelength, the mean of three independent measures is
plotted, and the error bars show the SD.
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Absorbance Cross Sections
To determine whether the pre-illumination-induced changes in 77K
fluorescence emission were representative of relative changes in the
antenna size of PSII, the PSII absorbance cross sections for excitation
of both Chl a and PBS in wild-type and
apcD cells of Synechococcus sp. PCC
7002 were measured at room temperature. PSII absorbance cross sections
were determined from Poisson fits to pump probe fluorescence flash
saturation curves generated for excitation of Chl a and PBS
as described in "Materials and Methods." Table
I shows a summary of the PSII absorbance
cross sections of the wild-type and apcD cells
in states 1 and 2 for excitation of both Chl a at 674 nm and
the PBS at 630 nm. Wild-type cells show significant decreases in the
absorbance cross sections for both PBS and Chl a excitation upon transition from state 1 to state 2. In agreement with the 77K
fluorescence emission data, the apcD cells do
not show a significant decrease in the PSII absorbance cross section
for PBS excitation upon transition from state 1 to state 2 conditions,
but do show a significant decrease in Chl a-sensitized
absorbance cross section.
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Table I.
Absorbance cross sections ( II) of
PSII in Synechococcus sp. PCC 7002 wild type and apcD mutant
Room temperature PSII absorbance cross sections ( II)
for wild-type and mutant cells in states 1 and 2 for excitation of Chl
a at 674 nm and for excitation of the PBS at 630 nm. The
percent change column shows the percent increase in II
on transition from state 2 to state 1. Cross sections were determined
from Poisson fits to pump probe Chl a fluorescence flash
saturation curves, as described in "Materials and Methods." Values
are mean ± SD, n = 4.
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DISCUSSION |
Our results confirm previous observations (Zhao et al., 1992;
Emlyn-Jones et al., 1999) that both the Synechococcus sp.
PCC 7002 apcD strain and the
Synechocystis sp. PCC 6803 rpaC strain are state transition
impaired. Furthermore, our results confirm the interesting observation
that the Synechocystis sp. PCC 6803 rpaC strain retains characteristic
changes in 77K fluorescence yield indicative of a state transition when
spectra are collected for excitation of Chl a but not for
excitation of the PBS. In addition, we show that the
Synechococcus sp. PCC 7002 apcD strain also retains changes in
emission spectra indicative of a state transition when spectra are
collected for excitation of Chl a. The
Synechococcus sp. and Synechocystis sp. mutants
are both impaired in PBS-associated proteins and had been characterized initially as lacking state transitions.
It is interesting that both of these state transition mutants appear to
be only impaired in state transitions when assayed for changes in the
distribution of excitation energy absorbed by the PBS. The observation
of clear changes in the distribution of Chl a in both
mutants clarifies an apparent contradiction in previous experimental
results with two Synechococcus sp. PCC 7002 mutants. A
mutant of Synechococcus sp. PCC 7002 lacking PBS
(apc cpc) was originally characterized
as being state transition competent (Bruce et al., 1989). In that
study, low-temperature fluorescence emission spectra characteristic of
the state transition were obtained by manipulating the oxidation state
of the plastoquinone pool, and it was concluded that the PBS was not
essential to the state transition mechanism. A later study with the
apcD strain showed it to be unable
to perform state transitions, as assayed by both room temperature
fluorescence induction and low-temperature fluorescence emission
spectroscopy (Zhao et al., 1992). In contradiction to the work with the
PBS-less mutant, the study with the
apcD strain concluded that the
AP-B subunit of the APC core of the PBS was
essential for the state transition mechanism. However, fluorescence
emission spectra used to assess state transitions in the experiments
with the PBS-less mutant were determined for excitation of Chl
a (Bruce et al., 1989), and the study with the
apcD strain used excitation of the
PBS (Zhao et al., 1992). Rather than contradicting each other, these
two studies support our present work and show that changes in the
distribution of Chl a-absorbed excitation energy can occur
independently of changes in the distribution of PBS-absorbed energy.
The Mechanism of the State Transition, Spillover, and Mobile
PBS
The spillover model (Murata, 1969; Dominy and Williams, 1987;
Biggins and Bruce, 1989; Bruce et al., 1989) predicts that "excess" energy absorbed by pigments associated with PSII (either the PBS or the
Chl a core antenna) "spills over" to PSI in state 2. The mechanism assumes that energy absorbed by the PBS is transferred to the
antenna Chl a of PSII, which forms the bridge for excitation energy transfer to PSI. In this way, energy absorbed by either the PBS
or the PSII core Chl a antenna will ultimately reach PSI and
any spillover-induced changes in the distribution of Chl
a-absorbed excitation must be correlated with changes in the
distribution of PBS-absorbed excitation. A change in only one rate
constant, the rate of energy transfer from the PSII Chl antenna to the
PSI Chl antenna, thus changes the distribution of both PBS- and
Chl-absorbed excitation. This basic feature of the spillover model is
not consistent with our observation that only changes in Chl
a-absorbed excitation energy are retained in both of the
"state transition-impaired" mutants used in this study.
What are the other options for the mechanism of the state transition?
The mobile PBS model (Allen and Holmes, 1986; Kruip et al., 1994; Bald
et al., 1996; Mullineaux, 1999) predicts that preferential binding of
the PBS to PSII in state 1 and to PSI in state 2 accounts for the
changes in excitation distribution. FRAP data indicate that the lateral
mobility of the PBS on the surface of the thylakoid membrane is much
higher than that of PSII within the membrane (Mullineaux et al., 1997).
However, there is also evidence that the redistribution of PBS-absorbed
energy requires a fluid thylakoid membrane (El Bissati et al., 2000), suggesting the requirement for mobile PSII and/or PSI. Regardless of
the details, the idea of a shift in a dynamic equilibrium between two
states, one characterized by preferential transfer from the PBS to PSII
and the other by transfer from the PBS to PSI, remains a strong
candidate for the explanation of changes in distribution of
PBS-absorbed excitation. The major limitation of this mechanism is the
failure to explain redistribution of Chl a excitation energy associated with the state transition.
It has been suggested that the state transition-associated changes in
fluorescence yield observed at 77K for excitation of Chl a
may only exist at low temperature and, thus, may not be indicative of
changes in the functional absorbance cross sections of PSII and PSI at
room temperature (Mullineaux, 1999). Uncertainty about this point is
reinforced by the lack of direct measurements of functional absorbance
cross sections accomplished under physiological conditions. However,
the room temperature PSII absorbance cross section measurements in our
present report show that the functional antenna size of PSII decreases
in state 2 for excitation of either the PBS or Chl a antenna
pigments. More importantly, our data show that changes in PSII cross
section for Chl a excitation are retained in the
apcD mutant even though the changes in PSII cross section
for PBS excitation are not. Changes in the distribution of Chl
a excitation do accompany the state transition in
cyanobacteria and must be accounted for.
The preceding point has been addressed by models for the state
transition in cyanobacteria, which combine elements of spillover and
mobile PBS models (Koblízek et al., 1998; Mullineaux, 1999). Combination models are consistent with the observed redistribution of
both PBS- and Chl a-absorbed excitation energy and with the observation that the relative amplitude of the change in distribution of PBS-absorbed excitation energy is often larger than that of the
change in Chl a-absorbed excitation energy (Salehian and
Bruce, 1992). However, as discussed above for the spillover model,
combination models cannot easily explain how changes in Chl
a distribution can occur in the absence of changes in PBS distribution.
A Structure-Based Model for the Light State Transition in
Cyanobacteria
In light of recent studies on excitation energy transfer within
the PSII core complex of cyanobacteria, we believe the spillover model
can be modified so that changes in the distribution of Chl a-absorbed excitation energy can be independent of changes
in the distribution of PBS-absorbed excitation. The x-ray structure of
PSII has revealed a physical "gap" between the antenna Chl a associated with both CP43 and CP47 and the reaction center
chromophores associated with D1/D2 (Zouni et al., 2001). Kinetic
modeling and simulation of excitation energy transfer based on the
x-ray structure has shown that the six reaction center chromophores are
not in equilibrium with the antenna Chl a molecules of CP43
or of CP47 (Vasil'ev et al., 2001). The PSII core complex is best
thought of as a three-compartment system, the central D1/D2 reaction
center chromophores being flanked on one side by antenna Chl
a in CP43 and on the other by those in CP47. Excitation
energy transfer between the chlorin pigments within any one of the
compartments is very fast, and the chromophores within a compartment
are effectively in equilibrium within a few picoseconds after
excitation. However, energy transfer between the compartments is
considerably slower and is also slower than the rate of charge
separation in D1/D2 (Vasil'ev et al., 2001).
This "compartmentalization" of excitation energy within PSII makes
the transfer of energy from the PBS to PSII and the transfer of energy
from PSII to PSI more complex than assumed in the original spillover
model. It becomes important which compartments of PSII are involved in
energy transfer with the PBS and PSI. For example, energy transferred
from the PBS to the chromophores in D1/D2 would have a relatively low
probability of reaching Chl a in either CP43 or CP47. In
addition, energy transferred from the PBS to CP43 would have a
relatively low probability of reaching CP47 and vice versa. Taking this
energetic compartmentalization of PSII into account, it is possible to
construct a model for the state transition in cyanobacteria that can
explain the independent changes in distribution of PBS- and
Chl-absorbed energy (Fig. 5).
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Figure 5.
A model for the organization of chromophores
associated with the cyanobacterial thylakoid membrane and for the state
transition based on the x-ray structures of PSII, PSI, and APC. A,
"Side view" in the plane of the thylakoid membrane showing possible
associations between the chromophores in one PSII complex, one PSI
complex, and one APC core cylinder in states 1 and 2. Antenna
chromophores in PSII and PSI are shown in green and reaction center
chromophores are shown in red. Chromophores in PSII are shown divided
into three compartments, D1/D2, CP47, and CP43, to reflect the
energetic isolation of the two Chl a-containing antenna from
the reaction center pigments. Energy transfer from the APC core to PSII
occurs from the ApcE subunit to D1/D2. Energy transfer from the APC
core to PSI is via the ApcD subunit and is defined by the rate constant
KD-I. Energy transfer from PSII to PSI,
spillover, is assumed to occur from CP47 to PSI and is defined by the
rate constant KII-I. The state transition
is proposed to control both KD-I and
KII-I. Both rate constants are larger in
state 2 than state 1. B, "Top view" down onto the surface of the
thylakoid membrane and includes antenna chromophores (in green) and
reaction center chromophores (in red) associated with PSII dimers and
PSI trimers. APC core cylinders are shown associated with PSII dimers
and phycocyanin rods with the core cylinders. In state 2, trimeric PSI
is found in close contact with both CP47 of PSII and the ApcD subunits
of the core cylinders. In state 1, the PBS-PSII supercomplexes are
organized into rows that exclude the PSI trimer from the immediate
vicinity of ApcD and CP47. One of the PBS-PSII supercomplexes is shown
to remain associated with one of the PSI trimers via ApcD to represent
the idea that some complexes may be more involved than others in the
state transition. The organization of the PBS-PSII supercomplex and the
association of PSI trimers with ApcD and the terminal rod disc in this
figure has been adapted from Bald et al. (1996).
|
|
The model uses the x-ray structures of PSII (Vasil'ev et al.,
2001), PSI (Jordan et al., 2001), and APC (Reuter et al., 1999). A
minimal version of the model showing one APC core cylinder, one PSII,
and one PSI is displayed in side view in Figure 5A. The APC core
cylinder consists of four APC disc-shaped trimers, (
)3 (for review, see Sidler, 1994). In this
model, two sets of two trimers were assembled face to face in a manner
analogous to the assembly of trimers into hexamers proposed for
C-phycocyanin in PBS rods (Sauer and Scheer, 1988; Wang et al., 2001).
This assembly is similar to the somewhat "looser" association of
face-to-face trimers found in the x-ray structure of APC hexamers
(lacking linker polypeptides) from the red algae Porphyra
yezoensis (Liu et al., 1999). One of the two internal APC core
discs is assumed to contain the core membrane linker polypeptide (ApcE)
that binds a long wavelength emitting phycocyanobilin chromophore
(Gindt et al., 1994) and has a hydrophobic loop domain thought to
function in association of the PBS to PSII and/or the thylakoid
membrane. ApcE is believed to act as both a physical attachment point
and an energetic bridge from the APC core to PSII. One of the external trimeric discs is assumed to contain ApcD, a specialized -subunit of
APC, characterized by long-wavelength absorbance and fluorescence emission maxima, which is believed to be responsible for excitation energy transfer from the APC core to PSI (Maxson et al., 1989; Zhao et
al., 1992).
The model shows the core associated with a "three-compartment"
version of PSII (showing CP43, D1/D2, and CP47) and PSI. The core
cylinder was placed on top of the x-ray structure of PSII and oriented
in a manner consistent with electron microscopy of cyanobacterial
membranes and PSII core particles as suggested previously by Bald et
al. (1996). Arrows show excitation energy transfer from the presumed
long-wavelength chromophore in ApcE to chromophores in D1/D2 in PSII.
Excitation energy transfer from the APC core to PSI is shown occurring
through the presumed long-wavelength chromophore in ApcD. Changes in
the distribution of PBS-absorbed excitation energy between PSII and PSI
are represented in this model by changes in the association of PSI with
the PBS-PSII supercomplex, which affects the value of the rate constant
KD-I. In state 2, KD-I is high and the absorbance cross
section of PSI would increase at the expense of PSII for PBS-absorbed
light. Changes in the distribution of Chl a-absorbed light
are accomplished in this model by a spillover mechanism characterized
by excitation energy transfer from only one component of PSII (CP47) to
PSI. Changes in the rate constant for spillover,
KII-I, in this model would predominantly
affect the distribution of excitation energy absorbed by the Chl
a in CP47 between PSII and PSI. Energy reaching the PSII
reaction center from the PBS either directly or via CP43 would have a
relatively low probability of visiting CP47 pigments and, thus, would
not be "lost" via spillover. The compartmentalization of PSII could
effectively serve to separate energy transferred from the PBS to PSII
from energy lost from PSII to PSI via spillover. Thus, the two
processes are more independent than predicted by the classic spillover
mechanism, which assumed all antenna and reaction center chromophores
in PSII to be in equilibrium. The choice of CP47 as the PSII component
involved in spillover is consistent with state 1 minus state 2 fluorescence emission difference spectra, such as those reported by
Salehian and Bruce (1992), which show a dominant peak at 695 nm,
characteristic of the long-wavelength Chl a associated with
CP47. However, because both CP47 and CP43 are somewhat energetically
isolated from D1/D2, either or both could be involved in spillover. It
is interesting to note that because of the strong distance
dependence for excitation energy transfer, a relatively small change in
distance between PSI and the PBS-PSII supercomplex would be sufficient
to significantly affect both KD-I and
KII-I. The relative magnitude of changes in
PBS- and Chl a-absorbed energy with the state transition
will depend not only on the relative changes in these two rate
constants, but also on the distribution of PBS-absorbed excitation
energy between the two terminal emitters of the PBS, ApcE, and ApcD.
Our model is consistent with results obtained from the two mutants used
in this study. The model predicts that the observation of changes in
PBS distribution requires control of the association of the PBS core
with PSI and the presence of ApcD. Control of the association of the
PBS with PSI is presumably lacking in the Synechocystis sp.
rpaC strain and ApcD itself is lacking in the
Synechococcus sp. apcD
strain. Loss of either would not necessarily affect spillover from CP47
to PSI.
Figure 5B is a top view of the model and shows the association of PSII
dimers with two APC core cylinders (from one PBS) and PSI trimers. This
view also shows two PBS rods (each consisting of two hexameric
phycocyanin discs) associated with the core lying on the surface of the
thylakoid membrane. This view of the model is a modified and more
detailed version (including chlorin chromophores of PSII and PSI) of a
structural model previously presented by Bald et al. (1996) based
primarily on electron microscopic data. This figure reflects the likely
oligomerization states of PSII (Morschel and Schatz, 1987) and PSI
(Kruip et al., 1994) found in cyanobacterial membranes. In addition,
Figure 5B is fairly consistent with the relative numbers of PSII, PSI,
and PBS usually found in Synechococcus sp. PCC 7002 (Zhao et
al., 2001), although the PSII/PSI ratio is higher than that found in
Synechocystis sp. PCC 6803. In state 2, PSI trimers are
shown in close proximity to both ApcD and CP47, which would facilitate
energy transfer from both the presumed long-wavelength ApcD chromophore
and CP47 Chl a to PSI Chl a. Although a
relatively small displacement of PSI from the PBS-PSII supercomplex
would result in large changes in the distribution of both PBS- and Chl
a-absorbed excitation, this figure shows a large-scale
change in organization consistent with electron microscopic data (Olive
et al., 1986). As discussed previously (Bald et al., 1996), the
alignment of PSII and PBS in rows in state 1 may exclude PSI from the
vicinity of PSII and of ApcD. Therefore, this alignment would serve as
an effective means of decreasing both KD-I
and KII-I in state 1. In states 1 and 2, the PSI trimer is shown associated with a terminal rod disc, consistent
with the idea that cyanobacterial ferredoxin NADP reductase (FNR)
serves to "link" PSI to the PBS rod. The cyanobacterial FNR has an
N-terminal extension homologous to the rod terminating linker protein
CpcD (Schluchter and Bryant, 1992). Although evidence is lacking in
cyanobacteria, cross-linking studies have shown FNR to be associated
with the stromal-exposed PsaE subunit of PSI in higher plants (Andersen
et al., 1992).
State 1 is characterized by a relative decrease in the contribution of
PBS-absorbed excitation to PSI, but not a complete loss of all PBS
contribution. Although this is fairly easily represented by fine-tuning
the two rate constants in Figure 5A, it is more difficult to envisage
in Figure 5B if all PSI complexes are excluded from access to the APC
core. Thus, the representation of state 1 in Figure 5B includes one
PBS-PSII supercomplex that remains associated with a PSI trimer via
ApcD. This was included to represent the idea that some complexes may
be "more involved" in the state transition than others. Although
the state transition may be represented by a small change in the two
rate constants characterizing the association of PBS-PSII
supercomplexes with PSI as shown in Figure 5A, it could also be
represented by a much larger change in these constants for a subset of
PBS-PSII supercomplexes involved in the formation of rows as shown in
Figure 5B.
It has been suggested previously that changes in the oligomerization
state of both PSII and PSI may accompany the state transition (Bald et
al., 1996; Mullineaux, 1999). However, because there is no strong
direct evidence for changes in oligomerization with the state
transition, and they are not required to effect significant changes in
energy distribution for either PBS- or Chl a-absorbed excitation energy, we have not included them in this model.
The two APC core cylinders are "antiparallel" and the ApcE occupies
an internal disc position in the core. The reaction center chromophores
of PSII, shown in red, are seen to lie directly under the
ApcE-containing core discs. Although the phycocyanobilin chromophores are not shown in this figure, in our model we have rotated the two
cylinders to allow the two presumed ApcE-associated long-wavelength chromophores to come close to the thylakoid membrane. In this orientation, each of the presumed long-wavelength chromophores is
located almost directly above each of the two D1/D2s in the PSII dimer.
The transition dipoles of the presumed ApcE chromophores are oriented
at an angle of approximately 15° to the normal of the
thylakoid membrane plane. The transition dipoles of the two pheophytins
of the reaction center are also oriented closer to the normal to the
thylakoid membrane plane than the majority of nearby Chl a
molecules in CP43 or CP47 whose dipoles are mostly aligned parallel to
the membrane plane. We have made some initial calculations (data not
shown), based on Förster theory and analogous to those described
in (Vasil'ev et al., 2001), of the relative efficiency of energy
transfer from the presumed ApcE chromophore to the nearest chromophores
in D1/D2 and CP43 and CP47. Although specific results are dependent on
the exact details of placement of the PBS core onto PSII, it is clear
that as long as the presumed ApcE chromophore is somewhere above D1/D2,
the pheophytins would carry the largest proportion of energy from the
ApcE chromophore to the reaction center. This confirms that it is
possible to envisage a "direct connection" between the PBS and PSII
reaction center that would be, to some extent, energetically
independent of spillover from CP47 to PSI.
Our model for the state transition is consistent with most of the
experimental data for state transitions in cyanobacteria. The most
notable exception is the FRAP data, which have indicated a higher
lateral mobility of the PBS compared with PSII-associated Chl
a molecules after photobleaching with a laser flash
(Mullineaux et al., 1997). Our model suggests less drastic changes in
association of PSII, PSI, and the PBS than the mobile PBS model and
cannot easily be reconciled with the FRAP data. However, it seems
unlikely that the FRAP technique is completely noninvasive. The
apparent mobility of the PBS that has been observed could reflect
changes in its association with the thylakoid membrane that arise from local heating generated by the high-energy laser flashes required to
bleach the pigments.
Our model does not address the underlying forces responsible for
inducing the proposed changes in association between the PBS-PSII
supercomplexes and PSI trimers. The state transition is clearly
triggered by the redox state of intersystem electron carriers and the
action spectra in this work show that changes in both PBS and Chl
distribution are triggered by the same environmental light conditions.
However, what makes the complexes move? Early reports of reversible
phosphorylation of PBS-associated components (Allen et al., 1985)
championed a mechanism based on electrostatic interactions and changes
in charge density associated with differential phosphorylation.
However, state transition-associated reversible phosphorylation has not
been supported in the subsequent literature and is unlikely to be
involved (for review, see Biggins and Bruce, 1989; Mullineaux, 1999)
and strong evidence for an alternative has not been presented.
Unfortunately, after more than 30 years of investigation, this aspect
of the state transition in cyanobacteria is still a mystery.
| |
CONCLUSIONS |
We have shown that regulation of the distribution of Chl
a-absorbed excitation energy with the state transition is
independent of the regulation of energy absorbed by the PBS in two
different cyanobacterial mutants impaired in state transitions. Despite this independence, the regulation of both Chl a and
phycobilin pigments are triggered by very similar environmental light
conditions. We have constructed a model for the state transition using
the x-ray structures of PSII, PSI, and APC that is consistent with our
results and most previous work. Our model combines regulation of direct
energy transfer from the PBS core to PSI with regulation of the
spillover of excitation energy from one of the core antenna complexes
of PSII, CP47, to PSI. Key to the mechanism is the idea that a
significant proportion of excitation energy from the PBS is transferred
directly to the reaction center chromophores of PSII. Because these
chromophores are not in energetic equilibrium with CP43 and CP47,
changes in spillover from CP47 to PSI will affect the distribution of
Chl a-absorbed excitation energy much more than the
distribution of PBS-absorbed excitation energy. Our model predicts that
relatively small movements of PSI with respect to the PBS/PSII complex
would be sufficient to change the distribution of both Chl
a- and PBS-absorbed energy. However, the model is also
consistent with the previously proposed larger scale changes in the
distribution of PBS/PSII complexes in the thylakoid membrane, from
ordered rows in state 1 to a more randomized pattern in state 2. Our
model is presented as a work in progress. We believe this model
currently offers the simplest explanation for our data and for most of
the data found in the literature on state transitions in cyanobacteria.
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MATERIALS AND METHODS |
Cyanobacteria Used and Culture Conditions
Synechococcus sp. PCC 7002 wild-type and
apcD cells were a generous gift from Don
Bryant (Pennsylvania State University, University Park).
Synechocystis sp. PCC 6803 wild-type and
rpaC cells were a generous gift from
Conrad W. Mullineaux (University College, London).
The wild-type and apcD cells of
Synechococcus sp. PCC 7002 were grown
photo-autotrophically in 100-mL batch cultures at 32°C in A+ growth
medium, containing vitamin B12, at a light intensity of 50 µmol m2 s1. Batch cultures were bubbled
with air. All cultures of Synechocystis sp. PCC 6803 were grown under similar conditions with the exception of light
intensity and growth medium. In this case, BG11 growth media with 10 mM NaHCO3 and 10 mM TES was used to
grow both wild-type and mutant cells photo-autotrophically at a light
intensity of 25 µmol m2 s1. For all
experiments, cells were harvested at mid- to mid-late exponential
growth phase.
Room Temperature Fluorescence
A 1-cm optical path fluorescence cuvette was filled (3 mL) with
cells at a Chl a concentration of approximately 10 µg
Chl mL1 and was dark adapted to state 2 (Rouag and
Dominy, 1994). The dark adaptation drives the transition to state 2 as
a result of a dark reduction of the plastoquinone pool (Mullineaux and
Allen, 1990). The cuvette was placed in the PAM Chl fluorometer (model PAM 101, H. Walz, Effeltrich, Germany) and variable fluorescence measured for a dark interval followed by exposure to blue light (425-nm
interference filter, 10-nm bandpass, 30 µmol m2
s1) used to drive the cells to state 1. During the course
of the experiment, cells were exposed to saturating 600-ms flashes of white actinic light (150-W tungsten halogen, approximately 10,000 µmol m2 s1) spaced 80 s apart, which
were used to determine the Fm.
77K Fluorescence Emission Spectra
Samples were assayed for 77K fluorescence emission with a
purpose-built fluorescence spectrophotometer previously
described by Salehian and Bruce (1992).
Samples for analysis were prepared in the following way. About 100 mL
of cells were kept in a stirred flask in their respective growth medium
at a Chl a concentration of 10 µg Chl
mL1 and illuminated with room light (approximately 25 µmol m2 s1). A small volume of cells (35 µL) was taken from this flask and placed in a Pasteur pipette with a
heat-sealed end. The cells in the Pasteur pipette were then immediately
exposed to a particular light wavelength, intensity, and duration
(details dependent on the experiment), quickly frozen by plunging into
liquid nitrogen, and kept in liquid nitrogen until emission spectra
were collected.
77K fluorescence emission spectra were collected for every sample for
two different excitation wavelengths: 435 nm to excite Chl
a and 580 nm to excite the PBS. The intensity of
fluorescence emission at 695 nm (F695), arising from the
long-wavelength Chl of the PSII core antenna polypeptide CP47 of PSII,
was divided by the intensity of emission at 715 nm (F715)
in Synechococcus sp. or at 725 nm (F725) in
Synechocystis sp., arising from the long-wavelength Chl
of PSI. F695/F715 and
F695/F725 were used as relative indicators of
the distribution of excitation energy between PSII and PSI, and,
therefore, the "state" of the cells in Synechococcus
sp. and Synechocystis sp., respectively.
Action Spectra for the State Transition-Induced Changes in 77K
Fluorescence Emission
To generate an action spectrum for the state transition, 14 samples were illuminated with 14 different wavelengths selected between
420 and 680 nm with a 75-W Xenon Arc lamp dispersed by a 0.25-m
monochromator (Sciencetech, London, Canada). Each 35-µL sample was
illuminated at room temperature in a Pasteur pipette as described in
the previous section for 90 s at one wavelength and then quickly
frozen in liquid nitrogen. The bandwidth for all illumination
wavelengths was about 6 nm and the intensity of the illumination was 30 µmol m2 s1. For each illumination
wavelength, three repeats using independent samples of cyanobacteria
from each of the four strains were used. As described above, 77K
emission spectra were obtained for excitation at both 435 nm (Chl
a) and 580 nm (PBS) for each frozen sample and the
F695/F715 or F695/F725,
depending on species, were calculated for each spectrum. The average
ratio for each room temperature illumination wavelength was determined
for the three repeats and plotted to give two action spectra (one for
435-nm excitation, the other for 580-nm excitation) for each of the
four sample types investigated. The action spectra show the resulting
F695/F715 (for Synechococcus sp.
PCC 7002 wild type and mutant) or F695/F725 (for Synechocystis sp. PCC 6803 wild type and mutant) as
a function of the room temperature illumination wavelength chosen to
drive the state transition. A high ratio shows a transition to state 1, and a low ratio shows a transition to state 2.
Absorbance Cross Sections
PSII absorbance cross sections were determined from flash
saturation curves generated with a pump probe fluorescence spectrometer as described by Samson and Bruce (1995). Cross sections were determined for excitation of Chl a at 674 nm and for excitation of
the PBS at 630 nm. Flash saturation curves were fit with a single-hit Poisson distribution as described by Mauzerall and Greenbaum
(1989).
Distribution of Materials
Upon request, all novel materials described in this publication
will be made available in a timely manner for noncommercial research
purposes, subject to the requisite permission from any third party
owners of all or parts of the material. Obtaining any permissions will
be the responsibility of the requestor.
Received June 11, 2002; returned for revision July 17, 2002; accepted July 30, 2002.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.009845.
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ABSTRACT
INTRODUCTION