(Received for publication, June 30, 1994; and in revised form, October 17, 1994)
From the
The D1 protein subunit of the photochemical reaction center II
(RCII) turns over rapidly in oxygenic photosynthetic organisms exposed
to the light. At high photon flux densities (PFD), photoinactivation of
RCII precedes the degradation of the D1 protein. We found that the
apparent quantum yield for the D1 protein degradation in Chlamydomonas cells is severalfold higher at low PFDs
(10-100 µmol m s
) as
compared to that observed at PFDs which induce photoinactivation of
RCII (1.5-3
10
µmol m
s
). Relative high levels of reduced RCII
secondary plastoquinone acceptor, Q
, are
induced in cells exposed to low PFDs as determined by
thermoluminescence measurements. The probability of generating elevated
levels of Q
which may recombine with the
S
oxidized states of the oxygen evolving complex
decreases with increase in the light intensities at which consecutive
double reduction of Q
and exchange with the plastoquinone
pool prevail. We have used light flashes to test if a correlation
exists between the degradation of D1 protein and the relative level of
Q
. D1 protein degradation could be
induced in dark-incubated cells exposed to a series of 1.4
10
single light flashes given at intervals compatible with
generation of elevated levels of Q
and
its decay by charge recombination. Oscillations of the
Q
level in cells exposed to 960-1440
series of 1 to several flashes correlated with oscillations of the D1
protein degradation in Chlamydomonas cells and in the Scenedesmus wild type but not in the LF-1 mutant lacking
photosystem II donor side activity. In this mutant the ``S state
cycle'' and Q
oscillations are
abolished. We propose that the process of recombination of long lived
RCII-Q
with the S
states
may involve damaging events related to the D1 protein degradation
induced by light flashes or continuous low light in vivo.
The photoinactivation of photosystem II and the degradation of
the D1 protein subunit of its reaction center (RCII) ()have
been extensively studied in photosynthetic oxygen evolving organisms
exposed to light intensities higher than those required for saturation
of photosynthetic electron flow (reviewed by Prasil et
al.(1992), Barber and Andersson (1992), Aro et al.(1993),
and Ohad et al. (1994)). It is now well established that
photoinactivation induces irreversible changes in RCII-D1 targeting the
D1 protein for degradation both, in vitro (Aro et
al., 1990) and in vivo (Ohad et al., 1990b;
Kirilovsky et al., 1990; Zer et al., 1994).
Two
mechanisms have been proposed to explain the degradation of the D1
protein induced by high light. Generation of high pH across the
thylakoid membrane and acidification of the thylakoid lumen may lead to
release of Ca
from the oxygen evolving complex and
inactivate the photosystem II donor side (Ono and Inoue, 1989; Krieger et al., 1992; Krieger and Weis, 1993). Under such conditions
oxidizing cation radicals, Y
and
P
may be formed within RCII with a high
quantum yield (Eckert et al., 1991), which may induce
irreversible modifications of its protein subunit(s) and thus target
the D1 protein for degradation. Alternatively, alteration of the RCII
acceptor side activity may lead to a block in the electron transfer
from Q
, the first quinone acceptor, to
the secondary quinone Q
, followed by double reduction of
Q
, and eventually its release from the binding site located
within the D2 protein subunit of RCII (Andersson and Franzen, 1992).
This may in turn cause charge recombination of the RCII radical pair
P
/pheophytin
,
resulting in formation of
P
, which may
interact with oxygen and generate harmful singlet oxygen (Van Mieghem et al., 1989; Durrant et al., 1990; Vass et
al., 1992b; Hideg et al., 1994). Accelerated
light-dependent degradation of the D1 protein in cyanobacterial mutant
cells defective in Q
/Q
electron flow due to replacements of single amino acids in the
Q
binding niche of the D1 protein
indicates that this mechanism may operate in vivo (N. Ohad et al., 1990a; Perewoska et al., 1994). Mutants
impaired in the photosystem II donor side activity exhibit high
sensitivity to light (Mayes et al., 1991; Burnap et
al., 1992, Rova et al., 1994) and enhanced degradation of
the RCII-D1 protein as compared to the wild type cells (Gong and Ohad,
1991).
The mechanisms of acceptor or donor side inactivation are mutually exclusive and account for the intermediary steps in RCII inactivation preceding D1 protein degradation and occurring only at high light intensities (Ohad et al., 1994). However, turnover of the RCII-D1 protein occurs also in vivo at light intensities well below those inducing photoinactivation (Greenberg et al., 1987). Thus the mechanisms based on the donor or acceptor side photoinactivation preceding the degradation of the D1 protein do not apply in this case, since no detectable loss of photosystem II activity can be measured unless de novo synthesis of D1 protein is inhibited, thus preventing replacement of the degraded protein.
Most of the studies of D1 protein turnover in vivo are based on radioactive pulse-chase experiments (Greenberg et al., 1987; Jansen et al., 1993; Sundby et al., 1993; Aro et al., 1994). Measurements of the rate of D1 protein degradation relative to light intensity over a wide range of PFDs and assayed by quantitative immunodetection of the remaining total D1 protein are not available for the same organism under standard experimental conditions. When such experiments were carried out in this work, low light was unexpectedly found to be more efficient in inducing D1 protein degradation as compared to high light.
At low light intensities the probability of accumulating long-lived
(seconds) RCII-Q increases as compared to
that of other intermediate steps in RCII electron flow (less than
milliseconds). Q
may decay by charge
recombination (back reaction) with the S
oxidized states
of the Mn cluster in the oxygen evolving complex (Rutherford, 1989).
Back reactions in photosystem II in low light-exposed chloroplasts have
been considered in the past to reduce photosynthetic efficiency (Radmer
and Kok, 1977). In the process of back reaction from
Q
to the S
states, triplet
Chl may be generated during the transient formation of the radical pair
P
/pheophytin
(Van
Gorkom et al., 1986; Rutherford, 1989; Volk et al.,
1993).
Chl may interact with oxygen forming harmful singlet
oxygen (Hideg et al., 1994). Here we propose that accumulation
of RCII-Q
in low light-exposed thylakoids
may be related to the low light-induced rapid turnover of the D1
protein in vivo. To test this hypothesis advantage was taken
from the fact that oscillations in the Q
level may be induced by series of single or multiple light
flashes. We have thus exposed dark-incubated algal cells to series of
short duration light flashes and were able to demonstrate oscillations
of D1 protein degradation related to the oscillations of the
Q
level induced by the various flash
regimes. Thus a correlation is established between the degradation of
the D1 protein in vivo and the generation of
Q
/S
states induced by light
flashes or low light. This may explain the low light-induced
degradation and ensuing replacement (turnover) of the D1 protein in
vivo. The damaging mechanism whereby charge recombination between
Q
and the S
states induces
triggering of the D1 protein for degradation remains to be established.
Figure 1:
Degradation of D1 protein as a function
of light intensity. Chlamydomonas cells were exposed to
different PFDs indicated by numbers (µmol m s
) or incubated in the dark (D) for 4
h. The remaining amount of D1 was detected by immunoblotting. A, equal exposure of the autoradiograms; B, shorter
exposure of the low light intensity range to visualize better the
degradation of the D1 protein at low PFDs. Separate cultures were used
to cover the low, medium, and high light intensity range, respectively.
Photosynthetic electron flow measured as oxygen evolution saturated at
about 10
µmol m
s
.
Densitometric measurements of the residual D1 protein level in
thylakoids of cells exposed to increasing light intensities show a
non-linear increase in the degradation with increase in light intensity (Fig. 2A). Only a small additional loss of D1 protein
was observed in cells exposed to a light intensity of 3000 µmol
m s
as compared to 2000 µmol
m
s
. This could be partially due
to the saturation of the degradation system activity (N. Ohad et
al., 1990a; Sundby et al., 1993).
Figure 2: Apparent quantum yield of D1 protein degradation at different light intensities. A, degradation of the D1 protein as a function of light intensity. Densitometric quantification, average of several independent electrophoretic separations, and immunoblotting of samples from the experiments presented in Fig. 1have been used to calculate the remaining amount of D1 protein in cells exposed to different light intensities for 4 h. B, relation between the ratio of D1 protein degraded/incident light intensity as a function of light intensity (apparent quantum yield); insets, expanded scale to show the effect at low PFDs. Data are normalized to the D1 protein level before onset of exposure to various light intensities (0). Arrow (A) indicates the light intensity at which electron flow measured as oxygen evolution was saturated under the experimental conditions used.
When the efficiency of D1 degradation relative to the light intensity was plotted against light intensity using the above data (Fig. 2B), it became obvious that low light is highly efficient in inducing D1 protein degradation. The apparent quantum yield, defined as D1 protein degradation/incident light intensity plotted against the light intensity, shows an unexpected steep decline with increasing light intensity. The apparent quantum yield is 5-7-fold higher at low light intensity supporting less than 5% of the light saturated electron flow rate as compared with that observed at light intensities severalfold higher than that required for electron flow saturation (Fig. 2B).
Figure 3:
Schematic representation of the S states
and Q reduction cycle as a function of consecutive light
excitations of RCII. Following light excitation by consecutive
absorption of photons, Q
, the secondary RCII-quinone
acceptor, is reduced to Q
while the water
splitting manganese complex (``S'') is oxidized (S
to S
). Q
is protonated
and exchanged with plastoquinone from the PQ pool
(S
Thermoluminescence measurements of the
RCII-Q levels as a function of light
intensity in cells illuminated for 30 s and thus reaching a steady
state level of electron flow are shown in Fig. 4A. A
high relative level of Q
is induced at
light intensities much below saturation of photosynthetic electron
flow. The apparent quantum yield of RCII-Q
accumulation (Fig. 4B) shows a transient increase
occurring at a light intensity in the range of 0.5-1% of that at
which Q
reaches the maximal steady state
level (Fig. 4B, arrow). In this range of light
intensity, it is probable that the dissipation of the
Q
state occurs primarily by charge
recombination. At light intensities approaching saturation of electron
flow and beyond, a drastic decrease of the apparent quantum yield of
Q
steady state level is evident,
compatible with increase in the forward electron flow relative to the
back reaction. Since it has been reported previously (Greenberg et
al., 1989) that the D1 protein is degraded in Spirodela plants exposed to low light of different wavelengths, it was of
interest to test whether low intensity light of different qualities may
induce accumulation of high levels of Q
as well. The steady state level of Q
was thus determined as described above also in cell suspensions
exposed to light of various wavelengths. The results of such
experiments are included in Fig. 4and show that blue, green,
and red light induce levels of Q
comparable to those induced by similar low intensities of white
light.
Figure 4:
Steady state level of
Q (A) and apparent quantum yield
of its formation (B) at various light intensities. Chlamydomonas cell suspensions containing 60 µg of Chl
ml
were layered over a thin disc of lens cleaning
paper (Kodak) mounted on the thermoluminescence measuring stage (2 cm
diameter). The paper was used to ensure equal spreading of the cell
suspension (suspension depth, 1 mm). The cells were dark-adapted for 2
min and then exposed to continuous illumination at light intensities as
indicated for 30 s at 6 °C. The cells were then immediately frozen,
and the relative Q
content of the sample
was assayed by photon counting of the thermoluminescence emission.
Illumination was provided by a projector delivering 250 µmol
m
s
of unfiltered light.
Attenuation of light intensity to the desired levels was achieved by
appropriate neutral density filters. The TL signal intensity obtained
from a similar cell suspension dark-adapted for 3 min, then frozen in
the dark and illuminated at -20 °C for another 30 s (white
light, 100 µmol m
s
) was
taken as 100%. Open symbols, unfiltered light; closed
symbols, light filtered through interference filters (20-nm
half-bandwidth) as follows: square, 430 nm; circle,
490 nm; diamond, 560 nm; star, 604 nm; triangle, 640 nm. Arrow, light intensity at which the
level of Q
reaches the steady state
level.
Figure 5: Induction of D1 protein degradation by series of single light flashes. Chlamydomonas cell suspensions were incubated for 4 h at 25 °C in the dark, low light (LL), or in the dark but subjected to a series of 1440 single light flashes given at intervals of 10 s. A, immunoblots showing the remaining amount of D1 protein at the end of the incubation; B, densitometric measurement of the amount of D1 protein, average of two separate runs of the same experiment. 0, D1 protein level before the onset of the incubation.
Figure 6:
Temperature dependence of t for Q
and
Q
decay in the dark. Chlamydomonas cell suspensions were placed on the measuring stage of the
thermoluminescence apparatus as in Fig. 4and maintained at
temperatures as indicated. The suspensions were dark-adapted for 3 min
and then exposed to a single excitation flash. Incubation was continued
in the dark for various times as indicated up to 130 s, and then the
cells were rapidly frozen and the residual TL signal arising from
recombination of Q
/S
states
was recorded. The Q
signal was recorded
in the presence of 5 µM Diuron added before the dark
adaptation. Values of t
were calculated using
one- and two-exponent fits for Q
and
Q
, respectively; vertical lines represent TL emission peak temperature for the recombination of
Q
(Q
band) and
Q
(Q
band),
respectively (Rutherford et al., 1982). Due to limitations in
the cooling rate of the sample holder in our thermoluminescence
apparatus, we could not obtain values for the decay of the
Q
/S
states level at
temperatures above 5 °C.
The relative level of
Qin vivo for a specific flash
regime can be simulated on the basis of: 1) a 1/1 ratio
of Q
/Q
and 1/3 ratio for
S
/S
in dark-adapted thylakoids (Rutherford et al., 1982), as well as in intact cells (Ohad et
al., 1988); 2) back reaction (charge
recombination) occurring primarily between Q
and the S
or S
states (Fig. 3); 3) the fraction of Q
decay by back
reaction (recombination) at the time of flashing. As stated above, the
level of remaining Q
at the onset of a
flash series can be theoretically set by adjusting the temperature of
the cell suspension and the dark time lapse between the series of
flashes.
For ideal conditions, i.e. saturation by every
light flash of all RCII population in the sample and no double
reduction of Q (double hits), the simulation predicts that
different steady state levels of Q
related to the number of flashes in a series will be established after
about 50 series of flashes. For a dark time interval between the series
compatible with about 50% decay of Q
by
charge recombination, a 60% level is predicted immediately after the
last flash in each series for a regime of single or 5 consecutive
flashes in a series. However, no Q
will
be accumulated in cells exposed to regimes of 2 or 6 flashes in a
series. For regimes of 3 or 4 consecutive flashes in a series, 57% and
50% Q
are predicted, respectively.
Furthermore, 40% and 28% of the total Q
population may recombine with the S
or S
states in the samples exposed to 1 and 5 or 3 flashes,
respectively. The simulation also predicts the presence of
S
states at all even flash regimes with one
exception. After a series of 4 flashes, each center returns to its
initial condition before the flash series. Since only the S
and S
states that cannot recombine with
Q
are present after the dark
adaptation of the cell sample before onset of the flash regime, no
recombination is expected in cells exposed to series of 4 flashes.
Thus, taken together, recombination may not occur in cells exposed to
multiple series of 2, 4, and 6 flashes.
The predictions of the
simulation program are based on ideal conditions as described above.
However, these conditions cannot be met experimentally and a certain
degree of double hits and misses, which may cause scrambling of
Q oscillation, is expected especially
after numerous series of multiple flashes. Despite the intrinsic
difficulty in obtaining synchrony in the Q
level in a cell population exposed to several hundred flash
series, we have attempted to measure the oscillations of the D1 protein
degradation induced by 960 series of 1-6 flashes. We have reduced
the temperature of the cell suspension to 6 °C at which the t
of the Q
decay
is about 30 s (Fig. 6). Under these conditions the decay of
Q
in the time interval between the
flashes in a series (300 ms) is also minimized, thus partially reducing
``scrambling'' of
Q
:Q
/S state cycle.
Spacing the series of flashes at 30-s intervals permits prolonged exposure of the cells to the action of the damaging event after the flash has induced the charge separation and allows the degradation of the putative irreversibly triggered protein, which is a slow process (Schuster et al., 1988). The degradation process proper may be further slowed down at 6 °C. As an indication for the continuation of cell metabolism at this temperature, we have measured oxygen evolution activity in cells incubated at 6 °C. The results indicated that oxygen evolution persists at 6 °C and is about 5-10% of that at 25 °C. As a control, cells were also exposed to low light or darkness at the same temperature and for the time duration as that used for the 960-flash series (8 h).
The results of these experiments including examples of immunoblots are shown in Fig. 7. Significant D1 protein degradation occurred in cells exposed to series of 1 flash in agreement with the data shown in Fig. 5. Slightly less D1 protein degradation was obtained in cells exposed to series of 3 flashes as compared to that induced in cells exposed to series of a single flash.
Figure 7: Oscillation of the D1 protein degradation in dark-incubated Chlamydomonas cells induced by series of single or multiple light flashes. A, cell suspensions were incubated at 6 °C for 8 h in the dark (D), low light (LL), or in the dark but subjected to 960 series of 1-6 light flashes (1F-6F) with 30 s of dark interval between the flash series. The amount of remaining D1 protein was estimated by immunoblotting as described under ``Materials and Methods.'' T0, D1 protein level at time zero of the incubation. For each set of flashing regimes, a different culture was used. B, calculation of the amount of the remaining D1 protein based on densitometric measurements and averaging of three independent immuno-blots. The data are normalized to the D1 protein content at the time zero (T0) of the incubation.
Strikingly, virtually no
D1 protein degradation was observed in cells exposed to series of 2
consecutive flashes. Thus, a correlation could be established between
the oscillations of the D1 protein degradation and those of the
Q levels in cells exposed to series of
1-3 consecutive light flashes.
As mentioned above the
flash-induced oscillations of Q/S
are expected to be scrambled due to double hits and misses
(Burnap et al. 1992; Etienne and Kirilovsky, 1993; Meunier,
1993). Such a situation may occur to an increasing extent with the
increase in the number of flashes in a series. In such a case the D1
protein degradation may not correlate well with the number of flashes
in series. Indeed, this is the case in cells exposed to series of
4-6 flashes. In this case the D1 protein degradation data deviate
from those theoretically predicted by the simulation program. However,
it is noteworthy that the amount of D1 protein degraded was highest in
cells exposed to series of 1 flash as compared to no degradation in
cells exposed to twice the amount of energy. Also, no significant
increase in the D1 protein degradation was obtained in cells exposed to
up to 6-fold higher amounts of light energy (6 flashes as compared to 1
flash in a series). Thus, the degradation of the D1 protein in
flash-exposed cells does not correlate with the amount of total
absorbed energy, but rather with the level of
Q
/S
induced by the flash
regime.
Presently we have no direct measurements for the extent of
misses and double hits. A simulation of the
Q level using the computer program
mentioned above and taking into account various possible degrees of
double hits and misses is shown in Fig. 8. A good correlation
between the simulated oscillation of the
Q
/S
level and the measured
degradation of the D1 protein is evident for the cells exposed to
series of 1-4 flashes (correlation coefficient, r = 0.82). As the number of flashes in a series increase, the
correlation decreases as expected but is not abolished (r = 0.56 for series of 5 flashes using 3% double hits and 10%
misses). Even for the entire range of 1-6 flashes, the
correlation coefficient is 0.47 if one assumes that 3% double hits and
20% misses are generated by the flash regime. Thus, we interpret the
oscillation in the D1 protein degradation shown in Fig. 6Fig. 7Fig. 8as experimentally valid and
related to the levels of the RCII-Q
induced by the flash regime. No significant changes in the level
of the D2 protein have been observed in cells exposed to low light or 1
and 2 consecutive light flashes (data not shown).
Figure 8:
Flash-induced oscillations of the
Q level and the degradation of D1
protein. Lower panel, predicted oscillation in the relative
level of the Q
immediately after the last
flash in a series, which may recombine with the S
states,
calculated after 200 or more series of 1-6 flashes. The
calculations were obtained using the simulation program for an interval
between the series of flashes equivalent with 50%
Q
decay in the dark and possible amounts
of double hits and misses; squares, triangles, and open circles, simulation for 3% double hits and 5%, 10%, and
20% misses, respectively. Upper panel, ratio of D1 protein
degradation in cells exposed to series of flashes to that induced by
low light and normalized to the D1 protein level at the zero time of
the experiment (T0) using the same data as in Fig. 7; 1F-6F, number of flashes in a series. The correlation
coefficient for the observed oscillations of the D1 protein degradation
and that calculated for series of 1-4 flashes using 3% double
hits and 5% misses is 0.82.
Figure 9: D1 protein degradation does not oscillate with the number of flashes in Scenedesmus LF-1 mutant cells impaired in photosystem II donor side activity. Chlamydomonas y-1 and Scenedesmus wt and LF-1 mutant cell suspensions were incubated in the dark (D), low light (LL), or in the dark but subjected to 960 series of 1 or 2 light flashes (1F, 2F) given under the same experimental conditions as in Fig. 7. The remaining amount of the D1 protein was assayed by immunoblotting. Lower panel, immunoblot; T0, the level of D1 protein at time zero of the experiment. Upper panel, quantitative scanning (percent of D1 protein), relative to the dark control.
The D1 protein degradation induced by series of 1 flash is higher than that induced by series of 2 flashes in both Chlamydomonas and Scenedesmus wt cells, thus following the oscillation pattern described above (compare with Fig. 7). However, practically no degradation of the D1 protein occurs in the LF-1 mutant cells exposed to series of 1 flash, while significant degradation is induced by series of 2 flashes (Fig. 9).
Prompted by the molecular and physiological relevance of the process of photoinhibition to the dynamics of photosystem II as well as by the economical aspects of the damage to crops, extensive efforts have been devoted to the elucidation of its mechanism. In contrast, little is known about the mechanism of the low light-induced degradation of proteins in the process of the light-induced rapid D1 protein turnover (Greenberg et al., 1989; Prasil et al., 1992; Ohad et al., 1994). Most of the lower part of canopy of crops and forests as well as of the population of photosynthetic aquatic organisms is exposed daily to light intensities considerably lower than those saturating photosynthetic electron flow. Thus the physiological significance of the low light-induced degradation of the D1 protein and the need for its replacement to maintain photosynthetic activity appear to have been underestimated. The importance of the back reaction in photosystem II in low light-exposed plants to the overall photosynthetic capacity has been considered in the past (Raven and Beardall, 1982). In the present work we provide evidence in support of a hypothesis that explains the degradation of the RCII-D1 protein induced by low light as well as the new observation, namely that flash trains may induce oscillations in the D1 protein degradation process.
The data presented in this work
demonstrate a correlation between the high efficiency of D1 protein
degradation in low light-exposed cells and the generation of high
relative levels of Q which may recombine
with the S
states of the oxygen evolving complex. The
total light energy delivered by about 10
single light
flashes is equivalent to that delivered in about 3 s to the cells
exposed to continuous low light as used in this work. Thus it is clear
that in the cells exposed to light flashes an efficient damaging event
occurs inducing the observed loss of D1 protein. The hypothesis
presented here predicts that the apparent quantum efficiency of the
flash-induced degradation related to accumulation of
Q
may be higher than that of the low
light, which permits a relatively significant proportion of the
Q
to be further reduced to plastoquinol.
Following the same reasoning, low continuous light is considerably more
efficient in inducing the degradation of the D1 protein as compared
with light intensities saturating or exceeding saturation of electron
flow. With increasing light intensity an increasing fraction of the
reduced Q
is dissipated by double
reduction, formation of PQH
, and exchange with a PQ
molecule from the plastoquinone pool. As light intensity increases
beyond saturation of electron flow, RCII photoinactivation and the
related degradation of D1 can be induced via the acceptor or donor side
mechanisms (Vass et al., 1992b; Barber and Anderson, 1992; Aro et al., 1993; Ohad et al., 1994; Hideg et
al., 1994).
The relative low apparent quantum yield of D1
protein degradation induced by light intensities exceeding saturation
of electron flow may be related to the low frequency of charge
recombination when the forward electron flow prevails. The double
reduction of Q before photoinactivation of
Q
/Q
electron flow may also
have a low quantum yield. Loss of Q
(Vass et al.,
1992b; Koivuniemi et al., 1993) is a prerequisite for an
increase in charge recombination of the radical pair
P
/pheophytin
(Vass et al., 1992b). The decrease in the apparent quantum
efficiency of D1 protein degradation with increasing light intensity
may indeed be explained by such a gradual transition from the low light
to the photoinhibitory light-induced D1 protein degradation.
In this
work it is demonstrated for the first time that oscillations of the
Q/S states induced in cells exposed to
series of short light flashes correlate with oscillation in the D1
protein degradation. In evaluating the data presented in support of
this conclusion, one must keep in mind that the degradation of the D1
protein is a complex multistep process including 1) the
generation of the damaging factor/species; 2) the ensuing
irreversible modification of RCII triggering the D1 protein for
degradation, and 3) the actual cleavage of the protein.
Maintenance of synchronization of this multistep process in a cell
population for a prolonged time so as to detect the oscillation pattern
is bound to be limited, and damping of the degree of oscillation by
double hits and misses is inevitable (Meunier, 1993; Etienne and
Kirilovsky, 1993). The fact that oscillations could be detected in the
degradation of the D1 protein is therefore considered as evidence in
support of the proposed hypothesis.
Exposure of the cells to series of flashes did not induce significant degradation of the D2 protein, as is also the case in low light-exposed cells. Degradation of the D2 protein follows extensive damage and degradation of the D1 protein in cells exposed to high light generating photoinhibition (Schuster et al., 1988; Prasil et al., 1992).
The absence of
oscillations in the D1 protein degradation observed in the Scenedesmus LF-1 mutant and the increased degradation in cells
exposed to series of 2 flashes as compared to that generated by series
of a single flash is in agreement with the observed increased
sensitivity to light of cells in which the photosystem II donor side
activity is impaired (Seibert et al., 1989; Mayes et
al., 1991; Burnap et al., 1992; Rova et al.,
1994). In the dark-adapted LF-1 mutant cells, all of the RCII
population is expected to be in the oxidized Q state and no
S states are available. Repeated series of 4 electrons can be delivered
sequentially to the reducing side of RCII in wt cells exposed to
continuous light or to multiple series of flashes. However, only 2
electrons may be transferred to the RCII acceptor side in the LF-1
mutant. Following a single photon excitation of RCII, 1 electron is
transferred to Q
to form Q
and the oxidized P
is reduced by
extracting an electron from the RCII-Y
donor (tyrosine 161
residue of the D1 protein) generating the cation radical
Y
. Back electron flow may occur from
Q
to Y
.
However, in mutant cells exposed to series of 2 consecutive flashes,
Q
may be doubly reduced by 2-electron transfer from
P
and Y
, respectively, and exchange with an
oxidized PQ molecule from the plastoquinone pool. However, both Y
and P
will remain in the oxidized cation radical
state. Formation of such harmful cation radicals will occur in any
reaction center after two consecutive light excitations. In the LF-1
cells, the Y
and
P
cation radicals cannot be reduced by
the normal pathway of electron donation from the manganese cluster and
thus electrons may be taken by oxidizing surrounding RCII proteins or
pigments. In the LF-1 cells exposed to numerous series of either 1 or 2
flashes, there will be no synchronization of the
Q
/Q
cycle. However, the
chances for generating the cation radicals mentioned above will
increase with the number of flashes. Thus more damage and related
degradation of the D1 protein is expected in the LF-1 cells exposed to
series of 2 flashes as compared with those exposed to series of a
single flash. In this respect the results presented here open a new
avenue for the investigation of the processes leading to the
degradation of the D1 protein in donor-defective photosystem II by use
of light flashes.
The question arises as to what is the mechanism of
damage induced by the high relative levels of Q leading to D1 protein degradation. Several alternative
explanations for the observed flash-induced degradation of D1 protein
could be considered.
In the process of charge recombination between
radical pairs
(P/pheophytin
),
P
may be generated. Triplet P
could be formed also during charge recombination between
S
/Q
, which may occur via
intermediate regeneration of the radical pair
P
/pheophytin
(Levanon
and Norris, 1982; van Gorkom, 1985). In this case,
Chl
formation will occur with a high probability due to spin uncoupling
during generation of the S
/Q
states (Volk et al.,
1993).
Chl may interact with oxygen leading to formation of
singlet oxygen, which may be the major damaging species in the case of
low light-induced D1 protein degradation as in the case of acceptor
side photoinactivation (Hideg et al., 1994). It is noteworthy
that light-induced D1 protein degradation does not occur under
anaerobic conditions (Arntz and Trebst, 1986; Hundal et al.,
1990). Alternatively, during the process of charge recombination, one
could expect that the equilibrium
S
/S
Y
/Y
:P
/P
may occur (van Gorkom, 1985) and damage of RCII may be due only to the
transient presence of the cation radicals Y
or P
. However, this equilibrium is
also generated in the cells exposed to series of more flashes
irrespective of whether Q
is reduced to the semiquinone or
quinol state. Thus, if Y
and
P
generated by the above equilibrium
were the damaging species in the normal reaction centers in which
Q
/S states may oscillate, one would
expect a similar amount of damage and subsequent D1 protein degradation
in all flash regimes independent of the number of flashes in a series.
However, this was not the case, more D1 being degraded in cells exposed
to series of 1 flash than in cells exposed to 2 or more flashes.
The
possibility that the Q semiquinone
radical may interact directly with oxygen and induce damage to
photosystem II was proposed before (Kyle, 1987). Presently, no
experimental evidence in support of this hypothesis is available.
Finally, one should also consider that the level of S
and
S
states of the manganese cluster may cause oxidative
damage directly. However, as calculated by the simulation program used
in this work, the relative level of the S
states remains
constant at about 50-60% of the total RCII population in all
flash regimes used, irrespective of the degree of misses and/or double
hits. Therefore, the level of S
states may not explain
the observed oscillation of the flash-induced D1 protein degradation.
Among all the above mentioned possibilities, generation of
Chl during Q
/S
charge recombination seems to be the best candidate as the cause
of damage resulting in RCII-D1 protein degradation in cells in which a
high relative level of Q
have been
induced by flashes or low light. This hypothesis predicts involvement
of
O
in the process and thus can be tested by
performing similar experiments under aerobic or anaerobic conditions.
Recently, it was demonstrated that generation of
O
is indeed involved in the process of light-induced damage to
photosystem II (Hideg et al., 1994).
Based on the
experimental results presented above, we propose that a correlation
exists between generation of RCII in the Q state and the low light-induced degradation of the RCII-D1
protein. This proposal is supported by the oscillations of the D1
degradation induced by series of light flashes. It has been reported
previously (Greenberg et al., 1989) that rapid D1 protein
turnover in the aquatic Spirodela plants exposed to visible
low light intensities of different wavelengths as well as UV light does
not correspond to the rate of electron flow elicited by various light
qualities, and it was proposed that different photosensitizers may be
responsible for the degradation of the D1 protein. The results obtained
in this work indicate that the level of Q
induced by visible low light of different wavelengths may explain
the reported action spectrum for the low PFD-induced D1 degradation.
Further experiments can be devised to test the working hypothesis
presented in this work. Measurements of the Chl (Vass et al., 1992b), which may be generated by single or
consecutive flashes in dark-incubated anaerobic cells, and the effect
of oxygen on the flash-induced D1 protein degradation may provide
additional information on the nature of this process. Characterization
of the nature of the damage induced to the various components of
photosystem II is now in progress. The results of such experiments may
contribute toward the elucidation of the mechanism whereby the
RCII-Q
prevailing in cells exposed to low light promotes
the D1 protein degradation and its replacement (turnover) in
vivo.