©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Oscillations of Reaction Center II-D1 Protein Degradation in Vivo Induced by Repetitive Light Flashes
CORRELATION BETWEEN THE LEVEL OF RCII-Q(B) AND PROTEIN DEGRADATION IN LOW LIGHT (*)

(Received for publication, June 30, 1994; and in revised form, October 17, 1994)

Nir Keren Huashi Gong (1)(§) Itzhak Ohad (¶)

From the Department of Biological Chemistry, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Department of Biology, University of Oslo, Blindern, 0316 Oslo 3, Norway

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 times 10^3 µmol m s). Relative high levels of reduced RCII secondary plastoquinone acceptor, Q(B), are induced in cells exposed to low PFDs as determined by thermoluminescence measurements. The probability of generating elevated levels of Q(B) 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(B) 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(B). D1 protein degradation could be induced in dark-incubated cells exposed to a series of 1.4 times 10^3 single light flashes given at intervals compatible with generation of elevated levels of Q(B) and its decay by charge recombination. Oscillations of the Q(B) 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(B) oscillations are abolished. We propose that the process of recombination of long lived RCII-Q(B) with the S states may involve damaging events related to the D1 protein degradation induced by light flashes or continuous low light in vivo.


INTRODUCTION

The photoinactivation of photosystem II and the degradation of the D1 protein subunit of its reaction center (RCII) (^1)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 DeltapH 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(z) 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(A), the first quinone acceptor, to the secondary quinone Q(B), followed by double reduction of Q(A), 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 ^3P, 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(A)/Q(B) electron flow due to replacements of single amino acids in the Q(B) 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(B) increases as compared to that of other intermediate steps in RCII electron flow (less than milliseconds). Q(B) 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(B) 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). ^3Chl may interact with oxygen forming harmful singlet oxygen (Hideg et al., 1994). Here we propose that accumulation of RCII-Q(B) 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(B) 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(B) 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(B)/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(B) and the S states induces triggering of the D1 protein for degradation remains to be established.


MATERIALS AND METHODS

Cell Growth and Exposure to Different PFDs

Chlamydomonas reinhardtii y-1 and Scenedesmus obliquus wt and LF-1 mutant cells were grown as described before in a mineral medium containing acetate or glucose as carbon source, respectively (Ohad et al., 1967; Gong and Ohad, 1991). The LF-1 Scenedesmus mutant is impaired in the donor side activity of photosystem II (Diner et al., 1988). The mutant is highly light-sensitive and RCII is inactivated in light-grown cells. To maintain equal growth conditions, both the wt and LF-1 mutant were thus grown in the dark. Dark-grown wt and LF-1 Scenedesmus cells contain normally developed thylakoids and wt cells exhibit normal levels of photosynthetic activity when transferred to the light. Photosystem II is also present and functional in the dark-grown LF-1 mutant as measured with an artificial electron donor. Cultures were harvested in the exponential phase of growth. Cell suspensions in fresh growth medium (30 µg of chlorophyll ml) were exposed to various PFDs provided by white fluorescent (0-100 µmol m s) or tungsten halogen lamps (200-3000 µmol m s) filtered through a water-cooled glass container (Gong and Ohad, 1991). Chloramphenicol (200 µg mlD-threo form, Sigma) was added to prevent chloroplast protein synthesis and D1 protein replacement (Schuster et al., 1988). The rate of the D1 protein degradation is not affected by chloramphenicol in Chlamydomonas (Schuster et al., 1988; Zer et al., 1994).

Thermoluminescence Measurements

Thermoluminescence was measured using a home-made, computer-controlled apparatus (Zer et al., 1994). Excitation was provided by continuous illumination (up to 250 µmol m s) or short (3 µs) light flashes delivered by a xenon discharge lamp (0.3 joules/flash) placed 1 cm above the sample. The light emitted due to charge recombination was measured by photon counting.

Flash-induced Degradation of the D1 and D2 Proteins

Cell suspensions as above were incubated in complete darkness in a covered glass Petri dish mounted on an aluminum plate maintained at temperatures as indicated, by water circulation. The cell suspension (0.5 cm depth) was stirred by a magnetic bar. The cells were first dark-adapted for 5 min and then exposed to short duration light flashes (about 6 µs for 66% discharge) delivered by an EG& FE-132 xenon arc lamp connected to a 2.6-microfarad capacitor charged at 1.5 kV and placed 3 cm above the cell suspension, which was protected from UV light by a broad band pass filter (380 nm to 620 nm transmission). The cell suspension was exposed to 960-1440 series of single or multiple flashes (up to 6 in a series, spaced at 300-ms intervals within a multiple flash series). Dark periods equivalent to or longer than the t of the charge recombination time for the Q(B)/S states were interspersed between the light flash series as indicated in the legends to figures. At the end of the flashing regime (4-8 h), the cells were rapidly cooled in ice and thylakoid membranes were prepared as described below. As a control for each experiment, similar cell suspensions were incubated at the same temperature in the dark or exposed to continuous white fluorescent light (low light, 25 µmol m s, optical path, 2 cm) for the same time duration.

Assay of D1 and D2 Proteins

Thylakoid membranes were prepared as described previously (Gong and Ohad, 1991), and all steps were carried out in the cold. The cells were washed by centrifugation in 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl(2) and 10 mM NaCl. The cell pellet was resuspended in 5 ml of the same buffer and broken by passing through an ice-cooled French pressure cell operated at 5000 p.s.i. The homogenate was diluted 10-fold in the same buffer, and cell debris and unbroken cells were removed by centrifugation for 2 min at 2000 times g. Thylakoid-enriched membrane fractions were isolated by differential centrifugation at 10,000 times g for 20 min and were either used immediately or stored at -80 °C until use. The D1 and D2 proteins were detected and quantified by immunoblotting using I-protein A as described by Schuster et al. (1988). Monospecific antibodies against the D2 protein were kindly donated by R. Berzborn (Geiger et al., 1987), and those against the D1 protein were prepared by overexpressing the corresponding gene using a plasmid kindly donated by J. Barber as described by Adir and Ohad(1988). Thylakoid membrane samples on equal chlorophyll content basis were loaded on the gels. The membrane proteins were resolved by SDS-polyacrylamide gel electrophoresis as described (Laemmli, 1970) and were electrotransferred to nitrocellulose paper. The transfer efficiency was assayed by Ponceau red staining of the paper and was found to be linear with the amount of protein in a range equivalent with 0.5-1.5 µg of Chl. The autoradiograms were exposed for various times to obtain a linear response of the photographic film and were quantified by computer-programmed densitometric scanning using NIH Image programs.


RESULTS

Degradation of the RCII-D1 Protein as a Function of Light Intensity

The D1 protein degradation in Chlamydomonas cells incubated in presence of chloramphenicol was linear with time for a period of up to 4 h for PFDs from 10 to 2000 µmol m s (data not shown). Linearity of D1 protein degradation with time for similar duration at high light intensity has been demonstrated in vivo in other organisms (N. Ohad et al., 1990a; Schnettger et al., 1994). The remaining amounts of D1 protein after 4 h of exposure of Chlamydomonas cells to various light intensities are shown in Fig. 1. Degradation of D1 protein was detected in cells exposed to all light intensities. The degradation increases with increasing light intensity. No significant degradation was observed for the D2 protein in cells exposed to the low range of light intensities (data not shown). However, degradation of the D2 protein in Chlamydomonas occurs at high light intensities, although at rates significantly lower than that of the D1 protein (Schuster et al., 1988).


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 10^3 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).

Low Light Intensity Induces Relatively High Levels of Q(B) in Vivo

The photoinactivation and irreversible damage preceding the degradation of the RCII-D1 protein is ascribed to generation of harmful cation or anion radicals within RCII (Prasil et al., 1992; Aro et al., 1993). However, in low light-exposed cells only Q(B) and the oxidative S(1) to S(3) states of RCII can be assumed to persist for a long time relative to the lifetimes of the other intermediate steps in RCII electron flow (Vass et al., 1981; Rutherford, 1989; Rutherford et al., 1982; Ohad et al., 1988). At light intensities saturating electron flow, the relative steady state level of Q(B) should reach a maximum of 50% and a high probability exists for its further reduction to Q(B), protonation to PQH(2) and exchange with a PQ molecule from the plastoquinone pool (forward electron flow). The relation between the forward and back electron flow within RCII are schematically represented in Fig. 3. At low light intensities the probability of an RCII in the Q(B) state to be excited a second time before charge recombination with the S states by back electron flow is expected to be significantly lower as compared to light intensities saturating electron flow. Under such conditions charge recombination and possible harmful effects related to this process may prevail relative to forward electron flow. The question thus arises as to whether the relative steady state level of RCII-Q(B) at low light intensities correlates with observed degradation of D1 protein.


Figure 3: Schematic representation of the S states and Q(B) reduction cycle as a function of consecutive light excitations of RCII. Following light excitation by consecutive absorption of photons, Q(B), the secondary RCII-quinone acceptor, is reduced to Q(B) while the water splitting manganese complex (``S'') is oxidized (S(0) to S(4)). Q(B) is protonated and exchanged with plastoquinone from the PQ pool (S



Thermoluminescence measurements of the RCII-Q(B) 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(B) is induced at light intensities much below saturation of photosynthetic electron flow. The apparent quantum yield of RCII-Q(B) accumulation (Fig. 4B) shows a transient increase occurring at a light intensity in the range of 0.5-1% of that at which Q(B) reaches the maximal steady state level (Fig. 4B, arrow). In this range of light intensity, it is probable that the dissipation of the Q(B) 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(B) 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(B) as well. The steady state level of Q(B) 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(B) comparable to those induced by similar low intensities of white light.


Figure 4: Steady state level of Q(B) (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(B) 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(B) reaches the steady state level.



Correlation between the Q(B) Level Induced by Series of Single Light Flashes and the Degradation of D1 Protein in Vivo

The information so far obtained indicates that a significant level of RCII in the Q(B) state which may recombine with the S states can be accumulated in vivo in low light-exposed cells. Q(B) may also be transiently accumulated in dark-incubated cells exposed to series of single saturating light flashes given at time intervals sufficiently long (seconds) to allow its decay by charge recombination. The flash discharge must be of sufficient short duration (<10 µs) to avoid two consecutive charge separation events in the same RCII and thus minimize further reduction of Q(B) to plastoquinol. To test whether a correlation can be found between formation of Q(B) and degradation of the D1 protein, cells were incubated in the dark at 25 °C and exposed to 1440 series of 1 flash given at time intervals of 10 s, thus allowing decay of the Q(B) by charge recombination. Chloramphenicol was added to prevent de novo synthesis of D1 protein. As a control cells were exposed for a similar period of time (4 h) to low light (25 µmol m s) or were incubated in the dark. The remaining amount of the D1 protein was assayed by immunoblotting. The results of such experiments (Fig. 5) show that 1440 flashes induce 30-40% loss of the D1 protein as compared to only about 25% in cells exposed for 4 h to low light as also shown in Fig. 2. Variable loss of D1 protein (20%) also occurred in the dark-incubated cells. The degradation of D1 protein induced by series of 1 flash and normalized to the dark control in each experiment (average of five independent experiments) was found to be 24 ± 5% in cells exposed to light flashes as compared to 7 ± 5% in cells incubated in low light (Fig. 5). Thus, a 3-fold higher degradation of the D1 protein is induced in cells in which relatively high amounts of Q(B) are accumulated following exposure to light flashes that do not result in forward electron flow as compared to cells exposed to continuous low light supporting low rates of photosynthetic electron flow.


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.



Correlation between Oscillation of D1 Protein Degradation and Q(B) Levels Induced by Series of Multiple Flashes

To further test whether the level of the RCII-Q(B) is related to the degradation of the D1 protein, we took advantage of the fact that the Q(B) level oscillate with the number of flashes (Rutherford et al., 1982; Ohad et al., 1988; Vass et al., 1992a). If our hypothesis is correct, we would expect that the amount of degraded D1 protein will oscillate in direct relation to the oscillation of the Q(B) level. To test if such a correlation exists, several factors have to be considered. The steady state level of Q(B) generated by a given flash regime depends on: 1) the initial Q(B)/Q(B) ratio, 2) the S state distribution in the RCII population of the sample at the onset of the flashing regime, and 3) the rate of charge recombination, i.e. the lifetime of the Q(B) and the S states, which is temperature-dependent. We have thus determined by thermoluminescence measurements (Demeter and Govindjee, 1989) the t values for Q(B)/S charge recombination as a function of temperature for the C. reinhardtii cells. The results (Fig. 6) show that the lifetime decreases exponentially with increasing temperature, being in the range of less than 2 s at growth temperature (25 °C) and about 35 s at 5 °C. For comparison, the t values for the Q(A)/S charge recombination measured in presence of the photosystem II inhibitor Diuron to prevent electron transfer from Q(A) to Q(B) are also given (Fig. 6).


Figure 6: Temperature dependence of t for Q(B) and Q(A) 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(B)/S states was recorded. The Q(A) 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(A) and Q(B), respectively; vertical lines represent TL emission peak temperature for the recombination of Q(A) (Q(A) band) and Q(B) (Q(B) 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(A)/S states level at temperatures above 5 °C.



The relative level of Q(B)in vivo for a specific flash regime can be simulated on the basis of: 1) a 1/1 ratio of Q(B)/Q(B) and 1/3 ratio for S(0)/S(1) 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(B) and the S(2) or S(3) states (Fig. 3); 3) the fraction of Q(B) decay by back reaction (recombination) at the time of flashing. As stated above, the level of remaining Q(B) 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(B) (double hits), the simulation predicts that different steady state levels of Q(B) 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(B) 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(B) 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(B) are predicted, respectively. Furthermore, 40% and 28% of the total Q(B) population may recombine with the S(2) or S(3) 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(1) and S(0) states that cannot recombine with Q(B)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(B) oscillation, is expected especially after numerous series of multiple flashes. Despite the intrinsic difficulty in obtaining synchrony in the Q(B) 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(B) decay is about 30 s (Fig. 6). Under these conditions the decay of Q(B) in the time interval between the flashes in a series (300 ms) is also minimized, thus partially reducing ``scrambling'' of Q(B):Q(B)/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(B) levels in cells exposed to series of 1-3 consecutive light flashes.

As mentioned above the flash-induced oscillations of Q(B)/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(B)/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(B) 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(B)/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(B) 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(B) level and the degradation of D1 protein. Lower panel, predicted oscillation in the relative level of the Q(B) 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(B) 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.



D1 Protein Degradation Does Not Oscillate in Cells in Which the Photosystem II Donor Side Activity Is Impaired

As an additional test for the validity of the working hypothesis presented here, we have measured the effect of series of light flashes on the degradation of the D1 protein in wt Scenedesmus cells as well as the LF-1 mutant lacking the manganese cluster of the donor side of photosystem II (Diner et al., 1988). The wt cells are expected to show the same pattern of D1 protein degradation and oscillation of the Q(B) level as the Chlamydomonas cells. However, a different situation is expected to occur in the LF-1 mutant. In absence of an active manganese cluster and water oxidation activity, single or multiple flashes may not induce oscillation of the Q(B) level as in control, wt cells. Results of experiments in which wt and LF-1 Scenedesmus mutant were exposed to low light or 960 series of 1 or 2 flashes are shown in Fig. 9. We have earlier observed that wild type Scenedesmus cells are less sensitive to light as compared with Chlamydomonas (Gong and Ohad, 1992). However, as expected for a donor side-inactivated mutant, the degradation of the D1 protein induced by low light is more pronounced in the LF-1 mutant as compared with the wt Scenedesmus cells.


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).


DISCUSSION

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(B) which may recombine with the S states of the oxygen evolving complex. The total light energy delivered by about 10^3 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(B) may be higher than that of the low light, which permits a relatively significant proportion of the Q(B) 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(B) is dissipated by double reduction, formation of PQH(2), 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(A) before photoinactivation of Q(A)/Q(B) electron flow may also have a low quantum yield. Loss of Q(A) (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(B)/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(B) 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(B) to form Q(B) and the oxidized P is reduced by extracting an electron from the RCII-Y(z) donor (tyrosine 161 residue of the D1 protein) generating the cation radical Y(z). Back electron flow may occur from Q(B) to Y(z). However, in mutant cells exposed to series of 2 consecutive flashes, Q(B) may be doubly reduced by 2-electron transfer from P and Y(z), respectively, and exchange with an oxidized PQ molecule from the plastoquinone pool. However, both Y(z) 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(z) 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(B)/Q(B) 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(B) 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), ^3P may be generated. Triplet P could be formed also during charge recombination between S/Q(B), which may occur via intermediate regeneration of the radical pair P/pheophytin (Levanon and Norris, 1982; van Gorkom, 1985). In this case, ^3Chl formation will occur with a high probability due to spin uncoupling during generation of the S(n)/Q(B) states (Volk et al., 1993). ^3Chl 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(3)/SY(z)/Y(z):P/P may occur (van Gorkom, 1985) and damage of RCII may be due only to the transient presence of the cation radicals Y(z) or P. However, this equilibrium is also generated in the cells exposed to series of more flashes irrespective of whether Q(B) is reduced to the semiquinone or quinol state. Thus, if Y(z) and P generated by the above equilibrium were the damaging species in the normal reaction centers in which Q(B)/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(B) 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(2) and S(3) 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 ^3Chl during Q(B)/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(B) have been induced by flashes or low light. This hypothesis predicts involvement of ^1O(2) in the process and thus can be tested by performing similar experiments under aerobic or anaerobic conditions. Recently, it was demonstrated that generation of ^1O(2) 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(B) 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(B) 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 ^3Chl (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(B) prevailing in cells exposed to low light promotes the D1 protein degradation and its replacement (turnover) in vivo.


FOOTNOTES

*
This work was supported by a grant (to I. O.) from the Germany-Israel Foundation in cooperation with D. Godde, Bochum; a grant from the Deutchesforschungsbereiche (to I. O.) in cooperation with R. Herrmann and W. Rudiger, München; a grant from the Binational Agricultural Research and Development Fund in cooperation with H. Pakrasi, St. Louis; and a grant (to I. O.) from the Israeli Academy of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a grant from the Norwegian Research Council.

To whom reprint requests should be addressed.

(^1)
The abbreviations and trivial names used are: RCII, reaction center II; Chl, chlorophyll; Diuron, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; Q(A) and Q(B), the primary and secondary quinone electron acceptors of reaction center II, respectively; ^3P, triplet state of the primary electron donor chlorophyll of RCII; TL, thermoluminescence; PFD, photon flux density; PQ and PQH2, plastoquinone and plastoquinol, respectively; P and P, the reduced and oxidized form of the primary electron donor of RCII, respectively; Y(z), the oxidized form of the electron donor to P, the Tyr-161 amino acid residue of the D1 protein; wt, wild type.


ACKNOWLEDGEMENTS

The construction of the TL apparatus used in this work is based on the TL equipment developed at the Solar Energy Research Group, The Institute of Physical and Chemical Research, Riken, Wako, Saitama, Japan, and the Institute of Plant Physiology, Biological Research Center, Szeged, Hungary. We are grateful to S. Malkin, The Weitzmann Institute of Science, Rehovot, Israel for providing the high light intensity flash apparatus and to H. Pakrasi for reading the revised version of this manuscript.


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