Plant Physiol. (1998) 116: 1053-1061

Heterogeneity and Photoinhibition of Photosystem II Studied with Thermoluminescence¹

Simone Andrée, Engelbert Weis, and Anja Krieger^{2, *}

Institut für Botanik, Universität Münster, Schlossgarten 3, 48149 Münster, Germany (S.A., E.W.); and Lehrstuhl für Botanik I, Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Mittlerer Dallenbergweg 64, 97082 Würzburg, Germany (A.K.)

	ABSTRACT

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Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone Q_B with the S₂ state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S₂Q_A recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50°C in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a Tyr_D⁺-Q_A recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42°C. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52°C) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45°C (C-band) slightly increased. It is proposed that the 42 to 52°C band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII Q_B-nonreducing centers.

	INTRODUCTION

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There exists a lateral heterogeneity in the distribution of thylakoid protein complexes (Anderson and Andersson, 1988; Albertsson et al., 1990). It is known that all PSI complexes and ATP synthases are located only in the nonappressed membrane domains. The Cyt b₆/f complex occurs in both membrane domains. Most PSII centers are located in the grana stacks, but a minor population is located in the stroma-exposed thylakoid region. Additionally, there exists a functional heterogeneity among PSII centers that is correlated with their localization.

Based on PSII heterogeneity with respect to fluorescence induction, the concept of PSII and PSII has been introduced (Melis and Homann, 1976; for review, see Black et al., 1986; Lavergne and Briantais, 1996). PSII, which is PSII associated with the light-harvesting complex, is located in the grana stacks and represents the active state of PSII. PSII located in the nonappressed thylakoids has a smaller antenna than granal PSII, since it requires higher light intensities for saturation of O₂ evolution (Mäenpää et al., 1987). This fraction of PSII is further divided into two types: PSII, which is characterized by a smaller antenna but otherwise normal functioning of the center, and PSII-Q_B-nonreducing, which, in addition to the smaller antenna, has lost the ability to reduce plastoquinone (Melis and Schreiber, 1979; Melis, 1985; Graan and Ort, 1986). The latter shows further differences with respect to the redox potential of Q_A (Horton and Croze, 1979; Thielen and van Gorkom, 1981) and the ability to oxidize water (Henrysson and Sundby, 1990).

PSII, inactive in linear electron transport and photosynthetic water splitting, seems to occur under different physiological conditions, such as prior to or after photoinactivation. Prior to photoactivation, the Mn cluster is not assembled and a shift of 150 mV toward a more positive potential is found for the Q_A/Q_A redox couple (Johnson et al., 1995). The same "high-potential" form of Q_A is observed after Ca²⁺ depletion (Krieger and Weis, 1992; Krieger et al., 1995), Ca²⁺ being an obligatory cofactor for photosynthetic water splitting (Debus, 1992). Under light stress Ca²⁺ release has been suggested to be involved in the reversible inactivation of PSII and, by the dissipation of excess energy, in the protection of PSII against photodestruction. Evidence has been presented that the formation of a large proton gradient across the thylakoid membrane may lead to a reversible Ca²⁺ release from the donor side of PSII and, as a consequence, to an inactivation of the water-splitting complex and a shift in the redox potential of Q_A (Krieger and Weis, 1993; Krieger et al., 1993).

Photodestruction of PSII will occur under prolonged light stress. Photoinhibition occurs in two steps: (a) inhibition of PSII activity and (b) degradation of the D1 protein (for reviews, see Prasil et al., 1992; Aro et al., 1993). During photoinhibition the extent of D1 degradation is higher than its rate of resynthesis and an overall loss of PSII activity occurs.

There is evidence that photoinhibition and reactivation of PSII occur in sequential steps in the PSII damage-repair cycle. These steps include light-induced impairment of electron transport, irreversible damage of the PSII reaction center, triggering of the D1 protein for degradation, resynthesis of the D1 protein, and reassembly and photoactivation of PSII (Guenther and Melis, 1990; Prasil et al., 1992; Aro et al., 1993). It has been proposed by Guenther and Melis (1990) that PSII Q_B-nonreducing centers are involved in the PSII damage-repair cycle.

To characterize PSII and PSII in thylakoid membranes, we performed TL measurements, which can be used as a probe of the behavior of PSII reaction centers, both in isolated systems and in whole leaves (for reviews, see Sane and Rutherford, 1986; Vass and Inoue, 1992). Samples are illuminated to generate charge pairs within the PSII reaction center and are then rapidly cooled down to trap these charge-separated states. Alternatively, samples are illuminated in the frozen state. Subsequent warming leads to the emission of light (luminescence) at characteristic temperatures. The emitted light originates from recombination of trapped charge pairs, and the emission temperature is characteristic of the charge pair involved. The peak position of a TL-band strongly depends on the redox potential of the involved charge pair. For example, recombination of the semiquinone Q_B with the S₂ state of the water-splitting complex yields a TL-band at approximately 30°C, the so-called B-band (Rutherford et al., 1982). In the presence of DCMU, which inhibits the electron transfer from Q_A to Q_B, charge recombination between Q_A and S₂ and/or S₃ state yields a TL-band at approximately 10°C, the so-called Q-band (Rutherford et al., 1982). These bands are found in active PSII.

A high-temperature TL-band (C-band) with a temperature maximum between 45 and 55°C is observed in PSII lacking water-splitting activity. This band has been observed after Tris washing (Inoue et al., 1977) and in Ca²⁺-depleted PSII (Ono and Inoue, 1989; Krieger et al., 1993; Johnson et al., 1994). The TL-band in Ca²⁺-depleted PSII, inactive in water splitting and showing a shift in the redox potential of Q_A, is thought to arise from recombination of the charge pair Tyr_D⁺-Q_A (Johnson et al., 1994). TL emission at a similar temperature has been observed in PSII prior to photoactivation (Inoue et al., 1976).

TL is a useful technique with which to study the heterogeneity of PSII in the thylakoid membrane. In the present study we followed two different strategies to characterize PSII and PSII present in the thylakoid membrane. First, we addressed the question of whether there exists a lateral heterogeneity in the localization of PSII and PSII. We separated PSII and PSII using a biochemical approach: fractionating dark-adapted thylakoid membranes into grana stacks (without margins), margins, and stroma lamellae. By measuring the TL bands of these fractions, we demonstrated the occurrence of functionally different types of PSII and their distribution among the compartments of the thylakoid membrane. Second, we increased the relative amount of inactive PSII centers by photoinhibition of thylakoid membranes. During photoinhibition the amount of PSII decreases, as seen by the quenching of the TL intensity, while the amount of inactive PSII remains essentially constant.

	MATERIALS AND METHODS

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Spinach (Spinacia oleracea L., cv Polka) was grown hydroponically in nutrient solution (Randall and Bouma, 1973) at 15°C at a light intensity of 250 µmol quanta m² s¹ and a light period of 10 h d¹.

Preparation of Thylakoids

Grana, Margin, and Stroma Preparation

1

1

1

1

PSI electron transport activity of the different fractions was measured with a Clark-type electrode under saturating white light (4000 µmol quanta m² s¹) in the same buffer used for the final resuspension in the presence of 1 µm nigericin, 20 µm DCMU, 40 µm 2,6-dichlorphenylindophenol, 5 mm ascorbate, 100 µm methyl viologen, and 1 mm sodium azide.

PSII activity was measured in the same buffer in the presence of 1 µm nigericin and 1.5 mm 2,6-dimethylbenzoquinone. Linear electron transport was measured in thylakoid membranes using 100 µm methyl viologen as electron acceptor in the presence of 1 µm nigericin and 1 mm sodium azide. The average rate was 256 ± 47 µmol O₂ mg¹ Chl h¹. Chl a, Chl b, and pheophytin (PSII) were quantified by reverse-phase HPLC. P₇₀₀ (PSI) was calculated from the amplitude of the absorption signal at 703 and 820 nm, respectively.

Photoinhibition Treatment

2

1

TL Measurements

The sample was cooled down and heated via the Peltier element with a heating rate of 0.5°C s¹, controlled by a temperature-control box (Marlow Instruments). Temperature was measured with a thermistor at the highest step of the Peltier element (temperature controlling) and with a thermocouple on top of the sample. Sample incubation and temperature regulation, data acquisition, handling, and graphical simulation were performed as described by Ducruet and Miranda (1992).

The samples (200 µg Chl mL¹) were incubated for 2 min in the dark at 20°C, and, unless otherwise stated, were then rapidly cooled to 0 or to 30°C and illuminated with a single-turnover flash. After a short, dark incubation time (20 s at 0°C), the sample was warmed to 70°C with a heating rate of 0.5°C s¹ and light emission was measured during the heating. The measurements shown in Figure 2 were made with another apparatus described by Ducruet and Miranda (1992).

View larger version (14K): [in this window] [in a new window]   Figure 2. TL signals from dark-adapted fractions of thylakoid membranes in the presence of 10 µm DCMU. TL was charged by a single-turnover flash at 30°C. A, Grana stacks; B, margins; and C, stroma lamellae. The ordinate was expanded by a factor of 2 for margins and by 14 for stroma lamellae. For all signals, a baseline was subtracted. The baseline was obtained by measuring the sample holder without the sample. a.u., Arbitrary units.

	RESULTS

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Heterogeneity of PSII

View larger version (12K): [in this window] [in a new window]   Figure 1. TL signals from dark-adapted fractions of thylakoid membranes. TL was charged by a single-turnover flash at 0°C. For the TL measurements the sample was heated from 0 to 70°C with a heating rate of 0.5°C s1. A, Grana stacks; B, margins; and C, stroma lamellae. The ordinate was expanded by a factor of 2 for margins and by 7 for stroma lamellae. The same Chl content was used for all measurements (200 µg Chl mL1). a.u., Arbitrary units.

The TL signal of the margin fraction (Fig. 1B) consists of two peaks, one with a maximum at approximately 15°C (52% of the total emission) and the other one with a maximum at approximately 47°C. We consider this high-temperature band as a C-band, which is thought to reflect recombination of the charge pair Tyr_D⁺- Q_A in PSII (Johnson et al., 1994). Fitting the signal by graphical and numerical analysis (as described by Ducruet and Miranda, 1992) showed that the emission at approximately 15°C consists of two bands, a Q-band arising from Q_AS₂ recombination and a B-band arising from Q_BS₂ recombination. In one preparation of this kind, the loss of the B-band was even more pronounced; no B-band at all could be charged after a single-turnover flash (data not shown). Detergents were used for the fractionation of thylakoid membranes (see ``Materials and Methods''). These detergents might affect the Q_B-binding site and result in a (partial) loss of Q_B, so that a Q_AS₂ recombination occurs. A higher-temperature TL-band can be seen clearly in the fraction of stroma lamellae (Fig. 1C). The maximal temperature is approximately 42°C, and the B-band seems to be missing. The overall TL emission obtained with the stroma fraction is very low relative to the Chl concentration.

To determine whether the 42°C band was caused by the detergent treatment of the thylakoid membranes, we performed TL measurements on grana and stroma fractions that were prepared by the mechanical disruption technique using a Yeda press. The signals obtained were very similar to those shown in Figure 1. The maximal emission temperature for the band obtained with the stroma fraction was 43°C; the grana fraction showed a B-band with the maximal emission at 30°C (data not shown), indicating that the high-temperature TL-band observed in the stroma fraction was not caused by the detergent treatment.

To determine whether this high-temperature band formed in the margin and stroma fractions could be assigned to a C-band, we repeated the TL measurements in the presence of DCMU (Fig. 2). In the presence of DCMU, the electron transport from Q_A to Q_B was blocked and the B-band was suppressed and replaced by the Q-band in active centers. The TL signal of grana stacks (Fig. 2A) consists mainly of a Q band (peak temperature at approximately 2°C) and a small C-band at 48°C, which was not observed in the absence of DCMU. In the absence of DCMU this band might be hidden by the B-band. TL measurements of the margin fraction in the presence of DCMU, (Fig. 2B) clearly show a Q-band and a C-band at 51°C. In the margin fraction (Fig. 2B), the Q-band has its maximum at 2°C, as in grana stacks (Fig. 2A).

The TL signal obtained in the presence of DCMU with the fraction of stroma lamellae (Fig. 2C) shows two bands, one corresponding to a Q-band at about 4°C and the second corresponding to a C-band at 52°C. The appearance of the Q-band shows that even in the stroma fraction a small amount of active PSII is present. Some light emission in the temperature range of a Q-band is also seen in the absence of DCMU. By the addition of DCMU, the peak temperature of the high-temperature band was shifted from 42 to 52°C. In the literature, peak temperatures between 45 and 55°C have already been reported for the C-band. The TL intensity of the stroma fraction (Fig. 2C), which is already very low in the absence of DCMU, is lowered by 50%. This observation is in contrast to the results obtained with margin fractions (Fig. 2B). A quenching effect of DCMU on stroma thylakoid preparations has also been reported by Hideg and Demeter (1988).

To obtain more information about the high-temperature band in stroma lamellae, we investigated whether the intensity of this band oscillates with the number of flashes in the absence of DCMU. As shown in Figure 3 for one and two flashes, no oscillation pattern was found. After three flashes, exactly the same intensity of the band was found. Without flash excitation no such TL-band was observed (not shown). This implies that the high-temperature band observed in the absence of DCMU is also a C-band and does not originate from a recombination with one of the S-states of the water-splitting complex.

View larger version (19K): [in this window] [in a new window]   Figure 3. TL signals from the fraction of stroma lamellae in the absence of DCMU. TL was charged at 0°C by one () or two () flashes. a.u., Arbitrary units.

For comparison, we measured the dependence of the TL signals of the grana and margin fraction on the number of excitation flashes. The B-band formed in grana stacks showed the typical period-four oscillation (Rutherford and Inoue, 1984). The maximum emission intensity was observed either after the first flash, when the sample was dark-adapted (5 min), or after the second flash, with a shorter time of dark adaptation (data not shown). The maximum was observed after the first flash, when most Q_B is in its oxidized state prior to excitation. The intensity of the TL signal of the margin fraction also oscillated with a period of four, showing the maximum emission after the second flash, but the oscillation was much more damped (data not shown). As already described, the TL signal consists of three bands: the B-, Q-, and C-bands. Only the B-band undergoes changes in intensity, depending on the number of excitation flashes.

Photoinhibition Studies

View larger version (13K): [in this window] [in a new window]   Figure 4. TL signal from dark-adapted thylakoid membranes in the presence of nigericin. The Chl concentration was 200 µg mL1. A, TL was charged by a single-turnover flash at 0°C. B, Fit of the curve shown in A. Smooth curve, measured signal; middle line, residuals; and top line, fit with two components (dotted lines). Maximal emission temperature of the components was 35 and 48°C. Bottom, Fit with one component, maximal emission temperature at 36°C. The curve was fit from 5 to 60°C. C, TL was charged by approximately 15 s of continuous illumination (2100 µmol quanta m2 s1) during cooling the sample from 20 to 0°C. a.u., Arbitrary units.

To study the effect of photoinhibition, the sample was first subjected to a photoinhibitory light treatment. TL was then excited by continuous illumination instead of using a single-turnover flash. The reason for this is that, during the photoinhibition treatment, long-lived charges may be formed that do not relax after a short time of dark adaptation; therefore, after a single-turnover flash, quite different charges might be present depending on the degree of photoinhibition of the sample. In Figure 4C, a TL curve is shown that was obtained by exciting nonphotoinhibited, uncoupled, dark-adapted thylakoid membranes with continuous illumination (2100 µmol quanta m² s¹) during cooling from 20 to 0°C. This short illumination does not lead to any photoinhibition. Charging TL by continuous illumination during cooling results in a more complex TL curve, due to the increased number of combinations of charge pairs that can be formed under these conditions. The distribution of S-states from a dark-adapted sample is different; higher S-states are formed under continuous illumination, whereas in a dark-adapted sample only the S₁- and the S₂-state are reached after a single-turnover flash.

Figure 5 shows TL signals of thylakoid membranes that were photoinhibited at 500 µmol quanta m² s¹ for the times indicated in the presence of an uncoupler. After only 2 min of this relatively moderate illumination, a more pronounced TL-band occurred at 45 to 50°C compared with the TL curve measured before photoinhibitory treatment. We considered this high-temperature band to be a C-band. Prolonged photoinhibitory illumination led to a decrease of the B- and Q-bands (TL emission between 0 and 40°C), whereas the C-band increased during the first 15 min of photoinhibition treatment. Prolonged light treatment (65 min) led to an overall loss of TL emission, including a decrease of the relative intensity of the C-band. During the photoinhibitory treatment, photosynthetic O₂ evolution was inhibited with comparable kinetics with respect to the decrease of the TL intensity of the B- and Q-bands (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 5. TL signals from photoinhibited thylakoid membranes. Photoinhibition treatment was performed by illuminating thylakoid membranes with white light (500 µmol quanta m2 s1) at 20°C in the presence of nigericin. After the photoinhibition treatment the samples were transferred into the TL apparatus and darkened for 2 min at 20°C. TL was charged by continuous illumination (2100 µmol quanta m2 s1) during cooling the sample from 20 to 5°C. For clarity, the curves were displaced vertically; the ordinate is the same for all curves. A baseline correction was performed by subtracting a signal that was obtained from photoinhibited thylakoids that were not illuminated during freezing. a.u., Arbitrary units.

	DISCUSSION

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The data presented in this paper reveal a distribution of functionally different PSII in the thylakoid membrane. As shown in Figures 1 and 2, there is a lateral heterogeneity in the localization of PSII and PSII. The B-band (or Q-band), characteristic of PSII, was dominant in the grana stack preparation (without margins). TL measurements of the fraction containing the margin region showed that it consists of both B- and Q-bands, characteristic of PSII, and a C-band, characteristic of PSII. The stroma lamellae gave rise to a high-temperature band in TL, which was considered to be a C-band (Figs. 1-3).

In stroma lamellae in the absence of DCMU, the peak temperature (42°C) was relatively low for a C-band. By the addition of DCMU, the TL-band was shifted to a higher peak temperature (52°C). The intensity of this band did not oscillate upon varying the number of excitation flashes (Fig. 3), which indicates that it cannot be attributed to a B-band and that the S-states are not involved in the formation of this band. Based on these observations we considered this TL-band to be a C-band. We propose that the up-shift of the peak temperature of the C-band was caused by DCMU. Herbicides are known to influence the peak position of TL-bands. By the addition of different herbicides the peak temperature of the Q-band can change by more than 15°C (Vass and Demeter, 1982). Herbicides that bind in the Q_B pocket might have a stabilization effect on the redox potential of Q_A. In the margins, the peak temperature of the C-band was lower in the absence of DCMU, although the effect was less pronounced than in stroma lamellae. An up-shift of the peak temperature of the C-band by 8 to 10°C upon the addition of DCMU was shown recently for Ca²⁺-depleted PSII membranes (Krieger et al., 1998). In most reports in the literature the C-band was measured only in the presence of DCMU (Johnson et al., 1994).

TL measurements of grana stacks (BBY particles) and stroma lamellae have previously been published by Hideg and Demeter (1988), who demonstrated that in stroma lamellae no emission, either in delayed luminescence or in TL, was associated with recombination from Q_B. In stroma lamellae, they observed a weak Q-band in both the presence and absence of DCMU, but they did not see an emission at high temperature in the stroma lamellae because they measured only up to 50°C.

A C-band can also be seen in preparations of whole thylakoid membranes (Fig. 4) but is more obvious after photoinhibitory illumination. After photoinhibitory treatment of uncoupled thylakoid membranes, the overall intensity of TL emission is decreased (Fig. 5). Photoinhibition decreased the ability to form the B-band (and Q-band) in TL, whereas the intensity of the C-band increased during prolonged photoinhibitory treatment (up to 15 min). This indicates that PSII become photoinhibited and no longer emit light, whereas PSII are less affected by light.

TL studies have previously been performed on photoinhibited, isolated thylakoid membranes, both in the absence (Vass et al., 1988; Farineau, 1990) and in the presence of uncouplers (Farineau, 1990), and in whole cells of Chlamydomonas reinhardtii (Ohad et al., 1988) and pea leaves (Briantais et al., 1992). In thylakoid membranes, the intensities of the B- and Q-bands were reduced after photoinhibitory illumination and no shift in the maximal emission temperatures of the TL-bands was observed. These observations are in agreement with the data shown here in Figure 5. However, Ohad et al. (1988) reported a different phenomenon for the in vivo system. In algae cells the maximal emission temperature of the B-band was shifted by 15°C toward a lower temperature after photoinhibitory treatment in addition to the decrease in the intensity. This shift of the B-band observed might be due to the presence of a proton gradient across the thylakoid membrane during the photoinhibitory treatment in whole cells, whereas this gradient might have been less stable in the thylakoid membranes. We performed the photoinhibitory treatment in the presence of an uncoupler to avoid the additional complications caused by a proton gradient. Similar effects as observed in algae have been reported for pea leaves (Briantais et al., 1992), in which an increase of a high-temperature band in addition to the reduction of the intensity of the B-band after photoinhibition was also shown. This agrees with results reported here but was not studied in more detail.

From the data presented here it is not possible to decide whether the C-band reflects PSII that were already present prior to photoinhibitory illumination and were not susceptible to damage by light or, less likely, whether these PSII were degraded and a fraction of PSII was converted into inactive centers at the same time. Under our experimental conditions, both donor-side- and acceptor-side-induced photoinhibition may occur (Prasil et al., 1992; Aro et al., 1993). Even in uncoupled thylakoid membranes, at least a fraction of PSII can become inactivated via an impairment of the donor side of PSII, as has also been shown previously by Barényi and Krause (1985).

The C-band is observed in PSII complexes unable to oxidize water, such as Tris-washed (Inoue et al., 1977), Ca²⁺-depleted (Ono and Inoue, 1989; Krieger et al., 1993; Johnson et al., 1994), and nonphotoactivated PSII (Inoue et al., 1976). Ca²⁺-depleted PSII (Krieger and Weis, 1992; Krieger et al., 1995) and nonphotoactivated PSII (Johnson et al., 1995) show an up-shift in the redox potential of Q_A (high-potential form) in addition to its inability to oxidize water. Furthermore, fluorescence measurements indicated that in such centers no efficient electron transfer from Q_A to Q_B is possible (Andréasson et al., 1995; Johnson et al., 1995). Our data indicate that the C-band is present before photoinhibition (margins and stroma lamellae from dark-adapted thylakoid membranes) as well as after photoinhibition of thylakoid membranes.

The similar characteristics of the TL signals presented in this paper compared with the observations mentioned above lead us to the conclusion that the C-band reflects inactive centers that are equivalent to the so-called PSII Q_B non-reducing centers, which have no functional water-splitting complex, possess Q_A in the "high-potential form," and are localized mostly in the margin and stroma fraction of thylakoid membranes. These centers play an important role in the turnover of PSII described in the PSII damage-repair cycle (Guenther and Melis, 1990; Prasil et al., 1992; Aro et al., 1993).

	FOOTNOTES

   Received August 29, 1997; accepted November 20, 1997.

	ABBREVIATIONS

Abbreviations: Chl, chlorophyll. PSII and PSII, active and inactive PSII centers, respectively. TL, thermoluminescence. TyrD, a Tyr residue (in PSII) that can be photooxidized.

	ACKNOWLEDGMENTS

A.K. would like to thank J.-M. Ducruet for his advice and help with building the TL machine and for providing her with his software for data acquisition, handling, and graphical simulation; U. Heber for giving support for building the machine and for this study; and U. Schreiber and U. Schliwa for help with building the TL machine. We would also like to thank A.W. Rutherford and C. Jegerschöld for stimulating discussions and T. Mattioli for the critical reading of the manuscript.

	LITERATURE CITED

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