Photosynthesis Research 62: 55–66, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
55
Regular paper
Molecular mechanism of high-temperature-induced inhibition of acceptor side of Photosystem II Pavel Posp´ıšil1,2 & Esa Tyystjärvi1,∗ 1 Department
of Biology, Plant Physiology and Molecular Biology, Biocity A, University of Turku, FIN-20014 Turku, Finland; 2 Permanent address: Department of Experimental Physics, Faculty of Science, Palacky University, Olomouc, Czech Republic; ∗ Author for correspondence, e-mail:
[email protected] Received 16 February 1999; accepted inr evised form 18 August 1999
Key words: bicarbonate, chlorophyll fluorescence, electron transfer, heat, QA , redox potential
Abstract High-temperature-induced inhibition of the acceptor side of Photosystem II (PS II) was studied in tobacco thylakoids using oxygen evolution, chlorophyll a (Chl a) fluorescence and redox potential measurements. When thylakoids were heated at 2 ◦ C/min from 25 to 50 ◦ C, the oxygen evolving complex became inhibited between 32 and 45 ◦ C, whereas the acceptor side of PS II tolerated higher temperatures. Variable Chl a fluorescence decreased more slowly than oxygen evolution, suggesting that transitions between some S-states occurred even after heatinduced inhibition of the oxygen evolving activity. 77 K emission spectroscopy reveals that heating does not cause detachment of the light-harvesting complex II from PS II, and thus the heat-induced increase in the initial F0 fluorescence is due to loss of exciton trapping in the heated PS II centers. Redox titrations showed a heat-induced increase in the midpoint potential of the QA /QA − couple from the control value of –80 mV to +40 mV at 50 ◦ C, indicating a loss of the reducing power of QA − . When its driving force thus decreased, electron transfer from QA − to QB in the PS II centers that still could reduce QA became gradually inhibited, as shown by measurements of the decay of Chl a fluorescence yield after a single turnover flash. Interestingly, the heat-induced loss of variable fluorescence and inhibition of electron transfer from QA − to QB could be partially prevented by the presence of 5 mM bicarbonate during heating, suggesting that high temperatures cause release of the bicarbonate bound to PS II. We speculate that both the upshift in the redox potential of the QA /QA − couple and the release of bicarbonate may be caused by a heat-induced structural change in the transmembrane D1 or D2 proteins. This structural change may, in turn, be caused by the inhibition of the oxygen evolving complex during heating. Abbreviations: Chl – chlorophyll; DCBQ – 2,6-dichlorobenzoquinone; F0 , FM , and FV – minimum, maximum and variable chlorophyll a fluorescence, respectively; kAB and KAB – rate constant for electron transfer from QA − to QB and the equilibrium constant of this reaction, respectively; kBA – rate constant for electron transfer from QB − to QA ; OEC – oxygen evolving complex; Pheo – pheophytin; P680 – primary electron donor of PS II; PPFD – photosynthetic photon flux density; QA and QB – primary and secondary quinone electron acceptor of PS II, respectively; HP QA and LP QA – high- and low-potential form of QA , respectively; [QA − ]50ms – proportion of QA − remaining reduced 50 ms after a single turnover flash; TyrZ – tyrosine, secondary electron donor of PS II Introduction Photosynthesis is one of the most heat sensitive processes in higher plants, electron transfer in the thylakoid membrane being more susceptible than en-
zyme reactions occurring in the stroma of chloroplasts (Santarius 1975; Krause and Santarius 1975). The donor side of PS II is more heat sensitive than the acceptor side of PS II or PS I (Berry and Björkman 1980; Weis and Berry 1988; also see Coleman et al.
56 1988). The high-temperature-induced inhibition of the acceptor side has been localized on the reaction center of PS II (Schreiber and Armond 1978) and data from triazine-resistant plants suggests that this inhibition may be related to a shift in the redox potential of QA (Ducruet and Lemoine 1985). Chlorophyll (Chl) a fluorescence measurements by Bukhov et al. (1990) support this suggestion, but direct measurements of the redox properties of QA upon heat stress have not been reported. Cao and Govindjee (1990) suggested that the inhibition of QA –QB electron transfer might result from a structural change in D1 and D2 proteins upon a high-temperature-induced reorganization of the thylakoid membranes (Gounaris et al. 1983; 1984). Indeed, FTIR measurements show that major conformational changes of the D1 and D2 proteins occur during heating between 35 and 50 ◦ C (De Las Rivas and Barber 1997). In this work, we applied redox titrations and analysis of the decay of Chl a fluorescence yield after a single turnover flash to analyze heat-induced changes in the function of the acceptor side of PS II. We observed an upshift in the midpoint redox potential of the QA /QA − couple and present evidence suggesting that bicarbonate is released from PS II during heating. These phenomena may cause the inhibition of electron transfer from QA − to QB . This is of direct relevance to the role of bicarbonate on the acceptor side of PS II studied by Govindjee and coworkers (see reviews by Blubaugh and Govindjee 1988, and Govindjee and Van Rensen 1993).
Materials and methods Thylakoid membrane preparation and heat treatments Thylakoid membranes were isolated from tobacco (Nicotiana tabacum L. cv. Samsun) grown under the photosynthetic photon flux density (PPFD) of 300 µmol m−2 s−1 , in a 12 h light/dark rhythm and temperature 22 ◦ C. Leaves were rapidly ground with a homogenizer (Ultra-Turrax, Janke and Kunkel, Staufen, Germany) in 40 mM Hepes-KOH (pH 7.4), 300 mM sorbitol, 10 mM MgCl2 , 1 mM EDTA, 1 M glycine betaine and 1% (w/v) BSA. The homogenate was filtered through Miracloth (Calbiochem) and centrifuged for 5 min at 1000 × g. The chloroplasts were resuspended in 5 mM sorbitol, 10 mM Hepes-KOH (pH 7.4) and 5 mM MgCl2 to cause an osmotic shock and thylakoids were then collected by centrifugation for 5 min at 2000
× g, resuspended in storage buffer solution containing 10 mM Hepes-KOH (pH 7.4), 500 mM sorbitol, 5 mM NaCl and 10 mM MgCl2 , and stored at –80 ◦ C. Chlorophyll was determined according to Porra et al. (1989). Unless otherwise indicated, all heat treatments and subsequent measurements were done with thylakoids suspended in a reaction medium containing 330 mM sorbitol, 40 mM Hepes-KOH (pH 7.6), 5 mM NaCl, 5 mM MgCl2 and 1 M glycine betaine. For the heat treatments, thylakoids were first equilibrated at 25 ◦ C and then heated at a constant rate of 2 ◦ C/min to the desired temperature. All heating was done in the dark. Finally, the thylakoids were incubated at the desired temperature (35–50 ◦ C) for 5 min, and the subsequent redox titrations and measurements of Chl a fluorescence and oxygen evolution were also done at the final treatment temperature. Oxygen evolution measurements Oxygen evolution was measured with an oxygen electrode (Hansatech, Kingþs Lynn, UK) in red light (6500 µmol m−2 s−1 ) using a slide projector with a red plexiglass filter (a cutoff at 600 nm) as a light source. Thylakoids (10 µg Chl ml−1 ) were suspended in reaction medium supplemented with 1 mM KH2 PO4 and 5 mM NH4 Cl; 250 µM 2,6-dichlorobenzoquinone (DCBQ) was added as an electron acceptor. To measure the temperature dependence of the oxygen evolution rate, the thylakoid suspension (10 µg Chl ml−1 ) was first linearly heated at 2 ◦ C min−1 to the desired temperature (25–45 ◦ C) in the absence of DCBQ and kept at this temperature during the subsequent measurement of oxygen evolution. Decay of Chl a fluorescence yield after a single turnover flash The decay of Chl a fluorescence yield after a single turnover flash was measured with a PAM-101 fluorometer (Heinz Walz GmbH, Effeltrich, Germany), using a blue measuring beam (450 nm, 5 µmol m−2 s−1 , US-L450, Walz GmbH) filtered through a Walz SP 580 filter. Fluorescence was measured at right angle to the excitation light with a photodiode detector (ED101US/D, Walz GmbH) protected with a Schott RG 645 red filter. The measuring beam was applied during the first 20 ms after the flash and only 10-ms bursts of the measuring beam were periodically applied during the rest of the decay to avoid an actinic effect. Single turnover flashes (10 µs) were generated by a
57 Walz XST-103 xenon flash lamp. Ten replicate measurements with a 20 s dark period between successive measurements were averaged. All analyses of Chl a fluorescence decay refer to those PS II centers that still contribute to variable fluorescence and all amplitudes were calculated relative to the total variable fluorescence measured at the treatment temperature. The proportion of PS II centers inactive in QA − to QB electron transfer, [QA − ]50ms, was calculated from the relative intensity of variable fluorescence remaining 50 ms after the single turnover flash, taking into account the curvilinearity of the relationship between [QA − ] and FV (Cao and Govindjee 1990). The connectivity factor p (Joliot and Joliot 1964) was taken to be equal to 0.5. The fluorescence decay curves were analyzed by least-square curve fitting, using four exponential components. The behaviour of the lifetimes and amplitudes of the components was first evaluated allowing them run free in the fitting. Thereafter, the lifetimes were fixed to the mean values of each treatment, obtained in the freerunning fits, and a new fit was calculated to obtain the amplitudes. The FIP fluorescence software (QAData Oy, Turku, Finland) was used to control the measurements and analyze the results. Measurements of 77 K emission spectra The 77 K emission spectra of control and heated thylakoids were measured with a diode array spectrophotometer (S2000, Ocean Optics Europe, Amsterdam). Thylakoids suspended in the reaction medium were first mixed with an equal volume of glycerol and then frozen in liquid nitrogen. The sample volume was 0.12 ml and the final Chl concentration was 5 µg/ml. Illumination was with a slide projector through a Balzers K-1 filter (50 nm HWHM; center wavelength 450 nm).
titrations by the addition of 100 µM potassium ferricyanide (K3 [Fe(CN)6 ]). The redox state of QA was followed by Chl a fluorescence measurements with a PAM-101 fluorometer using a red measuring beam (650 nm, 1 µmol m−2 s−1 ).
Results Oxygen evolving complex is inhibited before the acceptor side of PS II When tobacco thylakoids were heated from 25 to 50 ◦ C, their ability to evolve oxygen was gradually lost during heating between 32 and 45 ◦ C (Figure 1A, squares). Heating between 42 and 50 ◦ C caused a strong decrease in variable Chl a fluorescence (Figure 1A, triangles) indicating that an increasing number of PS II reaction centers lost the ability to photoreduce QA . Among the PS II centers still capable of photoreducing QA , the proportion of QA − remaining reduced 50 ms after a single turnover flash, [QA − ]50ms , increased, indicating a decrease in the number of PS II centers active in QA − to QB electron transfer (Figure 1A, circles). The 77 K fluorescence spectrum of thylakoids heated to 45 ◦ C showed a decrease in the fluorescence yield of PS II components emitting at 685 and 694 nm, compared to the 734 nm emission peak originating in PS I (Figure 1B). However, heating did not cause increase in fluorescence yield around 680 nm which is the emission maximum of the light-harvesting complex of PS II (LHC II) (Figure 1B). Thus, the 1.8-fold increase in the ratio of F0 to maximum fluorescence FM (data not shown) was not due to emission from detached LHC II. Heating to 45 ◦ C also caused a shift of the emission maximum of PS I from 734 to 732 nm.
Redox potential measurements
The effect of high temperature on Chl a fluorescence decay after a single turnover flash
The midpoint redox potential of the QA /QA − couple was measured with a redox combination platinum – silver/silver chloride electrode (MettlerToledo GmbH, Switzerland) connected to a Knick pH/millivolt meter. The electrode was calibrated using standard solutions provided by the manufacturer of the electrode. Thylakoid suspension (10 µg Chl ml−1 ) was kept in a temperature-controlled chamber and maintained at all times under argon. Reductive titrations were performed by the gradual addition of 100 µM sodium dithionite (Na2 S2 O4 ), and oxidative
To study the effect of heating on different reactions involved in the reoxidation of QA − in PS II centers still capable of photoreduction of QA , we further analyzed the kinetics of the decay of Chl a fluorescence yield after a single turnover flash in heated thylakoids (Figure 2A). Only PS II centers contributing to variable fluorescence show a decay of fluorescence yield, and therefore all fluorescence decay data refers to PS II centers that are able to photoreduce QA at the temperature of the treatment. Heating induced a considerable increase in F0 (Figure 2A), whereas FM was 5–10%
58
Figure 1. (A) Temperature dependence of the rate of oxygen evolution (squares), the relative concentration of PS II centers active in electron transfer from QA − to QB , calculated as 100-[QA − ]50ms , (circles) and variable Chl a fluorescence FV (triangles), measured in tobacco thylakoids in the temperature range of 25 – 50 ◦ C. Thylakoids (10 µg Chl ml−1 ) were linearly heated in the dark from 25 ◦ C to the desired temperature and kept at this temperature during the measurements. Oxygen evolution was measured with an oxygen electrode in saturating red light (6500 µmol m−2 s−1 ). DCBQ (250 µM), was added just before the measurement of oxygen evolution. [QA − ]50ms was calculated from Chl a fluorescence yield 50 ms after a single turnover flash taking into account the curvilinearity of the relationship between [QA − ] and FV . The connectivity factor p was taken to be equal to 0.5. PAM-101 fluorometer was used in the fluorescence measurements. (B) Emission spectrum of control thylakoids (dotted line) and thylakoids linearly heated to 45 ◦ C (solid line). The spectra were measured at 77 K with a diode array spectrophotometer, using 450 nm broad-band light for excitation.
lower in all heated samples than in the 25 ◦ C control sample (data not shown). The decay curves of untreated control thylakoids could be fitted to a sum of four exponential components with half life-times of 200–400 µs, 2–4 ms, 60–80 ms and 1.2–1.5 s. The fit improved with the
number exponentials, but this improvement was marginal if more than four components were used. In thylakoids heated to 45 ◦ C or higher temperatures, no significant improvement was seen when more than three exponentials were used (Figure 2B). The shortest component (200–400 µs) represents forward electron
59
Figure 2. (A) Effect of high temperature on the decay of Chl a fluorescence yield after a single turnover flash in tobacco thylakoids. Thylakoids (5 µg Chl ml−1 ) were linearly heated in the dark from 25 ◦ C (closed circles) to 42 ◦ C (open circles), 47 ◦ C (closed triangles) and to 50 ◦ C (open triangles) and kept at this temperature during the measurement. The lines show the best fit of the data to a sum of four exponentials and the F0 value measured at each temperature is shown at the left edge of the figure (large symbols). The fluorescence decay was measured after application of a single turnover flash (10 µs), and a blue measuring beam (450 nm, 5 µmol m−2 s−1 ) was used to excite the fluorescence. The traces shown represent the mean of 10 individual traces obtained with a 30 s dark delay between the measurements. The measuring beam was switched on for the first 20 ms and then periodically switched on for 10 ms burst at a time, and each small symbol shows the fluorescence level recorded during one such burst. The single turnover flash was fired at the 10−4 s data point of the logarithmic time scale. The fluorescence traces have been divided by FM and multiplied with the FM of the 25 ◦ C control sample to facilitate comparison. (B) Weighted sum of squared residuals, Chi2 , calculated from the best fit of the decay of variable Chl a fluorescence yield to a sum of 1, 2, 3, 4 or 5 exponential components. Control thylakoids (solid circles) and thylakoids heated to 45 ◦ C (open squares).
transfer from QA − to QB (Crofts and Wraight 1983; Dau 1994). In non-treated thylakoids, the amplitude of the 200–400 µs component was 70% of variable Chl a fluorescence, whereas the amplitudes of the 2–4 ms,
60–80 ms and 1.2–1.5 s components were 24%, 4% and 2%, respectively (Table 1). When thylakoids were heated from 25 up to 42 ◦ C, a moderate decrease in the amplitude and a slight increase in the life-time of
60 Table 1. Effect of high temperature on the percent amplitudes of individual exponential components of the decay of chlorophyll a fluorescence yield after a single turnover flash. Thylakoids were linearly heated from 25 ◦ C to the desired temperature and kept at this temperature during the measurement of the fluorescence decay with the PAM-101 fluorometer. The components were obtained by least-square curve fitting and they are named by their half life-times. The half life-time (t1/2 ) of the fastest component changed considerably during heating and is therefore shown; the half life-times of the other components were between the limits shown for each component. The amplitudes are calculated as a percentage of the variable fluorescence measured at each temperature. The data represents the mean ± S.E. of data from ten decay curves Component
200–400 µs %
t1/2
2–4 ms %
60–80 ms %
1.2–1.5 s %
Temperature ◦ C 25 32 42 45 47 50
70 ± 1.7 67 ± 1.6 64 ± 1.4 45 ± 1.1 23 ± 0.9 9 ± 0.4
321 µs 353 µs 481 µs 1246 µs 6 ms 30 ms
24 ± 0.8 19 ± 0.7 8 ± 0.3 – – –
4 ± 0.3 8 ± 0.4 13 ± 0.4 27 ±0.6 39 ±1.1 43 ±1.2
2 ±0.2 6 ± 0.3 15 ± 0.4 28 ± 0.7 38 ± 1.0 48 ± 1.3
Component designation
QA − to QB ; QA − to QB −
The binding of ‘fast’ or granal PQ molecules to PS II
The binding of ‘slow’ or stroma thylakoid PQ molecules molecules of PS II
Back reaction of S2 QA − to S 1 QA
the shortest component occurred. Heating above 42 ◦ C resulted in a rapid drop of the amplitude and increase in the half life-time of the shortest component of the decay of chlorophyll a fluorescence yield (Table 1). These results show that the acceptor side of PS II remains functional during heating of thylakoids up to the temperature of 42 ◦ C and that an almost complete inhibition of electron transfer from QA − to QB occurs between 42 and 50 ◦ C. The millisecond components (2–4 ms and 60–80 ms) are related to the binding of a PQ molecule to the QB -binding site. These millisecond components correspond to the ‘fast’ PQ pool of the grana thylakoids and the ‘slow’ PQ pool of the stroma thylakoids discussed by Joliot et al. (1992). The half reduction-time of the ‘fast’ PQ pool (6 PQ molecules per PS II) was estimated to vary between 2 and 5 ms (t1/2 = 25–60 ms for the whole pool), whereas that of the ‘slow’ PQ pool (3–6 molecules per PS II) was shown to take 66– 83 ms (t1/2 = 0.8–1 s for the whole PQ pool assuming 6 molecules per PS II) (Joliot et al. 1992). The amplitude of the 2–4 ms component was found to decrease when thylakoids were heated above 32 ◦ C and this component was completely abolished at 45 ◦ C (Table 1; see also Figure 2B), whereas the amplitude of the 60–80 ms component increased during heating of thylakoids between 32 and 50 ◦ C. This observation reflects the
heat-induced destacking of the thylakoid membranes which results in the loss of the ‘fast’ PQ pool. The slowest component (1.2–1.5 s) reflects charge recombination from the S2 QA − state to the S1 QA state (Cao and Govindjee 1990). The relative amplitude of the 1.2–1.5 s component increased when thylakoids were heated above 32 ◦ C indicating an increase in the probability of charge recombination of QA − on the acceptor side with the S2 state of the oxygen evolving complex (Table 1). The effect of high temperature on the midpoint redox potential of the QA /QA − couple The rate constant kAB of electron transfer from QA − to QB is determined by the free energy gap between the QA − QB and QA QB − states, which in turn depends on the redox potentials of the two quinones. To test whether the high-temperature-induced inhibition of electron transfer from QA − to QB is related to a change in the reducing power of QA − , the midpoint redox potential of the QA /QA − couple was measured by a direct redox titration of Chl a fluorescence yield in non-treated and heat-treated thylakoids (Figure 3A). The midpoint redox potential of the QA /QA − couple was found to be –80 mV (low-potential form LP Q ) in non-treated thylakoids, and heating caused a A
61
Figure 3. (A) Redox titration of Chl a fluorescence yield in non-treated (25 ◦ C) (triangles) and heat-treated (50 ◦ C) (circles) tobacco thylakoids. Thylakoids (10 µg Chl ml−1 ) were linearly heated in the dark from 25 to 50 ◦ C at 2 ◦ C min−1 and kept at this temperature during the measurements. Reductive titration of chlorophyll fluorescence yield was carried out by the addition of dithionite (open symbols) and the oxidative by the addition of ferricyanide (closed symbols). The PAM fluorometer with a red measuring beam (650 nm, 5 µmol m−2 s−1 ) was used to measure Chl a fluorescence. The fluorescence yield is normalized by subtracting the constant value obtained at high redox potential and then dividing by the maximum value obtained at low potential. The redox potential is expressed relative to a standard hydrogen electrode. (B) Increase in the midpoint redox potential of the QA /QA − couple during heating. The midpoint redox potential of the QA /QA − couple is the point at which the fluorescence yield, measured as in A, has the value 0.5.
temperature-dependent upshift in the redox potential (Figure 3B). The final value obtained in thylakoids heated to 50 ◦ C was +40 mV which is characteristic of the high-potential form HP QA (Krieger et al. 1993).
Addition of bicarbonate partially restores PS II reactions in heated thylakoids Bicarbonate plays an essential role in electron transfer from QA − to QB (see reviews by Blubaugh and Govindjee 1988; Govindjee and Van Rensen 1993). In order to test the importance of bicarbonate in the high-temperature-induced inhibition of electron transfer from QA − to QB , the decay of Chl a fluorescence
62
Figure 4. The effect of bicarbonate on Chl a fluorescence decay after a single turnover flash in heat treated thylakoids. Thylakoids (5 µg Chl ml−1 ) were linearly heated in the presence or the absence of 5 mM NaHCO3 from 25 ◦ C to the desired temperature as indicated and kept at this temperature during the measurement. The F0 value measured at 42◦ C (open circle), 47◦ C (closed circle) and 50◦ C (open triangle) is indicated, and the arrows point to the respective F0 value measured in thylakoids heated in the presence of 5 mM NaHCO3 . The fluorescence traces have been divided by FM and multiplied with the FM of the 25 ◦ C control sample to facilitate comparison.
yield after a single turnover flash was measured in thylakoids heated in the presence and absence of 5 mM NaHCO3 (Figure 4). The addition of bicarbonate partially prevented the heat-induced loss of variable fluorescence, as indicated by its effect on the F0 level (Figure 4). Furthermore, in thylakoids heated to 47 ◦ C in the presence of 5 mM NaHCO , the decay of 3 Chl a fluorescence yield contained a 41% contribution from a fast component with a half-time of less than 1 ms (Figure 4); the kinetics was much slower in thylakoids heated in the absence of bicarbonate (Table 1). These data may suggest that release of bicarbonate from its binding site between QA and QB may be one reason for the high-temperature-induced inhibition of electron transfer from QA − to QB . Discussion Inhibition of acceptor side of PS II occurs at 42–50 ◦C Heating of thylakoids caused a sequence of events starting with the gradual loss of oxygen evolution between 32 and 45 ◦ C and proceeding with inhibition of electron transfer from QA − to QB between 42 and 50 ◦ C (Figure 1A). These results confirm that the oxygen evolving complex is inherently more sensitive to heating than the acceptor side of PS II (Katoh and
San Pietro 1967; Yamashita and Butler 1968; Cheniae and Martin 1970). Variable Chl a fluorescence was lost between 32 and 50 ◦ C more slowly than oxygen evolution (Figure 1A), indicating that a fraction of PS II centers had lost their ability to evolve oxygen but were still capable of photoreducing QA . Capability to photoreduce QA , indicated by variable fluorescence, is apparently lost at a late state of inhibition. We also tested whether the increase in F0 during heating indicates detachment of LHC II from the PS II reaction center complex. The fluorescence emission spectrum of thylakoids heated to 45 ◦ C does not show higher 680 nm emission as compared to the emission spectrum of control thylakoids (Figure 1B), indicating that LHC II remains in contact with the rest of PS II in heated thylakoids. Detached LHC II would show a high fluorescence yield at 680 nm (Guiamet et al. in preparation). The increase in F0 is most probably simply a consequence of loss of the capacity to trap excitons by the heated PS II reaction centers. We speculate that the blue-shift of the emission maximum of PS I may result from heat-induced partial denaturation of PS I proteins. High-temperature-induced inhibition of electron transfer from QA − to QB Chl a fluorescence measurements were performed to further analyze the heat-induced modifications in the
63 Table 2. The equilibrium constant KAB of the reaction QA − QB QA QB − calculated from Chl a fluorescence and redox potential measurements. In the case of fluorescence, the half life-time of the forward reaction was taken from the first column of Table 1 and that of the back reaction was assumed to remain at 6.5 ms. In the case of redox potential measurements, KAB was calculated from Equation (1) and Em (QB /QB − ) was assumed to remain at 0 V Temperature ◦ C
KAB fluorescence measurements
KAB redox potential measurements
25 45 47 50
20 5 1 0.2
20 4 1 0.2
acceptor side of PS II before its ability to photoreduce QA is lost. The increase in the half-life time of the shortest component of the fluorescence decay after a single turnover flash indicates a decrease in the rate constant of the forward electron transfer from QA − to QB (kAB ). Assuming kAB = 2159 s−1 (t1/2= 321 µs) at 25 ◦ C (Table 1) and the equilibrium constant KAB = 20 (Diner 1977; Crofts and Wraight 1983), the rate constant of back electron transfer from QB − to QA is kBA = 107 s−1 (t1/2= 6.5 ms). After heating to 45 ◦ C, the rate constant of forward electron transfer decreased to kAB = 556 s−1 (t1/2 = 1246 µs) (Table 1) and thus the equilibrium constant becomes KAB = 5 (Table 2), assuming that the rate constant of back electron transfer kBA remains unchanged. The most severe heat treatments shift the equilibrium significantly towards QA indicating a complete inhibition of QA –QB electron transfer at 50 ◦ C (Table 2). Heating to 35 – 45 ◦ C is known to cause destacking of thylakoids (Gounaris et al. 1983, 1984) and this destacking is expected to abolish differences between PS II centers located in grana and stroma thylakoids. The 2–4 ms component of the decay of Chl a fluorescence yield after a single turnover flash has been suggested to originate from interaction between PS II and granal plastoquinone while the 60–80 ms component reflects the reduction of plastoquinone located in stroma thylakoids (Joliot et al. 1992). Our data suggests that heat-induced destacking of thylakoids leads to merging of these two components (Table 1). Furthermore, the slow kinetics of the merged fluorescence decay component may suggest that diffusion of plastoquinone is slower in non-appressed thylakoids than in grana stacks. The increase in the amplitude of the slowest component of fluorescence decay suggests that the prob-
ability of back electron transfer from QA − to S2 increases at the expense of forward electron flow above 32 ◦ C (Table 1). Conversion of QA to the high-potential form inhibits electron transfer from QA − to QB The kinetic properties of the acceptor side of PS II are determined by the energetic parameters of PS II electron transfer reactions. To elucidate heat-induced changes in the energetics, we measured the midpoint redox potential of the QA /QA − couple. Untreated thylakoids showed a value of Em (QA /QA − ) of –80 mV which is typical for the ‘normal’ low-potential form of LP QA (Krieger et al. 1993, 1995). Heating induced an increase in the midpoint redox potential of the QA /QA − couple, which can be described as a conversion of LP QA to a high-potential form HP QA (Krieger et al. 1993; Andréasson et al. 1995; Johnson et al. 1995). In the presence of HP QA , electron transfer from QA − to QB is inhibited because the equilibrium constant for electron sharing between QA − and QB − shifts towards QA − . Taking into account the relationship between the free energy difference 1G and the equilibrium constant KAB (1 G = -RT ln KAB ), we can write: − Em (QB /Q− B ) − Em (QA /QA ) =
(1)
1/F(−RT ln KAB ), where F and R are the Faraday and gas constants, respectively, and T is the absolute temperature. Using the value of Em (QB /QB − ) = 0 mV for the redox potential of the QB /QB − couple (Crofts and Wraight 1983; Diner et al. 1991) and the value of Em (QA /QA − ) = –80 mV at 25 ◦ C (Figure 3B), one can determine the equilibrium constant KAB to be 20. Similar calculations at
64
Figure 5. A thermodynamic scheme of PS II. In the presence of LP QA the free energy difference between the QA − QB and QA QB − states is 1 G = –80 meV and the equilibrium of electron sharing favours the QA QB − state. The upshift in the midpoint redox potential of the QA /QA − couple results in an inversion of the free energy difference up to 1 G = +40 meV at 50 ◦ C.
45, 47 and 50 ◦ C yield KAB values which are in good agreement with those estimated above from the half life-time of the shortest component of the decay of Chl a fluorescence yield after a single turnover flash (Table 2). Figure 5 illustrates the free energy changes related to the conversion of LP QA to HP QA . Does the upshift in Em (QA /QA − ) serve a protective function? An upshift of the midpoint redox potential of the QA /QA − couple has been shown to accompany lowpH-induced inactivation of OEC (Krieger et al. 1993). Also in the heat-treated thylakoids of the present study, conversion of LP QA to HP QA was preceded by a decrease in oxygen evolution (Figure 1A, squares, and Figure 3B), suggesting that both heat-induced and low-pH-induced inactivation of OEC cause a similar conversion. A conformational change originating at the donor side of PS II and mediated by one of the transmembrane helices of the D1 or D2 protein is a possible mechanism for this conversion of LP QA to HP Q . Heat-induced conformational changes in the A D1 and D2 proteins have been described (De Las
Rivas and Barber 1997) and functional changes on the donor side of PS II may have a significant influence on the electron transfer properties of the acceptor side (Andréasson et al. 1995; Rova et al. 1998). Donor-side-mediated regulation of the acceptor side of PS II may have relevance upon heat stress, as in the presence of HP QA , recombination of the [P680+ Pheo− ] radical pair is not likely to produce the potentially dangerous triplet state of P680 (Woodbury et al. 1986, Johnson et al. 1995). A protective function has also been assigned to the presence of HP QA in PS II when OEC is inactivated at low pH (Krieger et al. 1993) and during the photoactivation process (Johnson et al. 1995). High-temperature-induced depletion of bicarbonate The larger amplitude of the fastest component in the decay of Chl a fluorescence yield after a single turnover flash in thylakoids heated in the presence of 5 mM NaHCO3 , as compared to thylakoids heated in absence of added bicarbonate (Figure 4), leads us to suggest that the inhibition of QA –QB electron transfer may be related to the release of bicarbonate
65 from the acceptor side of PS II. We suggest that the same conformational change in D1 or D2 proteins that causes the conversion of LP QA to HP QA , also causes a depletion of bicarbonate from PS II, thereby further disturbing electron flow from QA − to QB . Protection by bicarbonate against thermoinactivation of PS II has been earlier reported but it was concluded that bicarbonate protects the oxygen evolving complex (Klimov et al. 1997). In the present experiments, the protective effect of bicarbonate on the acceptor side of PS II was seen at 42–50 ◦ C (Figure 4) while the oxygen-evolving activity was already lost at lower temperatures (Figure 1A, squares). We conclude that electron transfer from QA to QB − was partially protected by addition of bicarbonate, but we cannot completely exclude the possibility that the protective effect on the acceptor side of PS II is a consequence of protection of the donor side. The lower F0 values obtained in thylakoids heated in the presence of NaHCO3 (Figure 4) also suggest that bicarbonate partially prevents the heat-induced damage to PS II from proceeding to the complete loss of stable exciton trapping and photoreduction of QA . Protection against thermoinactivation of PS II resembles the protective effect of bicarbonate against the detergent-induced loss of some acceptor-side functions of PS II (Kashino et al. 1992).
Acknowledgements This work was supported by Academy of Finland. PP was supported by a fellowship from the Centre for International Mobility (CIMO). The authors would like to thank Marja Hakala for preparation of the thylakoid membranes and Mika Keränen for help with measurements and analysis of the data. Dr Taina Tyystjärvi is thanked for critically reading the manuscript.
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