Photosynthesis Research 72: 271 284, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands. 271 Regular paper Experimental and theoretical studies on the excess capacity of Photosystem II Radek Kaňa 1,,DušanLazár 1,Ondřej Prášil 2 &JanNauš 1 1 Palacký University, Faculty of Science, Laboratory of iophysics, tř. Svobody 26, 771 46 Olomouc, Czech Republic; 2 Institute of Microbiology, Academy of Sciences of the Czech Republic, Photosynthesis Research Center, Opatovickýmlýn, 379 81 Třebo n, Czech Republic; Author for correspondence (e-mail: kana@prfnw.upol.cz; fax: +420-68-5225737) Received 29 October 2001; accepted in revised form 8 April 2002 Key words: Chlorella, model, oxygen flash yield, photoinhibition, steady-state rate of oxygen evolution Abstract It has been recently suggested that compensatory changes in Photosystem II (PS II) electron turnover rates can protect photosynthesis from photoinhibition [ehrenfeld et al. (1998) Photosynth Res 58: 259 268]. We have further explored this feature of PS II using a rate electrode for simultaneous measurements of the steady-state rate of oxygen evolution and the oxygen flash yield depending on the background irradiance in both control and photoinhibited algal cells of Chlorella öhm. Theoretical simulations based on the two-electron gate model agree qualitatively with experimental data if we assume an increase of the electron turnover rate in the remaining functional PS II centers of the photoinhibited sample. Our results confirm the hypothesis that the compensatory effect enables cells to maintain the maximal rates of photosynthesis even in the presence of moderate photoinhibition (decrease of up to 50% in the number of functional centers) and that the effect originates from the inner capacity of electron transport through PS II. The origin of the compensatory effect is briefly discussed. Abbreviations: PS II Photosystem II; F V variable fluorescence (F V = F M F 0 ); F M maximal fluorescence; F 0 minimal fluorescence; P max maximal rate of photosynthesis (the photosynthetic capacity); α initial slope of the P(E) curve; PAR photosynthetically active radiation; PQ oxidized plastoquinone molecule; P(E) curve photosynthetic rate versus irradiance curve (the photosynthesis curve); Y(E) curve oxygen flash yield versus irradiance curve; CAP chloramphenicol; k exch rate constant of exchange of double reduced Q with oxidized PQ molecule from the PQ pool Introduction The physiological importance and possible molecular mechanisms of photoinhibition have been studied and discussed many times (reviewed by, e.g., Prášil 1992; Aro 1993; Anderson et al. 1997, 1998). It was observed that sensitivity of algae or higher plants to photoinhibition depends on many factors such as temperature, growth light, nutrients, and acclimation of photosynthetic apparatus (Kyle et al. 1987; Falkowski 1992; aker and owyer 1994; Park et al. 1997; Marshall 2000). ecause photoinhibition is caused by light, which is simultaneously used as a source of energy for photochemical reactions, higher plants and algae have developed mechanisms to balance photochemical usage of light and its possible harmful effects on photosynthetic apparatus. These mechanisms generally operate by one of two modes (Long et al. 1994; Osmond 1994): by diverting excess light from PS II (e.g. state transitions, xanthophyll cycle, re-arrangement of the thylakoid membrane) or they can intensify compensatory responses to the harmful effects of light (e.g. increasing the capacity of D1 turnover, increase in scavenging of free radicals, etc.).
272 The photoinhibitory damage of PS II induces changes in photosynthetic rate that can be described in terms of the parameters of the photosynthesis versus irradiance curve (see e.g. Henley 1993; Kirk 1993; Leverenz et al. 1994; Sakshaug 1997). The P(E) curve can be typically divided into two major regions. At low irradiances, the photosynthetic rate is nearly linearly proportional to irradiance. This initial linear slope of photosynthetic rate (α) is limited mainly by the rate of photon absorption. As irradiance increases, photosynthetic rate becomes non-linear and rises to a saturation level that is denoted as photosynthetic capacity (P max ) andislimitedmainlybytherateofco 2 fixation (Sukenik et al. 1987; Heber et al. 1988). An intercept between the initial slope and the saturation rate of photosynthesis is denoted as the light saturation index (E k ) that characterizes irradiance, at which point limitation of photosynthesis shifts from light absorption and photochemical energy conversion to utilization of reductants (see, e.g., Sakshaugh 1997). It has been shown that the exposure of algal cells or higher plants to high irradiance causes a decrease both in α and P max of the P(E) curve (see e.g. Henley 1993). However, it has been also observed that in some cases P max remains the same for photoinhibited and non-photoinhibited samples (Kok 1956; Leverenz 1990; Falk 1992; ehrenfeld et al. 1998). This unexpected behavior has been studied by changing the number of functional PS II centers and monitoring respective changes in the P(E) curve (Leverenz 1990; Falk 1992). In this way, it has been found that P max remained constant until more than half of PS II centers were photoinhibited. The insensitivity of overall photosynthetic capacity to partial photoinhibition of PS II is accompanied by an increase in the turnover rate of the rest of non-photoinhibited PS II centers (ehrenfeld et al. 1998). The authors have shown that as soon as the electron turnover rate reached its maximum, P max started to decrease. The decrease is due to the impossibility of the rest of non-photoinhibited (functional) PS II centers to further compensate for any decrease in their number caused by the photoinhibition resulting from an accelerated electron turnover rate. The capacity to increase the electron turnover rate through functional PS II centers after photoinhibitory treatment has been called the excess capacity of the photosynthetic electron transport rate relative to the Calvin cycle reactions (ehrenfeld et al. 1998). The decrease in the photosynthetic capacity (after 40 60% of PS II centers were photoinhibited) was also discussed with respect to the heterogeneity of PS II (Leverenz et al. 1990). This hypothesis has been sustained by observation that the PS IIα or the Q reducing PS II centers are more sensitive to photoinhibition than the PS IIβ and the Q non-reducing PS II centers (riantais 1988; Neale and Melis 1991; Falk 1992; Park 1995; Andree et al. 1998). To better understand the mechanism of the excess capacity of PS II during photo-inhibition, we have compared the steady-state rate of oxygen evolution and oxygen flash yield in dependence on the background irradiance in partially photo-inhibited and control cells of green alga Chlorella öhm. The experimental data were then theoretically simulated on the basis of a simple two-electron gate model. Very good qualitative agreement of the theory with experiments was obtained assuming an increase of PS II electron turnover rate in the rest of non-photoinhibited PS II centers in the photoinhibited sample. Materials and methods Organisms The experiments were carried out with chlorococcale algae Chlorella öhm (culture collection of the Institute of otany, Academy of Sciences, Třeboň, Czech Republic). Cells were grown in batch mode in glass tubes at 35 C, using the growth medium of Šetlík (Šetlík et al. 1981). Cells were continuously bubbled with 2.5 3.5% CO 2 -enriched air. The growth irradiance of 100 µmol photons of PAR m 2 s 1 was provided by halogen bulbs. The culture was kept optically thin (absorbance A 750 = 0.07 0.15) by periodic dilution with fresh medium. Measurement of chlorophyll concentration Concentration of chlorophyll (a+b) was determined spectrophotometrically with a double beam spectrophotometer Shimadzu UV3000 (Japan) in extracts of 80% acetone according to Lichtenthaler and Wellburn (1983). Photoinhibitory and post-photoinhibitory treatment and control sample For photoinhibitory treatment, algal suspension at 3.9 mg chlorophyll (a+b) l 1 was transferred into plateparallel cuvette in temperature-controlled bath (35 C) and bubbled with air containing 2.5 3.5% CO 2. Photoinhibitory irradiance of 500 µmol photons of PAR
273 m 2 s 1 was provided by filament bulb (Osram 500 W). The degree of photoinhibition was assessed by variable fluorescence. Following 1-h of photoinhibition, synchronous measurements of oxygen evolution were made on the sample, using the rate and Clark type electrodes. The remaining volume of the sample was exposed to 100 µmol photons of PAR m 2 s 1 in order to follow the recovery from photoinhibition. The value of irradiance is above the E k for Chlorella öhm, which we determined to be about 60 µmol photons of PAR m 2 s 1 (see the legend to Figure 2). The control sample was kept under similar conditions but the irradiance was maintained at 100 µmol photons of PAR m 2 s 1 during the whole duration of the experiment. To exclude the possible effects of protein synthesis and recovery of activities during the time necessary for measurements of oxygen evolution (approx. 210 min), the cells were treated with protein synthesis inhibitor chloramphenicol (CAP) at final concentration of 100 µg ml 1. CAP was added 20 min before the photoinhibitory treatment (photoinhibited sample) and 20 min before the start of oxygen evolution measurements to the control sample. Chlorophyll fluorescence measurements Chlorophyll fluorescence parameters F 0 and F M were measured using the Double-Modulation Fluorometer FL-100 (Photon Systems Instruments, rno, Czech Republic; for description see Trtílek et al. 1997). efore each measurement, samples were kept for 5 min in darkness at room temperature. F 0 was evaluated as a mean value of four weak measuring flashes (duration of 2.5 µs) given 200 µsapart.f M was determined subsequently as a signal 50 µs after application of single turnover saturating flash from red LEDs (duration of 20 µs). Oxygen evolution measurement oth the steady-state rate of oxygen evolution (P) and the oxygen flash yield (Y) were measured using the rate electrode (Haxo and links 1950), the design of which was similar to that described by Wang and Myers (1976) with the addition of possibility of temperature control of the sample holder. One hundred µl of concentrated cell suspension was left to settle on platinum surface of the rate electrode and then optically thin film of cells was created using the tight cellophane membrane. Fresh medium enriched with 2.5 3.5% of CO 2 was flowed continuously in the space above the cellophane membrane. The whole electrode system and the medium were maintained at constant temperature of 35 C. Continual actinic light of desired intensities was provided by 150 W halogen bulb in combination with neutral density filters. The intensity of actinic light was measured by PAR quantum sensor (LI-COR L189, Lincoln, Nebraska). Single turnover saturating flashes were provided at 1 Hz frequency by the xenon flash lamp (Hamamamtsu L 4634, Japan). Since the rate electrode cannot be calibrated in absolute units (Gorkom and Gast 1996), values of steady-state rate of oxygen evolution and of the oxygen flash yield at different irradiances are presented as relative, within the interval <0, 1>. The oxygen flash yield at given irradiance was measured after the steady-state rate of oxygen evolution was reached. For each irradiance, this value has been normalized to the average value measured 3 min after the continuous actinic irradiance has been switched off. The absolute values of the steady-state rate of oxygen evolution were measured with Clark type electrode (YSI Model 5793, YSI Inc., Yellow Spring, USA). The measurements were performed at 35 Cin temperature-controlled laboratory-built chamber (artoš et al. 1975) and continual actinic light was provided by projector with 250 W halogen lamp equipped with series of neutral density filters. The gross photosynthesis was calculated from the net photosynthesis after a correction for oxygen consumption due to dark respiration. Verification of the correlation between F V /F 0 and a number of functional PS II centers The alga culture with CAP (final concentration 100 µg ml 1 ) was exposed to photoinhibitory light of 1000 µmol m 2 s 1 for 2 h. Every half hour the fluorescence parameter and the PS II activity were detected simultaneously. The fluorescence parameters (F 0 and F M ) were measured after 5 min of dark adaptation by PAM 2000 (Waltz, Effeltrich, Germany). A number of functional PS II centers was accessed for PS II activity measured as changes in the rate of O 2 evolution using a Clark type electrode (YSI Model 5793, YSI Inc., Yellow Spring, USA) at background irradiance of 1200 µmol m 2 s 1 of white light. efore start of each measurement, an acceptor of electrons from PS II dimethyl p-benzoquinone (final concentration of 8 mm) was added into the chamber of the electrode.
274 Mathematical modeling The steady-state rate of oxygen evolution and the oxygen flash yield were mathematically simulated using the two-electron gate model (e.g., Renger and Schulze 1985; Hsu 1992a, b; see Figure 4) that incorporates the function of the primary and secondary plastoquinone molecules at the acceptor side of PS II (for details see, e.g., Crofts and Wraight 1983; Lazár 1999). ut because the two-electron gate model does not include the donor side of PS II, we had to make some assumptions leading to formulation of Equations (1) and (2) for the steady-state rate of oxygen evolution and the oxygen flash yield, respectively. First, since oxygen evolution leads to the reduction of Q A,therate of Q A oxidation is proportional to the rate of oxygen evolution at steady-state. Thus, we assumed that the steady-state rate of oxygen evolution in the model is proportional (by a constant value of 1/4 corresponding to four electrons necessary to be transferred through PS II to oxidize two H 2 O molecules and evolve one O 2 molecule) to a sum of the products of steadystate concentrations of PS II centers with Q A (i.e., Q A Q,Q A Q,andQ A Q2 ) and corresponding rates of draining of electrons from Q A or Q2 by successive electron acceptors (Q and PQ pool), i.e., the rate constants of electron transport from Q A to Q (in the Q A Q state), or to Q (in the Q A Q state) and the rate constant of exchange of Q 2 /PQ molecules (in the Q A Q2 state). The photosynthetic rate, P, for given irradiance, E, can then be expressed by Equation (1): P E =(k A1 [Q A Q ] E,ss + k A2 [Q A Q ] E,ss + k exch [Q A Q2 ] E,ss)/4 (1) where subscript E indicates irradiance and subscript ss indicates steady-state level. Second, it is known that the oxygen flash yield reflects relative number of functional PS II centers (see, e.g., Falkowski and Raven 1997). In the following text and for the purpose of mathematical modeling, functional PS II centers are considered as virtually active in Q A reduction and thus their number is determined by a number of open PS II centers, i.e., centers with Q A oxidized. Thus, we assumed that the oxygen flash yield in the model is proportional to the concentrations of PS II centers with Q A (i.e., Q A Q, Q A Q, and Q A Q 2 ). As the oxygen flash yield was measured after the steady-state rate of oxygen evolution was reached (see above), the concentrations of PS II centers with Q A at the steady-state were considered. Thus, the oxygen flash yield can be described by Equation (2) as: Y E =[Q A Q 2 ] E,ss +[Q A Q ] E,ss +[Q A Q ] E,ss (2) where the meaning of subscripts E and ss is the same as in Equation (1). However, defined in such a way, oxygen flash yield reflects the absolute fraction of the functional PS II centers (Figure 5). Only after normalization of the Y(E) curves calculated according to Equation (2) to 1, does the oxygen flash yield reflect the relative number of the functional PS II centers (Figure 6 below). A change in actinic irradiance was accessed by an alternation of the value of the rate constant k L in the model (see Figure 4) in accordance with, e.g., Hsu (1992a) and Tomek et al. (2001). Values of the rate constants of the model and the initial amounts of particular model forms (both summarized in the legend to Figure 4) were the entrance parameters for each simulation. The simulations were done with the help of mathematical program Gepasi 3.21 (P. Mendes, The University of Wales, UK) that was designed for simulation of chemical and biochemical kinetics (Mendes 1993, 1997). The convexity equation (see, e.g., Leverenz 1994) was fitted to the simulated and experimentally measured P(E) curves to obtain their parameters (α, E k ). Results The photoinhibition and subsequent recovery in Chlorella cells were followed from relative changes of fluorescence parameter F V /F 0 because F V /F 0 is linearly related to a fraction of functional PS II reaction centers (Crofts et al. 1993). This assumption was also checked (data not shown; see Materials and methods ) according to methodology published by Komenda et al. (1992) who also found linear correlation between PS II activity and F V /F 0 for algae cells exposed to photoinhibitory light. Figure 1 shows that photoinhibition causes gradual decrease in the number of functional PS II centers. After 1 h at 500 µmol photons of PAR m 2 s 1, the fraction of functional (i.e., non-photoinhibited) centers dropped to about 33%. At this point the photoinhibitory irradiance was decreased to 100 µmol photons of PAR m 2 s 1.The addition of CAP (blocks protein synthesis) to the algal suspensions of both photoinhibited and control cells
275 Figure 1. Time course of relative values of F V /F 0 ((F V /F 0 ) photoinhibited /(F V /F 0 ) control ) are measured before, during and after 1 h of photoinhibitory treatment of Chlorella öhm cells at irradiance of 500 µmol photons of PAR m 2 s 1 and at temperature of 35 C. The cells were kept in dark before the photoinhibitory treatment (indicated by black horizontal bar near the x-axis) and at optimal irradiance of 100 µmol photons of PAR m 2 s 1 (see Materials and methods ) after the photoinhibitory treatment (indicated by gray horizontal bar near the x-axis). The inset shows the time course of the F V /F M ratio before, during and after the photoinhibitory treatment of photoinhibited cells (open circles) and the ratio of the control (not-photoinhibited) cells (closed squares). Relative F V /F 0 ratio and the F V /F M ratios were calculated from the same determination of the F 0 and F M values that were measured after 5-min dark adaptation at room temperature. oth, photoinhibited and control cells were treated with CAP (100 µgml 1 ; see Materials and methods ). prevented any significant increase in the number of functional PS II centers during the time after the photoinhibitory treatment that was necessary to complete the whole set of measurements of oxygen evolution under all irradiances. The inset of Figure 1 shows that the maximal quantum yield of PS II photochemistry expressed by the F V /F M ratio (Kitajama and utler 1975) decreased due to the photoinhibitory treatment from about 0.65 to about 0.38. ecause of the blocked protein synthesis, the maximal quantum yield of PS II photochemistry has only partially recovered (to 0.44) during the subsequent 4-h incubation. On the other hand, the F V /F M ratio fluctuated between 0.55 0.67 for the control sample during all the time necessary for measurement of the photoinhibited sample. Photoinhibition induced specific changes in the P(E) curves measured by either the rate electrode (Figure 2) or by Clark type electrode (Figure 3 below) and also in the oxygen flash yield versus irradiance curve (Y(E) curve) (Figure 2). The P(E) curve shows a decrease in the initial slope, α, for the photoinhibited sample, as compared to the control sample (inset of Figure 2 and Figure 3). The decrease was by 72% (41%) as revealed by fitting of the convexity equation (see Materials and methods ) to the experimentally measured P(E) curves using the rate electrode (Clark type electrode) (see Figures 2 and 3). Ley and Mauzerall (1982) defined that α = nσ PSII,wheren is the number of functional PS II reaction centers and σ PSII is their effective absorption cross section. Thus, the decrease in α obtained from the fitting of our experimental P(E) curves suggests, in agreement with fluorescence measurements (Figure 1), a decrease in the relative number of functional PS II centers after photoinhibitory treatment, assuming σ PSII is unchanged by the photoinhibition. On the other hand, there is no change in absolute value of the maximal photosynthetic rate (P max ) measured by Clark type electrode, as can be seen from Figure 3 (note that even if the same values of P max are also shown for both the photoinhibited and control samples in Figure 2, these are only relative values; see Materials and methods ). This insensitivity of P max to photoinhibition was suggested to be a demonstration of the excess capacity of electron transport rate related to Calvin cycle reactions (ehrenfeld et al. 1998; see Introduction ). Thus, the cells of Chlorella öhm also exhibit the excess capacity of the electron transport rate. The fluorescencemeasurements and changes in the steady-state rate of oxygen evolution suggest (Figure 1,
276 Figure 2. Dependencies of the rate of photosynthesis P (squares) and of the oxygen flash yield Y (circles) on irradiance measured by the rate electrode with the control (closed symbols) and photoinhibited (open symbols) Chlorella öhm cells in the logarithmic scale of irradiance. Inset shows the P(E) curve for the control (closed squares) and photoinhibited (open squares) cells on linear scale of the x-axis with marks (dotted lines) of the initial slope α of the curves. All values of P(E) and Y(E) at different irradiance are presented as relative (see Materials and methods ). The convexity equation was fitted to the experimental P(E) curves to obtain the initial slope α and light saturation index E k.the fitting revealed that there was 72% decrease of α for photo-inhibited sample (α = 0.0046, E k = 61.5 µmol photons of PAR m 2 s 1 )when compared with the control sample (α = 0.0167, E k = 238 µmol photons of PAR m 2 s 1 ). Figure 3. Dependencies of the rate of photosynthesis P on irradiance measured by Clark type electrode with the control (closed squares) and photoinhibited (open squares) Chlorella öhm cells with marks (dotted lines) of the initial slope α of the curves. Absolute values of P are presented on y-axis. There was 41% decrease of α for photoinhibited sample (α = 0.05905) when compared with the control sample (α = 0.03466) as resulted from fitting of the convexity equation (see Materials and methods ) to the P(E) curves.
277 inset of Figure 2 and Figure 3) that photoinhibition causes a decrease in the relative number of functional PS II centers. The oxygen flash yield should also be measured, as it reflects the same quality (see, e.g., Falkowski and Raven 1997). The Y(E) curves of both the photoinhibited and control samples (Figure 2) show a decrease in oxygen flash yield with increased background irradiance. The findings indicate a decrease in a number of the functional PS II centers (i.e., open PS II centers; see Materials and methods ) due to increased background irradiance. The oxygen flash yield was higher for the photo-inhibited sample (Figure 2, open circles) than for the control (Figure 2, closed circles) samples for most of the used background irradiances. This means that a higher number of the open PS II centers is present in the photoinhibited sample than in the control at a given irradiance, but only relative to their initial number in darkness. The higher availability of the open PS II centers in the photoinhibited sample at a given background irradiance must be caused by the increased turnover rate of the functional PS II centers in the photoinhibited sample, as compared to the control, because the measurement of fluorescence indicates (Figure 1) that there was a decrease in the initial number of the functional PS II centers in the photoinhibited sample in contrast with the control sample. This finding is in agreement with the results mentioned above and with previous conclusions by ehrenfeld et al. (1998). Thus, in additiontothesamep max measured for the photoinhibited and control samples, the higher oxygen flash yield also detected in the photoinhibited sample reflects the excess capacity of the photosynthetic electron transport rate initiated by photoinhibition. To describe numerically the higher availability of the functional PS II centers in the photoinhibited sample when compared to the control sample, a ratio Y P E /YC E can be calculated, where Y P E and YC E is the oxygen flash yield of photoinhibited and control sample, respectively, measured at irradiance E. When this ratio is calculated for irradiance of about 80 µmol of PAR m 2 s 1,thatis irradiance at which Y C E = 0.5, there is about 1.78 (= 0.891/0.5) times higher relative number of the functional PS II centers in photoinhibited sample than in the control sample. To simulate the Y(E) and P(E) curves of both photoinhibited and control samples, we used the twoelectron gate model describing sequential electron transport from Q A to Q and to the PQ pool (Figure 4; see also Materials and methods ). The initial conditions of the model and values of the rate constants used for simulations are given in the legend to Figure 4. The correctness of usage of the two-electron gate model for simulation of the photosynthetic curves was verified from theoretical dependencies of the maximal photosynthetic rate P max on number of functional (non-photoinhibited) PS II centers for different PS II turnover rates (expressed by k exch ). The obtained theoretical result (data not shown) corresponded with previously known fact that photosynthetic capacity is linearly dependent on the number of functional PS II centers (Herron and Mauzerall 1971; Myers and Graham 1971). When the two-electron gate model (e.g., Renger and Schulze 1985; Hsu 1992a, b) or its derivatives (e.g., Lazár et al. 1997; Tomek et al. 2001) were used for theoretical simulations of chlorophyll a fluorescence rise, the exchange of Q 2 with the PQ molecule from the PQ pool and also the oxidation of the reduced PQ molecules from the PQ pool were considered as reversible reactions. This assumption was important in those cases because a detailed kinetics of fluorescence rise was studied. However, in our case, we use the model for evaluation of the steady-state concentrations of model forms only (see Materials and methods ), and thus it is sufficient to consider the previous two reactions as irreversible (see Figure 4) where values of the rate constants k exch and k ox represent final overall rates of, in principle, reversible reactions. The results presented in Figure 1 indicate that 1-h photoinhibitory treatment caused a decrease of relative fraction of the functional PS II centers to about 0.33 and subsequent increase to about 0.49 during the time after the photoinhibitory treatment that was necessary for measurements of oxygen evolution under all irradiances. For simplicity, we assumed in theoretical simulations that the relative fraction of functional PS II centers in photoinhibited sample during the measurements of whole P(E) and Y(E) curves was unchanged and equaled to 0.45 (thus [Q A Q ] 0 = 0.45; see the legend to Figure 4) whereas all PS II centers were functional during measurements of whole P(E) and Y(E) curves with the control sample (thus [Q A Q ] 0 = 1; see the legend to Figure 4). Furthermore, we assumed that the number of PQ molecules in the PQ pool was unchanged by photoinhibition ([PQ] 0 = 7; see the legend to Figure 4). Theoretical simulations of both P(E) and Y(E) curves for the photoinhibited (open symbols) and control (closed symbols) samples based on the above assumptions are shown for different values of k exch in Figures 5A and, respectively.
278 Figure 4. A scheme of the two-electron gate model (adapted from Lazár 1999). Q ( ) A Q(,2 ) means different redox forms of primary and secondary quinone electron acceptors while PQ and PQH 2 denote oxidized and reduced plastoquinone molecules, respectively. The meaning of rate constants is as follows: k L irradiance dependent rate constant for Q A reduction; k A1, k A2 rate constants of forward electron transport from Q A to Q or Q, respectively; k A1, k A2 backward rate constants of the previous reactions; k exch rate constant of exchange of double reduced Q with oxidized PQ molecule from the PQ pool (the rate constant also includes a protonation of Q 2 before it is exchanged with an oxidized PQ molecule; see, e.g., Renger and Schulze 1985; Hsu 1992a, b); k ox rate constant of oxidation of reduced PQ molecules (PQH 2 ) from the pool. Values of the rate constants used for the simulations were those summarized by Lazár (1999): k A1 =3500 s 1, k A2 =1750 s 1, k A1 =175 s 1, k A2 =35 s 1, k ox =200 s 1, k exch =50, 100, 300, 441, 500, and 600 s 1 (see the text). Rate constant k L equaled to 10, 50, 100, 200, 400, 800, 1600, 3200, 6400, 12 800, and 25 600 s 1 (higher is the rate constant, higher was the irradiance during the measurements). Initial amounts of the model forms used for the simulations were: [Q A Q ] 0 = 1; 0.7; 0.45 or 0.2 (see the text), [PQ] 0 =7 (it corresponds to 7 PQ molecules per one PS II), and [Q A Q ] 0 =[Q A Q ] 0 =[Q A Q ] 0 =[Q A Q 2 ] 0 =[Q A Q2 ] 0 =[PQH 2 ] 0 = 0. It means that all PS II acceptors were oxidized in darkness. All the Y(E) curves simulated for the photoinhibited sample (Figure 5, open symbols) start at 1/0.45 lower value than the Y(E) curves simulated for the control sample (Figure 5, closed symbols). This reflects our definition of the oxygen flash yield as an absolute fraction of the functional PS II centers (see Equation (2) in Materials and methods ) and the assumption of 1/0.45 less functional PS II centers for the photoinhibited sample than for the control sample. Figure 5 also shows that the Y(E) curves shifts to higher irradiances with increased value of k exch used in the simulations. On the other hand, 1/0.45 decrease in functional PS II centers in the photoinhibited sample relative to the control sample resulted in a proportionally similar decrease in the lowest irradiances of the steady-state rate of oxygen evolution for the photoinhibited sample (Figure 5A, open symbols) when compared to the control sample (Figure 5A, closed symbols) in agreement with experimental results (Figure 2) no matter which value of k exch was used in the simulations. However, this proportional difference between the P(E) curves simulated with the same value of k exch for the photoinhibited and control sample is present during the whole course of the P(E) curves (Figure 5A, compare curves presented by the same types of open and closed symbols). Thus, for no pair of such P(E) curves the same maximal rate of photosynthesis P max was reached. It should be noted that here the decrease in functional PS II centers from 1 to 0.45 during photo-inhibition causes an apparent 1/0.45-fold increase in the turnover rate in the remaining functional PS II centers (because the number of PQ molecules remains constant as the number of functional PS II centers decreases). Thus, this mechanism is, in principle, similar to the one suggested by ehrenfeld et al. (1998) as a cause of the excess capacity of photosynthetic electron transport rate relative to Calvin cycle reactions (see Introduction ). ut results of Figure 5A further suggest that when we assume only an apparent increase in the turnover rate in the remaining functional PS II centers, then the model is unable to generate the same value of P max for photoinhibited and control samples. Thus, the excess capacity of photosynthetic electron trans-
279 Figure 5. Theoretically simulated (on the basis of the two-electron gate model presented in Figure 4) dependencies of the rate of photosynthesis P (A; the P(E) curves) and of the oxygen flash yield Y (; the Y(E) curves) on irradiance (expressed by irradiance-dependent rate constant k L of the model; see Figure 4, and Materials and methods ) for 100% (closed symbols) and 45% (open symbols) of functional PS II centers and for different values of the rate constant k exch of exchange of Q 2 with oxidized PQ molecules from the PQ pool (see Figure 4). k exch equaled to 50 s 1 (down triangles), 100 s 1 (up triangles), 300 s 1 (circles), and 441 s 1 (squares). The data are the raw calculated data without any normalization, and k L is given in logarithmic scale. Thus, the oxygen flash yield reflects, according to its definition (Equation (2)), the absolute fraction of the functional PS II centers. port rate relative to Calvin cycle reactions cannot be caused only by an increase in the apparent turnover rate in the remaining functional PS II centers. Some additional mechanism must be involved. The possible mechanism is revealed by the theoretical simulations of P(E) curves for control and photoinhibited samples represented by the closed triangles and open squares in Figure 5A. In these simulations, variability in k exch was assumed (k exch equaled to 100 and 441 s 1,respectively), and, as a result, both the P(E) curves reached the same value of P max. The two theoretical P(E) curves mentioned above and corresponding theoretical Y(E) curves with maximal values of all the curves normalized to 1 are presented in Figure 6, so that they can be compared with the corresponding experimental P(E) and Y(E) curves of Figure 2. Due to the normalization, the Y(E) curves now reflect the relative number of the functional PS II centers (compare with the previous paragraph). A very good qualitative agreement of the theory with the experiment is achieved not only for the P(E) curves but also for the Y(E) curves. Fitting of the convexity equation (see Materials and methods ) to the two theoretical P(E) curves shown in Figure 6 revealed that there is a 58% decrease of α due to photoinhibition that is in the range determined from the experimental P(E) curves measured with the rate (72%) and Clark (41%) type electrode. When the higher availability of the functional PS II centers in the photoinhibited sample compared to the control sample is calculated from the theoretical Y(E) curves shown in Figure 6, just as it was done for the experimental Y(E) curves (see the third paragraph in Results ), it results in about 1.36 (= 0.681/0.5) times higher relative number of the functional PS II centers in the photoinhibited sample than in the control sample. The difference between this value and the value of the parameter calculated from the experimental Y(E) curves (1.78) of Figure 2 is probably caused by the fact that we assumed an unchanged initial number of functional PS II centers when simulating theoretical Y(E) curve for the photoinhibited sample. In fact, the number of functional PS II centers can slightly change during the measurement of experimental Y(E) curves (Figure 1). However, generally good correspondence between the theoretical and experimental P(E) and Y(E) curves enables us to suggest that the excess capacity of photosynthetic electron transport rate relative to the Calvin cycle reactions results, in fact, from an ability of PS II, initiated by photoinhibition, to actively increase the turnover rate through the rest of functional PS II centers of the photoinhibited sample. This phenomenon is expressed in our theoretical approach by the increase of the value of the rate constant of exchange k exch between the double reduced Q and the oxidized PQ molecule from the PQ pool. Discussion Our experimental results clearly show (Figure 3) that even if Chlorella öhm cells are severely photo-
280 Figure 6. Another presentation of theoretically simulated P(E) and Y(E) curves for control (closed symbols) and photoinhibited (open symbols) samples that are presented by up closed triangles and open squares in (A) of Figure 5 (i.e., the P(E) curves for the control and photoinhibited samples, respectively) and by the same symbols in () of Figure 5 (i.e., the Y(E) curves for the control and photoinhibited samples, respectively). To facilitate comparison of the courses of the theoretically calculated P(E) and Y(E) curves with the P(E) and Y(E) curves experimentally measured by the rate electrode as shown in Figure 2, the maximal values of the theoretical curves are normalized to 1 and k L is given in logarithmic scale. Thus, the oxygen flash yield reflects the relative number of the functional PS II centers. The convexity equation (see Materials and methods ) was fitted to the theoretical P(E) curves to obtain the initial slope α and light saturation index E k. The fitting revealed that there was 58% decrease of α for photoinhibited sample (α = 0.1042, E k = 1662 s 1 ) when compared with the control sample (α = 0.2465, E k = 635.6 s 1 ). inhibited, they can attain the same maximal photosynthetic rate P max as the control cells. This feature of photosynthetic cells was suggested by ehrenfeld et al. (1998) to be due to the excess capacity of photosynthetic electron transport rate relative to the Calvin cycle reactions. The authors explained this excess capacity as resulting from the ability of photosynthetic cells to increase turnover rate in the rest of non-photoinhibited PS II centers. To propose a more exact mechanism for the increase in the turnover rate and because the slowest event in the linear electron transport chain through PS II is related to the redox reactions of PQ molecules in the PQ pool, we have focused on the PQ pool reactions in our theoretical simulations, namely on an effect of a rate constant k exch of Q 2 exchange with the PQ molecule from the pool. However, the overall rate of the exchange reaction can also be influenced by an increased concentration of reduced plastoquinone (PQH 2 ) during increased irradiance, as shown by Cleland (1998). As the photosynthetic rate became limited by Calvin cycle reactions involving high irradiances (see Introduction ), this limitation could cause the accumulation of reduced PQ molecules. Thus, a decrease of our k ox (reflects oxidation of reduced PQ molecules by all reactions of electron transport behind PQ pool, including Calvin cycle reactions) should be taken into account. ut our theoretical simulations (data not shown) have revealed a qualitatively similar increase in the steadystate concentration of PQH 2 in increased irradiance as described by Cleland (1998) but without any changes in the rate constant k ox. Thus, kinetic properties of our model and increased irradiance themselves can cause an increased number of reduced PQ molecules without consideration of a decrease of our k ox upon increased irradiance. Hence, we considered the rate constant k ox of oxidation of reduced PQ molecules in increased irradiation to be unchanged. As photoinhibition causes a decrease in the fraction of functional PS II centers (a decrease of F V /F 0 in Figure 1 but also diminished slope of the P(E) curves of photoinhibited sample compared to the control sample in Figures 2 and 3), it results in a smaller fraction of the remaining functional PS II centers served by the unchanged number of PQ molecules in the PQ pool. This leads to an apparent increase of the turnover rate in the remaining functional PS II centers that, according to the suggestion of ehrenfeld
281 et al. (1998), should result in the same value of P max for control and photoinhibited samples. However, our theoretical simulations show (Figure 5A) that the apparent increase of PS II turnover rate is not enough per se to maintain the same P max in photoinhibited samples as in controls. On the other hand, the theoretical simulations show (Figure 5A) that a real increase of the turnover rate in the remaining functional (i.e., non-photoinhibited) PS II centers, expressed in our model as an increase in the rate constant k exch, is necessary to attain the constant value of P max during photoinhibition. We can only speculate about the molecular origin of this increase of the rate constant. As k exch in our model describes the rate of Q 2 exchange with PQ molecules from the pool (i.e., the rate of PQ pool reduction), it includes both physical properties of the Q pocket in D1 protein of PS II and also an availability of PQ molecules from the pool, i.e., diffusional properties of PQ molecules in thylakoid membrane. Thus, a conformational change of PS II and/or rearrangement of thylakoid membrane caused by photo-inhibition can be the possible reason for the increase of k exch. It is noteworthy that different values in the rate of PQ pool reduction were even found for physiological (non-photoinhibited) conditions (Joliot et al. 1992; Lavergne et al. 1992; Kirchhoff et al. 2000) demonstrating that variability of k exch really exists in vivo. An ability of PS II to compensate for the decreased fraction of functional PS II centers by increasing its turnover rate is only possible to the degree to which it is physiologically possible to increase the PS II turnover rate. When the saturation level of PS II turnover rate is reached, then further decrease in the fraction of functional PS II centers leads to decrease in P max (ehrenfeld et al. 1998). This fact is also demonstrated in Figure 7 where we plotted the theoretically calculated (on the basis of our model) values of P max in relation to the values of the rate constant k exch (represents the PS II turnover rate) for different fractions of functional PS II centers. For example, when we consider 100% of functional PS II centers and a k exch equal to 100 s 1 for the control sample (i.e., the P(E) curve represented by closed up triangles in Figure 5A), to maintain the same value of P max, k exch must be increased to about 180 s 1 when 70% of the PS II centers are functional, to 441 s 1 when 45% of the PS II centers are functional (i.e., the P(E) curve represented by the open squares in Figure 5A), but no increase of k exch is possible to compensate for the decrease of functional PS II centers to only 20%, if k exch equalled to 600 s 1 is assumed as the maximal physiological value (see Figure 7). The value of k exch equal to 600 s 1 (corresponds to the turnover time of PS II, i.e., the exchange of Q 2 with PQ molecules from the pool, equal to 1.7 ms) seems to be a reasonable estimate of the maximal turnover rate of PS II (see, e.g., Crofts and Wraight 1983). Our theoretical simulations show that both the increased oxygen flash yield of the-photoinhibited sample and also the insensitivity of P max to photoinhibition can be caused by an increase in the k exch. On the other hand, P max represents the rate of whole photosynthesis limited by the Calvin cycle reactions (Sukenik et al. 1987). This means that, under steady state photosynthesis, the maximal achievable rate of PS II turnover is adapted to (and thus lowered by) the slower Calvin cycle reactions. This kinetic difference between the slower Calvin cycle reactions and the faster PS II turnover represents in fact the excess capacity, which has been many times described and discussed in the literature (Dietz and Heber 1984a, b; Stitt 1986; ehrenfeld et al. 1998). A sample exposed to photoinhibition can exploit this excess capacity and increase its PS II turnover rate. ut this capacity of electron turnover rate is not in fact an excess; it is rather an inner capacity that can manifest itself due to photoinhibition. One can only speculate about the evolutionary origin of differences in turnover rates of major photosynthetic enzymatic complexes, i.e., of PS II, cytochrome b 6 /f,psiorruisco.inany case, this mismatch in maximal turnover rates seems to be an important mechanism for maintaining the maximal rate of the overall photosynthesis under high light conditions, when photoinhibition can temporarily decrease the number of functional PS II centers. Our theoretical simulations agree very well with experimental results, when we assume that the excess capacity of photosynthetic electron transport rate is caused by an increase in the constant rate of Q 2 exchanged with oxidized PQ molecules from the PQ pool. However, an opposite effect of photoinhibition on PS II turnover rate was reported by Laisk and Oja (2000) on the basis of measurements of oxygen evolution in response to multiple-turnover light pulses of different length. The authors found that the PS II turnover rate is about two times smaller when reversible photoinhibitory fluorescence quenching was activated. However, in their experiments, the authors always used far-red light to completely oxidize the PQ pool before measurement of the oxygen evolution. On the other hand, we simulated and measured both the
282 Figure 7. Theoretically simulated (on the basis of the two-electron gate model presented in Figure 4) dependencies of the maximal photosynthetic rate P max (the photosynthetic capacity) on PS II turnover rate (expressed by the rate constant k exch of Q 2 exchange with oxidized PQ molecule from the PQ pool; see Figure 4) for different relative numbers of functional (non-photoinhibited) PS II centers, indicated in the figure. For explanation of the dotted lines in the figure see the text. The data are the raw calculated data without any normalization. photosynthetic rate and the oxygen flash yield at the steady-state of photosynthesis, i.e., in cases when the redox equilibrium between oxidized and reduced PQ molecules from the PQ pool was achieved. Thus, different conditions used for measurements are probably the reason for our different conclusions from Laisk and Oja (2000). Conclusion Measurement of the photosynthetic rate by a Clarktype electrode shows that photoinhibited Chlorella öhm cells can reach the same maximal photosynthetic rate P max as the control, non-photoinhibited cells. Further, the oxygen flash yield measured by the rate electrode is higher for the photoinhibited than for the control cells for most of the background irradiances used. We explored, by using theoretical simulations, if the above mentioned features of the photoinhibited cells could be caused by the excess capacity of the PS II turnover rate, as suggested by ehrenfeld et al. (1998). Assuming in our theoretical simulations that the PS II turnover rate is determined by the constant constant (k exch )ofq 2 exchange with oxidized PQ molecules from the PQ pool in the two-electron gate model, a good qualitative agreement between P(E) and Y(E) curves for photoinhibited and control samples measured with the rate electrode and the theoretically simulated P(E) and Y(E) curves was obtained, so long as k exch increased from 100 s 1 for control to 441 s 1 in the photoinhibited samples. Even if during different extents of photoinhibition, values of the rate constant k exch different from those determined by us are probably involved, the agreement between theory and experiment suggests that PS II is able to increase its turnover rate to compensate for the decreased number of functional PS II centers caused by photoinhibition. A molecular mechanism for this ability of PS II is, however, not available and further study is necessary to solve this question. Acknowledgements This research in the laboratory of R.K., D.L., and J.N. has been supported by grant number MSM 153100010 of Ministry of Education of the Czech Republic. D.L. wishes to thank the Grant Agency of the Czech Republic for financial support (grant number 204/02/P071). The research in the laboratory of O.P. has been supported by the Grant Agency of the Czech Republic (project 206/98/P110) and by the Ministry of Education of the Czech Republic (project LN00A141). The Institute of Microbiology was also supported by The Institutional Research Concept no. AV0Z5020903.
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