Photosynthesis Research 68: 247–257, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
247
Technical communication
Determination of the antenna heterogeneity of Photosystem II by direct simultaneous fitting of several fluorescence rise curves measured with DCMU at different light intensities Dušan Laz´ar1,2,∗ , Pavel Tomek1 , Petr Il´ık1 & Jan Nauš1 1 Palack´ y
University, Faculty of Science, Department of Experimental Physics, tˇr. Svobody 26, 771 46 Olomouc, ˇ e Czech Republic; 2 Photosynthesis Research Center, University of South Bohemia, Branišovsk´a 31, 370 05 Cesk´ ∗ Budˇejovice, Czech Republic; Author for correspondence (e-mail:
[email protected]; fax: +420-68-5225737)
Received 12 September 2000; accepted in revised form 9 May 2001
Key words: connectivity, DCMU, fluorescence induction, model, PS II antenna heterogeneity
Abstract Several methods for determination of the antenna heterogeneity of Photosystem II from fluorescence rise curves measured with DCMU have been developed so far. Using these methods, two, three or four types of Photosystem II with respect to the antenna heterogeneity were determined. However, the accuracy of some of these methods is under debate. Here, we present a new method for the determination of the antenna heterogeneity of Photosystem II. The method is based on direct simultaneous fitting of several fluorescence rise curves measured with DCMU at different intensities of light excitation. As several curves measured under different light conditions are fitted simultaneously by the same model, reliability and accuracy in determination of model parameters increase. Our method was applied to two plant materials with different structure of the thylakoid membrane: wheat leaves and cells of green alga Chlamydomonas reinhardtii. Abbreviations: CA – complementary area; DCMU – 3-(3 ,4 -dichlorophenyl)-1,1-dimethylurea; DCMU-FI – fluorescence induction measured in presence of DCMU; F0 – minimal fluorescence intensity; FM – maximal fluorescence intensity; FR – fluorescence rise; F(t) – fluorescence intensity at time t; PAR – photosynthetically active radiation; PS II – Photosystem II Introduction It is well known that Photosystem II (PS II) is heterogeneous in many aspects. One of the heterogeneities is the PS II antenna heterogeneity, which involves the PS II antenna size heterogeneity and the heterogeneity in energetic connectivity between PS IIs. Determination of the antenna heterogeneity of PS II from fluorescence rise (FR) curve measured with 3-(3 ,4 dichlorophenyl)-1,1-dimethylurea, (DCMU) was first introduced by Melis and Homann (1975, 1976). The method is based on the evaluation of the time course of the complementary area (CA; area between FR curve and a horizontal line at the level of the maximal fluorescence FM ). The authors found a biphasic increase
of the CA. This finding was attributed to two different types of PS IIs denoted as PS IIα and PS IIβ. PS IIβ has a smaller rate constant (kβ ), characterising the overall rate of PS IIβ photochemistry, than the kα of PS IIα. As the rate of light utilisation by PS II photochemistry is directly proportional to the functional size of PS II antenna (Glazer and Melis 1987; Melis 1989) and as both PS IIα and PS IIβ have about the same photochemical quantum yield (Thielen and Van Gorkom 1981a, b), the smaller kβ in comparison to kα expresses smaller antenna size of PS IIβ than of PS IIα (Melis and Duysens 1979; Thielen and Van Gorkom 1981a). Furthermore, PS IIβ were characterised by an exponential rise of the time course of CA, whereas PS IIα showed a non-exponential rise (Melis and Homann
248 1976). The exponential shape of this rise for PS IIβ was suggested to reflect mutual energetic separation of these PS IIs (Melis and Duysens 1979; Anderson and Melis 1983; Melis and Anderson 1983). On the other hand, the non-exponential (sigmoidal) FR of PS IIα is generally believed to reflect energetic connectivity between these PS IIs (see latest review on fluorescence induction by Lazár 1999) as originally suggested by Joliot and Joliot (1964). Using the method introduced by Melis and Homann, three (denoted as α, β, γ , all exponential; Sinclair and Spence 1988) or even four (denoted as α, β, , δ, only α non-exponential; Sinclair and Spence 1990) PS II types with regard to the antenna heterogeneity were determined. However, the determination of the PS II antenna heterogeneity by this method suffers from uncertainty in the determination of the FM value that can lead to significant distortion of the results (see Bell and Hipkins 1985; Sinclair and Spence 1988). To eliminate this problem, Hsu et al. (1989) developed a new method in which the FM value is first determined by a mathematical method and then the same approach as used by Melis and Homann is applied. Three types of PS II with regard to the antenna heterogeneity were determined in such a way: one exponential (α) and two non-exponential (β, γ ) (Hsu et al. 1989; Hsu and Lee 1991). Meunier and Bendall (1992) used the Isenberg method of moments for evaluation of decrease of a curve defined as the difference between total CA and CA at any time of measurement. The method ensures an evaluation of the correct number of exponential components. Three types of PS II denoted as α, β, γ (all exponential; condition for usage of the method) were determined using this method. In contrast to the previous methods evaluating the time course of CA, Lavergne and Trissl (1995) and Trissl and Lavergne (1995) fitted the parameters of the reversible radical pair model directly to the time course of the DCMU-FR curve. Using this method, two types of PS II were determined: PS IIα (nonexponential rise) and PS IIβ (exponential rise). As noted by Lavergne and Trissl (1995), the direct fitting of the time course of the DCMU-FR curve enables a more accurate determination of PS II antenna heterogeneity than the evaluation of the heterogeneity from the time course of CA because the former method avoids possible mistakes resulting from the erroneous determination of FM . The determination of PS II antenna heterogeneity was also done without application of DCMU. In the flash fluorescence induction measurement, a very
strong 100 µs flash is applied which causes transient closure of PS II (Nedbal et al. 1999). Analysis of FR during the flash fluorescence induction using the method of Melis and Homann revealed exponential β and non-exponential α types of PS II (Nedbal et al. 1999). In addition to DCMU-FR measurement, the PS II antenna size can be also evaluated from measurement of the light induced absorbance change at 320 or 550 nm (McCauley and Melis 1986) or at 518 nm and by evaluating its light saturation curve (Chylla and Whitmarsh 1990) or from measurement of the light saturation curves of the variable fluorescence (Samson and Bruce 1996), CO2 fixation rate (Malkin et al. 1981), steady state oxygen evolution rate (Henrysson and Sundby 1990), flash oxygen yield (Wang and Myers 1973) or flash proton release yield (Nedbal et al. 1991). Furthermore, it should be noted that the same denotation used for PS II types with respect to antenna heterogeneity by different authors need not really mean the same types of PS II. PS II antenna heterogeneity is also connected to lateral heterogeneity of the thylakoid membrane. It was found that PS IIα is predominately located in the grana of the thylakoid membrane, whereas PS IIβ is predominantly located in the stromatal thylakoid membrane (Anderson and Melis 1983; Ghirardi et al. 1986). A connection of the PS II antenna heterogeneity to other types of PS II heterogeneity, mainly to the QB -reducing versus QB -non-reducing PS II heterogeneity, is under debate (see, e.g., Govindjee 1990; Guenther and Melis 1990). For further details on PS II heterogeneity, see review articles by Black et al. (1986), Melis (1991), Karukstis (1992), and Lavergne and Briantais (1996). In the present work, we introduce a method for the evaluation of PS II antenna heterogeneity from the DCMU-FR curve. Novel in our method is the simultaneous direct fitting of several DCMU-FR curves at different light intensities by one model. The simultaneous fitting of several FR curves increases reliability and accuracy of the determination of model parameters.
Materials and methods Wheat seedlings (Triticum aestivum cv. Samantha) were cultivated at 20 ◦ C in perlit substrate supplied with Knop solution and exposed to light–dark cycles (16 hours white light / 8 hours dark) with a light intens-
249 ity of about 90 µmol m−2 s−1 of PAR at the top of the seedlings. The leaves were harvested after 8 days of growing (growth phase 11 according to Zadoks et al. (1974)) in the dark phase of the growth cycle. A 1-cm long tip of the leaf blade was detached and the following 2-cm long leaf segment was used for measurement of FR from the adaxial side of the leaf segment. To inhibit electron transport between QA and QB acceptors of PS II in native leaves, the leaves were submerged in a 1-mM water solution of DCMU and exposed to low pressure (40 kPa) for 35 min. Asynchronous cultures of the green algal cells Chlamydomonas reinhardtii (UTEX 2246/137c) were grown at 25 ◦ C in 0.5 l of liquid medium (Setlik et al. 1981). The algal cultures were illuminated with continuous white light (80 µmol m−2 s−1 of PAR) and the cells were mixed by bubbling with air containing 3% CO2 . The cells were harvested in the log phase of their optimal growth conditions. To inhibit electron transport between QA and QB acceptors of PS II, a DCMU solution was added to the algal culture to obtain a final concentration of 10 µM of DCMU. Chlorophyll content was determined spectrophotometrically in 80% acetone according to Lichtenthaler (1987). Absorbance spectra were recorded with a spectrophotometer Beckman DU-8B (Beckman Instruments, Inc., USA). The chlorophyll content of the wheat leaves and the suspension of Chlamydomonas reinhardtii was about 35 µg cm−2 and 12 µg ml−1 , respectively. The Chl a/b ratio was about 3 for wheat leaves and about 1.8 for the culture of Chlamydomonas reinhardtii. The chlorophyll FR curves were measured on the segments of wheat leaves and in the algal suspension at room temperature using a shutter-less portable chlorophyll fluorometer PEA (Hansatech Instruments Ltd, UK) with different excitation light intensities. The excitation light was provided by light-emitting diodes (emission maximum at about 650 nm). The maximal intensity (100%) was about 12 000 µmol m−2 s−1 of PAR (measured with Quantum Radiometer LI-189, LI-COR, USA) at the sample surface. The measured leaf region or algal suspension was dark-adapted for 15 min. The experiments with green alga were done with aliquots of 0.5-ml cell suspension in 1-cm vials. The optical thickness of the sample was 5 mm, and the diameter of the sample area irradiated was 4 mm. Fluorescence signal at a time of 70 µs is considered as the minimal fluorescence F0 . The fluorescence signal at lower times was mainly for higher intensities of excitation, distorted by the fluorometer as was checked
with acetone chlorophyll extract. Thus, FR curves are presented from 70 µs in the figures. Fitting of theoretical expressions to FR curves measured with DCMU was performed using the Solver function in MS Excel ’97. The solver uses the Generalized Reduced Gradient (GRG2) non-linear optimisation algorithm to find a minimum of the sum of squared deviations between the experimental data and model calculations (the χ 2 value). To evaluate the error ranges for the optimal set of fitted parameters (values of the parameters for which the χ 2 is minimal), we adopted the unidimensional search procedure as described by Roelofs et al. (1992). In this procedure, the error ranges for a parameter are found by a stepwise change of the parameter from its optimal value to both sides until a significantly worse χ 2 value (χ 2 > χ 2 min ) is obtained. A value of the χ 2 that is significantly worse than the χ 2 min is found on the basis of the fact that a ratio of two χ 2 values is distributed according to the F-distribution. Thus, for a pre-set confidence level (of 67% in our case, roughly corresponding to one standard deviation) and number of degrees of freedom (number of data points minus number of free running fit parameters, 2017 in our case) the F-factor is calculated by which the χ 2 min can be increased without significant loss in quality of fit. In such a way determined error ranges could be asymmetric (that is, in general, the case in non-linear systems (Johnson 1983)) in contrast to commonly used ± standard error computed from the curvature matrix. For further details on the unidimensional search procedure, see Roelofs et al. (1992) and Holzwarth (1996) and references therein.
Results and discussion FR curves measured at different light intensities with DCMU-treated wheat leaves and the algal suspension are shown in Figure 1. The curves are presented by means of relative variable fluorescence rFV (t), which is defined as (F(t) − F0 )/(FM − F0 ), where F0 , FM , and F(t) are the minimal and maximal measured fluorescence intensities and fluorescence intensity at time t, respectively. The DCMU-treatment should, once QA is reduced, keep it in its reduced state at all times during the measurement of DCMU-FR (assuming that no recombination of QA − with a positive charge occurs). Thus, a slow decrease of the fluorescence signal after the FM value measured in the presence of DCMU probably does not reflect photochemical reactions. Be-
250
Figure 1. Time courses of the DCMU-FR curves measured by the PEA fluorometer at different light intensities with wheat leaves (A) and with Chlamydomonas reinhardtii cells (B). The curves are presented in term of the rFV (t) (see ‘Results and discussion’) and start from 70 µs (F0 , see ‘Material and methods’) and finish at 100 ms (FM , see ‘Results and discussion’). Results of the best fit are shown as full lines. Total χ 2 value (sum of χ 2 values of particular curves) was 0.105 and 0.228 for wheat leaves and Chlamydomonas reinhardtii cells, respectively. Light intensities in percentages used for the measurements are shown in the key and are related to the maximal light intensity of the PEA fluorometer.
cause of this reason, we plotted DCMU-FR curves in Figure 1 only to a time of 100 ms and also used these curves for subsequent fitting. This is in agreement with Lavergne and Trissl (1995) who found that there is no significant change between results of PS II antenna heterogeneity when the DCMU-FR curve is directly fitted to 50 ms or to 200 ms or to the whole experimental range. Figure 1 clearly demonstrates that the maximal fluorescence is reached at shorter times with increasing light intensity. As originally suggested by Duysens and Sweers (1963) that the fluorescence signal is related to the amount of reduced QA , the increasing light intensity causes a faster reduction of QA . Thus, the individual DCMU-FR curves measured with wheat leaves (or algal suspension) should only differ in the rate of QA reduction and other parameters (connectivity parameter p and amounts of particular PS II types) should remain the same for all measured curves. Hence, the rationale of our method is a direct fitting of the DCMU-FR curves measured with wheat leaves (or algal suspension) at different light intensities altogether. The advantage of this treatment is that a fitting of several curves by the same model should increase the reliability and accuracy in the determination of
the model parameters (see review on data analysis by Holzwarth 1996). A direct fitting of the courses of the DCMU-FR curves should also ensure better evaluation of the PS II antenna heterogeneity (see ‘Introduction’). As mentioned in the Introduction, two, three or even four types of PS II with respect to the antenna heterogeneity were distinguished by particular methods. As the most exact methods by Hsu et al. (1989) and Meunier and Bendall (1992) distinguished three types of PS II with respect to the antenna heterogeneity, we also assumed that three types of PS II are enough to sufficiently describe the DCMU-FR curves measured with one plant material at all light intensities. As for the value of the connectivity parameter p of connected PS IIs, we restricted it in the fitting procedure to its maximal value equalled to 0.7. This is in agreement with the reported values (p = 0.5–0.7 for connected PS IIs, see Lazár 1999 and references therein). Furthermore, we did not assume a strict energetic separation of PS IIs that have an exponential or near to exponential FR. This is based on the results of Lavergne and Leci (1993), who found that if a small amount (up to 40%) of one type of the PS II is energetically connected with the rest of the PS IIs, the former have an exponential FR, even though these
251 PS IIs have a p parameter equalling to 0.55, which is a typical value for the connected units. Furthermore, Lavergne and Trissl (1995) found that the p parameter equalled 0.4 for PS IIβ. It is also known that the time course of the FR with the p parameter in the range of 0 up to 0.33 is more exponential than sigmoidal in shape (Paillotin 1976; Den Hollander et al. 1983; Sorokin 1985; France et al. 1992). Thus, taken all together, the fluorescence rise measured with DCMU for one plant material and under all light intensities was fitted by the following equation: rFv (t) =
3 (1 – pi )PS IIclosed (t) i
i=1
1 – pi PS IIclosed (t) i
,
(1)
where pi is the connectivity parameter for the particular type of PS II, according to Joliot and Joliot (1964) and PS IIi closed(t) is the relative amount of closed PS IIs of a particular type at time t (PS IIs in which QA is reduced; also denoted in the literature as e according to Joliot and Joliot (1964), or as B according to Strasser (1978)). The pi parameters were assumed to be unchanged by light intensities used for particular DCMU-FRs. The closing of PS IIs in the presence of DCMU was supposed to be described by the reaction: open
PS IIi
kL,i −→ PS IIclosed , i = 1, 2, 3, i
(2)
where PS IIi open and PS IIi closed are the amounts of open and closed PS IIs of a particular type (i.e., PS IIs with QA oxidised and reduced, respectively). The above reaction leads to the amount of closed PS II presenting a solution in the form: open
(t) = PS II 0,i , (1 -e (−kL,i t) ), i = 1, 2, 3, PS II closed i (3) where t is time and PS IIopen0,i is a relative initial amount of PS II of a particular type ( PS IIopen0,i = 1) and it was enabled to fit. kL,i is an overall rate constant for PS II photochemistry of a particular type in the presence of DCMU and depends on the amount of excitation supplying PS IIs and thus was enabled to change in the fitting procedure depending on the light intensity used for the measurements. However, according to the theory of connected PS IIs, increasing closure of PS IIs with time causes an increase in the effective antenna size that serves the remaining open PS IIs, leading thus to an increasing rate of QA reduction in the remaining open PS IIs (see Lazár 1999). Thus, kL,i is a function of time according to the following
equation: kL,i (t) =
k0L,i 1 – pi PS IIclosed (t) i
, i = 1, 2, 3,
(4)
where k0 L,i is the initial rate constant (at t = 0) for PS II closure (i.e., for QA reduction) of a particular type and PS IIi closed(t) is the amount of closed PS II of a particular type at time t. The equations above describe energy utilisation leading to PS II photochemistry (closure) in the connected photosynthetic units (PSU). Similar expressions were derived for other types of PSUs: the energetically separated PSUs (also called the puddle model of PSUs) (Malkin 1966, 1974), the statistical model (also called the lake model) of PSU (Vredenberg and Duysens 1963; Clayton 1966, 1967), and the domain model of PSUs (Paillotin et al. 1979, 1983; Den Hollander et al. 1983; Valkunas et al. 1997). As the latest results show that the DCMU-FRs are best described by the model of connected PSUs (Lavergne and Trissl 1995; Trissl and Lavergne 1995; Vavilin et al. 1998; Lazár and Pospíšil 1999), we have also used the theory of the connected PSUs in our mathematical descriptions. In principle, however, any other theory could be used. However, a comprehensive discussion about mutual relationships between parameters of particular theories and about results of the fitting procedure using another theory are beyond the scope of this work. Graphical results of the best fit of FI curves measured with DCMU with both materials and at all used light intensities are shown in Figure 1 (full lines), whereas numerical values of the best fit model parameters are shown in Figure 2 and the results of error analysis (see ‘Materials and methods’) are listed in Table 1. As any value of measured or derived quantity is only as good as the knowledge about its error ranges (Holzwarth 1996), we briefly comment on results obtained from error analysis before further discussion of the results in detail. The error analysis revealed (see Table 1) that for both materials the amount of PS II1 is determined with the smallest error, whereas the amount of PS II3 is determined with the largest error. Interestingly, the amount of PS II3 could be higher by 27% in the case of wheat leaves (resulting in 5% of PS II3 from all PS IIs), or even higher by 53% in the case of Chlamydomonas reinhardtii cells (resulting in 43% of PS II3 from all PS IIs) without a significant loss in quality of fit. In the case of the pi parameters, the largest error in their determination was found for p3 parameters for Chlamydomonas reinhardtii cells. However, even in the case of the increase of this para-
252
Figure 2. Values of the parameters obtained from the best fit of DCMU-FR curves (top of figures) and a linear dependence of the k0 L,1 and k0 L,2 values (see keys) on light-intensity and light-independent values of k0 L,3 (insets) found for wheat leaves (A) and Chlamydomonas reinhardtii cells (B). k0 L,1 , k0 L,2 , and k0 L,3 are the initial rate constants (see the text) for the closure of PS II1 , PS II2 , and PS II3 , respectively, according to Equation (4). p1 , p2 , and p3 are the values of the connectivity parameters for PS II1 , PS II2 , and PS II3 , respectively, according to Equations (1) and (4). Correlation coefficients R (P < 0.0001) for the linear dependence of k0 L,i values on the light intensity were 0.994 (k0 L,1 ) and 0.969 (k0 L,2 ) for wheat leaves and 0.999 for both k0 L,1 and k0 L,2 of Chlamydomonas reinhardtii cells. For results of the error analysis, see Table 1.
Table 1. Results of the error analysis of the fitted model parameters for wheat leaves and Chlamydomonas reinhardtii cells. Changes to lower/higher values are maximal relative changes (in %) of the parameters to lower/higher values from their optimal best-fit values (presented in Figure 2) without a significant loss in quality of fit (see ‘Materials and methods’). In the case of k0 L,i , the maximal changes represent average values calculated from maximal changes of particular k0 L,i values determined for particular light intensities. The change to higher values for both materials in the case of p1 parameters and the change to lower values in the case of the p3 parameter for wheat leaves and the p2 parameter for Chlamydomonas reinhardtii cells were not determined (n.d.) because these parameters reached limiting values assumed by our model (0.7 and 0) already as the best-fit values. The changes to higher values in the case of, p3 parameter for wheat leaves and the p2 parameter for Chlamydomonas reinhardtii cells are not relative changes (because of division by zero) but absolute values
Wheat leaves Change to higher values (%) Change to lower values (%) Chlamydomonas reinhardtii cells Change to higher values (%) Change to lower values (%)
PS II1
PS II2
PS II3
p1
p2
p3
k0 L,1
k0 L,2
k0 L,3
0.2 1.0
1.5 1.9
27 5.0
n.d. 0.8
1.9 12
0.05 n.d.
2.5 2.4
3 2.9
42 38
0.6 2.0
7.0 3.8
53 3.5
n.d. 0.9
0.03 n.d.
46 4.0
2.4 2.5
7.4 8.2
48 41
253 meter by 46% (Table 1; resulting in p3 = 0.11), the appropriate conclusions presented in the next paragraphs are still valid. For both materials, the smaller the rate constant k0 L,i (the slower is the process of QA reduction), the larger the error (see Table 1) and probably longer measurements would be necessary for more exact determination of these slow processes. Figure 1 shows a very good agreement of the theoretical model with the experimental results. As for wheat leaves, 64% of PS II (marked by subscript 1) is characterised by high values of k0 L,1 (Figure 2A) and a high value of the connectivity parameter p1 (0.7). Thus, we think that PS II1 is PS IIα. Another 32% of PS II (marked by subscript 2) is characterised by smaller values of k0 L,2 (Figure 2A) and a smaller value of the connectivity parameter p2 (0.35). The value of p2 lies in the upper limit, indicating that the fluorescence rise can be described by an exponential function (see above); however, it is still smaller than 0.4, which was found for PS IIβ (see above). Thus, we think that these PS IIs are mutually energetically separated (however, see above for the description of the results of Lavergne and Leci (1993)) and we denoted them as PS IIβ. The remaining 4% of PS II (marked by subscript 3) is characterised by very small values of k0 L,3 (Figure 2A) and zero value of the connectivity parameter p3 . Zero value of p3 indicates that PS II3 could be another type of PS IIβ. However, values of k0 L,3 seem to be randomly distributed (inset of Figure 2A) and do not linearly depend on light intensity as is the case of k0 L,1 and k0 L,2 (Figure 2A). This means that PS II3 is not limited only by light absorption but by some other process, e.g., slow electron donation from the donor side of PS II as found by Hsu and Lee (1991). On the other hand, because the values of k0 L,3 are two to three orders of magnitude smaller than k0 L,1 and k0 L,2 , the photochemistry in PS II3 can be considered as negligible or more strictly as blocked and thus these PS IIs can represent some ‘inactive’ PS IIs. But as these PS IIs contribute to the fluorescence rise even if their photochemistry is hypothetically blocked, the inactive PS IIs must have QA always reduced, even in the dark (see also Lazár et al. 1997). Such inactive PS II can be understood as PS IIs repairing in the cycle of PS II as suggested by Guenther and Melis (1990). However, only on the basis of our analysis of the DCMU-FR curves, it is hard to conclude which real PS II type is represented by our PS II3 and additional measuring techniques should be carried out for a final decision. One way or another, PS II3 is different from PS II1 and PS II2 and we denoted the former as PS IIγ . As men-
tioned above, values of k0 L,1 and k0 L,2 linearly depend on light intensity (Figure 2A) and thus it seems that PS II1 and PS II2 are limited only by light absorption. The average ratio k0 L,1 / k0 L,2 is about 2.5, indicating that the antenna size of PS IIα is about 2.5 times larger than the antenna size of PS IIβ (see ‘Introduction’), which is in agreement with the literature (Thielen and van Gorkom 1981a; Melis and Anderson 1983). As for Chlamydomonas reinhardtii cells, 63% of PS II (marked by subscript 1) are characterised by high values of k0 L,1 (Figure 2B) and a high value of the connectivity parameter p1 (0.7). Thus, we think, as for the wheat leaves, that PS II1 is PS IIα. Nine percent of PS II (marked by subscript 2) are characterised by smaller values of k0 L,2 (Figure 2B) and zero value of the connectivity parameter p2 , and the remaining 28% (marked by subscript 3) are characterised by very small values of k0 L,3 and a very small value of the connectivity parameter p3 (0.07). Because of the same reasons as mentioned above for PS II2 in the case of wheat leaves, we think that PS II2 is PS IIβ in the case of Chlamydomonas reinhardtii cells. The small amount of PS II2 can represent a minor fraction of PS II that can reduce QA , and PS II1 can be considered as the major part of PS II with the ability to reduce QA . The suggestion of one dominant functional type of PS II (i.e., PS II1 ) in our algal culture is not surprising as there is no or only very little lateral heterogeneity of the thylakoid membrane: about three membranes are stacked throughout the whole cell (see e.g. Anderson 1999 and references therein). Hence, one type of PS II with respect to antenna heterogeneity should predominate in this alga, which we suggest to be our PS II1 . Values of k0 L,3 , again, do not depend on light intensity (Figure 2B) and are even four orders of magnitude smaller than values of k0 L,1 . Thus, PS II3 in the case of Chlamydomonas reinhardtii cells probably represents the same type of PS II as PS II3 in the case of wheat leaves (see above). The relatively high level of ‘inactive’ PS II3 can be simply explained by an asynchrony of alga culture (some cells were dead, some were dividing), which was used for our measurements. On the other hand, values of k0 L,1 and k0 L,2 linearly depend on light intensity (Figure 2B) and thus it seems that both PS II1 and PS II2 are limited only by light absorption. The average ratio k0 L,1 / k0 L,2 is about 2.3, which is, again, as in the case of wheat leaves, in agreement with the literature. Contributions of individual PS II types to overall fluorescence for 40% intensity of light excitation are shown in Figure 3. For wheat leaves, mainly PS II1
254 Table 2. Results of the PS II antenna heterogeneity determined either by different methods or with different results with respect to the amount of PS II types Authors / material
PS II types Amounts (%)
Types of the FR
Values of the rate constants (s−1 )
Values of the p parameters
Melis and Homann (1976) / tobacco chloroplasts
α; β
60; 40
non-exp; exp 0.1a ; 0,9
n.d.; n.d.
Dohnt and Renger (1984) / spinach chloroplasts
α; β
70; 30
non-exp; exp 160; 20
0.54; 0
Sinclair and Spence (1988) / spinach chloroplasts
α; β; γ
33; 28; 39
exp.; exp.; exp.
n.d.; n.d.; n.d.
Sinclair and Spence (1990) / spinach chloroplasts
α; β; ; δ
13.5; 13.5; non-exp.; 25.5; 47.5 exp.; exp.; exp.
9; 1.3; 0.25
n.d.; n.d.; n.d.; n.d.; 8.5; 1.65; 0.18 n.d.
Hsu and Lee (1991) / spinach chloroplasts α; β; γ
63; 12; 25
non-exp.; exp.; exp.
39.8; 10.1; 1.37
n.d.; n.d.; n.d.
Meunier and Bendall (1992) / spinach thylakoids 1; 2; 3
33; 36; 31
exp.; exp.; exp.
130; 31; 5.4
0.15; 0; 0
Lavergne and Trissl (1995) / spinach thylakoids
α; β
68; 32
non-exp.; exp.
n.d.; n.d.
0.7; 0.4
Nedbal et al. (1999) / Scenedesmus q. cells
α; β
76; 24
non-exp.; exp.
4.8; 2,9
0.57; 0
This work / wheat leaves
α; β; γ
64; 32; 4
8.4; 3.4; 1.9
0.7; 0.35; 0
This work / Chlamydomonas r. cells
α; β; γ
63; 9; 28
5.1; 2.2; 0.3
0.7; 0; 0.07
non-exp.; exp.; exp. non-exp.; exp.; exp.
The values of our rate constants and those of Nedbal et al. (1999) are re-calculated values to the light intensity of 10–15 µmol m−2 s−1 of PAR (see the text). a 0.1 is not the value of the rate constant but a half time (in seconds) of the growth of the CA of PS IIα. exp. – exponential; non-exp. – non-exponential; n.d. – not determined.
and to a lesser extent PS II2 contribute to the DCMUFR curve. The contribution of PS II3 to the DCMU-FR curve is only minor. Similar results were obtained for Chlamydomonas reinhardtii cells. However, a rather small amount of PS II2 (9%) in the case of Chlamydomonas reinhardtii contributes to a rather high extent to the overall DCMU-FR curve as compared to the high amount of PS II1 (63%). The reason lies in the different values of their p parameters. Zero value of p2 results in a linear relationship between PS II2 fluorescence and the amount of reduced QA in these centres. On the other hand, the high value of the p1 parameter causes a strong non-linear dependence of fluorescence
on the amount of reduced QA of PS II1 , which results in a lowering of the contribution of PS II1 fluorescence to overall fluorescence. The non-linearity between the number of reduced QA and the fluorescence signal originating in PS II1 is valid for the whole time range of DCMU-FR (and not just for the time range before the saturation of PS II1 fluorescence) because PS II1 is only a fraction of all the PS IIs (see Equation (1)). Even if we found a significant amount of PS II3 (28%) in Chlamydomonas reinhardtii, their contribution to the overall DCMU-FI curve is only minor as in the case of wheat leaves. The reason lies in a very small value of k0 L,3 . It is also interesting to note that due
255
Figure 3. Contribution of fluorescence originating from particular PS II types (see key) to overall fluorescence as determined from the best fit of the DCMU-FR curve measured at 40% intensity of light excitation by the PEA fluorometer with wheat leaves (A) and with Chlamydomonas reinhardtii cells (B). Measured DCMU-FR curves are presented using the square marks and the best fits are shown as solid lines.
to a higher value of k0 L,1 , compared to k0 L,2 , the fluorescence signal coming from PS II1 reaches its maximal value at a shorter time than the signal from PS II2 . This can have consequences for the interpretation of the O-J-I-P fluorescence transient (see Strasser et al. 1995; Lazár 1999) measured at high intensity of light excitation without DCMU, i.e., different PS IIs with different rates of their photochemistry can cause different steps in the O-J-I-P fluorescence transient. The direct fitting of the DCMU-FR curve by a model for the purpose of determination of the antenna heterogeneity of PS II is not a new idea. Dohnt and Renger (1984) fitted the DCMU-FR curve by an exponential rise and one non-linear function (as described by Equation (1) for i = 1). However, the authors did not consider the time dependency of the rate constants for PS II closure (our Equation (4)) and fitted only one DCMU-FR curve. Their results, together with our results and the results of the antenna heterogeneity of PS II determined by different methods, are summarised in Table 2. In almost all the cited works, a light intensity of 10–15 µmol m−2 s−1 of PAR was used, which is about one thousandth of our maximal intensity. Thus, to be able to compare our results with the other results, we re-calculated the values of our light-dependent rate constants k0 L,1 and k0 L,2 found for a particular light
intensity to the light intensity of 10–15 µmol m−2 s−1 of PAR and then evaluated their mean value, while values of the light-independent rate constant k0 L,3 are presented as the mean values found in our analysis for all light intensities. Such corrected values are presented in Table 2 as our results. The values of the rate constants found by Nedbal et al. (1999) were corrected in the same way (they also found a linear dependence of the values of the rate constants on light intensity). Even if Lavergne and Trissl did not (1995) determined only two types of PS II, and did not consider a small amount of PS II3 , our results agree best with their results in terms of amounts of particular types of PS II and the values of the p parameters for wheat leaves (see Table 2). This is not surprising because the authors also fitted the DCMU-FR curve directly, and mathematical expressions used by us are generalised formulas of the ones derived and used by Lavergne and Trissl (1995) on the basis of the reversible radical pair model for connected PS IIs. In terms of the values of the rate constants determined for wheat leaves, our results agree best with the results by Sinclair and Spence (1988). However, we did not determine such slow PS IIs as found by them for their PS IIγ (see Table 2) and our the fastest PS IIs are characterised by non-exponential FR, unlike those of Sinclair and
256 Spence (1988). On the other hand, Hsu and Lee (1991) determined, as we did, three PS II types, the fastest with the non-exponential FR, and the other PS II types with the exponential FR. However, the value or the rate constant of their PS IIα is about 5 times faster than the appropriate value determined by us. The differences between our results and the results by Sinclair and Spence (1988) and Hsu and Lee (1991) are, in addition to a different method used, most probably due to different sample material used. As for the values of the rate constants determined by us for Chlamydomonas reinhardtii cells, our results agree best with those of Nedbal et al. (1999) who also used algae cells in their determination (see Table 2). However, using the method by Melis and Homann (1975, 1976), the authors determined, only two PS II types. Using the same method, Guenther et al. (1990) also determined only two PS II types for light incubated Chlamydomonas reinhardtii cells with respect to the antenna heterogeneity with very similar results (78% of PS IIα with kα = 5.3 s−1 and 22% of PS IIβ with kβ = 1.4 s−1 ; the values of the rate constants are after the light intensity correction mentioned above) to those by Nedbal et al. (1999). The difference between our results and those of Guenther et al. (1990) and Nedbal et al. (1999) are most probably due to the use of different methods. Acknowledgements The authors thank J. Whitmarsh for his suggestions during the final editing of the manuscript and to P. Davis for correction of the English. This work was supported by grant number MSM 153100010 of Ministry of Education of Czech Republic and by grant number 3150 3012 by Palacký University in Olomouc. References Anderson JM (1999) Insights into the consequences of grana stacking of thylakoid membranes in vascular plants: A personal perspective. Aust J Plant Physiol 26: 625–639 Anderson JM and Melis A (1983) Localization of different Photosystems in separate regions of chloroplast membranes. Proc Natl Acad Sci USA 80: 745–749 Bell DH and Hipkins MF (1985) Analysis of fluorescence induction curves from pea chloroplasts. Photosystem II reaction centre heterogeneity. Biochim Biophys Acta 807: 255–262 Black MT, Brearley TH and Horton P (1986) Heterogeneity in chloroplast Photosystem II. Photosynth Res 8: 193–207 Chylla RA, Whitmarsh J (1990) Light saturation response of inactive Photosystem II reaction centers in spinach. Photosynth Res 25: 39–48
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