Photosynthesis Research 29: 1-9, 1991. © 1991 KluwerAcademic Publishers. Printed in the Netherlands. Regular paper

The appearance of quenching centres in photosystem II John Sinclair 1, Sandra K. Spence & Linda J. Langille

Biology Department, Carleton University, Ottawa, Ontario, Canada K I S 5B6; 1To whom all correspondence should be sent

Received 19 December 1990; acceptedin revised form 23 April 1991

Key words: fluorescence induction, oxygen flash yield, photosystem II, photosynthetic electron transport, photoinhibition Abstract

The variable fluorescence quenching found in the presence of DCMU with isolated chloroplasts which have been exposed previously to a prolonged low light intensity (Sinclair and Spence 1988), is accompanied by a loss of the sigmoidal appearance of the fluorescence induction transient. About 80% of the fluorescence decrease is due to the PS II,~ units and 50% of the a centres are inactivated by light exposure. Light incubation slows the PS II partial reaction while the PSI partial reaction is unaffected. We propose that in the light, normal PS II a centres change into quenching centres which degrade excitation energy to thermal energy. This change can be reversed by 30 min of darkness. A higher flash intensity is needed to saturate the steady state 0 2 flash yield from light-incubated chloroplasts indicating a light-induced decrease of the average photosynthetic unit size as would happen if PS IIa units were preferentially inactivated. These light-induced changes may relate to an adaptation in leaves to increasing light intensity. Abbreviations: Chl-Chlorophyll; DCMU-3-(3',4'-dichlorophenyl)-l,l'-dimethylurea; D C P I P - 2 , 6 Dichlorophenol-Indophenol; EDTA - ethylaminediaminetetraacetic acid; F v - Level of variable fluorescence emission; Fo -Initial level of fluorescence; Hepes b u f f e r - N-[2-Hydroxyethyl]piperazine-N'-[2ethanesulfonic acid]

Introduction

Recently we found that fluorescence emission from isolated spinach chloroplasts which had been exposed to 650nm light ( 6 / ~ E m - 2 s -1, 30min) and then inhibited with DCMU, was about 15% lower than the dark control (Sinclair and Spence 1988). This light-induced quenching of fluorescence occurred in the presence of uncouplers and also of hydroxylamine, an inhibitor of the backflow of electrons around PS II. It could also be demonstrated with both broken and intact chloroplasts. In this paper, we present the results of a more detailed investigation of this fluorescence quen-

ching along with observations on the changes in the electron transport properties of the chloroplasts which occur following exposure to low light intensities.

Methods

Chloroplasts for this study were isolated from 4-week-old, greenhouse-grown spinach. Relatively intact chloroplasts which were isolated using a modification (Heber 1973) of the procedure given by Jensen and Bassham (1966), were stored on ice in the dark in a medium containing 0.33M sorbitol, 50mM Hepes buffer, pH7.5,

l mM MgCI2, 2 m M NaNO3, 2 m M EDTA, l mM MnCI2, 0.5mM K 2 H P O 4 (Medium A). For all experiments except the oxygen flash yield experiments, these organelles were osmotically shocked immediately prior to use by immersion in 50mM Hepes buffer (pH7.5) for 10s. A concentrated solution was added so that the final solution (Medium B) was identical to Medium A, except that the concentration of MgCl 2 was 10mM and 0.1 mM methyl viologen was included. Chlorophyll concentrations were determined by the method developed by Vernon (1960). All experiments were performed at room temperature (23 - I°C). In a typical fluorescence induction experiment, a chloroplast suspension (8/zg Chlm1-1 of Medium B) was incubated for 30 min either in the dark or in light (10/zE m -2 s -1) which had passed through a 650nm interference filter (Baird-Atomic, B-l). Afterwards, the electron acceptors to PS II were allowed to oxidize by leaving the chloroplasts in the dark for 5 min. The chloroplasts were inhibited with D C M U (50/zM) and then left for 1 min in the dark before the fluorescence induction curve was observed. The D C M U (E.I. du Pont de Nemours & Co.) was 99.7% pure. The apparatus used to observe the fluorescence induction curve and the methods of analys~s are described in Sinclair and Spence (1988). We characterise the early rise of the variable fluorescence with a sigmoidicity parameter (o-) which is the difference between the maximum and initial values of the gradient of the variable fluorescence curve. The gradient is determined by measuring the change in variable fluorescence between successive data points, i.e., for constant time intervals. Initially, the gradient of the variable fluorescence curve will increase if there is a sigmoidal component present but will decrease if the curve is purely exponential. To measure the P S I partial reaction, samples of broken chloroplasts were incubated in medium B in the light or dark as described for the fluorescence induction experiments. Light and dark incubation experiments were performed alternately. Then photosystem I activity was measured as the rate of oxygen consumption under light-limiting conditions after the addition of 0.1 mM DCPIPH 2, 3 mM sodium ascorbate,

superoxide dismutase (200/zg m1-1) and 5/xM DCMU. For PS II, the the chloroplast samples were incubated alternately in the light or dark for 30min in medium B modified to include 2 m M potassium ferricyanide instead of the methyl viologen. The light and dark incubation experiments were performed alternately. The rate of oxygen evolution was then measured under light-limiting conditions with the addition of 0.1 mM DCPIP to the suspension. All samples were left for 5 min in the dark before the rates of oxygen consumption or evolution were measured. The oxygen concentration changes were followed with an oxygen electrode (Yellow Springs Instruments, model 53) whose temperature was controlled at 23 +--0.5°C by a Lauda constant temperature water bath (Model K-2). The apparatus used to measure the oxygen flash yield was a Joliot-type oxygen electrode (Joliot and Joliot 1968) and is described in Arnason and Sinclair (1976). The polarization of the electrode was provided by a potentiostat (Meunier and Popovic 1988) so that the potential difference between the platinum and silver chloride electrodes was held constant at -0.743 volts. The results were stored and analysed with a Raven 10 computer (Science Technology Centre, Carleton University). Intact chloroplasts were used and the experimental medium contained 0.33 M sorbitol, 100 mM NaCI, 10 mM MgCI 2, 5 mM NH4CI, 10 mM Hepes buffer, pH 7.5, and 0.5mM oxaloacetate (Medium C). These uncoupled, intact chloroplasts gave a large oxygen signal when immersed in this solution. In a typical experiment, a sample of chloroplast suspension (300/zg Chl m1-1) was injected into the electrode and allowed to settle in the dark for 5 min. The thick suspension is necessary because only a thin (300/z) layer is present over the platinum electrode. The absorbance of this layer is very similar to that of the samples used in the fluorescence experiments. The chloroplasts were then illuminated with 30 saturating light flashes and the resulting oxygen flash yields recorded. The light flashes were delivered once per second to maximise the oxygen yield. The polarization of the oxygen electrode was then reduced to zero, and the chloroplasts were either kept for 30 min in the dark or exposed for 30 min to light (15/~E m - 2 s -1) which had passed

through a 660nm interference filter (BairdAtomic, B-I). Polarization was resumed during 5 min of darkness which preceded the onset of a second sequence of light flashes. Once a steady oxygen flash yield was achieved, the flash intensity was altered by inserting neutral density filters (Kodak, VII) into the light path and the decreased oxygen flash yields were measured after a new steady state was established. The steady state flash yield at any given flash intensity was normally taken as the average of ten flash yields.

Results

In Fig. 1 we show the variable fluorescence rise during the first four hundred milliseconds of three fluorescence induction experiments. The

F

curves have been separated vertically to assist comparison. The three straight lines are the bestfitting straight lines for the first 20 ms of the variable flurorescence and highlight the sigmoidicity of the curves. The top curve was obtained for isolated chloroplasts which had been illuminated for 30 min with 650 nm light while the chloroplasts which yielded the bottom curve had been in the dark for 30 min. The middle curve is from chloroplasts which had been in the dark for 5 min. Incubation in the light .greatly reduces the sigmoidal appearance of the fluorescence induction curve while incubation in the dark for 30 min enhances it relative to the 5 min dark experiment. The experiments involving 30min of either light exposure or darkness are analysed in Table 1. It can be seen that the variable fluorescence

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Fig. 1. The first four hundred milliseconds of the variable fluorescence from three samples of broken chloroplasts immersed in medium B and inhibited with DCMU. The straight lines are the best-fitting straight lines for the initial 20 ms of the variable fluorescence. The three curves have been separated vertically for purposes of clarity. Prior to the addition of DCMU (50/xM), the chloroplasts which gave the upper curve had been exposed to 30 min of 650 nm light (10/~E m -2 s -1) and 5 min of darkness while those which gave the bottom curve had been kept in the dark for 30 min. The middle curve comes from an experiment in which the chloroplasts were kept in the dark for 5 rain before the inhibitor was added. Chlorophyll concentration used was 8 ttg mi-~. Other conditions are described in the Methods section.

Table 1. Fluorescence induction curve parameters. These results are from the 30 min light exposure and 30 min dark exposure experiments shown in Fig. 1. All units are relative. Terms such as F. represent the part of the variable fluorescence which is calculated to be associated with a particular kinetic phase. The method of calculation is given in the text. The areas above the fluorescence induction curve associated with each phase were calculated as described in Sinclair and Spence (1988) Phase fluorescence

Dark Light

Phase area

Fo

Fv

tr

F~

Fo

Fr

F~

a

/3

F

6

2.72 2.57

6.57 4.26

0.092 0.008

4.06 2.19

1.68 1.16

0.63 0.55

0.19 0.36

66 35

71 55

129 116

242 414

from the light exposed sample is 35% smaller than that from the dark control and the sigmoidicity is much smaller. Four kinetic phases can be detected in the complimentary area above fluorescence induction curves (Sinclair and Spence 1990) and Table 1 contains the calculated contributions which each kinetic phase makes to the variable fluorescence signal. The contributions of the three exponential phases (i.e., /3, F and 8) to F v were calculated by multiplying the area for each phase by the rate constant. The contribution of the a phase was taken as the difference between F v and the total of the three exponential phase contributions. From these calculations it appears that most of the decrease (80%) in the variable fluorescence after light incubation is due to the a phase although the/3 phase is also significant in this respect. Since the contributions of the F and 6 phases to the variable fluorescence are so small, they cannot explain the decreased fluorescence emission we have observed. The areas above the fluorescence induction curve associated with the four kinetic phases are shown in Table 1. These areas are proportional to the amount of oxidized electron acceptors available for reduction before the DCMU block (Bennoun and Li 1973). However, since we do not know the relative quantum efficiencies for fluorescence emission of the different phases, it is not valid to compare their areas, as pointed out in Joliot et al. (1973). Instead, we will compare the area of the same phase in different experiments. Hence the results indicate that light exposure produces a decrease in the number of electron acceptors being reduced in the a and/3 phases by 47 and 22%, respectively. While the areas of the slowest two phases also change, they cannot assist us in explaining the fluorescence quenching as already noted.

Further evidence that there is a loss of activity by PS II as a result of light exposure, comes from measurements of the partial reaction rates. When measured under identical light-limiting conditions, the PS II partial reaction rates following 30 min of dark incubation or 30 min of 650nm light incubation are 2 4 + 2 and 16--3/xmoles 0 2 evolved mg Chl -a h -1 respectively (Mean---standard deviation, 8 determinations). Similarly, the P S I partial reaction rates are 19 4 and 19 +- 3/xmoles 0 2 consumed mg Ch1-1 h -1 after 30 min of dark or 650 nm light incubation (6 determinations). These experiments indicate that there is a decrease in the partial reaction rate for PS II but none for P S I following 650 nm light incubation. Far-red light (>715nm) is much more efficiently used by photosystem I than photosystem II (Joliot et al. 1968) and using it for the incubation light should mean that the redox components between the two phototsystems remain oxidized during light incubation. After 30 min of 70/zE m -2 s -a of far-red light (>715 nm, RG715 glass filter, Schott Optical Glass inc.) the sigmoidicity is smaller and the variable fluorescence has decreased because of the smaller contributions of the a and/3 phases (Table 2). The areas of these two phases are also smaller. Thus, although far-red light principally activates PS I, it still gives rise to the same type of fluorescence quenching as 650 nm light. Parallel experiments with the modulated oxygen electrode indicated that the rate of oxygen evolution with the far-red light would be much smaller than with the usual 650 nm light. The reversibility of the light-induced changes was tested by exposing chloroplasts to 30 min of light followed by 30 min in the dark before the addition of D C M U and the observation of the fluorescence induction curve. The initial rise of

5 Table 2. Fluorescence induction curve parameters. These results were obtained for an experiment in which samples of broken chloroplasts in medium B were exposed to far-red light (70/,~E m -2 s -1, >715 nm) for 30 s (control sample) or 30 min before inhibition with DCMU and the observation of the fluorescence induction curve. All units are relative Phase fluorescence

Control 30 min far-red light

Phase area

Fv

tr

F,

F~

a

/3

6.89 5.59

0.093 0.024

2.58 1.46

3.22 2.56

45 24

87 75

the variable fluorescence for such an experiment is shown in Fig. 2 along with the results for a light-incubated sample treated to the normal 5 min dark period. The more sigmoidal appearance of the curve from the chloroplasts which had been in the dark for 30 min is quite apparent. When these curves are analysed, the a phase area and the value of the variable fluorescence from this phase are larger following the

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30 min dark incubation. Thus, it is possible to reverse some of the light-induced changes in fluorescence emission. To maximise the signal in our study of oxygen flash yields, we employed uncoupled, intact chloroplasts in medium C which is significantly different from medium B used in our fluorescence experiments. However, such chloroplasts do exhibit the same light-induced fluorescence

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Fig. 2. The effect of prolonged darkness following light exposure on the variable fluorescence from isolated chloroplasts immersed in medium B and inhibitied with DCMU. Prior to the addition of DCMU (50/xM), the chloroplasts which gave the upper curve had been exposed to 30min of 650nm light (10/~E m -2 s -1) and 5 min of darkness while those which gave the bottom curve had been exposed to 30 rain of the same light followed by 30 rain of darkness. The two curves have been separated vertically for purposes of clarity. Other conditions as described for Fig. 1.

quenching and loss of sigmoidicity as is seen with broken chloroplasts in Medium B (results not shown). The bare platinum employed to detect oxygen in the Joliot type oxygen electrode is not well suited to making absolute measurements since its sensitivity can be altered e.g., by material adhering to the platinum. This makes it difficult to compare signal sizes between different chloroplast samples. However, in the experiments performed here, samples were introduced to the apparatus and left in the dark for 5 min and then exposed to 30 full-intensity light flashes. This permitted a steady state oxygen flash yield to be determined. The samples were then treated to 30 min of light or dark and a second sequence of light flashes to obtain the results shown in Fig. 3. Comparison of the steady state oxygen flash yields before and after

the 30 min treatments shows that the steady-state oxygen yield from the light-treated chloroplasts fell by 15%, while that from the dark-treated chloroplasts rose by 18%. Hence these results suggest that PS II is about 33% less active after the light treatment compared with the dark treatment. The relationship between the relative oxygen flash yield and the relative flash intensity is illustrated in Fig. 3 for chloroplast samples which had either been in the dark for 30min ('O' symbols) or exposed to light for 30min ( ' x ' symbols). The maximum flash intensity used is designated as 100% while the corresponding relative oxygen flash yield is 100%. Since reduction of the flash intensity by 50% produced a decrease of about 10% in the relative oxygen flash yield, the maximum flash intensity is a close

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30.0

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Fig. 3. The relative oxygen flash yield in the steady state versus the relative flash intensity for chloroplasts which had either been incubated in the dark ('O' symbols) or light ( ' x ' symbols) for 30 rain while immersed in medium C. The light passed through a 660 nm interference filter (Baird-Atomic, type B-l) and had an intensity of 15/~E m -2 s -1. The 100% flash intensity was that from the unfiltered flash lamp while the lower intensities were obtained by means of neutral density filters. The 100% oxygen flash was that produced by the unfiltered flash lamp. The results are the average of ten oxygen flash yields and the error bars are the standard deviations.

7 approximation to the saturating value. It can be seen that at each of the intensities shown in Fig. 3, the relative oxygen flash yield is lower for the chloroplasts which had been incubated in the light. This is consistent with the saturating flash intensity for such chloroplasts being larger than that for the chloroplasts which have been incubated in the dark.

Discussion

The detailed analysis of the fluorescence induction curves presented in Table 1 demonstrates that about 80% of the decrease in variable fluorescence caused by light exposure is due to decreased emission by PS li e units while /3 units account for almost all the remaining decrease. The different kinetic phases in fluorescence induction curves have been ascribed to PS II units containing different numbers of pigment molecules per reaction centre (Melis and Duysens 1979) so that units exhibit fast kinetics because they have a relatively large number of pigment moelcules per reaction centre. It follows from this that the fluorescence quenching we see here is associated mainly with the PS II~ units which have the largest numbers of pigment molecules per reaction centre. The area of the a phase is almost 50% smaller after 30 min of light incubation relative to that after an equally long dark incubation (Table 1). The corresponding decrease for the /3 phase is about 20%. The area of a phase is proportional to the number of electron acceptors which are oxidized at the onset of illumination and can be reduced by PS II during the light (Bennoun and Li 1973). If some electron acceptors are in a reduced state when the light is switched on, this will cause a decrease in the phase area and in the amount of variable fluorescence but there should also be a compensating increase in the value of F o. Since the value of F o is not highe r after light exposure (Table 1), we can conclude that the decreased a and /3 areas are not due to fewer oxidized electron acceptors but to the inability of PS II to reduce them. This loss of active PS II units was verified when the partial reactions of the two photo-

systems were measured. The PS II partial reaction rate was about 25% smaller as a result of light exposure while the P S I partial reaction rate was unaltered. These results also demonstrate that there was no apparent diversion of absorbed light energy from PS II to P S I since this would have caused the P S I partial reaction to speed up. This confirms our previous rejection (Sinclair and Spence 1988) of both the phosphorylation of light-harvesting chlorophyll proteins and the formation of a high energy state as possible mechanisms to explain the fluorescence quenching. This result also demonstrates that a transfer of light-harvesting chlorophyll proteins from PS II to P S I cannot explain the fluorescence quenching seen here, as suggested by Sinclair and Spence (1988). The quenching of fluorescence observed here seemed to be unaffected by the redox state of the intersystem electron transport chain. Thus, using a far-red light during the 30 min light incubation period still produced a decrease in sigmoidicity and fluorescence quenching due to the a and /3 phases (Table 2 ) . This suggests that, unlike the phosphorylation of light-harvesting chlorophyll proteins (Allen et al. 1981), we are not dealing with a mechanism which is concerned with the balance of light energy supply between the two photosystems. Since the rate of oxygen evolution with the far-red light was much lower than with the 650 nm light, it appears possible that the mechanism causing the light-activated fluorescence quenching may be triggered by either photosystem. In a theoretical study, Sorokin (1985) investigated the effects on fluorescence emission of transforming active PS II~ reaction centres into quenching centres. Quenching centres were defined as reaction centres which convert excitation energy to thermal energy but do not transport electrons. Sorokin's study predicted that the creation of quenching centres would decrease variable fluorescence emission and abolish sigmoidicity in the fluorescence induction curve. It also follows from Sorokin's model that there should be a decrease in the partial reaction for PS II when quenching centres are created. Although Sorokin (1985) discussed only the properties of multicentral photosynthetic units which yield sidmoidal induction kinetics, his findings do

appear to be relevant to our experiments. Thus, we see a 70% decrease in F v associated with the a phase, a loss of sigmoidicity, a 50% reduction in the active reaction centres in the a phase (Table 1) and a smaller partial reaction rate for PS II with no change in that for P S I following light incubation. This represents a good agreement between the theoretical predictions and our findings, and we conclude that exposure to light can cause the conversion of PS II~ reaction centres from an active to a quenching form. We note that a conversion of active reaction centres to quenching centres may also occur in the/3 phase (Table 1). It appears that the transformation of active reaction centres into quenching centres is partly reversible if light-exposed chloroplasts are kept in the dark for a sufficient time (Fig. 2). This indicates that the fluorescence quenching observed here does not signify damage to the chloroplasts but is more probably an adjustment made to their local environment. Our analysis of the fluorescence results suggests that the largest effect of light-exposure is manifested by the a phase where about 50% of the reaction centres are changed to quenching centres. We would expect therefore to see a smaller steady state saturated oxygen flash yield from light-exposed chloroplasts and we did find a 33% decrease relative to the yield from darkincubated chloroplasts, which confirms the results of the photosystem II partial reaction measurements. Also, we would expect to see an increase in the flash intensity needed to saturate the oxygen yield since there should be a smaller average PS II unit size remaining active after light exposure. As shown in Fig. 3, the relative oxygen flash yields are moved to higher flash intensities following light incubation of the chloroplasts as would occur if the saturating flash intensity is higher, so that our oxygen flash yield observations are consistent with the analysis of the fluorescence data. Previously, we have rejected photoinhibition as an explanation of the light-induced quenching of fluorescence (Sinclair and Spence 1988), but the similarity between our findings and those obtained in studies of photoinhibition (e.g., Cleland et al. 1986) require us to consider this possibility in more detail. Taken literally, photo-

inhibition means inhibition by light and our findings would fall within this definition. However, the normal working definition of photoinhibition is the inhibition of photosynthesis caused by super-saturating light power input to the chloroplasts. While our results can be produced by light intensities in the linear range, a survey of ten studies of photoinhibition in isolated chloroplasts (Barenyi and Krause 1983, Cleland et al. 1986, Satoh 1971, Krause et al. 1985, Demeter et al. 1987, Cleland and Critchley 1985, Cleland and Melis 1987, Satoh and Fork 1982, Powles and Bjorkman 1982, Nedbal et al. 1986) reveals that the light intensities used were at least two orders of magnitude higher than those used here. These studies also employed some other form of stress such as the absence of an electron acceptor or a low temperature or the exclusion of oxygen from the bathing medium during high light exposure. In contrast, our experiments were done at room temperature and in the presence of an electron acceptor. Also, the fluorescence quenching we have detected is at least partly reversible which is not thought to be the case with photoinhibitory damage to isolated chloroplasts (Krause 1988). We conclude that the fluorescence quenching seen here is not due to excessive power supply to PS II or some other form of stress, but more probably results from an adaptation of the chloroplasts to the light intensity in which a photosynthetic units with relatively large numbers of pigment molecules per reaction centre (Melis and Duysens 1979) are switched off. Such units are better adapted than smaller photosynthetic units to function at low light intensities but conversely are more in danger of being overloaded with power at high intensities. These results may have relevance to leaf behaviour during the hours after sunrise.

Acknowledgements This research was funded by the Natural Sciences and Engineering Research Council of Canada and Carleton University. The gift of D C M U from E.I. Du Pont de Nemours and Co. and the help of Ms J.L. Singleton and Dr P. Heytler is acknowledged.

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Joliot P, Bennoun P and Joliot A (1973) New evidence supporting energy transfer between photosynthetic units. Biochim Biophys Acta 305:317-328 Krause GH (1988) Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms. Physiol Plant 74:566-574 Krause GH, Koster S and Wong SC (1985) Photoinhibition of photosynthesis under anaerobic conditions studied with leaves and chloroplasts of Spinacia oleracea L. Planta 165: 430-438 Krause GH, Vernotte C and Briantais JM (1982) Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components. Biochim Biophys Acta 679:116-124 Melis A and Duysens LMN (1979) Biphasic energy conversion kinetics and absorbance difference spectra of photosystem II of chloroplasts. Evidence for two different photosystem II reaction centers. Photochem Photobiol 29: 373382 Melis A and Homann PH (1976) Heterogeneity of the photochemical centers in system II of chloroplasts. Photochem Photobiol 23:343-350 Meunier PC and Popovic R (1988) High-accuracy oxygen polarograph for photosynthetic systems. Rev Sci Instr 59: 486-491 Nedbal L, Setlikova E, Masojidek J and Setlik I (1986) The nature of photoinhibition in isolated thylakoids. Biochim Biophys Acta 848:108-119 Powles SB and Bjorkman O (1982) Photoinhibition of photosynthesis: Effect on chlorophyll fluorescence at 77 K in intact leaves and chloroplast membranes of Nerium oleander. Planta 156:97-107 Satoh K (1971) Mechanism of photoinactivation in photosynthetic systems IV. Light-induced changes in the fluorescence transient. Plant and Cell Phys 12:13-27 Satoh K and Fork DC (1982) Photoinhibition of reaction centers of photosystem I and II in intact Bryopsis chloroplasts under anaerobic conditions. Plant Phys 70: 10041008 Sinclair J and Spence SM (1988) The analysis of fluorescence induction transients from dichlorophenyldimethylureapoisoned chloroplasts. Biochim Biophys Acta 935:184-194 Sinclair J and Spence SM (1990) Heterogeneous photosystem 2 activity in isolated chloroplasts. Photosynth Res 24: 209-220 Sorokin EM (1985) The induction curve of chlorophyll fluorescence in DCMU-treated chloroplasts and its properties. Photobiochem Photobiophys 9 : 3 - 1 9 Vernon LP (1960) Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Anal Chem 32: 1144-1150