Journal of Photochemistry and Photobiology, B: Biology, 5 (1990) 481 - 494 481

EFFECTS OF STRONG LIGHT IRRADIATION ON ANTENNAE AND REACTION CENTRES OF THE CYANOBACTERIUM Anabaena variabilis: A TIME-RESOLVED FLUORESCENCE STUDY’

W. NULTSCH

Fachbereich Biologie-Botanik der Philipps-Universitiit, K. u. Frisch-Str., D-3550 Marburg (F.R.G.)

E. BITTERSMANN and A. R. HOLZWARTHS

Max-Planck Znstitut fiir Strahlenchemie, Stiftstr. 34 - 36, D-4330 Miilheim an der Ruhr (F.R.G.)

G. AGEL

Fachbereich Biologie-Botanik der Philipps-Universitiit, K. v. Frisch-Str., D-3550 Marburg (F. R.G.)

(Received August 16,1989; accepted November 10,1989)

Keywords. Reaction centre, cyanobacteria, photosystem II, time-resolved fluorescence.

Summary

Photoinhibition and photobleaching as a function of time at high fluence rates were studied using picosecond time-resolved emission spectra (TRES) and fluorescence induction in the cyanobacterium Anabaena uari- abilis. Photoinhibition and photobleaching can be distinguished using these techniques as phenomena which operate on different parts of the photo- synthetic apparatus with very different induction periods. Photoinhibition is a short-term irradiation effect (time scale of hours) under our irradiation conditions and operates on photosystem II reaction centres in agreement with earlier studies. On photoinhibition the minimum fluorescence intensity F, is increased and the maximum fluorescence F,,, is decreased. Both effects lead to a decrease in the variable fluorescence yield. In contrast, photobleaching is a long-term effect (time scale of days) and operates on the phycobilisome antenna. This finding is supported by experiments with sodium azide, a protective agent against photobleaching. Although sodium azide cannot fully prevent the formation of long-lived fluorescence components (detached phycobiliproteins), large negative amplitudes

*Dedicated to Prof. Dr. Dr. h.c. Hans Mohr on the occasion of his 60th birthday. $ Author to whom correspondence should be addressed.

loll-1344/90/$3.50 0 Elsevier Sequoia/Printed in The Netherlands

482

measured in the sodium-azide-protected samples indicate an efficient energy transfer to the reaction centre which is absent in the control.

1. Introduction

The exposure of the cyanobacterium Anabaena uariabilis to high fluence rates of white light causes photoinhibition which increases with prolonged exposure up to about 24 h [l]. If the irradiation is continued, photobleaching, characterized by a change in colour from blue-green to yellow-green, is observed, which becomes more pronounced as the period of exposure to strong light is extended [2]. Both phenomena are reversible by subsequent exposure to low fluence rates. Recovery from photoinhibition occurs within hours, whereas photobleaching effects are reversed within days only. The action spectra of both phenomena are very similar, indicating that the active radiation is absorbed by the same photosystem (PS) II pigments [ 1,2]. Photobleaching is largely prevented either by gassing with nitrogen, argon or COZ, or by treatment with the ‘02 quenchers sodium azide and crocetin, but photoinhibition is not. Therefore the mechanisms of photoinhibition and photobleaching seem to be different [ 31.

In a previous study the effects of strong white light (13.5 W mw2) exposures of either 6 h, causing about 50% photoinhibition, or 5 days, causing strong photobleaching, have been studied by measuring the pico- second time-resolved emission spectra (TRES). In addition, the effects of irradiation for 5 days with monochromatic blue light (432 nm, 110 W mV2) and red light (662 nm, 72 W mw2) on the TRES have been compared [4]. The TRES of the cyanobacteria photoinhibited by short-term irradiation and the TRES of those exposed to strong blue light for 5 days resemble the low fluence rate control in so far as short-lived components with life- times in the picosecond range prevail, indicating an intact energy transfer in the antenna pigments, i.e. both within the phycobilisomes (PBSs) and from the PBSs to chlorophyll (Chl). In contrast, long-term irradiation with both strong white and red light, causing photobleaching of the cells [2], induces a dramatic increase in the long-lived fluorescence components in the TRES originating from phycobiliproteins. These long-lived components with an emission maximum near 650 nm have been interpreted as a result of a func- tional decoupling of the PBS rods from the core. These results are consistent with those from the action spectra, i.e. ineffectiveness of blue light and strong effect of red light. Moreover, they indicate that the radiation effective in photoinhibition changes neither the composition nor the molecular organization of the PBS antennae.

However, some important questions still need to be answered. Does photobleaching commence only after full photoinhibition is reached, or do both processes develop in parallel? Is the generation of ‘02 the cause or the result of the functional decoupling of the PBS rods? Does sodium azide,

433

which partially prevents photobleaching, influence the TRES? To answer some of these questions various experiments were performed. In a first series of experiments the duration of pre-irradiation with white light was varied between 1 h and 2 days. For those samples exposed to high irradi- antes for a longer time an excitation wavelength h,,, of 620 nm (preferen- tially absorbed by the PBS rods) was used in the TRES experiments. In the short-term pre-irradiation experiments an excitation wavelength A,,, of 670 nm (preferentially absorbed by Chls) was used for the TRES measurements, as photoinhibition is a relatively fast process which does not affect the organization of the PBS antenna. Moreover, in order to characterize further the photoinhibition of PS II, fluorescence induction experiments were carried out in parallel with the picosecond TRES measurements. Finally, the picosecond TRES of A. uariabilis pre-irradiated in the presence of lop3 M sodium azide were also measured and compared with those samples exposed to the same conditions in the absence of quencher.

2. Materials and methods

2.1. Algal material The cyanobacterium A. variabilis (Kuetz.) B 377 (Indiana University

culture collection) was grown in an inorganic liquid medium at 30 “C in continuous fluorescent light (0.8 W m-*) and bubbled with a mixture of air and 2% COP. After 10 days the algal mass was harvested, spread over membrane filters covering agar plates and exposed to 0.54 W m-* white light at 20 “C to obtain a homogeneous distribution (for further details see ref. 4).

2.2. Irradiation Five days after inoculation the cyanobacterial layers were exposed to

white light produced by projectors (Prado, Leitz, Wetzlar, F.R.G.) equipped with quartz iodine lamps. In the photoinhibition experiments a fluence rate of 5.4 W m-* was used and the time was varied between 1 h and 2 days. In order to cause photobleaching, higher fluence rates (13.5 W m-*) were applied and the time was varied between 2 and 5 days.

2.3. Time-resolved emission spectra (TRES) measurements Picosecond TRES (often referred to as decay-associated spectra [ 5 -

71) were recorded using the single-photon timing method as described previously [ 4, 5, 71. For excitation a synchronously-pumped cavity-dumped dye laser system (Spectra Physics) was used. The fluorescence kinetics were fitted to a sum of exponentials with the aid of a global data analysis algorithm [ 51. A plot of the amplitude Ai (X,,,, X,,) as a function of the emission wavelength X,, represents a time-resolved emission spectrum for the kinetic component i [ 5,8]. A positive amplitude represents a decay term and a negative amplitude a rise term. The latter indicates an indirect popula- tion of chromophores due to an energy transfer between different types of

484

pigments. Average lifetimes at a particular emission wavelength h,, are defined as

The average lifetime r, is directly proportional to the total fluorescence intensity IF (and yield @v) at the same emission wavelength [ 81 according to

where c is an arbitrary but fixed apparatus constant and krad is the inverse of the radiative lifetime. It can thus be used to make a direct link and com- parison between the intensities observed in the fluorescence induction experiment on the one hand and the lifetime experiment on the other under the same conditions). This comparison provides a particularly useful in- sight into the detailed origins of the fluorescence changes observed on ir- radiation.

Details of the TRES measurements and the global data analysis pro- cedure are given in ref. 4. The TRES experiments were carried out as described previously on samples adapted to the dark for at least 20 min before the measurement. The algae were pumped at a high rate through a flow cuvette. The measuring light was sufficiently low to ensure F0 condi- tions, i.e. closing of PS II reaction centres was not induced by the measuring light.

2.4. Fluorescence induction experiments The fluorescence induction kinetics were recorded on a home-built

apparatus with narrow band excitation (halfwidth approximately 10 nm) centred at 665 - 670 nm. The fluorescence was detected at 695 nm through an interference filter (half bandwidth 10 nm; Balzers). The cells were adapted to the dark for 20 min before the measurements. After this adapta- tion 3-(3’,4’-dichlorophenyl)-1,1-dimethylurea (DCMU) was added in the dark to a final concentration of 20 PM. After 5 min the induction kinetics were recorded. The induction experiments were analysed in terms of a sum of a fast sigmoidal phase (a, phase) and a slow exponential phase (/.l phase) [ 7, 91. This new analysis method yields the equivalent parameters which can also be obtained from the complementary area analysis [lo]. However, this method avoids the difficulties involved in the conventional area analysis [ 111. Exact details of the analysis method will be published elsewhere [ 121. All measurements were carried out at 25 “C.

485

3. Results

3.1. TRES measured at X,,, = 620 nm In Fig. 1 the TRES of a control sample (Fig. l(A)) of A. uariabilis

(grown at a fluence rate of 0.54 W mm2 white light) is compared with the TRES of cells irradiated with high intensity white light (fluence rate, 5.4 W mP2) for 1 day (Fig. l(B)) and 2 days (Fig. l(C)). The excitation wavelength for the lifetime measurements was chosen to excite preferentially the

(-4)

1

I 0.5 3 c E E aO

- 0.5

((3

640 660 680

Wavelength, nm

1 I I I I I I I

640 660 680 700

Wavelength, nm

1

- 0.5

@I

0 Tq q 0.09 ns

6LO 660 680 700

Wavelength, nm

Fig. 1. TRES of Annbaena cells irradiated for various periods with white light (5.4 W mP2): (A) control 0 h; (B) 24 h; (C) 48 h. The excitation wavelength for recording the TRES was he = 620 nm. The TRES are plots of the amplitude Ai of the fluorescence decay component ri us. the fluorescence detection wavelength. The analysis is carried out using a global analysis procedure (see Section 2). A negative amplitude represents a rise term, indicating energy transfer between different parts of the antenna system,. The data are normalized to the amplitude of the 73 component at &., = 660 nm.

486

phycocyanin part of the PBS rods, i.e. h,,, = 620 nm. TRES obtained from samples irradiated for less than 1 day are not shown, as they are almost identical with that of the control. As reported previously, four kinetic decay components are necessary in the global fits to achieve a satisfactory description of the decay kinetics. One of the components has a negative amplitude, i.e. it represents a rise term. Up to and including 1 day, only small changes in the relative amplitudes of the long-lived components are observed in the TRES of high-light-intensity-treated cells (Fig. l(B)) com- pared with the control (Fig. l(A)), although photosynthetic oxygen produc- tion is inhibited by about 70% [ 11. However, after 2 days, when photoin- hibition has reached its maximum value of 80% [ 11, a substantial increase in the amplitude of the long-lived r1 component at A,, = 650 nm (approxi- mately 1.8 ns) is observed; this shows a further drastic increase if strong light irradiation is continued [4]. Smaller increases are observed at A,, = 680 nm for the r1 component and also for the r2 component (0.38 - 0.42 ns). These amplitude ratios (normalized to the amplitude A3 of the unchanging short-lived PS I component 7s) are collected in Table 1. The value of the average lifetime raV, which is proportional to the total fluores- cence yield and intensity (cf. eqns. (1) and (2)), is also given in Table 1 and can thus be used directly to make a comparison with the intensities mea- sured in the fluorescence induction experiments (see below). In conclusion, these experiments indicate that long-lived components appear after photo- inhibition of photosynthesis has reached its maximum value.

3.2. TRES measured at X,,, = 670 nm In another series of experiments (Fig. 2), the effects of high intensity

irradiation (5.4 W mM2) for 1 h and 3 h (data not shown), 6 h (Fig. 2(B)) and 12 h (data not shown) on the TRES excited at A,,, = 670 nm were measured. The control is shown in Fig. 2(A). An excitation wavelength A exe of 670 nm was used in this series because it excites the Chl antennae directly rather than the PBS. Thus this experiment measures very sensitively any changes which occur in the Chl antennae and/or the reaction centres of one of the photosystems. With increasing irradiation time the relative amplitude of the long-lived decay component at X,, = 685 nm with a life- time r1 of approximately 1.8 ns (and consequently the average lifetime (cf. eqn. (1))) increases substantially. A less pronounced increase is also observed for the r2 = 0.38 ns component. The amplitudes of the two long- lived components, normalized to the amplitude of the r3 component, are compiled in Table 1. Also given are the average lifetimes at emission wave- lengths of 685 nm (r,(6s5j) and 705 nm (~,(,~s)).

3.3. Fluorescence induction The fluorescence induction kinetics in the presence of 20 PM DCMU

for the control cells and cells irradiated with white light of 5.4 W rnA2 for various periods of time are shown in Fig. 3. With increasing irradiation time the ratio Fmax/Fo, i.e. the ratio of the maximum fluorescence yield

487

TABLE 1

Effects of white light irradiation (5.4 W m”) on the amplitudes Ai of long-lived fluores- cence components (relative to the amplitude A3 of component 3) and the average life- times 7, (ns) obtained from the TRES of Anabaena cells excited at Lx, = 620 nm or 670 nma. The range of values indicated for each lifetime 7i in the footnotes reflects the variation in the data corresponding to the different irradiation times

Irradiation A1/Asb AzIA3 b ~av(680) d

AIIA~~ AzIA~~ Tav(650) d

time (h)

0 0.015 0.06 0.43 0.05 0.08 0.26 12 0.079 0.10 0.76 0.19 0.16 0.49 24 0.066 0.08 0.77 0.19 0.11 0.48 48 0.20 0.10 1.04 0.61 0.11 0.86

120e = 0.70 mo.5 = 1.5 5.7 = 1.0 F== 1.7

Irradiation A1/Asf AzlA3 f 7,V(685) d

A1/Asg Az/A~~ 7av(705) d

time (h)

0 0.027 0.13 0.09 0.028 0.11 0.079 1 0.042 0.14 0.11 0.045 0.11 0.087 3 0.039 0.16 0.11 0.046 0.13 0.091 6 0.051 0.19 0.12 0.065 0.15 0.097

12 0.062 0.21 0.14 0.065 0.18 0.098

sThe top part of the table refers to &x, = 620 nm and the bottom part of the table refers to &xc = 670 nm. bAverage values; A, = 680 nm; rr= 1.7 - 1.8 ns; 72 = 0.38 - 0.42 ns. CAverage values; & = 650 nm; or = 1.7 - 1.8 ns; 72 = 0.38 - 0.42 ns. dAverage lifetime calculated at the emission wavelengths indicated. Calculations are based on eqn. (1). The average lifetimes should be compared with the fluorescence induction data (Table 2). eTaken from ref. 4 for comparison; these values are typical for photobleaching condi- tions. fAverage values; &, = 685 nm; r1 = 1.7 - 1.8 ns; 72 = 0.38 - 0.42 ns. aAverage values; h,, = 705 nm; 7r = 1.7 - 1.8 ns; 72 = 0.38 - 0.42 ns.

(closed PS II centres) to the minimum fluorescence yield (before closing PS II centres by the actinic light), decreases strongly. After 24 h irradiation time the variable fluorescence F,,,,, -F. has dropped to less than 10% of its value in the control. The results of the analysis of the fluorescence induc- tion data are compiled in Table 2. It should be noted that the limiting value for the F,,,,.JFo ratio is unity.)

3.4. Azide effects The effects of a very high light intensity (fluence rate, 13.5 W mp2)

in the presence and absence of sodium azide on the TRES of Anabaena are shown in Fig. 4. For better comparison with previous data, the excitation wavelength used to record the picosecond TRES was 580 nm. The TRES of the control and the sample irradiated for 5 days (without azide) have been

488

n T2 = 0.36 ns A T,=O.l5ns 0 Tq = 0.03 ns

680 700 720 680 700 720

(A) Wavelength, nm

a : 6 T, = 1.6 ns

n Tp = 0.36 ns 2- A T,=O.l6ns

0 TL = 0.031 ns

0) Wavelength, nm

Fig. 2. TRES of Anabaena cells irradiated with white light (5.4 W m-*): (A) control 0 h; (B) 6 h exposure. The excitation wavelength for the TRES was Lx, = 670 nm (see also Fig. 1). The amplitudes in (A) and (B) are normalized to the maximum of the short- lived 74 component at h, = 690 nm.

100 200 3:o Time, ms

Fig. 3. Fluorescence induction curves of Anabaena cells in the presence of 20 PM DCMU after exposure to white light (5.4 W m”) for various periods of time, Le. control 0 h, 1 h, 6 h and 12 h exposure. In the figure the curves are normalized to the Fmax value. h exe = 665 - 670 nm; hdet = 695 nm (interference filter (halfwidth, 10 nm) plus cut-off filter RG 695). The Fe value is indicated for each curve by short horizontal lines. The exciting light intensity was kept constant for the recording of all curves shown (see also Table 2) except for the 24 h curve which was recorded at a 50% higher exciting light intensity.

489

TABLE 2

Fluorescence induction parameters of white-light-irradiated (5.4 W m”) Anabaena cells as analysed by the fitting procedure described in refs. 7 and 9 (see also Section 2); hhxc = 665 - 670 nm; )hm = 695 nm recorded in the presence of 20 PM DCMU

Irradiation Fmax/Foa kl b kz b L~I/Az~ pd Foe F,,xe Fvar' time (ms-‘) (ms-l)

0 1.95 0.040 10.0 x 1O-3 2.8 0.23 1.0 1.95 0.95 1 1.5 0.048 8.3 x 1O-3 6.6 0.09 1.1 1.65 0.55 3 1.4 0.053 7.8 x 1O-3 6.0 0.00 1.15 1.61 0.46 6 1.4 0.053 8.3 x 1O-3 6.3 0.01 1.23 1.72 0.49

12 1.1 - 1.2 0.045 1.6 x 1O-3 4.0 0.00 1.24 1.36 0.12 24 1.08 -_g -_g 0.8 0.00

sAverage value with error of kO.2. hRates of fast (kl) and slow (k2) induction components; the error in these rates is +lO%. CAmplitude ratio of fast to slow induction component; error, +lO%. dconnectivity parameter as defined in ref. 13 error, kO.01. eDetermined from the 7,” values (Table 1) and the fluorescence induction data. The FO value for the control is set arbitrarily as unity. fFvar= F max -Fe sData not given because recording of this induction curve was at a higher actinic light intensity.

2.5-

2.0- + T, = 2.1"s

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2

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A TX= 0.23"s G i \

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II I ! ) I I I -0.5- y,< , , , , , , _

620 610 660 680 700 620 610 660 680 700

(A) Wavelength, nm (B) Wavelength, nm

Fig. 4. TRES of Annboena cells irradiated with high intensity white light (13.5 W m-*) for 2 days without azide (A) and for 5 days with added azide (B) (see also Fig. 1) ($,, = 580 nm).

given previously [ 41. Figure 4(A) shows the TRES after 2 days of irradiation in the absence of azide and Fig. 4(B) shows the corresponding spectrum after 5 days irradiation in the presence of azide. The most striking difference between these TRES is the pronounced negative amplitude component (rise term) in the control (pre-irradiation with low fluence rates; see ref. 4,

490

fig. 1) and the sample irradiated in the presence of azide (Fig. 4(B)); this component is not present in the TRES of samples irradiated for 2 days (Fig. 4(A)) or 5 days [ 41 in the absence of azide.

4. Discussion

In a previous study [4] we monitored the picosecond TRES excited at 580 and 620 nm of A. uariabiEis cells exposed to strong white light (fluence rate, 13.5 W mm2) for 6 h and 5 days, which caused photoinhibition and photobleaching respectively. These excitation wavelengths were chosen as they are preferentially absorbed by phycobiliproteins located in the PBS rods. In order to study possible changes in the antennae and/or reaction centres in the transition range from photoinhibition to photobleaching, the picosecond TRES of Anabaena cells exposed to lower fluence rates (5.4 W mm2) of white light for times between 1 h and 2 days were mea- sured in this investigation. In a first series of experiments (Fig. 1, Table 1) an exciting wavelength of 620 nm was used. Comparison with the control (Fig. l(A)) reveals that a 1 day exposure (Fig. l(B)) does not affect the antenna structure, the energy transfer kinetics and efficiency within the PBS antennae or the energy transfer kinetics from PBS to Chl. This can be concluded from the very small changes in the relative amplitudes of the long- lived decay components as compared with typical photobleaching conditions and the negligible changes in the lifetimes (see Table 1). However, after 2 days of light exposure to 5.4 W m-2 (Fig. l(C)), a significant increase in the relative amplitude of the long-lived decay component (7 = 1.8 ns) with an emission maximum around 650 nm is observed, similar to the changes in the TRES characteristic of photobleaching [ 41. Quantitatively, these effects are still very small compared with the drastic effects observed after irradia- tion with higher fluence rates (13.5 W mp2) (cf. Fig. 4(A)) and also with the data published previously [ 41 (cf. Table 1).

In contrast with the negligible effects on the picosecond fluorescence kinetics and TRES (when excited in the PBS rods), large effects on the fluorescence induction kinetics are observed after 1 - 6 h of exposure (Fig. 3 and Table 2). The most pronounced effects seen in the fluorescence induc- tion after exposure to high fluence rates are the reduction in the F,,,,,/F, ratio and the decrease in the rate k2 of the slow induction phase (Table 2). The rate k, of the fast induction phase is not significantly influenced. The data also seem to indicate a variation in the relative contribution (amplitude) of the fast induction phase compared with that of the slow component; however, we do not want to overemphasize these variations since this ratio sometimes depends significantly on the total time range analysed. It should be kept in mind that the fluorescence induction curves are only sensitive to changes in the PS II fluorescence properties [ 141.

Another variation that is worth mentioning is the observation that the fluorescence induction curve changes from a pronounced sigmoidal shape

491

(as indicated by a connectivity parameter p of approximately 0.2 for the control) to a purely exponential shape (connectivity parameter approxi- mately zero) in the irradiated samples. The connectivity parameter is a measure of the probability of energy transfer from one PS II unit to the neighbouring units [13]. A value of p = 0 indicates completely isolated units which are not capable of sharing their energy with neighbouring PS II units. This reduction is observed after 1 h of exposure and is completed within 3 h. The decrease in the Fmax/Fo ratio reflects a decrease in the amount of variable fluorescence, which could be caused by a change in either F. or F,,, or both. The constant ki value reflects the constant antenna size of those PS II units which give rise to the predominant fast phase of the fluorescence induction kinetics. This latter observation excludes any major changes in either the PS II antenna structure or the energetic coupling of the antenna to the PS II reaction centre, except for changes in connectivity between photosynthetic units (see above). Such a conclusion is not valid for those PS II units which give rise to the minor slow induction phase (rate k,).

The apparent discrepancy between the TRES experiments (excitation at 620 nm, i.e. PBS excitation), which indicate only minor changes for exposures up to 1 day, and the fluorescence induction experiments, which indicate large changes over the same exposure period, prompted us to carry out another series of TRES experiments in which the cells were pre- irradiated as in the first series but an excitation wavelength of 670 nm was used. This wavelength excites Chl directly and for this reason it was also used in the fluorescence induction experiments. Thus the TRES excited at this wavelength should emphasize any changes which occur in the Chl antenna and/or the PS I and PS II reaction centres compared with experi- ments using 580 nm or 620 nm excitation. These experiments can also answer the important question of whether a change occurs in F. or in F,,, (this cannot be decided on the basis of the induction experiments alone since the concentration of the pigments cannot be maintained constant in each sample). However, the average lifetime does not depend on the concentra- tion. The fluorescence intensity and yield are proportional to the average lifetime (see Section 2 and ref. 7). The data given in Table 1 on the average lifetime under F. conditions clearly indicate a large increase in F. with increasing time of exposure of the cells. This increased F. value can be cor- related directly with the reduced F,, /F. ratio found in the fluorescence induction. Thus the results of the two types of measurement agree quantita- tively and are complementary to each other. Furthermore, the lifetime data in combination with the induction experiments indicate that the change in the Fmax/Fo ratio is not caused exclusively by an increase in F,. A decrease of comparable percentage in Fmax occurs at the same time. Both effects lead to a decrease in the variable fluorescence F,,, = F,, - Fw The increase in F. is caused by the increased contribution of long-lived decays (Table 1). Since the variable fluorescence originates exclusively from PS II [ 141, these results are indicative of a light-induced process located in the PS II reaction centres and occurring on a time scale much faster than the photobleaching effects discussed earlier [ 2,4].

492

The TRES together with the fluorescence induction data clearly reveal the pronounced photoinhibition effects on PS II under conditions where photobleaching and changes in antenna structure are not significant. Photo- inhibition manifests itself as an increase in F0 (caused by an increased contribution of long-lived fluorescence components with lifetimes of approximately 1.8 ns and approximately 0.4 ns which have a fluorescence emission maximum around 680 - 685 nm) and a decrease in F,,. This is clearly different from the photobleaching phenomenon which also gives rise to a long-lived component with a similar lifetime at F, (r = 1.85 ns) but with an emission maximum near 650 nm, Le. phycocyanin and/or allophyco- cyanin emission [ 41. Although the relative amplitudes of these long-lived components at F, seem to be small, they contribute up to 45% (sum of yields of r1 and r2 components) to the fluorescence yield. The long-lived (7 = 1.8 ns and T = 0.4 ns) PS II fluorescence is reminiscent of the fluores- cence kinetics observed with closed PS II reaction centres of cyanobacteria [ 151. Since the measuring light is not effective in closing the reaction centres (cf. control experiment) we interpret our data in the following way. As a result of photoinhibition an increasing percentage of PS II reaction centres behave as if they are permanently closed, i.e. they lose the ability to return to the open (F,) state. The most probable cause of this inactivity is a func- tional defect on the acceptor side, giving rise to a qualitatively different fluorescence behaviour. In addition, the fluorescence quenching of the photoinhibited centres is increased relative to normal closed centres, thus leading to a parallel decrease in F,, and F,,,. Therefore, our findings may be in line with recent molecular models [16 - 181 which try to explain the photoinhibition phenomenon by a modification at or near the Qz binding protein of PS II.

The TRES recorded with an excitation wavelength A,,, of 670 nm (Fig. 2) show a pronounced component with a lifetime of about 30 ps. The maxima of the associated spectrum are located near 690 nm and at 710 - 720 nm. This is clearly not a PS II Chl spectrum. The features of this spec- trum point to PS I as the origin [19, 201. A pronounced amplitude of PS I fluorescence on excitation at 670 nm is to be expected on the basis of the fact that PS I has only a Chl antenna and is not fed substantially by energy transfer from phycobiliproteins. For this reason, this fast component is not observed when the PBS antenna is preferentially excited.

A comparison of the TRES of Anabaena uariabiZis exposed to high fluence rates (13.5 W me2) of white light for 5 days in the presence of 10m3 M NaN3 (this paper) with the TRES of cells exposed to the same condi- tions in the absence of azide [4] indicates similarities and differences. A striking similarity is the appearance of long-lived decay components (7 = 1.94 ns and r = 1.85 ns respectively) of high relative amplitude. However, the two spectra differ in that the short-lived component (T = 80 ps) with a large negative amplitude (indicating energy transfer) is significant in the azide sample but is not found in the sample irradiated in the absence of azide. Apparently azide prevents, at least in part, the functional decoupling

493

of the PBS rods from the core, thus allowing energy transfer from PBS rods to PS II, albeit to a smaller extent. The data seem to suggest that the transfer is inhibited from the PBS core to PS II but not from the PBS rod to the PBS core. In connection with this it is of interest that, compared with the control, the photoinhibition of photosynthetic oxygen production is decreased rather than increased by sodium aside [ 1, 31. This effect can also be understood on the basis of a decreased energy transfer efficiency from PBS to the PS II core. Since a large increase in the long-lived components (lifetime, approximately 1.8 ns) is observed in both samples, we may expect that photobleaching occurs in the absence and in the presence of azide. However, this is not the case. Whereas the untreated samples are strongly photobleached and appear yellow-brown, the aside-treated samples are still blue-green. Since, according to our former discussion [4], the long lifetime of the first excited singlet state of the disconnected PBS rods can result in the formation of triplet states by intersystem crossing, which in turn can give rise to the generation of singlet molecular oxygen, the protection of the PBS rods from photobleaching could be the result of a quenching effect exerted by azide. However, the question of whether this protection is the result of triplet state quenching or ‘02 quenching or both cannot be answered yet. In any case, these results support the hypothetical model of the phototactic reaction sign reversal [ 21 - 231. Our previous hypothesis [4] that photo- bleaching and photoinhibition are different phenomena, which act on dif- ferent parts of the photosynthetic apparatus and with very different rates, is further supported by the results presented here. The photoinhibition reaction saturates after 24 h at a fluence rate of 5.4 W m-* of white light, whereas photobleaching takes several days at even higher fluence rates [ 41. Moreover, our data show that photobleaching commences after full photo- inhibition is achieved and thus suggest that closed PS II reaction centres are a prerequisite for photobleaching.

References

1 G. Agel, W. Nultsch and E. Rhiel, Photoinhibition and its wavelength dependence in the cyanobacterium Anabaena variabilis, Arch. Microbial., 147 (1987) 370 - 314.

2 W. Nultsch and G. Agel, Fluence rate and wavelength dependence of photobleaching in the cyanobacterium Anabaena variabilis, Arch. Microbial., 144 (1986) 268 - 271.

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5 A. R. Holzwarth, J. Wendler and G. W. Suter, Studies on chromophore coupling in isolated phycobiliproteins. II. Picosecond energy transfer kinetics and time-resolved fluorescence spectra of C-phycocyanin from Synechococcus 6301 as a function of the aggregation state, Biophys. J., 51 (1987) 1 - 12.

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9 E. Bittersmann, H. Senger and A. R. Holzwarth, Synchronous cultures of the green alga Scenedesmus obliquus: comparison of picosecond decay associated emission spectra and fluorescence induction kinetics, J. Photochem. Photobiol., 1 (1987) 247 - 260.

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19 A. R. Holzwarth, Picosecond fluorescence spectroscopy and energy transfer in photo- synthetic antenna pigments, in J. Barber (ed.), Topics in Photosynthesis: The Light Reactions, Elsevier, Amsterdam, 1987, pp. 95 - 157.

20 A. R. Holzwarth, J. Wendler and W. Haehnel, Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgoris. Resolution of photosystem I fluorescence, Biochim. Biophys. Acta, 807 (1985) 155 - 167.

21 W. Nultsch and H. Schuchart, A model of the phototactic reaction chain of the cyanobacterium Anabaena variabilis, Arch. Microbial., 142 (1985) 180 - 184.

22 W. Nultsch, H. Schuchart and F. Koenig, Effects of sodium azide on phototaxis of the blue-green alga Anabaena variabilis and consequences to the two-photoreceptor systems-hypothesis, Arch, Microbial., 134 (1983) 33 - 37.

23 H. Schuchart and W. Nultsch, Possible role of singlet molecular oxygen in the control of the phototactic region sign of Anabaena variabilis, J. Photochem., 25 (1984) 317 - 325.