Photosynthesis Research 49:11-20, 1996. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light* Kaushal KumarSingh, RadheyShyam & PrafullachandraVishnu Sane National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India Received 18 July 1995; accepted in revised form 19 April 1996

Key words: D 1 protein synthesis, oxidative phosphorylation, photophosphorylation, recovery from photoinhibition, respiratory inhibitor, uncouplers

Abstract

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 #mol m -2 s -l. The recovery was partial (< 10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75/~mol m -2 s -l did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 #mol m -2 s - l ) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 #mol m -2 s -1) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.

Abbreviations: CCCP-carbonyl cyanide m-chlorophenylhydrazone; D1 - 3 2 kDa protein of PS II reaction center; FCCP-carbonyl cyanide p-(trifiuoromethoxy)phenylhydrazone;K C N - potassium cyanide; P B Q - phenyl -pbenzoquinone; PFD - photon flux density; SHAM- salicylhydroxamic acid Introduction

Photosynthetic efficiency decreases when algae and higher plants are exposed to light intensity higher than that experienced by them during their normal growth. Photoinhibition of photosynthesis is a reversible process and normal photosynthetic rate is restored when organisms are brought back to suitable reactivating conditions. In most of the studies on algae and higher plants it has been demonstrated that only a limited recovery takes place in dark and light is an essential element required for recovery (Ohad et al. 1984; Greer et al. 1986; Skogen et al. 1986; Lidholm et al. * NBRI Research Publication No. 431.

1987; Shyam and Sane 1989). Although light plays a major role in the process of reactivation, surprisingly fast and complete reactivation occurs invariably in the presence of low light (Greer et al. 1986, Skogen et al. 1986; Greer and Laing 1988). The light dependent steps in the process of recovery are not as yet clear. Recent in vitro studies on the molecular mechanism of reactivation revealed a role of light at various stages starting from the transcription ofpsbA gene coding for D1 protein to insertion and integration of this protein into the functional PS II complexes and their lateral migration from stroma exposed thylakoids to the functional site in the appressed thylakoid region (cf. Barber and Andersson 1992; Aro et al. 1993). In vivo studies carried out particularly on Chlamydomonas (Kyle et

12 al. 1984; Ohad et al. 1984; Kyle and Ohad 1986),

Spirodela (Mattoo et al. 1984) and Pisum sativum (Aro et al. 1994) showed that repair of photodamage involves degradation, removal and synthesis of D1 protein (cf. also Critchley and Russell 1994). The latter process is energy dependent and requires light to drive photophosphorylation to generate ATP (Mattoo et al. 1984; Ohad et al. 1984). A recent study carded out on Anacystis nidulans by Shyam et al. (1993) showed that mitochondrial respiration via oxidative phosphorylation may also serve as an alternate energy source for D1 protein synthesis. Interaction between photosynthesis and respiration has been documented in a number of studies in algae and higher plants (cf. Graham 1980; Graham and Chapman 1979). An increase in respiratory 02 uptake at high light intensity, and its suppression in low light or in the dark, has been reported in many studies (Brown and Webster 1953; Jones and Myers 1963; Hoch et al. 1963; Stokes et al. 1990). Changes in dark respiratory rate influence the photosynthetic efficiency of plants at lower light intensities (Sharp et al. 1984; Krischbaum and Farquhar 1987). The dark respiration may also serve as a source of CO2 for carbon assimilation. The discovery of the presence ofNADH oxidising respiratory chain in the chloroplast of eucaryotic green algae, that uses 02 as substrate and shares the plastoquinone pool of photosynthetic electron transport chain, suggests an intrinsic relationship between photosynthesis and respiration in this group of organisms (Godde and Trebst 1980; Bennoun 1982). A somewhat similar situation also exists in cyanobacteria (blue-green algae) where membrane bound respiratory electron transport proceeds in the thylakoid membrane (Hirano et al. 1980; Peschek 1983; Matthijs et al. 1984, 1985). The structural and functional coordination of photosynthesis and respiration indicates that the two processes are mutually beneficial. It is not as yet clear, how photosynthesis and respiration interact during the course of photoinhibition and its reactivation. However, studies of Sardadevi and Raghavendra (1992) on the mesophyll protoplasts of Pea and Shyam et al. (1993) on cyanobacterium Anacystis nidulans suggested a protective role of dark respiration in the process of photoinhibition of photosynthesis and its reactivation. In the present study, we have examined the role of quality, quantity and minimum duration of light required for complete recovery of photosynthesis in the photinhibited cells of the green alga Chlamydomonas reinhardtii. The kinetics of dark respiration in the overall process of photoinhibition of photosynthesis and

its reactivation were also critically studied to evaluate the quantitative significance of respiration in offering protection against photoinhibition as also the recovery from photodamage.

Material and methods

Chlamydomonas reinhardtii cwl 5 was grown in batch cultures in Tris-acetate-phosphate medium (Gorman and Levine 1966). The cultures were grown at 25 + 1 °C by providing a photon flux density (PFD) of 250 #mol m -2 s -l for 14 h daily from cool white fluorescent lamps (Philips, India). The density of culture was determined after subtracting the absorbance at 750 nm (A750) from the absorbance obtained at 663 nm. The chlorophyll was extracted in 80% acetone and estimated by monitoring absorbance at 652 nm according to Arnon (1949). Photoinhibitory treatment was given in a water jacketed glass vessel with a stirring device. The temperature of the vessel was maintained at 25 + 1 °C by connecting it with a thermostatic circulator (LKB, Sweden). Exponentially growing algal cultures were ?centrifuged and washed with fresh medium. Cells were resuspended in the treatment vessel to maintain an A663 of 1.0. Light for photoinhibitory treatment was provided with a 150W Xenophot, HLX lamp (Osram, Germany) of a slide projector (Kindermann, Germany). PFD was varied by placing the projector at different distances from the sample. At specific time intervals, algal samples were withdrawn from the photoinhibitory treatment and photoinhibition of photosynthesis was assayed by measuring photosynthetic 02 evolution using CO2 as the acceptor. Absorbance at A663 was measured in the control and treated samples after photoinhibitory treatment and during the course of recovery from photoinhibition. There was very little change in the absorbance at A663 during the course of experiment. PFD was measured, with quantum sensor (LI-1905A,, LiCor Inc., Lincoln, NE, USA). Photosynthetic 02 evolution and respiratory 02 uptake were measured in a water jacketed cell of variable capacity (< 3 ml) by using a Clark type 02 electrode (Hansatech Ltd., Norfolk, UK). For the purpose, a 2 ml sample was withdrawn, centrifuged and washed with the fresh medium. The cells were resuspended in 2 ml of fresh medium in the water jacketed cell maintained at 25 -4- 1 °C to monitor the 02 evolution/consumption. The rate of photosynthesis was measured at limiting light intensity (150 #mol m -2

13 s-1) as it is found to effectively express the impairment of photosynthesis (Powles 1984; Lidholm et al. 1987). Interference filters (Oriel, USA) with a band width of 10 nm were used to irradiate samples with different wavelengths (725 nm, 660 nm and 480 nm) of light. Chlorophyll fluorescence was also measured to express the photosynthetic performance during photoinhibition of photosynthesis and its reactivation by PAM 2000 fluorometer (Walz, Germany) in a laboratory-made cuvette at 25 + 1 °C. The fluorescence parameters studied were initial chlorophyll fluorescence (Fo), maximum total chlorophyll fluorescence yield (Fro) and the Fv/Fm ratio (Fv, the variable chlorophyll fluorescence yield defined as Fm-Fo). The cultures were dark adapted for 5-10 min prior to making measurements. For the measurement of Photosystem II electron transport, thylakoid membranes were isolated from photoinhibited, reactivating or control cells following the method of Shim et al. (1990). PS II electron transport activity from H20 to PBQ was measured in a Clark type 02 electrode described by Singh et al. (1990). Thylakoid membrane proteins were analyzed by denaturing SDS-PAGE (Laemmli 1970) by employing 12-16% gradient of acrylamide and 4 M urea in the separating gel. Reaction center DI polypeptide was identified by western blotting using the antibody of D1 protein, kindly provided by Dr A. K. Mattoo (USA). Electrophoretic transfer of the SDS-PAGE resolved thylakoid membrane polypeptides to nitrocellulose membrane and the subsequent incubations with the antibodies and with alkaline phosphatase conjugated antibodies were carried out as described by Harlow and Lane (1988). In all the experiments the data represent the averages of results from three measurements.

Results

Photoinhibition of photosynthesis Chlamydomonas reinhardtii showed time and intensity dependent inhibition of photosynthetic 02 evolution when exposed to PFDs higher than that of the growth light intensity. Exposure of cultures growing at saturating light intensity (250 #mol m -2 s-1) to photoinhibitory light of 2500 #mol m -2 s -I resulted in 50% loss of photosynthetic activity within 60 min (Figure 1A). PS II electron transport activity of

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Figure I, Time course of inhibition of photosynthetic 02 evolution (A), PS II electron transport activity (B) and Fv/Fm ratios (C) in Chlamydomonas reinhardtii exposed to PFD of 2500 #mol m - 2 s - I. Initial control values: (A.) 85 4- 4 #tool 02 evolved rag- I Chl h - I , (B) 128 4- 5/~mol 02 evolved mg - t Chl h - l , (C) 0,708 40.006.

the thylakoid membranes isolated from photoinhibited cells (Figure 1B) and chlorophyll fluorescence of photoinhibited intact algal cells (Figure 1C) followed a kinetics somewhat similar to inhibition of photosynthetic O2 evolution. Dark respiratory rate of cultures of C. reinhardtii growing exponentially ranged from 15-20 #mol 02 consumed mg -1 chl h -l . The magnitude of dark respiration recorded at different time intervals during the process of photoinhibition revealed a rapid increase during the first 20 min, reaching a rate of 24-32 #mol 02 consumed mg -1 chl h -1 followed by a gradual decline with longer period of photoinhibitory treatment (Figure 2A, open circles). The kinetics of dark respiration during photoinhibition suggested some specific physiological significance of this process. With a view to understand precisely the role of dark respiration, KCN was used as an inhibitor. In presence of 0.1 mM KCN, dark respiratory 02 uptake of exponentially growing Chlamydomonas cells was inhibited by

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Figure 2. (A) Effect of photoinhibitory light (2500 #mol m - 2 s - l) on photosynthesis and respiration in Chlamydomonas reinhardtii in the presence and absence of 0.1 mM KCN. Initial photosynthetic rate, 82 + 7 #mol 02 evolved mg - 2 Chl h - l ; initial respiratory rate, 20 5 : 2 pmol 02 consumed mg -1 Chl h - l . Photosynthesis: KCN (Q), + KCN (R); respiration: - KCN (O), + KCN (O). (B) Effect of photoinhibitory light (2500 #mol m - 2 s - l) on photosynthesis and respiration in Chlamydomonas reinhardtii in the presence and absence of uncouplers (FCCP, 1 /~M; CCCP, 2.5 #M). Initial photosynthetic rate, 76 + 4/zmol 02 evolved mg - 2 Chl h - l ; initial respiratory rate 21 4- 1 #mol 02 consumed mg -1 chl h -1. Photosynthesis: - uncoupler ($), + FCCP (A), + CCCP (11); respiration: - uncoupler (O), + FCCP (A), + CCCP ([[1).

40-50% while it had an insignificant effect on photosynthetic 02 evolution. It may, however, be mentioned that C. reinhardtii has both cytochrome c and alternative oxidase respiratory pathways (cf. Goyal and Tolbert 1989). The measured respiration presumably represents largely the alternative respiration rate (SHAM sensitive). The combination of KCN (0.1 mM) and SHAM (2.0 mM) did decrease the 02 uptake still further but at the same time inhibited the photosynthetic 02 evolution appreciably. Addition of KCN at the commencement of photoinhibitory treatment showed an increased susceptibility of alga to photoinhibition and resulted in a sharp decline in photosynthetic rate leading to 50% inhibition of photosynthetic 02 evolution within 15-20 min (Figure 2A, closed squares). The time required for 50% inhibition was otherwise 60 min in cultures exposed to photoinhibitory light in the absence of inhibitor (Figure 2A, closed circles). Cultures exposed to photoinhibitory light in the presence of KCN showed a remarkable decrease in their respiratory 02 uptake (Figure 2A, open squares) compared to cultures photoinhibited in absence of KCN

(Figure 2A, open circles). An increase in dark respiration from the initial rate was observed in the beginning of photoinhibitory treatment in the presence as also in the absence of KCN. This increase lasted for the first 10 min in KCN added cultures and 20 min in cultures without KCN. The degree of susceptibility of C. reinhardtii to photoinhibition was studied in the presence of uncouplers FCCP and CCCP. Both FCCP (1/zM) and CCCP (2.5 #M) increased the dark respiration rate by 40% without affecting the photosynthesis even after 90120 min of incubation (data not shown). Addition of 1 #M FCCP (Figure 2B, closed triangles) and 2.5 #M CCCP (Figure 2B, closed squares) in the beginning of photoinhibitory treatment resulted in 50% inhibition of photosynthesis within 30 min. In the absence of uncouplers, however, it took 60 min for this inhibition to occur (Figure 2B, closed circles). The pattern of respiration in the presence of uncouplers during the course of photoinhibition was similar but the rates were lower than in the absence of uncouplers.

Reactivation of photosynthesis in photoinhibited cells Reactivation of photosynthesis in photoinhibited Chlamydomonas cells was monitored by incubating 50% photoinhibited cultures at different PFDs ranging from 5 to 300/~mol m -2 s -1 and measuring the photosynthetic O2 evolution (Figure 3). It was found that PFD of 25/~mol m -2 s -1 was optimal in which complete recovery of photosynthetic 02 evolution took place within 60 min (Figures 3 and 4A, open circles). Recovery of electron transport activity of PS II and Fv/Fm was also monitored at optimal PFD of 25 #mol m -2 s -1. While the PS II electron transport activity recovered fully (Figure 4B open circles), the recovery in Fv]Fm w a s incomplete and could not attain 100% value (Figure 4C, open circles). In the absence of light, the recovery in all the parameters was incomplete and there was only a marginal increase (< 10%) in photosynthetic activity (Figure 4A-C, closed circles). In the subsequent studies, photosynthetic O2 evolution was the only parameter studied to assess the extent of recovery in photosynthesis. Reactivation was also monitored by incubating 50% photoinhibited cells in far red (725 nm), red (660 nm) and blue (480 nm) monochromatic light of different intensities (10, 25, 50 and 75 #mol m -2 s -1) to see if the quality of light had any specific effects on the process of recovery. The optimal PFD required for fast and complete reactivation in different qualities of

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Time ( m i n ) Figure 3. Effect of different PFDs on the time course of recovery of photosynthesis in Chlamydomonas reinhardfii. Reactivation of photosynthesis was carded out by incubating 50% photoinhibited cells at 5 #mol m - 2 s - t (@), 15 #tool m - 2 s - l (~)), 25 #tool m - 2 s - l (O), 75 #tool m - 2 s -1 (E]), 150 #mol m - 2 s -1 (A), 300 #mol m - 2 s - I (~7). Initial photosynthetic rate, 80 4- 5 #tool 0 2 evolved m g - l Chl h - 1. Table 1. The optimal PFD and minimal time required for complete reactivation of 50% photoinhibited cells of Chlamydomonas reinhardtii in White (40(0700 nm), Farred (725 nm), Red (660 rim) and Blue (480 rim) light. Light quality

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light is presented in Table 1. The fastest recovery was observed in white light. Although a complete reactivation took place in 25 #mol m -2 s-1 of far red light, the time required for this reactivation was 90 min. Reactivation in 25 #mol m -2 s - l of red and blue light

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Figure 4. Time course of reactivation of photosynthetic 02 evolution (A), PS II electron transport activity (B) and Fv/Fm ratios (C) in 50% photoinhibited cultures of Chlamydomonas reinhardtii incubated at 25/~mol m - 2 s - l (O) and in dark (@). Initial control values: (A) 81 -4- 5 #mol 02 evolved mg -1 Chl h - l , (B) I26 4- 6 #tool 02, evolved mg -1 Chl h - l , (C) 0.692 4- 0.011.

was considerably lower as compared to white and far red light. Complete reactivation in blue and red light required a longer time and higher PFD (Table 1). To define the effect of light more precisely we determined the minimum duration of light which could result in complete reactix;ation of photosynthesis in the photoinhibited cells. Cultures photoinhibited to 50%, when incubated in dark, showed less than 10% reactivation of photosynthesis. Recovery in such cultures was monitored by exposing them for different duration ranging from 1 min to 10 min in diffuse light (25 #tool m -2 s - j ) or 1 s to 60 s in strong light (2500 #mol m -2 s -1) followed by their reincubation in the dark. The results showed that complete reactivation in the dark incubated photoinhibited cultures occurred if they were exposed for a minimum period of 5 min to diffuse light followed by incubation in dark (Figure 5A, open triangles). Exposure of dark incubated photoinhibited cultures to even strong light from 1 s to 60 s showed that an exposure of 5 s to 2500 #mol

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Figure 5. (A) Requirement of minimum duration of light (25 #mol m -2 s -l) for reactivation of photosynthesis in Chlaraydomonas reinhardtii. 50% photninhibited cultures were initially incubated in

dark for 15 min and subsequently exposed to 25 #tool m-2 s -l of white light for 1 min (©), 5 rain (A),7.5 min (V) and 10 rain (D). (B) Requirement of minimum duration of light (2500 #mol m-2 s-l) for reactivation of photosynthesis in Chlamydomonas reinhardtii. 50% photoinhibited cultures were initially incubated in dark for 15 min and subsequendyexposedto 2500 #tool m- 2 s- 1of white light for I s (©), 5 s (A), 30 s (V) and 60 s (D).

m -2 s - I was optimal to bring out complete reactivation in the dark (Figure 5B, open triangles). However, in this case the cultures, after photoinhibitory treatment, were required to be incubated in dark for a minimum period of 15 min before the high light exposure was provided for reactivation. The process of reactivation of photosynthesis from photoinhibition was severely affected in the presence of respiratory inhibitor KCN. In all the above cases irrespective of the mode of exposure (continuous diffuse light, 5 min diffuse light or 5 s high light) reactivation was found to be inhibited by 0.1 mM KCN and no trace of recovery could be seen even after incubation for several hours (Figure 6A, closed squares). The cultures, reactivated in absence of KCN, showed a rapid recovery resuming normal photosynthesis within an hour (Figure 6A, closed circles). During the course of recovery, the dark respiration, in the absence of KCN, gradually increased by 25% from the initial value recorded in the beginning of reactivation (Figure 6A, open circles). This increase in dark respiration was, however, not observed in cultures reactivated in the presence of KCN. In this case the dark respiration fell abruptly to 70% of its initial value (Figure 6A, open squares) and it remained somewhat

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Figure 6. (A) Time course of recoveryof photosynthesis in Chlamydomonas reinhardtii in the presence and absence of 0.1 mM KCN

under continuous exposure to 25 #mol m-2 s -1 . Initial photosynthetic rate, 85 -4-3/~mo102 evolvedrag- l Chl h- l; initial respiratory rate, 20 -I-2 #mol 02, consumedrag- 1 Chl h- i. Photosynthesis: - KCN (O), + KCN (R), respiration: - KCN (C)), + KCN (D). (B) Time course of recovery of photosynthesis in Chlaraydomonas reinhardtii in the presence and absence of uncouplers (FCCP, 1/zM; CCCP 2.5/~M) under continuous exposure to 25/~mol m -2 s -1 . Initial photosynthetic rate; 81 -t- 6 #mol 02 evolved mg-1 Chl h-l; initial respiratory rate, 19 -4-2 #mol 02 consumedmg-1 Chl h -l . Photosynthesis:- uncoupler (O), + FCCP (IN), + CCCP (A); respiration: - uncoupler (©, + FCCP (El), + CCCP (A).

static or decreased a little more when no reactivation took place. Recovery of photosynthesis in the presence of FCCP and CCCP was slow and only a marginal gain in photosynthesis (10--20%) could be achieved after 2 h of incubation under reactivating condition (Figure 6B, closed squares and closed triangles respectively). During the course of reactivation, in the presence of FCCP, the rate of respiration increased slightly (Figure 6B, open squares). This increase was, however, less than what was observed during reactivation in the absence of uncouplers (Figure 6B, open circles). In case of CCCP the respiratory rate gradually decreased from the beginning (Figure 6B, open triangles). With a view to understand the role of protein synthesis, reactivation was carried out in the presence of drugs which inhibit protein synthesis. Addition of chloroplastic translation inhibitor chloramphenicol (100 #g m1-1) at the time of photoinhibitory treatment or before the incubation of photoinhibited cultures for reactivation almost completely stopped the process of recovery (data not shown). Addition of

17 cycloheximide (2 #g ml-l), a cytoplasmic translation inhibitor or rifampicin (250 #g ml-t), a transcription inhibitor did not stop the recovery although it took a little longer time to recover completely in the latter (data not shown). Immunodetection of D1 protein of the thylakoid membranes isolated from 50- 60% photoinhibited cultures showed a slight decrease (< 15%) in the level of this protein as compared to control. When such cultures were exposed to 5 rain of diffuse light or 5 s of strong light, complete reactivation of photosynthesis with an increase in the level of D1 protein equal to that of the control (data not shown) was observed. A similar trend in the increase of D1 protein was observed in the normal reactivation under continuous diffuse light. These results showed that the level of D1 protein increases with regain of photosynthesis in all the cases of reactivation irrespective of duration and intensity of light exposure.

Discussion

The increase in the susceptibility of Chlamydomonas reinhardtii to photoinhibition when dark respiration was partially inhibited by KCN, suggested a specific role of respiration in manifestation of photoinhibition. Increase in photoinhibition in presence of respiratory inhibitors has recently been reported by Sardadevi and Raghavendra (1992) in mesophyll protoplasts of pea and Shyam et al. (1993) in cyanobacterium Anacystis nidulans. The degree of photoinhibition varies with the concentration of oxygen in the cell (Krause et al. 1978; Cornic et al. 1982; Chaturvedi et al. 1992; Leitsch et al. 1994) and high concentration of 02 in the medium inhibits the photosynthesis of many algae (Nilsen and Johnsen 1982). Although the rate of dark respiration is low as compared to the rate of photosynthesis in most of the algae including Chlamydomonas reinhardtii, dark respiration may act as scavenger of excess 02 produced in the cell during photoinhibition that may otherwise damage the photosynthetic machinery particularly under CO2 limiting condition (Krause and Comic 1987). Another role of dark respiration may be attributed to oxidation of excess redox equivalents generated in photosynthetic electron transport (Kromer and Heldt 1991) during photoinhibition. A prerequisite of low light in the process of reactivation of photosynthesis in photoinhibited leaves and algal cells has conclusively been demonstrated in a number of studies (Ohad et al. 1984; Greer et al. 1986;

Skogen et al. 1986; Lidholm et al. 1987; Shyam and Sane 1989). However, reactivation at low levels does occur in certain cases in the dark. Intensity dependence of reactivation in C. reinhardtii in the present study revealed 25 #mol m -2 s- 1 as an optimal PFD for the reactivation of 50% photoinhibited cells within 60 min. Light intensity higher or lower than this resulted into incomplete and/or slow reactivation. Recovery in 25 #tool m -2 s-1 monochromatic far red light (725 nm) which excites mainly PS I, was somewhat slow than white light and took longer time (90 min) for completion. Complete recovery in monochromatic red (660 nm) and blue (480 nm) light required higher PFD (75 #mol m -2 s- 1) and longer time (90 rain) as compared to far red and white light. These results demonstrate that the excitation of a particular photosystem does not enhance the process of reactivation in photoinhibited Chlamydomonas cells. In the earlier studies a demand for PS I activity through the involvement of cyclic photophosphorylation in the process of reactivation had been suggested (cf. Chaturvedi et al. 1985; Skogen et al. 1986). The results obtained in the present study with reference to minimum duration of light, required for complete reactivation of photoinhibited C. reinhardtii cells indicated a triggering role of light in this process. Exposure to 25/~mol m -2 s -l for 5 min to dark incubated photoinhibited cells was sufficient to bring out complete reactivation within 60 min. Even 2500/zmol m -2 s- l of light which causes photoinhibition, if provided to dark incubated photoinhibited cultures, elicits the process of reactivation leading to full regain of photosynthesis. However, the duration of exposure could be as low as 1 s, the shortest tried in these studies. Longer exposures progressively decreased the extent of reactivation. Both types of reactivations in C. reinhardtii in the present study were inhibited by translation inhibitor chloramphenicol that substantiate the finding that protein synthesis was required for the recovery of photoinhibited cells (Ohad et ai. 1984; Samuelsson et al. 1985; Greer et al. 1986; Lidholm et al. 1987; Schuster et al. 1988; Vonshak et al. 1988; Shyam and Sane 1989; Tyystjarvi et al. 1992). Immunoblot analysis of D1 protein of thylakoid membrane isolated from photoinhibited C. reinhardtii cells in the present study gave an indication of increase in intensity of D1 bands with time under both the sets of reactivating conditions, suggesting thereby requirement of D 1 protein synthesis for recovery process. These results are consistent within the concept of a 'PS II repair cycle' in algae

18 and higher plants where recovery from photodamage requires de novo synthesis of D 1 protein (Ohad et al. 1984; Greer and Laing 1988; Guenther and Melis 1990; Prasil 1992). In the earlier studies the requirement of light was shown to be mandatory for the complete reactivation of photosynthesis. However, the results obtained in the present study, suggest that continuous light is not a necessity but even a short exposure of light during dark incubation is adequate to complete the process of reactivation. The triggering role of light, however, cannot be explained when one considers the demand of energy supply for D1 protein synthesis that seems to be obligatory for complete reactivation, in spite of the complex relationship drawn between D1 turnover and regain of PS II quantum efficiency. Photosynthesis during reactivation under continuous but low illumination may generate some ATP via cyclic photophosphorylation to meet out the partial energy requirement for D1 synthesis (Ohad et al. 1984). However, supply of adequate ATP through photophosphorylation, particularly when organism is exposed to such a short duration of low light (25 ttmol m -2 s -1 for 5 min) or high light (2500 #mol m -2 s -1 for 5 s) during dark incubation does not seem possible. Raven and Samuelsson (1986), on the basis of analysis of data available on photoinhibition and its repair in cyanobacterium Anacystis nidulans, computed the rate of protein synthesis in repairing process. They concluded that protein synthesis during reactivation used up to 1/5 of the ATP production rate from oxidative phosphorylation in darkness and up to 1/10 of the ATP production rate (including photophosphorylation) at an incident photon flux density of 5 #mol m -2 s -1. It therefore, appears that oxidative phosphorylation occuring in dark, contributes towards the energy need for D1 synthesis. Michaels and Herrin (1990), by using synchronous cultures of C. reinhardtii, demonstrated the availability of ATP as one of the regulators of translation of D1 protein. In the present context their observation is relevant as they also showed that an alternative energy source can substitute for light in promoting translation of D 1 during the light dark cell cycle of this alga. In our study, where a brief duration of light induced complete recovery of photosynthesis in photoinhibited C. reinhardtii cells in dark, role of dark respiration becomes unequivocal. Here it may be presumed that energy for protein synthesis during reactivation is almost being fully supplied by dark respiration via oxidative phosphorylation. This proposal is plausible since a partial inhibition of respiration

by respiratory inhibitor KCN resulted in complete loss of reactivation in this alga, irrespective of continuous low-light exposure or short exposure of low or high light during dark incubation. Furthermore, under both sets of reactivating conditions, the process of recovery was affected by FCCP and CCCP in the present study. Although actions of FCCP and CCCP are not very specific in vivo, these are known uncouplers of oxidative phosphorylation and photophosphorylation, respectively. Since photosynthesis under the reactivating condition of short exposure of light followed by dark incubation is negligible, uncoupling resulting in inhibition of ATP synthesis by oxidative phosphorylation leads to complete loss of reactivation. These results further strengthen the role of dark respiration in the process of reactivation. Kuroda et al. 1992 showed that the level of stromal ATP is an essential determinant in the regulation of the synthesis of D1 protein in isolated spinach chloroplasts. However, the level of ATP in chloroplast in vivo may be elevated by transport of ATP produced in mitochondria via adenine nucleotide translocators located on the chloroplast outer envelope (Heldt 1969; Robinson and Wiskich 1976). Decrease in intracellular ATP concentration in cyanobacteria under continuous anaerobiosis or in presence of uncouplers or respiratory electron transport inhibitors has been reported by Nitschmann and Peschek (1986). Increased rate of photoinhibition and lack of recovery in presence of respiratory inhibitor and uncouplers has also been reported in cyanobacterium Anacystis nidulans by Shyam et al. (1993). Uncouplers may also affect the phosphorylation of the light-harvesting complex of Photosystem II as well as phosphorylation of D1 to DI* protein during photoinhibition of photosynthesis. While the former process imparts protection from photoinhibition (Horton and Lee 1985) by redistributing the excitation energy in favour of Photosystem I, the latter protects D 1 from the degradation and involved in the regulation of repair cycle of photoinhibited PS II centers (Aro et al. 1993; Rintamaki et al. 1995). However, phosphorylation of D1 protein has not been reported so far in lower group of organisms (cf. Rintamaki et al. 1995). It is apparent from the above discussion that mitochondrial respiration may serve as an alternative source to supply energy required for D 1 protein synthesis during the process of reactivation in the dark. Recent studies on Scenedesmus obliquus (Gong and Ohad 1995) and Chlamydomonas reinhardtii (Zer and Ohad 1995) further demonstrated that degraded D1 protein during photoinhibition could be replaced by its synthe-

19 sis in the dark. This synthesis and replacement of D1 protein in dark, however, led to partial reactivation of photosynthetic activity in the photoinhibited cells. These findings, however, do not explain the obligatory requirement of short exposure of light prior to dark incubation which indeed accelerated the partially initiated process of reactivation towards completion. This triggering effect of light suggests that light has some specific functions in the overall process of reactivation. Light regulatory functions, apart from D 1 synthesis, also include various stages (as discussed earlier) in the assembly of functional PS II complex in the appressed thylakoid region (Aro et al. 1993). How can a short exposure of light perform these functions is required to be looked into critically. It can be argued that light may switch on the process as an activator to transduct certain signals which perform light regulatory functions even in dark (storage signal). In the whole process of reactivation, the possibility of ATP acting as a signaling and or regulatory molecule can not be ruled out. Another attractive possibility, which we favour, is that light as a short 5 s high intensity pulse may induce conformational and dynamic movements of the complete thylakoidal membrane system that may facilitate transport of the damaged D1 and movement in of repaired D1 into the granal appressed regions for reassembly of the PS II functional complex.

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