Systematic Analysis of the Relation of Electron Transport and ATP Synthesis to the Photodamage and Repair of Photosystem II in Synechocystis

Inhibition of Electron Transport in PSII and of ATP Synthesis Both Enhanced the Extent of Photodamage to PSII

The photosynthetic transport of electrons is inhibited by DCMU on the acceptor side of PSII. Figure 1A shows the effects of this inhibitor on the extent of photodamage to PSII, as monitored in terms of the photosynthetic evolution of oxygen with 1,4-benzoquinone (BQ) as the artificial acceptor of electrons. The extent of photodamage was significantly enhanced by 20 μm DCMU, which was sufficient to inhibit completely the electron transport in PSII. In this experiment, DCMU was removed by washing cells twice, by centrifugation and resuspension, prior to measurements of electron transport activity (see “Materials and Methods”). This treatment completely removed DCMU and its inhibitory effects.

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Figure 1.

Effects of DCMU, DCCD, and Nig/Val on the photodamage to PSII in Synechosystis in the absence of lincomycin (A) and in the presence of 250 μg mL⁻¹ lincomycin (B). At designated times during incubation in light at 500 μE m⁻² s⁻¹ at 34°C, aliquots of each suspension of cells were withdrawn and PSII activity was measured in terms of the photosynthetic evolution of oxygen in the presence of 1.0 mm BQ, as the electron acceptor, after DCMU and lincomycin had been removed by washing as described in “Materials and Methods.” Each point and bar represent the average ± se of results from four independent experiments. ○, No addition (control); •, 20 μm DCMU; ▪, 20 μm DCMU plus 10 μm DCCD; ▴, 20 μm DCMU, 2 μm nigericin plus 2 μm valinomycin.

To investigate the contribution of ATP synthesis to the extent of photodamage, we used N,N-dicyclohexylcarbodiimide (DCCD), which is an inhibitor of ATP synthesis, and a combination of two ionophores, namely, nigericin and valinomycin (abbreviated as Nig/Val), which reverses the energization of thylakoid membranes (Dilley et al., 2001; Ke, 2001). The results in Figure 1A demonstrate that this inhibitor and these ionophores enhanced the photodamage to PSII that occurred in the presence of DCMU.

Next we examined the effects of DCMU, DCCD, and Nig/Val on the photodamage that occurred in the absence of repair, using 250 μg mL⁻¹ lincomycin, an inhibitor of protein synthesis that blocks the repair of PSII (Tyystjärvi and Aro, 1996). The results in Figure 1B show that lincomycin enhanced the extent of photodamage and that further addition of DCMU, DCCD, or Nig/Val had no additional effect on the extent of photodamage. These observations suggested that inhibition of electron transport by DCMU and inhibition of ATP synthesis by DCCD or Nig/Val had no direct effect on photodamage. They also suggested that inhibition of electron transport and of ATP synthesis suppressed the repair of PSII, with resultant enhancement of the extent of photodamage.

Acceleration of Electron Transport in PSI and Inhibition of ATP Synthesis Affected the Extent of Photodamage to PSII

We examined the effects of the electron transport in PSI and the coupled synthesis of ATP using N-methyl phenazonium methosulfate (PMS), which mediates cyclic electron transport via PSI. In the presence of PMS, we detected no photodamage, even in the presence of DCMU (Fig. 2). The further addition of Nig/Val appeared to accelerate photodamage, as did lincomycin. DCCD had the same effects as Nig/Val. These observations suggested that the synthesis of ATP might be essential for the protection of PSII from the appearance of photodamage. It was of particular interest that the presence of 250 μg mL⁻¹ lincomycin completely eliminated the protective effects of PMS (data not shown).

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Figure 2.

Effects of DCMU, PMS, DCCD, and Nig/Val on the photodamage to PSII in Synechocystis. The experimental conditions were the same as described in the legend to Figure 1. ○, No addition (control); •, 20 μm DCMU; ▾, 20 μm DCMU and 20 μm PMS; ▪, 20 μm DCMU, 20 μm PMS, and 10 μm DCCD; ▴, 20 μm DCMU, 20 μm PMS, 2 μm nigericin, and 2 μm valinomycin; ♦, 250 μg mL⁻¹ lincomycin. Each point and bar represent the average ± se of results from five independent experiments.

Photodamage to PSII Was Unaffected by Electron Transport and the Synthesis of ATP

To investigate details of the effects of electron transport and ATP synthesis on photodamage to PSII, we determined the initial rate of photodamage in the presence of lincomycin. The initial rate of photodamage was proportional to light intensity, as has been observed also in the leaves of higher plants and in Synechocystis cells that had been treated with lincomycin (Tyystjärvi and Aro, 1996; Lee et al., 2001; Allakhverdiev and Murata, 2004).

We next examined the initial rate of photodamage to PSII under various conditions, namely, in the presence of DCCD, DCMU, DCMU plus DCCD, DCMU plus PMS, DCMU plus PMS plus DCCD, and DCMU plus PMS plus Nig/Val. However, none of these conditions affected the initial rate of photodamage by light at 1,000 and 2,000 μE m⁻² s⁻¹ (Table I), and the initial rate of photodamage to PSII under these conditions was proportional to the light intensity. These observations suggested that photodamage to PSII in vivo might be independent of electron transport in PSII, the energized state of thylakoid membranes, and the synthesis of ATP.

Inhibition of Electron Transport in PSII and Inhibition of ATP Synthesis Depressed the Initial Rate of Repair of PSII

When Synechocystis cells are exposed to strong light at 2,500 μE m⁻² s⁻¹ for 65 min to reduce the activity of PSII to 90% of the original level, the activity is restored during exposure to weak light at various intensities (Allakhverdiev and Murata, 2004).

Figure 3 shows the effects of light intensity on the initial rate of recovery of PSII activity during the repair of PSII. The initial rate of recovery reached a maximum of 0.01 min⁻¹ in light at an intensity of 300 μE m⁻² s⁻¹ and 50% of this level was achieved at approximately 70 μE m⁻² s⁻¹. These results suggested that a reaction might exist that limits the rate of the entire repair process and that the maximum turnover rate of this reaction was 0.01 min⁻¹ under our experimental conditions.

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Figure 3.

Effects of DCMU and DCCD on the initial rate of recovery of PSII activity after photodamage in Synechocystis. Cells were incubated for 65 min in light at 2,500 μE m⁻² s⁻¹ to induce 90% inactivation of PSII. Then cells were incubated in light at various intensities in the presence and absence of DCMU and DCCD. The initial rate of recovery was calculated from the time course of recovery experiments, as described in “Materials and Methods.” ○, No addition (control); □, in the presence of 20 μm DCMU; ▵, in the presence of 20 μm DCMU and 10 μm DCCD. Each point and bar represent the average ± se of results from six independent experiments.

Figure 3 also shows that inhibition by DCMU of the electron transport in PSII depressed the initial rate of recovery by 50% without changing the profile of dependence of light intensity, with this level being achieved at 300 μE m⁻² s⁻¹. This observation suggests that electron transport might be involved, in some way, in the repair of PSII and that inhibition of electron transport by DCMU might have repressed a reaction in the repair process. The further addition of DCCD completely abolished the capacity for repair. The addition of Nig/Val produced the same results as the addition of DCCD (data not shown). These observations suggested that ATP synthesis via PSI, which is assumed to occur in the presence of DCMU, might be important for the residual capacity for the repair of photodamaged PSII.

Acceleration of Electron Transport in PSI and Inhibition of ATP Synthesis Affected the Initial Rate of Repair of PSII

We examined the effects of DCMU, PMS, DCCD and Nig/Val on the initial rate of repair of PSII. The activity of PSII in Synechocystis cells that had been exposed to strong light at 2,500 μE m⁻² s⁻¹ for 65 min, to reduce the activity of PSII to 90% of the original level, was restored during exposure to weak light. Table II shows the effects of the various inhibitors and ionophores on the initial rate of recovery in light at 300 μE m⁻² s⁻¹ and 600 μE m⁻² s⁻¹. In the absence of inhibitors and ionophores, the initial rate yielded a “saturation-type” curve with a maximum rate of recovery of approximately 0.01 min⁻¹ at 300 and 600 μE m⁻² s⁻¹. The initial rate of recovery was depressed by 50% by DCMU. However, the further addition of PMS significantly increased the rate of recovery. Recovery was suppressed completely by DCCD and Nig/Val. These observations suggested that the recovery of PSII in vivo might be accelerated by the electron transport in PSI, via the accelerated synthesis of ATP.

Changes in Intracellular Levels of ATP

We measured levels of ATP directly in Synechocystis cells and examined the effects of DCMU and other reagents on the intracellular levels of ATP. Table III shows that after incubation of Synechocystis cells in light at 500 μE m⁻² s⁻¹ for 30 min the level of ATP was about 70 μmol (mg Chl)⁻¹. Inhibition of electron transport in PSII by DCMU reduced the level of ATP by approximately 20%. The further addition of PMS significantly increased the level of ATP. However, dissipation of the energization associated with thylakoid membranes by Nig/Val reduced the level of ATP to as little as 20% of the control level. Furthermore, DCCD also reduced the level of ATP to approximately 10% of the original level. Thus, the level of ATP was responsive to these various reagents. However, neither DCCD nor Nig/Val completely eliminated ATP in cells when PMS was present. Addition of DCCD or Nig/Val, in the presence of DCMU but in the absence of PMS, completely eliminated the intracellular accumulation of ATP.

Inhibition of Electron Transport in PSII and of ATP Synthesis Depressed the Levels of psbA Transcripts

The repair of PSII depends on the expression of the psbAII and psbAIII genes, which encode the precursor to the D1 protein (Mohamed et al., 1993). To clarify the mechanism responsible for the regulation of the repair of PSII, we examined the effects of DCMU on levels of psbA transcripts. In Synechocystis, the D1 protein is encoded by a small multigene family that consists of the psbAI, psbAII, and psbAIII genes (Jansson et al., 1987). However, psbAI is not expressed (Mohamed and Jansson, 1989), while psbAII and psbAIII, which are almost identical and encode only single form of D1, are both expressed (Mohamed and Jansson, 1989; Bouyoub et al., 1993; Mohamed et al., 1993) but at different levels. Approximately 95% of psbA transcripts originate from psbAII, while only 5% originate from psbAIII in Synechocystis cells that are grown under weak or strong light (Bouyoub et al., 1993; Mohamed et al., 1993). The reason for the differential expression of the psbA genes in Synechocystis is unknown.

Northern-blotting analysis of psbA transcripts revealed that the level of psbA transcripts reached a maximum plateau value after illumination for approximately 10 min (data not shown). Figure 4 shows that levels of the mRNA were not very different over a range of light intensities from 200 to 1,000 μE m⁻² s⁻¹. Figure 4 also shows that DCMU depressed the level of psbA transcripts by approximately 50% at all light intensities examined. These results are consistent with those in previous reports (Alfonso et al., 1999; Sippola and Aro, 2000; Zhang et al., 2000; Trebitsh and Danon, 2001) and they suggest that inhibition of electron transport by DCMU blocked, in part, the synthesis of the psbA transcripts. The further addition of DCCD eliminated the synthesis of psbA transcripts, suggesting that the 50% level of psbA transcripts, observed in the presence of DCMU, depended on the ATP that is essential for transcription.

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Figure 4.

Effects of DCMU and DCCD on the dependence on light intensity of the level of psbA transcripts in Synechocystis. Cells were incubated for 30 min in light at the indicated intensities in the presence and absence of DCMU and DCCD. A portion of each suspension of cells was withdrawn for extraction of RNA, which was subjected to northern-blotting analysis as described in “Materials and Methods.” ○, No addition (control); •, in the presence of 20 μm DCMU; ▵, in the presence of 20 μm DCMU and 10 μm DCCD. Each point and bar represent the average ± se of results from four independent experiments.

Effects of PMS, DCCD, and Nig/Val on Levels of psbA Transcripts

We monitored changes in levels of psbA mRNAs due to the presence of inhibitors and other reagents (Fig. 5). The presence of DCMU decreased the level of psbA transcripts, and PMS reversed this effect. The level of psbA transcripts was depressed by DCCD and by Nig/Val (Fig. 5; Table IV). However, neither Nig/Val nor DCCD completely eliminated the psbA transcripts.

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Figure 5.

Effects of DCMU, PMS, DCCD, and Nig/Val on the level of psbA transcripts in Synechocystis. Cells were incubated for 30 min in darkness and in light at 300 μE m⁻² s⁻¹ and at 600 μE m⁻² s⁻¹ in the presence of 20 μm DCMU, 20 μm PMS, 10 μm DCCD, and 2 μm nigericin plus 2 μm valinomycin (Nig/Val) as indicated or in their absence (control). A portion of each suspension of cells was withdrawn for extraction of RNA, which was subjected to northern-blotting analysis as described in “Materials and Methods.” The arrow labeled 1.2 kb indicates the position of psbA transcripts.

Effects of Electron Transport and ATP Synthesis on the Synthesis of Proteins de Novo

To identify the reaction(s) in the repair process that is inhibited by DCMU, we performed a labeling experiment with [³⁵S]Met in light at 300 μE m⁻² s⁻¹ after the induction of photodamage. Incubation of cells in light at 2,500 μE m⁻² s⁻¹ decreased the activity of PSII to approximately 10% of the original level. During subsequent exposure of cells to light at 300 μE m⁻² s⁻¹, the activity of PSII returned to the original level. At this light intensity, the initial rate of recovery was maximal (Fig. 3). Figure 6 shows the time courses of incorporation of radiolabeled Met into the D1 protein. In the presence of DCMU, the synthesis of the D1 protein was depressed by approximately 50% as compared to that under control conditions. These observations indicated that the synthesis of the D1 protein de novo was inhibited to some extent by the inhibition of electron transport. The further addition of DCCD completely eliminated the synthesis of D1 protein, suggesting that conditions that allow the synthesis of ATP might be essential for the synthesis of the D1 protein, which is essential for the repair of PSII.

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Figure 6.

Effects of DCMU and DCCD on the synthesis of the D1 protein de novo in Synechocystis. Cells were incubated at 34°C for 65 min in light at 2,500 μE m⁻² s⁻¹ to induce 90% inactivation of PSII. Cells were then incubated with 10 nm [³⁵S]Met (>1,000 Ci mmol⁻¹) for designated periods of time at 300 μE m⁻² s⁻¹ in the presence of 20 μm DCMU and 10 μm DCCD or in their absence (control) as indicated. At designated times, a portion of each suspension of cells was withdrawn for preparation of thylakoid membranes, which were subjected to PAGE as described in “Materials and Methods.” Proteins from thylakoid membranes corresponding to 0.8 μg of Chl were applied to each lane. Each point and bar represent the average ± se of results from four independent experiments.

To examine the effects of DCMU, PMS, and ionophores on the synthesis of proteins de novo during the exposure of Synechocystis cells to light, we monitored incorporation of [³⁵S]Met into the proteins of thylakoid membranes in light at 500 μE m⁻² s⁻¹ (Fig. 7). The synthesis of proteins during exposure of cells to light was markedly suppressed by DCMU. However, the further addition of PMS fully restored the synthesis of all proteins. Further addition of DCCD or Nig/Val completely abolished the synthesis of all proteins, including the D1 protein. These results suggested that exposure of cells to light in the presence of PMS induced the synthesis of proteins, including the D1 protein, even though electron transport in PSII was inhibited by the presence of DCMU and, moreover, that conditions that allowed the synthesis of ATP were essential for the synthesis of proteins, including the D1 protein, which is essential for the repair of PSII. It should be noted that protein synthesis was totally eliminated in the presence of DCCD or Nig/Val, in contrast to the synthesis of psbA transcripts, which occurred under the same conditions, albeit at a reduced rate.

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Figure 7.

Effects of DCMU, PMS, DCCD, and Nig/Val on the synthesis of proteins de novo in Synechocystis. Cells were incubated with 10 nm [³⁵S]Met (>1,000 Ci mmol⁻¹) for 30 min in light at 300 μE m⁻² s⁻¹ in the presence of 20 μm DCMU, 20 μm PMS, 10 μm DCCD, and 2 μm nigericin plus 2 μm valinomycin (Nig/Val) as indicated, or in their absence (control). After incubation, a portion of each suspension of cells was withdrawn for preparation of thylakoid membranes, which were subjected to PAGE as described in “Materials and Methods.” Proteins from thylakoid membranes corresponding to 0.8 μg of Chl were applied to each lane. The arrow indicates the position of the D1 protein (molecular mass 32 kD). The results shown are representative of the results of four independent experiments, each of which gave similar results.

Cyanobacterium and Culture Conditions

Synechocystis sp. PCC 6803 was kindly donated by Dr. J.G.K. Williams of DuPont de Nemours (Wilmington, DE). Cells were grown photoautotrophically in glass tubes (2.5 cm i.d. × 20 cm; 120 mL) at 34°C under constant illumination from incandescent lamps at 70 μE m⁻² s⁻¹ in BG-11 medium (Stanier et al., 1971) supplemented with 20 mm HEPES-NaOH, pH 7.5. Cultures were aerated with sterile air that contained 1% (v/v) CO₂ (Ono and Murata, 1981).

Conditions for Photodamage

Cells from 3-d-old cultures at a chlorophyll (Chl) concentration of 3 μg mL⁻¹ were incubated under strong light, as described below, to induce photodamage or under weak light (70 μE m⁻² s⁻¹) to induce repair in growth chambers at 34°C under the growth conditions described above. Small aliquots of cultures were withdrawn at designated times for measurements of PSII activity. Strong light, for induction of photodamage, was provided by two lamps (300 W; Toshiba, Tokyo) and attenuated to 250, 500, 1,000, 1,500, or 2,000 μE m⁻² s⁻¹ by passage through neutral density filters (PC-S380; 200 × 500 wide and 3 mm thick; Hoya Glass, Tokyo). The intensity of light was measured at the surface of glass tubes that contained suspensions of cells.

In some experiments, the synthesis of proteins was blocked by 250 μg mL⁻¹ lincomycin (Sigma Chemical, St. Louis), which was added to the culture medium 10 min before the start of incubation. In some other experiments, the photosynthetic transport of electrons through PSII was inhibited by 20 μm DCMU (Sigma Chemical). For inhibition of the formation of a proton gradient and synthesis of ATP, we used a pair of uncouples, 2 μm nigericin (Sigma Chemical) and 2 μm valinomycin (Wako Chemicals, Osaka), or 10 μm DCCD (Wako Chemicals). For inhibition of the cyclic transport of electrons via PSI, 20 μm PMS (Wako Chemicals) was added during the incubation of cells. After incubation for designated times, 5 mL of the suspension of cells were withdrawn and diluted 10-fold with BG-11 medium. Cells were collected by centrifugation at 5,000g for 6 min at 34°C and resuspended in 50 mL of BG-11 medium. The centrifugation and resuspension were repeated once. Finally, cells were collected by centrifugation as above and suspended in BG-11 medium at a density of 3 μg Chl mL⁻¹. This washing procedure completely removed DCMU and its effects on the activity of PSII in Synechocystis cells.

Measurement of Photosynthetic Activity

The activity of PSII in intact cells was measured by monitoring oxygen-evolving activity in the presence of 1.0 mm BQ, an artificial acceptor of electrons, with a Clark-type oxygen electrode (Hansatech Instruments, King's Lynn, UK). The sample, in a 3-mL cuvette, was illuminated by light from incandescent lamps that had been passed through a red optical filter (R-60; Toshiba, Tokyo) and an infrared-absorbing filter (HA-50; Hoya Glass). The intensity of light at the surface of the cuvette was 2,000 μE m⁻² s⁻¹. The initial rates of photodamage and recovery in the presence and absence of 250 μg/mL lincomycin, respectively, were calculated from the time courses of photodamage and recovery experiments and also by fitting the photodamage and recovery to a first-order reaction curve, as described previously (Allakhverdiev and Murata, 2004).

Measurement of Levels of ATP

A suspension of cells at a Chl concentration of 3 μg mL⁻¹ was exposed to light at 500 μE m⁻² s⁻¹ for 30 min with standard aeration at 34°C in the absence or presence of various reagents, such as 20 μm DCMU, 20 μm PMS, 10 μm DCCD, and 2 μm nigericin plus 2 μm valinomycin. Then cells were immediately disrupted by vortex mixing for 30 s with glass beads (diameter, 0.1 mm; BioSpec Products, Bartlesville, OK), which was repeated a total of three times, with 30-s pauses. A portion of the homogenate (with a Chl concentration of 3 μg mL⁻¹) was passed through a syringe-driven filter with 0.22-μm pores (Millipore, Bedford, MA) to separate the soluble fraction from the suspension. The resultant soluble fraction was subjected to assays for determination of levels of ATP. This assay was performed with a luminometer (MicroLumat LB 96 P, EG&G Berthold, Bad Wildbad, Germany) that had been designed for the measurement of bio- and chemiluminescence in 96-well microplates, using a luciferase-luciferin assay kit (ASR7921; Wako Pure Chemical Industries, Osaka). According to the protocol supplied with the kit, 100 μL of a solution of luciferase-luciferin in sterile distilled water were added to 200 μL of assay mixture and chemoluminescence was measured over 10 s. A standard curve was generated with ATP from Oriental Yeast (Tokyo).

Northern-Blotting Analysis

Total RNA was extracted from cells and northern-blotting analysis was performed as described previously (Los et al., 1997). An equal amount of RNA (4 μg) from each sample was loaded in individual wells of the gel and rRNA was visualized by staining with ethidium bromide. A 1.0-kb fragment of DNA that included the coding region of the psbA gene was amplified by the PCR with primers 5′-AACGACTCTCCAACAGCGCGAAA-3′ and 5′-CGTTCGTCATTACTTCAAAACCG-3′ and genomic DNA from Synechocystis as the template. The amplified fragment of DNA was ligated into the TA cloning vector pT7Blue-T (Novagen, Darmstadt, Germany). The plasmid was cleaved at the HincII and NcoI sites within the insert. The resultant 700-bp fragment of DNA was conjugated with alkaline phosphatase using an Alkphos Direct kit (Amersham Pharmacia Biotech, Piscataway, NJ) and the conjugate was used as the probe. After hybridization, blots were soaked in CDP-star solution (Amersham Pharmacia Biotech) and signals from hybridized mRNAs were detected with a digital camera system (LAS-1000; Fuji Photo Film, Tokyo).

Labeling of Proteins in Vivo

A suspension of cells, at a concentration corresponding to 5.00 ± 0.05 μg Chl mL⁻¹, was supplemented with 10 nm [³⁵S]Met (>1,000 Ci mmol⁻¹; Amersham Pharmacia Biotech), as described previously (Nishiyama et al., 2001; Allakhverdiev et al., 2002). Then the suspension was incubated at 34°C for designated periods of time in light at 300 μE m⁻² s⁻¹. The labeling was terminated by the addition of nonradioactive Met to a final concentration of 1.0 mm and immediate cooling of samples on ice. Cells were collected by centrifugation at 5,000g for 6 min at 4°C, and thylakoid membranes were isolated from these cells as described previously (Allakhverdiev et al., 1999, 2000). Thylakoid membranes were solubilized by incubation for 5 min at 65°C in 60 mm Tris (pH adjusted to 6.8 with HCl) that contained 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 10% (v/v) glycerol, and then proteins were separated by PAGE [12.5% (w/v) polyacrylamide] in the presence of 0.08% (w/v) SDS and 6 m urea, as described previously (Laemmli, 1970; Taguchi et al., 1993, 1995). Solubilized thylakoid membranes corresponding to 0.8 μg of Chl were loaded in each lane. Labeled proteins on the gel were visualized by exposure of the dried and fixed gel to x-ray film. Radioactivity of radiolabeled D1 was quantitated with the digital camera system.