Effects of 60Co γ radiation on thylakoid membrane functions in Anacystis nidulans

Available online at www.sciencedirect.com

Journal of Photochemistry and Photobiology B: Biology 91 (2008) 9–19 www.elsevier.com/locate/jphotobiol

Effects of

60

Co c radiation on thylakoid membrane functions in Anacystis nidulans

Rachna Agarwal a, S.S. Rane b, Jayashree Krishna Sainis a,* b

a Molecular Biology Division, Bhabha Atomic Research Center, Mumbai 400 085, India Control and Instrumentation Division, Bhabha Atomic Research Center, Mumbai 400 085, India

Received 19 October 2007; received in revised form 11 January 2008; accepted 13 January 2008 Available online 31 January 2008

Abstract In photosynthetic organisms oxidative stress is known to result in photoinactivation of photosynthetic machinery. We investigated effects of 60Co c radiation, which generates oxidative stress, on thylakoid structure and function in cyanobacteria. Cells of unicellular, non-nitrogen fixing cyanobacterium Anacystis nidulans (Synechococcus sp.) showed D10 value of 257 Gy of 60Co c radiation. When measured immediately after exposure, cells irradiated with 1500 Gy (lethal dose) of 60Co c radiation did not show any differences in photosynthetic functions such as CO2 fixation, O2 evolution and partial reactions of photosynthetic electron transport in comparison to unirradiated cells. Incubation of irradiated cells for 24 h in light or dark resulted in decline in photosynthesis. The decline in photosynthesis was higher in the cells incubated in light as compared to the cells incubated in dark. Among the partial reactions of electron transport, only PSII activity declined drastically after incubation of irradiated samples. This was also supported by the analysis of membrane functions using thermoluminescence. Exposure of cyanobacteria to high doses of 60Co c radiation did not affect the thylakoid membrane ultrastructure immediately after exposure as shown by electron microscopy. The level of reactive oxygen species (ROS) in irradiated cells was 20 times higher as compared to control. In irradiated cells de novo protein synthesis was reduced considerably immediately after irradiation. Treatment of cells with tetracycline also affected photosynthesis as in irradiated cells. The results showed that photoinhibition of photosynthetic apparatus after incubation of irradiated cells was probably augmented due to reduced protein synthesis. Active photosynthesis is known to require uninterrupted replenishment of some of the proteins involved in electron transport chain. The defective thylakoid membrane biogenesis may be leading to photosynthetic decline post-irradiation. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Cyanobacteria;

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Co c radiation; Lethal dose; Photosynthesis; Thylakoid membrane

1. Introduction Cyanobacteria represent a diverse group of most primitive oxygenic photosynthetic prokaryotes, which can resist variety of environmental stresses [1]. These organisms converted the reducing atmosphere of primitive earth into oxidizing one through oxygen evolution under harsh conditions of high cosmic and UV influx. Ionizing radiation is known to cause oxidative damage by generation of free radicals to all living cells. Their major and immedi-

*

Corresponding author. Tel.: +91 22 25595079; fax: +91 22 25505326. E-mail address: [email protected] (J.K. Sainis).

1011-1344/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2008.01.006

ate targets are believed to be DNA and also membranes. DNA undergoes strand breaks whereas membranes are known to lose their permeability and organization mainly because of free radical induced lipid peroxidation and protein modifications [2]. Gamma and UV-B/C radiation is known to affect photoautotrophic systems [3]. In plants, exposure to 5 kGy of radiation was shown to result in complete break down of middle lamella [4]. In case of banana fruits, exposure above 0.2 kGy of gamma radiation resulted in dilation between thylakoid membranes and a loss of grana stacks [5]. Studies on the effects of ionizing radiations on cyanobacteria have shown that some of the cyanobacteria can tolerate very high doses [6,7] as well as chronic low dose of ionizing

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radiation [8,9]. Chronic exposure to low dose (53.5 mGy/ year) of ionizing radiation in case of Synechococcus lividus was found to stimulate cell proliferation rate, activation of peroxidases and glucose catabolism via the oxidative pentose phosphate pathway [8,9]. In case of desiccation-tolerant Chroococcidiopsis, extreme radioresistance to X-rays was shown to be by virtue of efficient DNA damage repair system [7]. Exposure to X-ray up to 1200 Gy in Agmenellum quadriplicatum has previously been shown not to alter O2 evolution ability up to 5 h post-irradiation although nitrate reduction ability was reduced by 87% after 6 h post-irradiation [10]. Other oxidative stresses caused by UV, high light intensity and chemicals such as H2O2, rose Bengal affect photosynthesis in cyanobacteria [11–13]. In the present study, we explored effects of 60Co c radiation on photosynthetic functions of the thylakoid membranes of A. nidulans. These membranes showed extraordinary stability of the photosynthetic apparatus to oxidative stress created by exposure to c radiations. 2. Materials and methods 2.1. Culture conditions Culture of Anacystis nidulans (BD-1) was grown in BG11 medium [14] under continuous fluorescent white light of intensity 21 W/m2 at 30 °C. Log phase culture (7–10 day old) with O.D730nm 0.2–0.3 was used for all experiments. 2.2. D10 value determination Cells in 10 ml aliquots were exposed to different doses of Co c radiation ranging from 10 to 2500 Gy in a Gamma Cell 5000 (dose rate, 8.3 kGy h1) at 30 °C. The irradiated cells were plated on BG-11 agar plates containing 10 mM TES (pH 8.2) and 3% sodium thiosulphate and incubated under continuous fluorescent white light of intensity 21 W/m2 at 30 °C. Colonies were counted after one week for calculation of D10 value. 60

2.3. CO2 fixation Rate of CO2 fixation by irradiated and unirradiated cells was measured in fluorescent white light (24 W/m2). The assay mix (200 ll) contained cells with 5–10 lg chlorophyll in BG-11 medium along with 20 mM MgCl2, 10 mM K2HPO4 [15]. After 15 min incubation, the assay was started by addition of 20 mM NaH14CO3 (specific activity 0.5 mCi) and quenched after 10 min by addition of 100 ll of reaction mixture to 200 ll of 6 N acetic acid. The acid stable product was counted in a liquid scintillation counter in 0.4% BBOT in toluene containing 35% absolute ethanol. 2.4. Chlorophyll estimation Chlorophyll estimation was carried out according to Tandeau de Marsac [16].

2.5. Permeabilisation and substrate dependent CO2 fixation Log phase irradiated and unirradiated control cells of A. nidulans were harvested, washed and resuspended in 0.1 M HEPES pH 8.0. Cells were permeabilised by treating with 0.5 vol. of toluene for 10 min on ice. Substrate dependent CO2 fixation in light was measured using permeabilized cells as described previously [15]. Briefly, the permeabilised cells were incubated with assay buffer containing HEPES 50 mM pH 8.0, MgCl2 20 mM, DTT 10 mM and NaH14CO3 20 mM (specific activity 0.5 mCi/m Mole) for 15 min. The reaction was started by adding either R-5P + ATP or 3-PGA + NADPH + ATP. Total volume of reaction mixture was 1 ml and final concentration of ATP and substrates was 2 mM, whereas that of NADPH was 1 mM. RuBP dependent CO2 fixation activity was measured using cells, which were not permeabilized. For this assay, cells in 50 mM HEPES pH 8.0 were incubated as mentioned above without DTT and the reaction was started by addition of 1 mM RuBP. The reaction was terminated after 10 min in case of RuBP and R-5-P and after 30 min in case of 3-PGA containing assay mixture by transferring 100 ll of reaction mixture to scintillation vial containing 200 ll of 6 N acetic acid. The acid stable reaction product was counted as described previously in Section 2.3. 2.6. Assays of PSI and PSII Log phase unirradiated and irradiated cells of A. nidulans were sonicated for 4 min in pulse mode at 4 °C (6 cycles) in 50 mM Tricine–KOH buffer (pH 7.6) containing 5 mM KCl and 50 mM MgCl2. PSI activity was determined by measuring oxygen uptake in a Clark type oxygen electrode using ascorbate-DCPIP as donor and methyl viologen as acceptor of electrons [17]. PSII activity was determined in whole cell with 1 mM 2,6-dichlorobenzoquinone as the electron acceptor [18]. 2.7. Thermoluminescence studies Log phase cells were incubated in dark for 30 min. Cells equivalent to100 lg chlorophyll were resuspended on a planchet (with or without 25 lM DCMU) and exposed to actinic white light (200 W/m2 light intensity) for 7 min. The samples were frozen in liquid nitrogen. Frozen samples were placed on the pre-cooled stage of an in-house built thermoluminescence-measuring apparatus equipped with a Hamamatsu photomultiplier tube. The glow curves were recorded from 20 °C to +60 °C with a constant heating rate of 12.5 °C min1 [19]. 2.8.

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S-methionine uptake and de novo protein synthesis

Irradiated and unirradiated control cells were resuspended in BG-11 medium at 400–500 lg chlorophyll (ml)1. 35S-labeled L-methionine (10 lCi (ml)1) was added to the medium. Reaction was quenched after 30 min by

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addition of 10 mM unlabeled methionine. Cells were quickly washed with medium and 10 ll of the cells were mixed with 3 ml absolute alcohol and counted in liquid scintillation counter for determining methionine uptake. Rest of the cells were extracted by repeated freezing and boiling in lysis buffer (50 mM Tris pH 6.8, 100 mM DTT, 2% SDS and 10% glycerol). The lysed sample (20 ll) was spotted on nitrocellulose paper, washed twice with 10% chilled TCA followed by 50% methanol containing 10% acetic acid. Dried membranes containing TCA insoluble protein fraction were counted in liquid scintillation counter as described above. 2.9. ROS measurement The content of reactive oxygen species (ROS) was measured by DCHFDA assay [20]. Briefly, DCHFDA (10 lM final concentration) was added to the cells suspended in BG-11 medium (120 lg chlorophyll (ml)1) and samples were irradiated with 1.5 kGy of 60Co c radiation. The cells were incubated for 20 min on a rocker in dark at 25 °C. Fluorescence emission (kex = 490 nm, kem = 520 nm) of the unirradiated and irradiated cells was measured immediately or after incubation of cells in light or dark. 2.10. Lipid peroxidation measurement Thiobarbituric acid reactive substances, the product of lipid peroxidation, were estimated using TBARS assay [20]. 2.11. Transmission electron microscopy Sample preparation was done according to Dani et al. [15] with the following changes. Cells were washed with 100 mM sodium phosphate buffer pH 7.4 and fixed with 0.5% gluteraldehyde and 2% paraformaldehyde for 2 h at room temperature followed by treatment with 0.5% OsO4 (aqueous) for 1 h. Samples were washed thoroughly with water. Serial dehydration was carried out using 35–100% ethanol for 30 min each followed by incubation with propylene oxide. Samples were infiltrated and embedded in Araldite. Sections were prepared and stained with 4% aqueous uranyl acetate for 30 min and viewed under transmission electron microscope at 120 keV.

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Co c radiation on the process of photosynthesis in unicellular non-nitrogen fixing photoautotroph A. nidulans. 3.1. Survival curve of Anacystis nidulans irradiated with 60Co c radiation

Fig. 1 shows the survival curve of A. nidulans in response to exposure to 60Co c radiation. The D10 value (the amount of dose required for 90% killing) for this organism was 257 Gy. It has been reported that some sp. of cyanobacteria viz. Chroococcidiopsis isolated from desert and hyper saline environment can tolerate X-rays up to 15 kGy. The D10 value for A. nidulans compared well with the values reported earlier for Synechococcus sp. (6, 7). This value, though not as high as Chroococcidiopsis, was higher in comparison to other bacteria such as Escherichia coli (100 Gy). The higher radiation resistance in the cyanobacteria is supposed to be due to efficient DNA repair and their ability to use redundant genetic information (7). 3.2. Effect of

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Co c radiation on CO2 fixation

We were interested in studying the effect of 60Co c radiation on photosynthesis, a membrane dependent function. The rate of CO2 fixation was measured as a function of dose of 60Co c radiation immediately after irradiation (Fig. 2). Surprisingly, there was no effect on CO2 fixation up to a dose as high as 2000 Gy, which is almost 8 times the D10 value. Exposure of cells to doses up to 500 Gy showed slight stimulation in rate of CO2 fixation. However, when these irradiated cells were incubated for 24 h there was decrease in rate of CO2 fixation around D10 value (Fig. 2). The decline was more pronounced in light

3. Results and discussion Biological membranes are known to be susceptible to ionizing radiation-induced oxidative stress mediated by short-lived ROS [2]. This is mainly due to lipid peroxidation, cross-linking and breakage of C–C bond in membrane lipids, changes of membrane permeability and protein degradation. In animal cell lines and red blood cells such changes can be seen at doses as low as 10 Gy [2]. The process of photosynthesis, which occurs in thylakoid membranes, should therefore also be extremely susceptible to radiation damage. We investigated effects of

Fig. 1. Survival curve of Anacystis nidulans. Log phase culture of Anacystis nidulans in BG-11 medium was exposed to 0, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 2500 Gy of 60 Co c radiation. The culture (10 ll) was plated on BG-11 plates and colonies were counted after one week. D10 value was 257 Gy. Values represent mean ± SE (n = 3). No Surviving fractions were observed at doses above 600 Gy.

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R. Agarwal et al. / Journal of Photochemistry and Photobiology B: Biology 91 (2008) 9–19 Table 1 Light dependent CO2 fixation in unirradiated and irradiated cells of Anacystis nidulans Light dependent CO2 fixation lM of CO2 fixed, (mg Chl1) h1 Activity measured immediately after irradiation Activity measured after incubation for 24 h in light Activity measured after incubation for 24 h in dark

Fig. 2. Dose response of CO2 fixation in Anacystis nidulans. Log phase cells of Anacystis nidulans were irradiated with doses 0, 10, 100, 200, 400, 800, 1200, 1500, 1800 and 2000 Gy and CO2 fixation was measured as described in Section 2.3. Filled squares represent CO2 fixation rates measured immediately after irradiation; circles represent CO2 fixation rates measured after incubation for 24 h in fluorescent white light of intensity 21 W/m2; triangles represent CO2 fixation rates measured after incubation for 24 h in dark. All values represent mean ± SE (n = 3).

Unirradiated

Irradiated

50.3 ± 1.1

50.3 ± 0.5

48.7 ± 0.4

7.0 ± 0.6

35.0 ± 2.7

20.8 ± 1.4

Log phase cells of Anacystis nidulans in BG-11 medium were exposed to 1.5 kGy of 60Co c radiation. The cells were incubated for 24 h under continuous fluorescent white light of intensity 21 W/m2 or in dark at 30 °C. Light dependent CO2 fixation activity was measured in cells immediately or 24 h after incubation in light and dark as described in Section 2.3. All values represent mean ± SE (n = 3).

incubated cells as compared to cells incubated in dark. Results on light dependent O2 evolution showed similar trend (data not shown). The results indicated that the thylakoid membranes were resistant to 60Co c radiation damage. The reduction in CO2 fixation brought upon incubation in light or dark could be due to subsequent damage to photosynthetic machinery. 3.3. Effect of incubation after irradiation with 60Co c on CO2 fixation In order to analyze the effect of incubation time after exposure to 60Co c radiation on photosynthesis, the cells of A. nidulans were exposed to 1.5 kGy, a dose five times higher than the D10 value. No surviving fraction was observed even 2 months after exposure to this dose (hence called lethal dose). Since the dose rate was 8.3 kGy/h, the time needed for giving this dose was 10.5 min and therefore the changes in the photosynthetic metabolism could be studied. Table 1 shows the data on effect of incubation of unirradiated and irradiated cells in light and dark on photosynthetic CO2 assimilation. In unirradiated cells 97% and 70% of CO2 fixation was observed when cells were incubated in light and dark. This is in contrast to irradiated cells wherein after 24 h, only 13% CO2 fixation activity was observed in light incubated cells and 42% of activity was seen in cells incubated in dark. In cells exposed to lethal dose of 60Co c radiation (1.5 kGy) and incubated in light or dark, photosynthetic CO2 fixation started declining significantly after incubation for 3–4 h in light or dark (Fig. 3). The results suggested that light augmented the damage to photosynthesis caused by irradiation with 60 Co c radiation.

Fig. 3. Effect of incubation for different time periods after c irradiation on CO2 fixation in Anacystis nidulans. Log phase culture of Anacystis nidulans was irradiated with 1.5 kGy of 60Co c rays and incubated in fluorescent white light of intensity 21 W/m2 for the time mentioned on X-axis. Filled squares represent CO2 fixation rates measured in unirradiated cells incubated in light; circles represent CO2 fixation rates measured in unirradiated cells incubated in dark; stars represent CO2 fixation rates measured in irradiated cells incubated in light; triangles represent CO2 fixation rates measured in irradiated cells incubated in dark. All values represent mean ± SE (n = 3).

3.4. Effect of irradiation with cycle enzymes

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Co c on activities of Calvin

Partial activities of Calvin cycle enzymes were measured using cells permeabilized with toluene (Table 2). R-5-P dependent CO2 fixation involves three consecutive enzymes of Calvin cycle viz. phosphoriboisomerase, phosphoribulokinase and RuBP carboxylase where as 3-PGA dependent CO2 fixation involves all the 13 enzymes of Calvin cycle [15]. Comparable rates of CO2 fixation in R-5-P dependent CO2 fixation assays were observed in permeabilized cells immediately after irradiation. Upon incubating the cells for 24 h, linked activities of Calvin cycle enzymes decreased by about 20% and 35%, respectively, in unirradiated and

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Table 2 Activities of Calvin cycle enzymes in unirradiated and irradiated cells of Anacystis nidulans

Activity measured immediately after irradiation Activity measured after incubation for 24 h in light Activity measured after incubation for 24 h in dark

Unpermeabilised cells

Permeabilised cells

RuBP dependent CO2 fixation

R-5-P dependent CO2 fixation

3-PGA dependent CO2 fixation

Unirradiated

Irradiated

Unirradiated

Irradiated

Unirradiated

Irradiated

0.54 ± 0.04 1.61 ± 0.04 1.61 ± 0.02

0.56 ± 0.04 1.57 ± 0.04 1.1 ± 0.11

8.18 ± 0.08 6.54 ± 0.12 6.75 ± 0.07

8.79 ± 0.12 5.69 ± 0.05 5.91 ± 0.09

0.70 ± 0.03 0.53 ± 0.02 0.75 ± 0.03

0.56 ± 0.01 0.36 ± 0.05 0.39 ± 0.01

Log phase cells of Anacystis nidulans were irradiated with 1.5 kGy of 60Co c radiation. The cells were incubated in light and dark as mentioned in Table 1. Substrate dependent CO2 fixation activities were determined using R-5-P + ATP or 3-PGA + ATP + NADPH in permeabilised cells and RuBP dependent activity in unpermeabilised cells as described in Section 2.5. Values are expressed as lM of CO2 fixed, (mg Chl1) h1. All values represent mean ± SE (n = 3).

irradiated cells. There was no discernible difference in the extent of decrease in cells incubated in light and dark. In the case of 3-PGA dependent activity irradiated cells showed about 20% less activity when compared to unirradiated cells. This activity decreased by about 30% in irradiated cells after incubation in light and dark. There was about 25% decrease in this activity in the unirradiated cells incubated in light. The decrease in the activity of Calvin cycle enzymes in irradiated cells incubated in light was much less than the decrease observed in CO2 fixation rates in the cells under similar conditions (Table 1). We, therefore, examined the effect of irradiation on partial reactions of electron transport (see Section 3.5). In the same experiment we also measured RuBP dependent activity in whole cells, which were not permeabilized with toluene to examine the extent of permeability of plasma membrane after irradiation. Generally whole cells assays are done using cells permeabilized with toluene [15]. The aim of this experiment was to find out whether radiation resulted in increase in permeability of the cells. Table 2 shows that RuBP dependent CO2 fixation activity was very low in the unirradiated as well as irradiated cells indicating intactness in plasma membrane permeability to this solute. The low level of activity so obtained may be due to cytosolic RuBP regenerated through Calvin cycle. There was no significant difference in RuBP dependent CO2 fixation activity in irradiated and unirradiated cells incubated in light after exposure to 60Co c radiation. This activity declined to higher extent in irradiated cells incubated in dark. The results showed that the exposure to c

radiation probably do not affect the plasma membrane permeability in A. nidulans. 3.5. Effect of

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Co c radiation on PSII and PSI

Partial reactions of photosynthesis were monitored in cells irradiated with lethal dose and unirradiated cultures of A. nidulans. Table 3 shows that there were no significant differences in activities of PSI and PSII when assayed immediately after the exposure. However if the cells were incubated for 24 h in light after irradiation, 40% of PSII activity was observed. In unirradiated controls 89% of PSII activity was seen under similar conditions. When cells were incubated in dark for 24 h, PSII activity declined in both unirradiated and irradiated cells by about 60%. Thus incubation in light enhanced the PSII inactivation in irradiated cells. Similar decrease in PSII activity of unirradiated and irradiated cells in dark is interesting phenomenon. It is known that dark preincubation of Synechocystis inactivates PSII [21]. This has been shown to be due to role of light mediated activation of certain proteins in light recovery of photosystem II. PSI activity of unirradiated and irradiated cells was similar when monitored immediately as well as after incubation for 24 h in light (Table 3). In dark 18–22% of PSI activity was found in both cases suggesting that c irradiation does not specifically affect PSI. Earlier PSI activity was shown to decline after dark incubation in case of rye thylakoids [22]. Since there was decline in PSII activity in cells incubated in light after irradiation, PSII activity was monitored using whole cells at different

Table 3 PSII and PSI activities in unirradiated and irradiated cells of Anacystis nidulans PSII Activity lM O2 evolved, (mg Chl1) h1

Activity measured immediately after irradiation Activity measured after incubation for 24 h in light Activity measured after incubation for 24 h in dark

PSI Activity lM O2 consumed, (mg Chl1) h1

Unirradiated

Irradiated

Unirradiated

Irradiated

127.4 ± 2.0 113.6 ± 5.0 73.86 ± 2.5

118.7 ± 17.8 47.4 ± 5.8 70.7 ± 2.5

326.4 ± 30.6 301.5 ± 17.7 73.1 ± 10.1

358.1 ± 28.4 332.2 ± 28.9 67.0 ± 1.9

Log phase cells of Anacystis nidulans in BG-11 medium were exposed to 1.5 kGy of 60Co c radiation and incubated in light and dark as described in Table 1. PSII activity was measured in whole cells and PSI activity was measured in cell free extracts as described in Section 2.6. All values represent mean ± SE (n = 3).

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Fig. 4. Effect of incubation of irradiated cells for different time periods on PSII activity in Anacystis nidulans. Log phase cells of Anacystis nidulans were exposed to 1.5 kGy of 60Co c radiation. Cells were incubated under continuous white light of intensity 21 W/m2 for different times after irradiation and PSII activity was determined as described in Section 2.6. Filled squares represent activity in unirradiated control cells; circles represent activity in irradiated cells. All values represent mean ± SE (n = 3).

times after c irradiation. A gradual decline in PSII activity was seen in irradiated cells (Fig. 4). The results showed that there was about 60% loss of PSII activity after 6–7 h of incubation in irradiated cells in light. Experiments were also done to monitor effect of lethal dose of 60Co c radiation irradiation on thermoluminescence pattern of A. nidulans. Thermoluminescence glow peaks are known to arise in photosynthetic organisms as a result of charge recombination process between various components of PSII. The unirradiated cells showed one glow peak in the temperature range of 40–45 °C. The intensity of this glow peak was reduced when the cells were treated with DCMU suggesting this peak is due to QB (Fig. 5). The intensity of this peak remained unchanged in irradiated cells when measured immediately after irradiation signifying the intactness in various components of PSII, which is a prerequisite for origin of such peaks. The intensity of this glow peak was negligible after incubating the irradiated cells in light for 24 h suggesting a damage to PSII. Thus the results on thermoluminescence supported our contention that PSII was sensitive to damage due to exposure to gamma radiation. 3.6. Effect of tetracycline on photosynthesis We examined the effect of incubation on CO2 fixation and PSII activity in cells treated with 500 lM tetracycline. Table 4 shows that in cells incubated for 24 h with tetracycline, the rate of CO2 fixation was reduced to similar level as seen in case of irradiated cells (see Table 1). There was 85–95% decline in CO2 fixation in cells incubated in light as against 40–50% in cells incubated in dark in both treatments (compare Tables 1 and 4) indicating the role of light in enhancing the post-irradiation damage. Fig. 6 shows

Fig. 5. Thermoluminescence glow curves of irradiated and unirradiated cells of Anacystis nidulans. Log phase cells of Anacystis nidulans were exposed to lethal dose of 60Co c radiation. The cells were incubated in light and dark as mentioned in Table 1. Cells were harvested and TL glow curves were recorded in presence and absence of DCMU as described in Section 2.7. (A) and (C) Glow curves of unirradiated and irradiated cells monitored immediately after exposure. (B) and (D) Glow curves of unirradiated and irradiated cells monitored after incubation for 24 h in white fluorescent light (21 W/m2). Y-axis represents arbitrary values of TL intensity and X-axis represents temperature in °C.

that incubation of tetracycline treated cells in light resulted in 80% decrease in CO2 fixation activity in seven hours. Since PSII was more sensitive to radiation we also examined PSII activity in cells treated with tetracycline (Fig. 7). In seven hours about 20% PSII activity was observed in tetracycline treated cells as compared to untreated cells. The results suggested that tetracycline mimicked irradiation treatment. 3.7. Effect of irradiation on

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S-methionine incorporation

The photosynthetic machinery of thylakoid membranes comprise of pigment-protein complexes, which need continuous repair. 35S-methionine uptake and incorporation in TCA insoluble fraction was determined in unirradiated and irradiated cells immediately after irradiation and in cells incubated in light and dark for 24 h. Table 5 shows that in unirradiated cells there was over 50% incorporation of 35S-methionine in proteins where as this was reduced to 3.5% in irradiated cells when measured immediately after exposure. Irradiated cells incubated in light and dark showed around 4% and 2% incorporation of 35 S-methionine in TCA insoluble fraction. The drastic decline in 35S-methionine incorporation in TCA insoluble fraction in irradiated cells immediately after exposure suggested that the rate of de novo protein synthesis was decreased in irradiated cells (Table 5). The uptake of labeled methionine was affected only by 20% in irradiated

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Table 4 Comparison of CO2 fixation and PSII activity in untreated and tetracycline treated cells of Anacystis nidulans

Activity measured immediately after irradiation Activity measured after incubation for 24 h in light Activity measured after incubation for 24 h in dark

CO2 fixation lM of CO2 fixed, (mg Chl1) h1

PS II Activity lM O2 evolved, (mg Chl1) h1

Not treated with tetracycline

Treated with tetracycline

Not treated with tetracycline

Treated with tetracycline

50.3 ± 1.1 48.7 ± 1.4 35.0 ± 2.7

47.0 ± 3.6 2.3 ± 0.8 20.1 ± 2.1

112.9 ± 15.11 112.5 ± 17.2 73.9 ± 3.0

107.7 ± 1.52 0 52.5 ± 2.8

Log phase cells of Anacystis nidulans were incubated with 500 lM tetracycline and CO2 fixation and PSII activity was determined as described in Sections 2.3 and 2.6, respectively. All values represent mean ± SE, (n = 3).

Fig. 6. Effect of tetracycline on CO2 fixation by Anacystis nidulans. Log phase cells of Anacystis nidulans were incubated in light (21 W/m2) with and without 500 lM tetracycline for the time indicated on X-axis and CO2 fixation was determined as described in Section 2.3. Filled squares represent CO2 fixation activity in untreated cells; circles represent CO2 fixation activity in cells treated with 500 lM tetracycline. All values represent mean ± SE (n = 3).

Fig. 7. Effect of tetracycline on PSII activity by Anacystis nidulans. Log phase cells of Anacystis nidulans were incubated in light (21 W/m2) with and without 500 lM tetracycline for the time indicated on X-axis and PSII activity was determined. Filled squares represent PSII activity in untreated cells; circles represent PSII activity in cells treated with 500 lM tetracycline. All values represent mean ± SE (n = 3).

cells immediately after exposure indicating that the observed decline in protein synthesis was not due to reduced uptake of methionine by these cells (Table 5). The uptake was considerably reduced if irradiated cells were incubated in light as compared to cells incubated in dark after exposure. However, the percent of label incorporated in TCA insoluble fraction was similar in irradiated cells incubated in dark or light. This was also the case for unirradiated controls, where in there was about 8% decrease and 13% increase in methionine uptake in cells kept in light and dark, respectively. In this case also there was no significant change in percent incorporation of 35S-methionine in TCA insoluble fraction. The results indicated that irradiation decreased rate of de novo protein synthesis almost immediately after exposure.

3.8. Effect of irradiation on ROS and lipid peroxidation Exposure to ionizing radiation is known to generate ROS which are extremely short-lived species causing damage to biological macromolecules especially DNA and lipid bilayer. The reactive oxygen species (ROS) generated in irradiated and unirradiated cells were measured. Large increase in ROS content (20 times higher as compared to unirradiated cells) was detected in cells immediately after irradiation with lethal dose (Table 6). However, no ROS could be detected in the cells incubated for 24 h after irradiation in light as well as in dark suggesting that no long lived free radicals were produced in these cells. ROS content was measured in cells kept in light and dark for different times after irradiation. No ROS could be seen in cells 1 h after irradiation (data not shown). Thiobarbituric acid reactive substances (TBARS), the indicators of the amount of lipid peroxidation were measured in irradiated cells (Table 7). The results showed that there is no appreciable difference in the content of TBARS in the unirradiated and irradiated samples when measured immediately. After incubation in light and dark for 24 h there was slight decrease in thiobarbituric acid reactive substances in irradiated cells. The results suggested that ROS production did not result in significant increase in lipid peroxidation.

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Table 5 35 S-methionine uptake and incorporation into TCA insoluble fraction in unirradiated and irradiated cells of Anacystis nidulans Total uptake 35S-CPM (mg Chl1)  106 35

S-CPM measured immediately after irradiation S-CPM measured after incubation for 24 h in light 35 S-CPM measured after incubation for 24 h in dark 35

Incorporation in TCA insoluble fraction 35 S-CPM (mg Chl1) x106

Unirradiated

Irradiated

Unirradiated

Irradiated

36.8 ± 5.0 36.4 ± 1.7 43.8 ± 6.6

28.1 ± 2.8 13.9 ± 1.0 31.8 ± 1.5

18.8 ± 4.5 (51%) 17.8 ± 2.6 (49%) 21.4 ± 4.9 (49%)

1.0 ± 4.6 (3.5%) 0.6 ± 0.2 (4.3%) 0.7 ± 0.2 (2.2%)

Log phase cells of Anacystis nidulans were exposed to lethal dose of 1.5 kGy of 60Co c radiation and 35S-methionine uptake and incorporation in protein was determined as described in Section 2.8. Values in parenthesis represent incorporation in proteins as percent of respective uptake value. Light intensity during 24 h light incubation was 21 W/m2. All values represent mean ± SE (n = 3).

Table 6 Estimation of reactive oxygen species in unirradiated and irradiated cells of Anacystis nidulans ROS (A.U.) (mg Chl1)

ROS measured immediately after irradiation ROS measured after incubation for 24 h in light ROS measured after incubation for 24 h in dark

Control

Irradiated

33.7 ± 3.6 75.4 ± 3.5 0

677.5 ± 4.4 0 0

Log phase cells of Anacystis nidulans in BG-11 medium were exposed to 1.5 kGy of 60Co c radiation. Cells were incubated in light and dark as described in Table 1 and ROS content was estimated using DCHFDA method as described in Section 2.9. All values represent mean ± SE (n = 3).

Table 7 Estimation of lipid peroxidation in unirradiated and irradiated cells of Anacystis nidulans nM Tetramethoxy propane (mg Chl1)

TBARS measured immediately after irradiation TBARS measured after incubation for 24 h in light TBARS measured after incubation for 24 h in dark

Unirradiated

Irradiated

62.2 ± 3.9

71.2 ± 15.6

88.1 ± 12.6

83.7 ± 5.5

80.4 ± 7.1

77.8 ± 6.0

Log phase cells of Anacystis nidulans in BG-11 medium were exposed to 1.5 kGy of 60Co c radiation. Cells were incubated in light and dark as described in Table 1 and amount of thiobarbituric acid reactive substances (TBARS) produced were measured as described in Section 2.10. All values represent mean ± SE (n = 3).

3.9. Effect of irradiation on ultra structure Ultra structure of A. nidulans was also monitored using transmission electron microscopy. Fig. 8A and B shows the structure of unirradiated cells. The cells did not show significant alterations in ultra structure when fixed immediately after exposure (Fig. 8C and D). The typical concentric rings of thylakoids, cell wall and carboxysomes were observed in these cells as seen in normal cells. However, the structure of thylakoid membranes was altered when cells after exposure to lethal dose were incubated in light for 24 h (Fig. 8E and F). The membranes were oblit-

erated by electron dense proteins and with less denser, regularly deposited white granules. Such structures were observed previously in Synechocystis under nitrogen-limited photosynthetic conditions, which have been shown to be due to the presence of glycogen [23]. If the irradiated cells were incubated in dark, there was reduced damage to thylakoid ultra structure (Fig. 8G and H). These studies on the partial reactions of photosynthesis and ultra structure showed the structural and functional stability of thylakoid membranes after exposure to 60Co c radiation up to 1500 Gy, indicating robustness of these membranes to immediate damages by ROS. This may be an adaptive response of cyanobacteria to the conditions of the primordial earth where the cosmic influxes of ionizing radiations were very high. There was also no increase in lipid peroxidation products in cells irradiated with lethal dose. Presence of poly-triunsaturated fatty acids in the lipids is a prerequisite for ROS induced peroxidation. Such polyunsaturated lipids have not been detected in A. nidulans [24]. This may be one of the reasons for enhanced radioresistance of membrane localized photosynthetic functions to the lethal dose of 1.5 kGy 60Co c radiations. In contrast to photosynthesis, de novo protein synthesis was reduced drastically in cells exposed to lethal doses of 60 Co c radiation. This reduction in protein synthesis could not be attributed to reduction in uptake of methionine. The damage to the protein synthesis machinery in irradiated cells would result in impairment of thylakoid function. PSII is known to require continuous repair. One of its components, D1 protein has a very high turn over rate and is sensitive to photo damage by ROS produced through several stresses [25]. The photoinhibition of PSII by oxidative stress has been shown to be due to inhibition of repair of D1 protein [26–28]. Oxidative stress is known to affect the process of protein synthesis in Chlamydomonas renharditii [29], in rat liver [30] and in E. coli [31]. It has been shown previously that ROS produced in several stresses inhibits the synthesis of D1 protein and a new scheme of photoinhibition due to ROS mediated suppression of repair of damaged proteins has been proposed [32]. In the present study also, large quantities of ROS are produced which may be leading to irrecoverable photoinhibition according the above scheme. Incubation of the cells

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Fig. 8. Electron micrographs of Anacystis nidulans before and after irradiation. Longitudinal and transverse sections of unirradiated cells (A) and (B); cells fixed immediately after irradiation (C) and (D); cells incubated in light (E) and (F) and cells incubated in dark after irradiation (G) and (H) for 24 h after irradiation as described in Table 1. Cx: carboxysome; Thy: thylakoid membranes; Cw: cell wall; Gly: glycogen deposits. Bars represent 250 nm.

with tetracycline was found to produce similar effect as that of c radiation on photosynthetic CO2 assimilation. The results strongly suggest the robustness of the thylakoids to ROS mediated direct damage. Another interesting point was that the damage to photosynthetic apparatus was enhanced in light incubated cells, though protein synthesis was inhibited to same extent in cells incubated in light or dark after irradiation. Thus photoinhibition may be responsible for augmenting the effect of 60Co c radiation on thylakoid structure and function.

4. Conclusions Ionizing radiation is known to cause lethality by damaging the DNA and the membranes. Both these damages are the cause of concern in radiation biology. We decided to investigate the effects of 60Co c radiation on efficiency of photosynthesis, a process that needs integrity of thylakoid membranes and is very sensitive to free radical damage. The D10 value of A. nidulans was 257 Gy of 60Co c radiation. We assessed the damage to photosynthetic apparatus by exposing the cells of A. nidulans to different doses of

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60

Co c radiation. Functionality of photosynthetic apparatus was monitored by measuring CO2 fixation, PSII and PSI activities, thermoluminescence and activities of Calvin cycle enzymes. Ultra structure of cells exposed to 60Co c radiation was also monitored. Our results showed that the thylakoid membranes of A. nidulans were extremely tolerant to very high doses of c radiation up to 1500 Gy, if all these functions and structure of photosynthetic apparatus were monitored immediately after exposure. Photosynthesis declined and the thylakoid membranes were disorganized only when irradiated cells were further incubated. The decline was more in the cells incubated in light as compared to the dark incubated cells. The ultra structure of cells was drastically altered in irradiated cells, which were incubated in light. ROS levels were higher in irradiated cells as compared to unirradiated cells. Interestingly the de novo protein synthesis was reduced in irradiated cells immediately after exposure. Cells treated with tetracycline also showed reduction in rate of photosynthetic CO2 assimilation similar to the effect of exposure to 60Co c radiation. The results suggested that the decline in the photosynthesis after incubation of the irradiated cells may be due to inhibition of protein synthesis affecting repair of thylakoid membranes, which is obligatory for maintaining the photosynthetic apparatus functional. The reduction in protein synthesis following exposure to lethal dose may be due to direct effect on translation or due to extensive damage to DNA which needs to be further explored in these photoautotrophic bacteria. The cells incubated in light involved in active photosynthesis showed comparatively more reduction in photosynthesis as compared to the cells incubated in dark after exposure to 60Co c radiation. The complex interaction between the damage due to exposure to 60Co c radiation and the photoinhibition also needs to be explored. 5. Abbreviations BBOT 2,5-bis (5-tert-butylbenzoxazole-2-yl) thiophen DCBQ 2,6-dichloro-p-benzoquinone DCMU 3-(3,4-dichlorophenyl) 1,1-dimethyl urea DCHFDA 20 ,70 dichlorodihydro-fluorescein diacetate 3-PGA 3-phospho glyceric acid PSI photosystem I PSII photosystem II R-5-P ribose-5-phosphate ROS reactive oxygen species TBARS thio-barbituric acid reactive substances TL thermoluminescence RuBP ribulose 1,5-bisphosphate Acknowledgements We thank Dr. G.K. Dey, Dr. R. Tewary and Mr. Rajanikant C. for help in electron microscopy and Ms. A. Gupta for help in carrying out biochemical assays.

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