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Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity
Acta Astronautica 55 (2004) 953 – 957 www.elsevier.com/locate/actaastro
Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity Gen-bao Li, Yong-ding Liu∗ , Gao-hong Wang, Li-rong Song Institute of Hydrobiology, Chinese Academy of Sciences, 7 Donghunanlu, Luojiashan, Wuhan 430072, China Received 19 December 2002; received in revised form 29 March 2004; accepted 28 April 2004 Available online 3 August 2004
Abstract It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days’ experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity. c 2004 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are organisms that are capable of oxygenic photosynthesis. Due to higher rate of light-energy transformation and stronger adaptation to stress environment, microalgae are model organisms for the study of space biology, and are suitable to be the best members of the controlled ecological life support system (CELSS). Many studies reported that alteration of gravity a:ected the microalgal structure, population growth and physiological characteristics. Those e:ects included change of intracellular secretions, reduced photosynthesis and decreased starch number [1–3]. Chen et al. [4] reported that the rate ∗ Corresponding author. Tel.: +86-27-87647715; fax: +86-2787875132. E-mail address: [email protected] (Y. Liu).
of photosynthesis in spaceAight was lower than in ground control. However, strong adaptable ability of microalgae to spaceAight environment was found [5]. What is the cause of responses of microalgae to the microgravity environment? Which stress defence mechanisms enable algae to adapt to their particular environment? In previous studies, lipid peroxidation and membrane permeability in clinostat-treated microalgal cells were observed (we reported in another paper). Because many stress conditions cause cellular redox imbalances, it has been proposed that reactive oxygen species (ROS) exhibit important signaling function in response to both biotic and abiotic stress. We thought high level of ROS in microalgal cells maybe were responsible for abnormal physiological reaction. Thus, abilities in microgravity tolerance among algae can be correlated with the capacity to develop antioxidant systems, but there is still not much
c 2004 Elsevier Ltd. All rights reserved. 0094-5765/$ - see front matter doi:10.1016/j.actaastro.2004.04.014
954
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957
information about this topic. For this aim, ROS and some antioxidant enzymes activity were measured in order to explore the mechanism by which algae cells scavenged ROS and adapted to microgravity, which is valuable for us to select suitable plants as the member of CELSS [6].
Shimadizu corporation). The reduction of H2 O2 in reaction system was showed with units=g chl. 2.5. Measurement of gluathione (GSH) in cells
2. Materials and methods
GSH was assayed as described by Zeng [9]. The Oxidation reaction was recorded at 412 nm (UV-1601, Shimadizu corporation). The standard curve was performed with analytical GSH.
2.1. Organism and cultures
2.6. Determination of chlorophyll (chl)
Anabaena sp. PCC 7120 (Hlamentous cyanobacterium, blue-green alga) was obtained from the Freshwater Algae Culture Collection of Institute of Hydrobiology, China (FACHB). Cultures in logarithmic phase were transferred into tubes containing BG-11 medium on a horizontally slow-rotating clinostat (8 rmp) under simulated microgravity of 10−3 –10−4 g at 27 ± 1◦ C with white Auorescent irradiation of 36 E m−2 s−1 for 3 and 7 days.
Chlorophyll was extracted in 95% ethanol and characterized by the method of Wintermans and de Mots [10].
ROS assay kits was purchased from Jiancheng bioengineering institute, Nanjing China. Since Fenton reaction is a common chemic reaction producing OH− , the amount of H2 O2 is directly proportional to the amount of produced OH− , when given electron acceptor, stained with Gress, the red-color production is in proportion to OH− , the authors employed OH− expressed by H2 O2 as an indicator of ROS. One unit of ROS was deHned as 1 mmol=L reduction of H2 O2 concentration in the reaction system in a minute at 25◦ C. 2.3. Determination of superoxide dismutase (SOD) activity SOD activity was measured according to Droillard et al. [7]. One nitrite unit of SOD was deHned as the amount of the enzyme extracts which would produce a 50% inhibition. 2.4. Measurement of catalase (CAT) activity According to Jablonski [8], the change of OD240 was recorded in spectrophotometer (UV-160,
Chlorophyll Auorescence was measured using a plant eOciency analyzer (PEA, HansatechJ, UK). Measurements were performed in dark-adapted cultures after 15 min of dark period. 3. Results 3.1. E7ects of clinorotation on ROS in Anabaena cells The amount of ROS in clinostat-treated cells increased at both time points with a 44% (3 days) and 75% (7 days) increase in comparison with stationary control samples, while the amount of ROS in stationary control samples showed no changes during the 7 days of experiment (Fig. 1). The curves of both the 70
control
60 ROS U/µg.chl
2.2. Analysis of ROS in the cells
2.7. Chlorophyll 6uorescence analysis
50
clinorotation
40 30 20 10 0 7
3 Time (Day)
Fig. 1. The amount of ROS in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957 0.7
60
0.6
SOD activity NU/µg.chl
control clinorotation
OD665
0.5 0.4 0.3 0.2 0.1 00
control clinorotation
50 40 30 20 10
2
4
6
8
0
Time (Day)
Fig. 2. The growth curve of cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
Fig. 4. The SOD activity in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
clinorotation
clinorotation
CAT activity U/µg.chl
1.8
0.4 Fv/Fm
control
2
control
0.5
0.3 0.2 0.1 0
7
3
Time (day)
0.6
955
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2
0
3
6
9
Time (day)
0 3
7
Time (Day)
Fig. 3. The optimal quantum yield of cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
Fig. 5. The CAT activity in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
control and the clinostat-treated samples were shown in Fig. 2. Obviously, growth of the clinostat-treated Anabaena cells was slower than that of the control. Clinorotated Anabaena cells showed a reduction in their optimal quantum yield (Fv =Fm ) values. On the contrary, the Anabaena cells under normal condition did not change the value of Fv =Fm (Fig. 3).
increased by 61% in comparison with cultures under 1g on the third day. Longer clinorotation (7 days), there was an increase in about 75% (Fig. 4). The change of CAT activity was similar to that of SOD, but slight di:erences existed: prolonged clinorotation caused a decrease of CAT activity, however, it remained 45% higher than in controlled samples (Fig. 5). The contents of GSH in cells treated with clinostat were lower than in controlled samples. Clinorotated Anabaena cells showed a GSH content reduction of 45% on the third day, but there was a reduction in about 37% on the seventh day (Fig. 6). The above results indicate that the antioxidative ability in Anabaena cells obviously increased under simulated microgravity.
3.2. E7ects of clinorotation on the activities of SOD, CAT and the content of GSH in Anabaena cells The activities of SOD, CAT and the content of GSH in Anabaena cells were measured after culture of 3 days and 7 days under microgravity of lower than 10−3 g. The activity of SOD in clinostat-treated cells
956
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957
Content of GSH µg/mg.chl
25
control
clinorotation
20 15 10 5 0
3
7 Time (day)
Fig. 6. The contents of GSH in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
4. Discussion Normally, plant cells could produce ROS through a few pathways. For example, the electron transport chain in chloroplasts is a source of ROS [11]. Electrons are transported to oxygen to form activated oxygen in the acceptor site of PS I [12]. Superxoide anion radical (OD 2 ), the primary product, may be catalyzed to H2 O2 and O2 by dismutation reaction, or the hydroxyl radical (OH− ) is generated by the interaction of O2D , H2 O2 , and transition metal ion such as Fe and Cu ion [13,14]. Extracellular stress caused an enhanced production of ROS. If those ROS were not scavenged, superabundant ROS would be then able to cause oxidative damage to cells. After summarizing results from various experiments, Ray [15] thought that a large number of abnormal physiological response in space environment is related with oxygen radical accumulated in organism. The lipid peroxidation (LP) level in chloroplasts of pea grown in clinostat increased for 7 and 14 days [16]. The reduction of PS I activity and increased levels of LP could be interpreted as result of elevated production of ROS under these conditions. In our previous work, we also found an increase of LP level in microalgal cells under simulated microgravity, but no information about ROS metabolism in the condition was available. In the report here, the ROS in clinostat-treated Anabaena cells was examined, indicating that microgravity stress cause an increase of ROS level. The value of Fv =Fm is an indicator of photosystem II (PS II) function [17]. In fact, the clinorotated Anabaena cells showed a decreased values of Fv =Fm (Fig. 3), which may indicate an inhibition of
PS II function in such condition, consequently, clinorotated cells had a slower growth than the control. All results from both spaceAight and simulated microgravity experiments implicated that microgravity environment may break the metabolism balance of oxygen radical, which cause to elevation of ROS level resulting in a series of physiological changes in cells. The production of ROS is unavoidable in the oxygenic organisms. Superabundant ROS could cause damage to cells, but more and more evidences showed ROS may be the signal in abiotic and biotic stress on the other hand [18]. Little had been known about the accurate role of ROS in signal transduction. Whether ROS was involved in signals transduction in cells under microgravity, especially in cells without special gravireceptor, such as Anabaena cells. Maybe, the change in ROS level could alter signaling pathways or regulate enzymatic function, which play a role in transmitting gravity signal as a second messenger. The antioxidant ability of plant cells is a kind of self-protective mechanism. The tolerance to oxygen damage is correlated with the state of intracellular antioxidant system [11]. The ROS scavenging depends on the antioxidant enzymes such SOD to dismutate OD 2 to O2 and H2 O2 , CAT and antioxidants, e.g., GSH, AsA, which play an important role in plant resistance. Baranenko [16] reported that SOD activity of clinostat-treated pea increased by 31% as compared to control samples on the seventh day. We found the SOD activity in Anabaena cells treated with clinostat was 61% higher than that in the control on the third day, and 75% higher than the control on the seventh day. Activity of CAT, an enzyme scavenging H2 O2 , increased too, but on the seventh day CAT activity slightly decreased (Fig. 5). Compared to the control, the content of GSH decreased. More prolonged clinorotation caused a slight increase, however, it was still lower than in the control (Fig. 6). Such antioxidant enzyme behavior could be a defense response in order to adapt the unfavorable environment. However, the adaptive changes of antioxidant system activity seemly were not suOcient to prevent oxidative damage, as shown inhibition of PS II function during 7 days of experiment. Our research suggests that simulated microgravity environment provided by clinostat might break the oxidative/antioxidative balance, resulting in an increase of ROS in cells. As a protective mechanism, the total
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957
antioxidant system activity increased to scavenge elevated cellular ROS to a sublethal level. The changes of redox status of cells induced by microgravity environment could be an important cause of biological responses of microalgae under altered gravity. Acknowledgements The authors gratefully acknowledge the Hnancial support from the project of knowledge innovation program of CAS (Grant No. KSCX2-SW-322) and the project of Chinese manned spaceAight. References [1] Y.D. Liu, H.M. Lin, L.F. Dai, S. Yang, E:ects of spaceAight by retrievable satellite on Anabaena and Chlorella, Chinese Science Bulletin 38 (1993) 177–180. [2] H.F. Chen, Y. Fu, L.R. Song, Y.D. Liu, The cytological observation of Anabaena siamensis after spaceAight by retrievable satellite, Chinese Journal of Space Science 17 (1997) 102–106. [3] Z.L. Hu, Y.D. Liu, Cell responses of Dunaliella salina FACHB 435 (Green Alga) to microgravitational stimulation by clinorotation, Chinese Science Bulletin 43 (1998) 1737–1741. [4] H.F. Chen, L.R. Song, Y.D. Liu, H.M. Lin, Y.D. Zou, T.F. Wu, L.M. Lei, D.H. Li, E:ect of spaceAight on the population increase and physiological features of microalga Anabaena siamensis, Chinese Journal of Space Science 17 (1997) 67–72. [5] Z.L. Hu, Y.D. Liu, L.R. Song, E:ect of space Aight on the survivorship and adaptation of microalgae, Chinese Journal of Space Science 17 (1997) 95–101. [6] Y.D. Liu, X.M. Zhang, H.F. Chen, L.R. Song, Z.L. Hu, L.F. Dai, Study on the structure and function of a hydrobiological
[7]
[8] [9] [10]
[11] [12] [13] [14] [15] [16] [17] [18]
957
closed eco-system in space, Chinese Journal of Space Science 17 (1997) 73–77. M.J. Droillard, A. Paulin, J.C. Massot, Free radical production, calatase and superoxide dimutase activities and membrane integrity during senescence of petals of cut carnations (Dianthus caryophyllus), Physiologia Plantarum 71 (1987) 179–202. P.P. Jablonski, J.W. Anderson, Light-dependent reduction of dehydroascorbate by ruptured pea chloroplasts, Plant Physiology 67 (1981) 1239–1244. S.X. Zeng, Y.R. Wang, H.X. Liu, E:ects of chilling stress on ascorbic acid content of rice seedings, Acta Phytophysiologia Sinica 13 (1987) 365–370. J.F.G.M. Wintermans, A. de Mots, Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol, Biochimica et Biophysica Acta 109 (1965) 448–453. C. Bowler, M. van Obenland, I. Inze, Superoxide dismutase and stress tolerance, Annual Review of Plant Physiology and Plant Molecular Biology 43 (1992) 83–116. J.E. Thompsm, R.L. Legge, R.E. Barber, The role of free radicals in senescence and wounding, New Phytologist 105 (1987) 317–344. C.H. Foyer, M. Lelandis, K.J. Kunert, Photooxidative stress in plants, Physiologia Plantarum 92 (1994) 696–717. K. Asada, M. Takahashi, Production and Scavenging of Active Oxygen in Photosynthesis, Elsevier Science Publishers, Amsterdam, 1987, pp. 227–287. E.K. Ray, Introduction: are aging and space e:ects similar?, Experimental Gerontology 26 (1991) 123–129. V.V. Baranenko, Pea chloroplasts under clino-rotation: lipid peroxidation and superoxide dismutase activity, Advanced Space Research 27 (2001) 973–976. G.H. Krause, E. Weis, Chlorophyll Auorescence and phyotosynthesis: the basics, Annual Review of Plant Physiology and Plant Molecular Biology 42 (1991) 313–349. C. Bowler, R. Fluhr, The role of calcium and activated oxygen as signals for controlling cross-tolerance, Trends in Plant Science 5 (2000) 241–246.
Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity Gen-bao Li, Yong-ding Liu∗ , Gao-hong Wang, Li-rong Song Institute of Hydrobiology, Chinese Academy of Sciences, 7 Donghunanlu, Luojiashan, Wuhan 430072, China Received 19 December 2002; received in revised form 29 March 2004; accepted 28 April 2004 Available online 3 August 2004
Abstract It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days’ experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity. c 2004 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are organisms that are capable of oxygenic photosynthesis. Due to higher rate of light-energy transformation and stronger adaptation to stress environment, microalgae are model organisms for the study of space biology, and are suitable to be the best members of the controlled ecological life support system (CELSS). Many studies reported that alteration of gravity a:ected the microalgal structure, population growth and physiological characteristics. Those e:ects included change of intracellular secretions, reduced photosynthesis and decreased starch number [1–3]. Chen et al. [4] reported that the rate ∗ Corresponding author. Tel.: +86-27-87647715; fax: +86-2787875132. E-mail address: [email protected] (Y. Liu).
of photosynthesis in spaceAight was lower than in ground control. However, strong adaptable ability of microalgae to spaceAight environment was found [5]. What is the cause of responses of microalgae to the microgravity environment? Which stress defence mechanisms enable algae to adapt to their particular environment? In previous studies, lipid peroxidation and membrane permeability in clinostat-treated microalgal cells were observed (we reported in another paper). Because many stress conditions cause cellular redox imbalances, it has been proposed that reactive oxygen species (ROS) exhibit important signaling function in response to both biotic and abiotic stress. We thought high level of ROS in microalgal cells maybe were responsible for abnormal physiological reaction. Thus, abilities in microgravity tolerance among algae can be correlated with the capacity to develop antioxidant systems, but there is still not much
c 2004 Elsevier Ltd. All rights reserved. 0094-5765/$ - see front matter doi:10.1016/j.actaastro.2004.04.014
954
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957
information about this topic. For this aim, ROS and some antioxidant enzymes activity were measured in order to explore the mechanism by which algae cells scavenged ROS and adapted to microgravity, which is valuable for us to select suitable plants as the member of CELSS [6].
Shimadizu corporation). The reduction of H2 O2 in reaction system was showed with units=g chl. 2.5. Measurement of gluathione (GSH) in cells
2. Materials and methods
GSH was assayed as described by Zeng [9]. The Oxidation reaction was recorded at 412 nm (UV-1601, Shimadizu corporation). The standard curve was performed with analytical GSH.
2.1. Organism and cultures
2.6. Determination of chlorophyll (chl)
Anabaena sp. PCC 7120 (Hlamentous cyanobacterium, blue-green alga) was obtained from the Freshwater Algae Culture Collection of Institute of Hydrobiology, China (FACHB). Cultures in logarithmic phase were transferred into tubes containing BG-11 medium on a horizontally slow-rotating clinostat (8 rmp) under simulated microgravity of 10−3 –10−4 g at 27 ± 1◦ C with white Auorescent irradiation of 36 E m−2 s−1 for 3 and 7 days.
Chlorophyll was extracted in 95% ethanol and characterized by the method of Wintermans and de Mots [10].
ROS assay kits was purchased from Jiancheng bioengineering institute, Nanjing China. Since Fenton reaction is a common chemic reaction producing OH− , the amount of H2 O2 is directly proportional to the amount of produced OH− , when given electron acceptor, stained with Gress, the red-color production is in proportion to OH− , the authors employed OH− expressed by H2 O2 as an indicator of ROS. One unit of ROS was deHned as 1 mmol=L reduction of H2 O2 concentration in the reaction system in a minute at 25◦ C. 2.3. Determination of superoxide dismutase (SOD) activity SOD activity was measured according to Droillard et al. [7]. One nitrite unit of SOD was deHned as the amount of the enzyme extracts which would produce a 50% inhibition. 2.4. Measurement of catalase (CAT) activity According to Jablonski [8], the change of OD240 was recorded in spectrophotometer (UV-160,
Chlorophyll Auorescence was measured using a plant eOciency analyzer (PEA, HansatechJ, UK). Measurements were performed in dark-adapted cultures after 15 min of dark period. 3. Results 3.1. E7ects of clinorotation on ROS in Anabaena cells The amount of ROS in clinostat-treated cells increased at both time points with a 44% (3 days) and 75% (7 days) increase in comparison with stationary control samples, while the amount of ROS in stationary control samples showed no changes during the 7 days of experiment (Fig. 1). The curves of both the 70
control
60 ROS U/µg.chl
2.2. Analysis of ROS in the cells
2.7. Chlorophyll 6uorescence analysis
50
clinorotation
40 30 20 10 0 7
3 Time (Day)
Fig. 1. The amount of ROS in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957 0.7
60
0.6
SOD activity NU/µg.chl
control clinorotation
OD665
0.5 0.4 0.3 0.2 0.1 00
control clinorotation
50 40 30 20 10
2
4
6
8
0
Time (Day)
Fig. 2. The growth curve of cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
Fig. 4. The SOD activity in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
clinorotation
clinorotation
CAT activity U/µg.chl
1.8
0.4 Fv/Fm
control
2
control
0.5
0.3 0.2 0.1 0
7
3
Time (day)
0.6
955
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2
0
3
6
9
Time (day)
0 3
7
Time (Day)
Fig. 3. The optimal quantum yield of cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
Fig. 5. The CAT activity in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
control and the clinostat-treated samples were shown in Fig. 2. Obviously, growth of the clinostat-treated Anabaena cells was slower than that of the control. Clinorotated Anabaena cells showed a reduction in their optimal quantum yield (Fv =Fm ) values. On the contrary, the Anabaena cells under normal condition did not change the value of Fv =Fm (Fig. 3).
increased by 61% in comparison with cultures under 1g on the third day. Longer clinorotation (7 days), there was an increase in about 75% (Fig. 4). The change of CAT activity was similar to that of SOD, but slight di:erences existed: prolonged clinorotation caused a decrease of CAT activity, however, it remained 45% higher than in controlled samples (Fig. 5). The contents of GSH in cells treated with clinostat were lower than in controlled samples. Clinorotated Anabaena cells showed a GSH content reduction of 45% on the third day, but there was a reduction in about 37% on the seventh day (Fig. 6). The above results indicate that the antioxidative ability in Anabaena cells obviously increased under simulated microgravity.
3.2. E7ects of clinorotation on the activities of SOD, CAT and the content of GSH in Anabaena cells The activities of SOD, CAT and the content of GSH in Anabaena cells were measured after culture of 3 days and 7 days under microgravity of lower than 10−3 g. The activity of SOD in clinostat-treated cells
956
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957
Content of GSH µg/mg.chl
25
control
clinorotation
20 15 10 5 0
3
7 Time (day)
Fig. 6. The contents of GSH in cells of Anabaena sp. PCC 7120 under clinorotation compared to the control.
4. Discussion Normally, plant cells could produce ROS through a few pathways. For example, the electron transport chain in chloroplasts is a source of ROS [11]. Electrons are transported to oxygen to form activated oxygen in the acceptor site of PS I [12]. Superxoide anion radical (OD 2 ), the primary product, may be catalyzed to H2 O2 and O2 by dismutation reaction, or the hydroxyl radical (OH− ) is generated by the interaction of O2D , H2 O2 , and transition metal ion such as Fe and Cu ion [13,14]. Extracellular stress caused an enhanced production of ROS. If those ROS were not scavenged, superabundant ROS would be then able to cause oxidative damage to cells. After summarizing results from various experiments, Ray [15] thought that a large number of abnormal physiological response in space environment is related with oxygen radical accumulated in organism. The lipid peroxidation (LP) level in chloroplasts of pea grown in clinostat increased for 7 and 14 days [16]. The reduction of PS I activity and increased levels of LP could be interpreted as result of elevated production of ROS under these conditions. In our previous work, we also found an increase of LP level in microalgal cells under simulated microgravity, but no information about ROS metabolism in the condition was available. In the report here, the ROS in clinostat-treated Anabaena cells was examined, indicating that microgravity stress cause an increase of ROS level. The value of Fv =Fm is an indicator of photosystem II (PS II) function [17]. In fact, the clinorotated Anabaena cells showed a decreased values of Fv =Fm (Fig. 3), which may indicate an inhibition of
PS II function in such condition, consequently, clinorotated cells had a slower growth than the control. All results from both spaceAight and simulated microgravity experiments implicated that microgravity environment may break the metabolism balance of oxygen radical, which cause to elevation of ROS level resulting in a series of physiological changes in cells. The production of ROS is unavoidable in the oxygenic organisms. Superabundant ROS could cause damage to cells, but more and more evidences showed ROS may be the signal in abiotic and biotic stress on the other hand [18]. Little had been known about the accurate role of ROS in signal transduction. Whether ROS was involved in signals transduction in cells under microgravity, especially in cells without special gravireceptor, such as Anabaena cells. Maybe, the change in ROS level could alter signaling pathways or regulate enzymatic function, which play a role in transmitting gravity signal as a second messenger. The antioxidant ability of plant cells is a kind of self-protective mechanism. The tolerance to oxygen damage is correlated with the state of intracellular antioxidant system [11]. The ROS scavenging depends on the antioxidant enzymes such SOD to dismutate OD 2 to O2 and H2 O2 , CAT and antioxidants, e.g., GSH, AsA, which play an important role in plant resistance. Baranenko [16] reported that SOD activity of clinostat-treated pea increased by 31% as compared to control samples on the seventh day. We found the SOD activity in Anabaena cells treated with clinostat was 61% higher than that in the control on the third day, and 75% higher than the control on the seventh day. Activity of CAT, an enzyme scavenging H2 O2 , increased too, but on the seventh day CAT activity slightly decreased (Fig. 5). Compared to the control, the content of GSH decreased. More prolonged clinorotation caused a slight increase, however, it was still lower than in the control (Fig. 6). Such antioxidant enzyme behavior could be a defense response in order to adapt the unfavorable environment. However, the adaptive changes of antioxidant system activity seemly were not suOcient to prevent oxidative damage, as shown inhibition of PS II function during 7 days of experiment. Our research suggests that simulated microgravity environment provided by clinostat might break the oxidative/antioxidative balance, resulting in an increase of ROS in cells. As a protective mechanism, the total
G. Li et al. / Acta Astronautica 55 (2004) 953 – 957
antioxidant system activity increased to scavenge elevated cellular ROS to a sublethal level. The changes of redox status of cells induced by microgravity environment could be an important cause of biological responses of microalgae under altered gravity. Acknowledgements The authors gratefully acknowledge the Hnancial support from the project of knowledge innovation program of CAS (Grant No. KSCX2-SW-322) and the project of Chinese manned spaceAight. References [1] Y.D. Liu, H.M. Lin, L.F. Dai, S. Yang, E:ects of spaceAight by retrievable satellite on Anabaena and Chlorella, Chinese Science Bulletin 38 (1993) 177–180. [2] H.F. Chen, Y. Fu, L.R. Song, Y.D. Liu, The cytological observation of Anabaena siamensis after spaceAight by retrievable satellite, Chinese Journal of Space Science 17 (1997) 102–106. [3] Z.L. Hu, Y.D. Liu, Cell responses of Dunaliella salina FACHB 435 (Green Alga) to microgravitational stimulation by clinorotation, Chinese Science Bulletin 43 (1998) 1737–1741. [4] H.F. Chen, L.R. Song, Y.D. Liu, H.M. Lin, Y.D. Zou, T.F. Wu, L.M. Lei, D.H. Li, E:ect of spaceAight on the population increase and physiological features of microalga Anabaena siamensis, Chinese Journal of Space Science 17 (1997) 67–72. [5] Z.L. Hu, Y.D. Liu, L.R. Song, E:ect of space Aight on the survivorship and adaptation of microalgae, Chinese Journal of Space Science 17 (1997) 95–101. [6] Y.D. Liu, X.M. Zhang, H.F. Chen, L.R. Song, Z.L. Hu, L.F. Dai, Study on the structure and function of a hydrobiological
[7]
[8] [9] [10]
[11] [12] [13] [14] [15] [16] [17] [18]
957
closed eco-system in space, Chinese Journal of Space Science 17 (1997) 73–77. M.J. Droillard, A. Paulin, J.C. Massot, Free radical production, calatase and superoxide dimutase activities and membrane integrity during senescence of petals of cut carnations (Dianthus caryophyllus), Physiologia Plantarum 71 (1987) 179–202. P.P. Jablonski, J.W. Anderson, Light-dependent reduction of dehydroascorbate by ruptured pea chloroplasts, Plant Physiology 67 (1981) 1239–1244. S.X. Zeng, Y.R. Wang, H.X. Liu, E:ects of chilling stress on ascorbic acid content of rice seedings, Acta Phytophysiologia Sinica 13 (1987) 365–370. J.F.G.M. Wintermans, A. de Mots, Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol, Biochimica et Biophysica Acta 109 (1965) 448–453. C. Bowler, M. van Obenland, I. Inze, Superoxide dismutase and stress tolerance, Annual Review of Plant Physiology and Plant Molecular Biology 43 (1992) 83–116. J.E. Thompsm, R.L. Legge, R.E. Barber, The role of free radicals in senescence and wounding, New Phytologist 105 (1987) 317–344. C.H. Foyer, M. Lelandis, K.J. Kunert, Photooxidative stress in plants, Physiologia Plantarum 92 (1994) 696–717. K. Asada, M. Takahashi, Production and Scavenging of Active Oxygen in Photosynthesis, Elsevier Science Publishers, Amsterdam, 1987, pp. 227–287. E.K. Ray, Introduction: are aging and space e:ects similar?, Experimental Gerontology 26 (1991) 123–129. V.V. Baranenko, Pea chloroplasts under clino-rotation: lipid peroxidation and superoxide dismutase activity, Advanced Space Research 27 (2001) 973–976. G.H. Krause, E. Weis, Chlorophyll Auorescence and phyotosynthesis: the basics, Annual Review of Plant Physiology and Plant Molecular Biology 42 (1991) 313–349. C. Bowler, R. Fluhr, The role of calcium and activated oxygen as signals for controlling cross-tolerance, Trends in Plant Science 5 (2000) 241–246.