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I. Bertalan et al: Photosystem II Stress Tolerance in the Unicellular Green Alga Chlamydomonas reinhardtii under Space Conditions

Ivo Bertalan, Dania Esposito, Giuseppe Torzillo, Cecilia Faraloni, Udo Johanningmeier and Maria Teresa Giardi

Photosystem II Stress Tolerance in the Unicellular Green Alga Chlamydomonas reinhardtii under Space Conditions

Photosynthesis was established on the earth 3.5 billion years ago. Due to the absence of the ozone layer in the early atmosphere it was most likely adapted to the presence of ionizing radiation continuously emitted by solar and stellar flares. That complex radiation spectrum comprises protons, alpha particles, heavy charged particle-HZE, electrons, X-ray and neutrons. Such spectrum has a significant impact on biological systems which capture light energy for e.g. photosynthesis. Oxygenic photosynthesis of plants, algae and cyanobacteria initiates at the level of photosystem II (PSII), a multisubunit protein complex embedded in the thylakoid membrane inside chloroplasts. PSII uses sunlight to power the unique photo-induced oxidation of water to atmospheric oxygen which is indispensable for most life Authors Ivo Bertalan, Udo Johanningmeier Martin-Luther-Universität Halle-Wittenberg, Institute of Plant Physiology Weinbergweg 10, D-06108 Halle (Saale), Germany Maria Teresa Giardi, Dania Esposito, IC, CNR, Monterotondo Scalo, Rome, Italy Giuseppe Torzillo, Cecilia Faraloni, ISE, CNR, 50019 Sesto Fiorentino, Florence, Italy

Correspondence MT Giardi Present address: Institute of Crystallography, National Research Council via Salaria Km 29,3 00016 Monterotondo Scalo Rome, Italy. Phone: 0039-0690672704 Fax:0039-0690672630 E-mail:[email protected]

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forms. It is an especially sensitive component if exposed to space radiation and thus an important target for research aimed at improving bioregenerative life-support systems. The unicellular green algae Chlamydomonas reinhardtii is a long standing model organism for photosynthesis research. It was exposed to ionizing radiation in the ESA facility Biopan located in the Foton capsule brought to space by the Russian Soyuz for 15 days. The algae were tested in space under shielded conditions in the past, but they were never exposed to direct ionizing radiation such as in Biopan. Conditions for survival were identified. It was observed that the effect of space stress on the survival of the algae varied depending on the light conditions to which they were exposed during the flight. In some cases the flight experience caused a stimulation of the photosystem II oxygen evolution of the cells. 1 Introduction The primary event of photosynthesis in the chloroplast is a light-driven electron transfer, a redox reaction that sets in motion a chain of electron transfers upon which all life ultimately depends. The development of two photosystems, photosystem II (PSII) and photosystem I (PSI), and the ability of the ancestral algae and higher plants to evolve oxygen was the result of an endosymbiotic event that turned a primordial cyanobacterium into a cell organelle termed chloroplast. PS II is a supramolecular pigment-protein complex which catalyses the light-induced transfer of electrons from water to plastoquinone. The oxidation of water molecules generates the earth’s entire atmospheric oxygen. PSII consists of more than 25 polypeptides, which make up the oxygen-evolving complex, a light-harvesting chlorophyll protein complex and a reaction

© Z-Tec Publishing, Bremen Microgravity sci. technol. XIX-5/6 (2007)


center core composed of the two proteins D1 and D2, which are involved in the primary charge separation. Antennae chlorophyll-protein complexes capture light energy and transfer it to the reaction center chlorophyll P680. This excitation leads to primary charge separation and the formation of the radical pair P680+Pheo-. The radical pair is stabilized when Pheo- transfers its electron to QA, the primary quinone acceptor bound to the D2 protein, and then to the secondary quinone acceptor QB. The electron is then transferred via the cytochrome b6/f complex and plastocyanin to PSI for NADP reduction and the synthesis of organic compounds [1]. Environmental extremes like high light conditions have a negative effect on photosynthesis and seem to act primarily by damaging PSII at the level of the D1 protein turnover. D1 protein turnover appears to be the ultimate solution of nature to cope with the damage caused by reactive oxygen species generated within the reaction center. Radiation of many sources aggravate this damage [2, 3]. Space is permeated by ionizing radiation that consists of various charged particles from diverse origins and with varied energies. This complex radiation, emitted by the sun and the stars, comprise protons, alpha particles, heavy charged particleHZE, X-ray, electrons and neutrons. Galactic radiation, created outside the solar system, consists mainly of protons with some helium and heavier ions, produced and accelerated as a consequence of stellar flares, supernova explosions, pulsar acceleration and the explosion of galactic nuclei. Solar particle radiation contains low energy particles, although energetic particles are copiously emitted during magnetic disturbances. The energy of

Fig. 1: Photo probe container with 16 holes filled with medium and algae probes.

Microgravity sci. technol. XIX-5/6 (2007)

electrons reach several MeV and the energy of protons reach several hundred MeV. Trapped protons are more important than electrons for human missions in a low earth orbit. Moreover, the radiation field is modified by the interactions with the shielding of a spacecraft material generating neutrons and heavy charged particles. There are very significant temporal variations in both ionizing radiation composition and intensity that range from 1 MeV to 103 GeV (see Solar chart at http://hiraiso.crl.go.jp/ and ref. [4]). The majority of studies on the physiological effects of space stress on plants concerns the absence of gravity and its consequence on the root apparatus but little is known about the influence of space radiation on the photosynthetic apparatus of unicellular organisms. Organellar membranes appear to be strongly affected by space stress as evidenced by the destruction of grana thylakoids, hence the presence of ionizing radiation in space is thought to affect photosynthetic activity (G. Nechitailo personal communication). Here the observation is of special importance that complex space radiation is specifically damaging the PS II reaction centre, thus reducing in some cases the photosynthetic efficiency. Bioregenerative life-support systems in space are severely disturbed at this point. Identification and manipulation of photosynthesis check points offer the possibility to adjust biomass and oxygen production to changing environmental conditions. As the photosynthetic apparatus has adapted to terrestrial and not to space conditions, we are trying to adapt a central and particularly stress-susceptible element of the photosynthesis apparatus - the D1 subunit of PS II. We are using the model organism Chlamydomonas reinhardtii because this unicellular green alga is easily susceptible to genetic modification. The alga was already tested in space under shielded conditions in the past, but it was never exposed to direct ionizing radiation as it prevails in the experimental platform Biopan [5,6]. In our laboratory we have constructed a Chlamydomonas mutant with a defect D1 protein [7]. This mutant is rapidly and easily transformable with PCR fragments without purification and cloning steps. When generating a pool of randomly mutated DNA fragments by error prone PCR and transforming the mutant strain with this pool [8,9], it is possible to expose the resulting transformants to selective conditions, i.e. exposure to space radiation. In this particular project we preliminarily analyse Chlamydomonas reinhardtii algae after a Soyuz flight mission. The ESA Foton M2 Mission gave us the possibility to send the algae to space and to expose them directly to these extreme conditions. The Biopan platform was fixed at the exterior wall of the ESA-Russian Foton capsule and brought to space by Soyuz. This recoverable capsule is used for scientific experiments in near orbit. Biopan was especially developed for exposing scientific experiments and is successfully in use since 1992. The biological material in the open Biopan unit experiences quite different radiation intensities and qualities and therefore 123


quite different evolutionary pressure compared to earth. Thus, only organisms with a photosynthetic apparatus advantageous for these conditions will survive. In orbit the probes can be directly exposed to space conditions. Experiments in the bottom part are temperature controlled (5-15°C). Here, our Photo experiment was located in the probe container (Fig. 1). The algae were protected from vacuum, heat and cold as well as from too intensive visible and UV radiation, but were exposed to ionizing radiation with only 2 mm of filter glass between the algae lawn surface and open space. 2 Photo experiment Different algae strains were inserted into the Photo module (Fig. 1). In 16 positions every strain could be exposed to four different conditions concerning quality and intensity of visible and UV radiation using a combination of special filters (Table 1). The algae were filled into each of the 16 spaces on top of solid Tris-acetate-phosphate (TAP) medium. On TAP medium Chlamydomonas reinhardtii can grow photoautotrophically as well as heterotrophically because it contains acetate as carbon and energy source. In that way, mutants which possibly may be generated by space radiation and are unable to perform photosynthesis can be preserved. The final device was integrated into the Biopan platform and transported via Samara to Baikonur, Russian space station (Fig. 2, 3, 4). The results of Chlamydomonas in the various conditions are described here. On 31 May 2005 at 2 p.m. (CET) the Foton M2 spacecraft was successfully launched from Baikonur Cosmodrome in Kazakhstan. After reaching a low-earth orbit at an altitude between 260 and 305 km, Biopan was opened and the experiments were exposed to space. During this time the Photo module was only temperature controlled; the temperature range was between 5-15°C. Additionally, the algae were exposed to weightlessness as well as solar and ionizing space radiation. Concerning the solar and absolute radiation intensities during the flight we could refer to the results of the experiment R3D-B (D.P. Häder, University of Erlangen) and RADO-HTR (M. Hajek, T. Berger, M. Fugger, N. Vana; Vienna Univ./DLR), which were also part of Biopan. Accordingly, Photo experiment was exposed to a maximum peak of 54.8 SCh (1 SCh = 1 solar constant hour = absolute solar energy per hour = 4932 kJ/m2 x h). According to the position of Photo and the measured peaks of light during the rotation around the earth, the minimum and maximum light intensities reaching the samples are reported in Table 1. Our probes received a total dose of ionizing radiation of 50 mGy. The Foton M2 spacecraft moved with 7.8 km/s and orbited earth within 90 minutes. During this time it was exposed to the sun for 55 min and it stayed in darkness for 35 min. Because of the spacecraft’s self-rotation Biopan experienced a permanent change between light and dark during 55 min. The Biopan was opened for a total of 351 h; at the same time the Foton M2 spacecraft orbited earth 234 times. One day before landing 124

Table 1: Light filters in Photo experiment

Position

Attenuation of external light

Filter permeablity

MaximumMinimum peak of light (micromoles photon/m2 s)

1

99%

visible light-UV radiation (250-800 nm)

2-10

2

95%

visible light (400-800 nm)

30-80

3

90%

visible light-UV radiation (250-800 nm)

90-180

4

95%

visible light-UV radiation 30-80 (250-800 nm) Quality and intensity of visible and UV radiation received by the algae using a combination of special filters set on Photo experiment device

Biopan was closed by tele-control. On June 16, 2005 at 9.37 CET after nearly 250 orbiting and approx. 379 h in orbit the Foton capsule landed in an uninhabited area, 140 km south-east of Kostanay, Kazakhstan, near the Russian border. Subsequently, Biopan was transported to ESTEC in Noordwijk where it was opened. After that the Photo module was opened and the algae analysed. 2.1 Results of the Post Flight Analysis Different algae lawn were integrated in the Photo experiment container (Fig. 1): Con: reference strain IL. It is a mutant without introns in the psbA gene [3], but physiologically corresponds to the wild-type (WT). UV: Chlamydomonas reinhardtii wild type 11/32-b (from algae strain collection Göttingen) which was pre-exposed to UV radiation (40 minutes; 280-370 nm; 0,5 mW/cm2s) once a week for three months before flight. The algae lawns were protected against visible and UV light by the different filters (Table 1, Fig. 1). The Photo module was opened under sterile conditions. The probes were transferred together with the Agar medium to a 500

Fig. 2: Biopan open and closed with the Photo module..



ml flask filled with 200 ml TAP medium. We used TAP medium to also obtain mutants that are able to grow heterotrophically only because of a severe defect in the photosynthesis apparatus. The flasks with the probes were gently moved overnight on a rotator under constant light (40 μmol/m2s). In this way the liquid media removed the algae from the solid agar support. The next day algae were found to be completely suspended in liquid media. A volume of 1 ml was removed from each flask and plated onto a TAP plate. Only surviving cells could then form colonies. By counting these we were able to estimate the percentage of surviving cells: between 0.013% and 0.001% survivors were calculated (Table 2). After one week survivors in liquid media became clearly visible. We found surviving cells in five positions. We named them according to their position in the module: IL1, IL2, UV1, UV2 und UV4 (Fig. 4, Table 2).

3 Conclusions

Table 2: Surviving algae cells in Photo experiment

Algae probe

colonies on plate

surviving cells

surviving cells in %

IL1

1

ca. 200 cells

0,001 %

IL2

10

ca. 2000 cells

0,010 %

UV1

13

ca. 2600 cells

UV2

0

< 200 cells

0,013 % < 0,001 %

UV4 0 < 200 cells < 0,001 % 1 ml algae suspension (out of 200 ml TAP medium) was used to estimate how many of the algae exposed to space conditions could survive. Applied on TAP plates only surviving cells were able to form colonies. These colonies became visible after 14 days. By referring to the total volume of 200 ml the approximate number of surviving cells could be calculated and their percentage of the source material (2*107 cells) could be determined.

Fig. 3: Foton spacecraft, ESA facility for scientific experiments brought to Space by the Russian Soyuz, provided of the external experiment facility called Biopan.


2.2 Physiological tests First physiological tests on earth included the measurements of photoautotrophic growth and of photosynthetic oxygen production. The growth rates (Fig. 5) show that an increase of radiation intensity (controlled by different filters in Photo experiment) results in a slightly lower growth rate. The UV2 strain, which was exposed to 5% visible radiation, showed the lowest drop in growth rate. Measurement of oxygen production (Fig. 6) shows that strains UV2 and IL2 have improved their electron transport rate, which exceeds the rate of control algae kept on ground. The analyses were performed post-flight after 4 days landing. In that period the cells were maintained at 40 μmol/m2s light intensity as the control. The control was performed on ground in parallel to the mission and after the mission when the data of temperature and of light conditions were available.

Previous space experiment with Chlamydomonas reinhardtii are in accordance with our observations of a stimulation of the oxygen evolution activity in space. The microgravity effects were studied in Chlamydomonas at both cellular and population levels; in space the cell size was increased, stage of active growth of the culture was extended, it contained the juvenile vegetative motile cells in greater quantities [5]. The overt circadian rhythm and a short period strain of Chlamydomonas reinhardii has been studied in space using the photoaccumulation behaviour as the recorded parameter. The period of the wildtype was 29.6 h, in a short period strain was 21.4 h and did not

Fig. 4: Schematic representation of Photo experiment with exact position details of the algae strains and UV/VIS filters. Ticks indicate positions where survivors were found.

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deviate significantly from ground controls performed exactly at the same time. The phase was delayed in space by 4.2 h in the wild-type, but was not altered in the short period strain. In both strains the amplitudes were significantly higher in space than in the ground controls. During the recording period of 6.5 days the cell density increased in both strains. The survival rate, i.e. the ability to form colonies on agar, was higher in space than on ground [6]. The effects of light on gravitaxis and velocity in the bi-flagellated green alga Chlamydomonas reinhardtii has been investigated in space using a real time automatic tracking system. Three distinct light effects on gravitaxis and velocity with parallel kinetics were found. Photosynthetically active continuous red light reversibly enhanced the swimming velocity and increased or decreased the precision of gravitaxis, depending on its initial level. Blue light flashes induced fast transient increases in velocity immediately after the photophobic response, and transiently decrease or even reverse negative gravitaxis in a calcium dependent-manner. The third response, a prolonged activation of velocity and gravitaxis, was also induced by blue light flashes, which can be observed even in calcium-free medium [10, 11]. In the Photo experiment the unicellular green alga Chlamydomonas reinhardtii was – only protected by glass or quartz filters - exposed to space radiation for the first time. The new discovery is that photosynthetically active cells can principally survive under such extreme conditions. The light experienced by the cells was variable depending on the rotation movement of the spacecraft. However, it was always physiological in the range of 2-180 micromoles photon/m2s as minimum and maximum peak, respectively. The ultraviolet light experienced by the algae was about five times higher than that experienced in cells exposed on earth to solar light but still physiological for the algae (data not shown). It was observed that the effect of space stress on the survival of the algae varied depending on the light conditions to which they were exposed during the flights. These results are in accordance with our previous studies in which the effect of space stress on the quantum yield of PSII varied among the species tested depending on the light conditions to which they were exposed during the space flight. Ground studies indicated that dark and intense light irradiance of 120 μmol m-2 s-1 enhanced the effect of space radiation by reducing PSII fluorescence, while exposure to weak light irradiance of 20 μmol m-2 s-1 protected them against damage, indicating the probable involvement of a light-dependent protein turnover in the repair to stress [2,3, 12 and unpublished results]. Surprisingly, although the cell growth was inhibited in some cases the flight experience caused a stimulation of PSII oxygen evolution of the cells. Although we do not understand this phenomenon right now, it seems useful to pre-adapt the organisms on earth to high light and/or UV radiation for improved space survival. These observations raise intriguing questions about a potential role of Photosystem II in ionizing radiation energy capture and utilization.

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Fig. 5: A) growth rate in % of WT, UV1, UV2 and UV4 B) growth rate in % of IL, IL1 and IL2 at a light intensity of 40 μmol/m2s. The cells were grown post-flight after 4 days landing. These colonies became visible in liquid culture after 14 days. In that period the cells were maintained at 40 μmol/m2s light intensity as the control.

Fig. 6: A) O2-production of strains UV1,UV2 and UV4 in comparison with reference strain WT; B) O2-production of strains IL1 and IL2 with reference strain IL. Rates in μmol O2/mg Chlorophyll/h at light intensities of 100, 500 und 1000 μmol/m2s. The cells were grown post-flight after 4 days landing. These colonies became visible in liquid culture after 14 days. In that period the cells were maintained at 40 μmol/m2s light intensity as the control

Due to the high dependence to light conditions of the reported phenomena studied in space, we would expect some possible relationships. This will be tested in the future ESA space flight FotonSoyuz M3 expected on 2007. References [1]. Barber, J., Photosystem II: an enzyme of global significance. Biochem. Soc. Trans. vol 34, pp 619-631 (2006). [2]. Giardi, MT., Masojidek, J. and Godde, D., Effects of stresses on the turnover of D1 reaction centre II protein, a review, Physiol. Plant. vol 101, pp 635-642 (1997). [3]. Giardi, M.T. and Pace, E. Photosynthetic proteins for technological applications. Trends in Biotechnology vol. 25, pp 253-267 (2005). [4]. Ohnishi,T., Takahashi, A., Ohnishi, K. Studies about space radiation promote new fields in radiation biology. J Radiat Res (Tokyo) vol. 43, pp 712 (2002). [5]. Gavrilova, O.V., Gabova A.V., Goriainova LN, Filatova EV. An experiment with Chlamydomonas reinhardtii on the Kosmos-2044 biosatellite. Aviakosm Ekolog Med. Vol. 26(5-6), pp 27-30 (1992). [6]. Mergenhagen, D., Mergenhagen, E. The biological clock of Chlamydomonas reinhardii in space. Eur J Cell Biol. Vol. 43(2), pp 203-



7 (1987). [7]. Preiss, S., Schrader, S. and Johanningmeier, U. Rapid, ATP-dependent degradation of a truncated D1 protein in the chloroplast. Eur. Journal of Biochemistry vol. 268, pp. 4562-4569 (2001). [8]. Johanningmeier, U., Bertalan, I., Hilbig, L., Schulze, J., Wilski, S., Zeidler, E. and Oettmeier, W. Engineering the D1 subunit of photosystem II: application to biosensor technology. In: Biotechnological Applications of Photosynthetic Proteins: Biochips, Biosensors and Biodevices (Giardi and Piletska, eds.), pp 46-56, Landes Bioscience (2006). ISBN 0-38733009-7. [9]. Johanningmeier, U. and Heiß, S. Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA-gene. Plant Molecular Biology vol.22, pp 91-99 (1993). [10]. Häder, D.-P., R. Hemmersbach, and M. Lebert. Gravity and the Behaviour of Unicellular Organisms. Cambridge University Press, Cambridge (2005). [11]. Sineshchekov, O., Lebert, M., Hader, D.P. Effects of light on gravitaxis and velocity in Chlamydomonas reinhardtii. J Plant Physiol. Vol. 157(3), pp 247-54, 2000. [12]. Esposito, D., Faraloni, C., Margonelli, A., Pace, E., Torzillo, G., Zanini A., and Giardi, M.T. The effect of ionising radiation on photosynthetic oxygenic microorganisms for survival in space flight revealed by automatic Photosystem II-based Biosensors. Microgravity Sci. Technol. vol. XVIII-3/4, pp 215-218 (2006).


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