In Vitro Cell. Dev. Biol.ÐPlant 36:312±318, September±October 2000 q 2000 Society for In Vitro Biology 1054-5476/00 $10.0010.00

EFFECT OF SIMULATED AND REAL WEIGHTLESSNESS ON EARLY REGENERATION STAGES OF BRASSICA NAPUS PROTOPLASTS ELSE BERIT SKAGEN and TOR-HENNING IVERSEN*

The Plant Biocentre, Department of Botany, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway (Received 31 July 1999; accepted 15 May 2000; editor D. Dudits)

Summary Results from experiments using protoplasts in space, performed on the Biokosmos 9 satellite in 1989 and on the Space Shuttle on the IML-1-mission in 1992 and S/MM-03 in 1996, are presented. This paper focuses on the observation that the regeneration capacity of protoplasts is lower under micro-g conditions than under 1 g conditions. These aspects have been difficult to interpret and raise new questions about the mechanisms behind the observed effects. In an effort to try to find a key element to the poor regeneration capacity, ground-based studies were initiated focusing on the effect of the variable organization and quantity of corticular microtubules (CMTs) as a consequence of short periods of real and simulated weightlessness. The new results demonstrated the capacity of protoplasts to enter division, confirming the findings in space that this was affected by gravity. The percentage of dividing cells significantly decreased as a result of exposure to simulated weightlessness on a 2-D clinostat. Similar observations were made when comparing the wall components, which confirmed that the reconstitution of the cell wall was retarded under both space conditions and simulated weightlessness. The peroxidase activity in protoplasts exposed to microgravity was slightly decreased in both 0 g and 1 g flight samples compared with the ground controls, whereas activity in the protoplasts exposed to simulated weightlessness was similar to activity in the 1 g control. The observation that protoplasts had randomized and more sparse corticular microtubules when exposed to various forms of simulated and real weightlessness on a free-fall machine on the ground could indicate that the low division capacity in 0 g protoplasts was correlated with an abnormal CMT array in these protoplasts. This study has increased our knowledge of the more basic biochemical and cell biological aspects of g effects. This is an important link in preparation for the new space era, when it will be possible to follow the growth of single cells and tissue cultures for generations under microgravity conditions on the new International Space Station, which will be functional on a permanent basis from the year 2003. Key words: protoplasts; space experiments; microgravity environment. isolation of the protoplasts), callus tissue, and eventually mature plants. The few space-flight protoplast experiments performed under micro-g conditions by the present research group generally indicate that changes occur in the synthesis of cell wall material in higher plants developing under micro-g conditions, and that these will have an effect on the regeneration of a new cell wall. Using normal rapeseed hypocotyl protoplasts, a retardation of regeneration was observed on board Biokosmos 9 during a 14 d orbital period in 1989 (Rasmussen et al., 1992). Similar observations were made from protoplast experiments on the space shuttle, for example during the IML-1 mission in 1992, where the same authors obtained evidence that in plant protoplasts exposed to micro-g for 8 d, cell wall formation and division were significantly delayed compared with development under 1 g (Rasmussen et al., 1994). A few small cell aggregates formed under micro-g conditions, while the 1 g control samples, both on board the shuttle and on the ground, regenerated plants (Rasmussen et al., 1994). Contradictory results were obtained on the S/MM-03 mission in 1996, where the small calluses obtained in orbit developed either shoots or roots after

Introduction Several space experiments have been performed with the aim of gaining knowledge about the effect of gravity on basic cell biological processes such as growth, division, and differentiation, using protoplasts as a model system (Rasmussen et al., 1992, 1994; Iversen et al., 1999). Protoplasts are a convenient model system for comparative studies of the structural and functional aspects of cell wall formation under stationary growth conditions and under the effect of extreme environmental factors known as microgravity (micro-g) conditions on a space vehicle. It is generally accepted that after the enzymatic removal of the cell wall, protoplasts start to regenerate new cell wall material, facilitated by microtubule (MT) organization and structure (Skagen and Iversen, 1999). Regeneration of the cell wall is followed by cell division and formation of small cell aggregates (3±5 d after

*Author to whom correspondence should be addressed: Email [email protected]

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retrieval on the ground, but were not capable of regeneration to plants (Iversen et al., 1999). In parallel with the space experiments, the behavior of protoplasts exposed to simulated and short periods of real weightlessness on the ground has been followed. Both parabolic flights and use of the free-fall machine (FFM) are tools which give periods of seconds of real weightlessness on the ground. The freefall machine has been used to a limited extent to follow the effect of weightlessness on the organization of corticular microtubules (CMTs) in protoplasts. More extensive studies on CMT organization under simulated weightlessness have been performed using a fastrotating 2-D clinostat (Skagen and Iversen, 1999). The goal of the present study is to evaluate earlier and more recent results obtained when plant protoplasts are exposed to microg and simulated weightlessness, in order to increase our knowledge of more basic biochemical and cell biological aspects of g-effects. This will be an important link in preparation for the new space era, when it will be possible to follow the growth of single cells and tissue cultures for generations under micro-g conditions on the new International Space Station (ISS), which will be functional on a permanent basis from the year 2003. Materials and Methods Plant material, protoplast isolation and cultivation. For both on-theground studies and space experiments, protoplasts were isolated from 5-dold aseptically dark-grown hypocotyls of rapeseed (Brassica napus cv. Line), following standard procedures. Details of the procedures are given by Rasmussen et al. (1992) and Skagen et al. (1994). The protoplasts were

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suspended in A medium (Kao and Michayluk, 1981) or MS medium (Murashige and Skoog, 1962) with 2,4-dichlorophenoxy-acetic acid (2,4-D; 4.5 mM), benzyladenine (6-BA; 2.2 mM) and naphthaleneacetic acid (NAA; 0.5 mM) or only with NAA (5.4 mM) to a final concentration of 5:0  104 protoplasts per ml medium. The protoplasts were cultivated in liquid medium either in petri dishes sealed with Parafilm or in small polyethylene plastic bags (Fig. 1; Rasmussen et al., 1994) especially designed for the Biokosmos 9, International Microgravity Laboratory (IML-1), and Space Shuttle to MIR (S/MM-03) missions. Immunodetection of microtubules. For immunodetection of microtubules the protoplasts were fixed in 1% (w/v) glutaraldehyde (GA) in PIPES buffer (pH 6.9) containing ethylene glycol bis beta-aminoethyl ether (EGTA), magnesium and glucose, or in the MS medium. The cells were fixed for 20 min then washed in PBS. Details for the immunodetection procedure are given by Skagen and Iversen (1999). Briefly, the protoplasts were transferred to poly-l-lysine-treated cover slips, and the plasma membranes extracted by adding Triton X-100. The protoplasts were then incubated with a primary antibody raised against yeast tubulin in rats (MAS 078p, clone YOL 1/34, Harlan Sera-Lab Ltd., Loughborough, U.K.) before the secondary antibody, a rabbit anti-rat IgG fluorescein isothiocyanate conjugate (FITC), was added. Finally the slides were mounted in Citi Fluor (Alltech Associates, Baulkham Hills, Australia). Data collections of protoplast area and CMT quantity were carried out by analyzing digitalized images of protoplast surfaces attached to the cover slip (protoplast profile), using nih image 1.60 software. Fluorescence images were segmented according to a threshold based on grey level. The frequency (%) of protoplasts with parallel organized CMTs was assessed by analyzing 50±100 protoplasts on each slide, and the experiments were repeated at least three times. Only protoplasts with the whole CMT array organized in parallel were noted as protoplasts with parallel-oriented CMTs. Protoplasts and variable g-forces. Different facilities were used to simulate and create weightlessness: a horizontal one-axis 2-D clinostat (60 rpm); a three dimensional clinostat (3-D clinostat) and the FFM. Characteristic physical details for each of the instruments are given by Skagen (1998). For simulation of 0 g on the horizontal clinostat, the

Fig. 1. Calluses regenerated from protoplasts exposed to micro-g and 1 g centrifuge (flight 0 g; control flight 1 g) after retrieval from the Space Shuttle. Comparable calluses from the ground controls are shown below.

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protoplasts were kept in narrow polyethylene bags (2±3 mm diameter), while cryotubes filled with protoplast suspension were used in the 3-D clinostat and FFM experiments. In addition to the stationary 1 g control, the effect of bounce acceleration on the reorganization of CMTs in protoplasts was tested by adding a 1 g centrifugal force to the protoplasts in the FFM for 48 h. To obtain hyper-g effects, a special centrifuge was used to expose the protoplast samples to 7±10 g. The protoplasts (10 ml in suspension) were placed in plastic tubes on the centrifuge in darkness, with a static control under the same temperature conditions. Fixation for immunodetection (see above) was performed immediately after centrifugation and clinostat rotation. Cell wall analysis and enzyme activity. Methods for determining the cell wall components pectin, cellulose, hemicellulose, and intracellular polysaccharides were performed by incorporation of 14C-labeled glucose, which was added to flight and ground samples before launch of the space vehicle (Rasmussen et al., 1992). Cell wall components were also indirectly identified through Calcofluor White fluorescence, by viewing the cells in a Leitz Diavert inverted microscope with a Leitz G-filter (spectral range 350± 460 nm) or with a Zeiss epifluorescence microscope equipped with a UV filter (Zeiss no. 487702, BP 365/10 and LP 418). Guaiacol-dependent peroxidase (EC 1.11.1.7) activity was determined using standard procedures (Rasmussen et al., 1994). Flight and after-landing procedures: cultivation of protoplasts and microcallus in orbit and after retrieval. Protoplasts were grown in bags in European Space Agency (ESA) Type 1 containers in the biological facility called `Biorack' on the Space Shuttle. The test sample was left under micro-g conditions and the control sample on a 1 g control centrifuge. After defined periods of time the plant samples were prefixed for 45 min in 1% glutaraldehyde in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.8, with 0.4 M glucose, washed in the cultivation medium and stored at 48C until retrieval after landing. The living plant samples were left in Biorack in orbit, and immediately after the flight the free-floating microcalluses were transferred from plastic bags to petri dishes with fresh A medium. About 5 wk after isolation the microcalluses were sufficiently large to be transferred to solid medium (Kao and Michayluk, 1981) with agar (0.6%), 0.1 M sucrose containing 6-BA (4.4 mM) and NAA (0.5 mM). After another 2±3 wk of cultivation the calluses developed shoots, which were immediately transferred to solid MS medium containing sucrose (3%) and agar (0.6%) without any growth regulators, in order to develop roots.

TABLE 1 COMPARISON BETWEEN THE SIZE OF PROTOPLASTS MEASURED AS A RESULT OF EXPOSURE TO MICRO-g CONDITIONS Average dimensions (area) of protoplasts* Experimental conditions

IML-1² (mm2)

Ground experiments³ (mm2)

Flight, 0 g Flight, 1 g Ground, 1 g Simulated, 0 g

1064 ^ 1229 554 ^ 670 ± ±

± ± 777 ^ 370 1126 ^ 440

*Protoplasts measured as a result of exposure to micro-g conditions on the IML-1 mission, simulated 0 g on a 2-D clinostat and under normal 1 g conditions on the ground. In all experiments 1% glutaraldehyde was used as a fixative. ² Fixed after 3 d in orbit. ³ Fixed after 2 d on the ground.

Results and Discussion The results are divided into those obtained as a consequence of (i) variable periods under micro-g conditions and on a 1 g control centrifuge in space; and (ii) short periods of real and simulated weightlessness on the ground. Microgravity effects. In regeneration capacity, the size of protoplasts was apparently affected by the micro-g conditions. During the IML-1 mission one of the astronauts, Dr. Roberta Bondar, made a detailed microscopic analysis and found that after 4 d in orbit at 0 g the size of the single protoplasts was enlarged due to swelling (Rasmussen et al., 1994). A similar effect was demonstrated in protoplasts exposed to 0 g during the S/MM-03 mission (Iversen et al., 1999). More detailed figures confirming these observations are given in Table 1. Despite the increasing effect on size of the micro-g conditions, the protoplasts still showed the capacity to develop into calluses, as demonstrated in Fig. 1. However there is a clear difference in the size of calluses at the same age (3 wk) observed after retrieval on the ground. The same pattern is demonstrated 11 wk after isolation; calluses established in 0 g samples from orbit (Fig. 2A) were smaller than those on the ground (Fig. 2B), but both cultures grew well. Even though protoplasts that have experienced micro-g conditions for periods from 8±14 d are capable of establishing calluses, they have never been able to regenerate into an intact plant. Calluses with roots were developed after the IML-1 flight, whereas

Fig. 2. Calluses developed from protoplasts in both flight (A, 0 g) and ground (B) samples 11 wk after isolation of the protoplasts. Horizontal bars, 1 mm.

calluses with either roots or shoots, but never both, were obtained after the S/MM-03 flight (Iversen et al., 1999). Cell wall analyses and enzymatic activity. Regeneration of the cell wall is a prerequisite for the initial cell divisions in the establishment of microcalluses. The analysis of the cell wall compounds in rapeseed protoplasts exposed to micro-g conditions

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TABLE 4

RECONSTITUTION OF THE CELL WALL IN BRASSICA NAPUS PROTOPLASTS

DIVISION CAPACITY (PERCENTAGE OF DIVIDING CELLS) OF PROTOPLASTS

Wall components ECP Pectin HC1 HC2 Cellulose

Flight

Control

1:79 ^ 0:25 2:25 ^ 0:32 0:13 ^ 0:01 0:11 ^ 0:02 0:54 ^ 0:08

1:70 ^ 0:28 2:20 ^ 0:35 0:27 ^ 0:01 0:14 ^ 0:03 1:17 ^ 0:15

Percentage of divided cells Age of protoplasts* (d) 3 5

1 g protoplasts

0 g protoplasts

11 ^ 8 25 ^ 11

4^4 13 ^ 4

Extracellular polysaccharides (ECP), pectins, hemicellulose (HC) and cellulose were determined (given as mM [14C]glucose calculated on the basis of packed cell volume). Mean ^ SD.

*Protoplasts exposed to 3 d simulated weightlessness (0 g) on a 2-D clinostat or to simulated weightlessness for 3 d followed by 2 d cultivation under 1 g conditions (5-d-old protoplasts), compared to the respective 1 g control protoplasts. Mean ^ SD.

for 14 d on Biokosmos 9 demonstrated a significant decrease in the content of structural components, cellulose, and hemicellulose, whereas the non-structural element pectin and other extracellular components were virtually the same in the flight and ground samples (Table 2). Peroxidase activity in the regenerating protoplasts was also determined after both the Biokosmos 9 flight and the IML-1 mission. A statistical analysis of peroxidase activity was performed on the protoplasts flown on the IML-1 mission, but not on the results from Biokosmos 9. The latter results did, however, demonstrate decreased peroxidase activity (U ml21 packed cell volume; PCV) in flight samples compared to the ground controls. The opposite effect was obtained after the IML-1 flight; in this case peroxidase activity was increased in both 0 g and 1 g samples (flight) compared to the ground control experiments (Table 3). The specific activity (U mg21 protein), on the other hand, showed a slight, but not significant, decreased peroxidase activity in the flight samples, both when exposed to 0 g and in the on-board 1 g control, when compared to the ground controls. This indicates that factors other than weightlessness (such as cosmic radiation) may have caused the decreased activity. This interpretation has been confirmed by exposing protoplasts to simulated weightlessness on the 2-D clinostat for a period up to 3 d (see below; Table 3). Any differences in peroxidase isoenzyme (isoperoxidase) activity were detected in the protoplasts only after the Biokosmos 9 flight. These results demonstrated that distinct bands were missing in the flight samples compared to the ground controls when determined by isolectric focusing (see Fig. 1 of Rasmussen et al., 1992). Differences in isoperoxidase activity are found to be correlated with protoplast regeneration (Siminis et al., 1993). Based on this, the differences in isoperoxidases may be interpreted as an

indication of differences in the developmental status of the protoplasts. It should, however, be noted that a 1 g flight control was not present on board the Biokosmos satellite. Effects on the ground of simulated and real weightlessness. Regeneration capacity in experiments with protoplasts exposed to simulated weightlessness (0 g) on the 2-D clinostat also resulted in increased protoplast size, as in the 0 g flight samples (Table 1). The capacity of the protoplasts to enter division was affected by gravity; the percentage of dividing cells was reduced to less than half after 3 d g-treatment on the clinostat compared to the 1 g control (Table 4). After transferring the 0 g protoplasts to 1 g conditions for further cultivation for 2 d, the division capacity of the 0 g protoplasts was still retarded compared to the 5-d-old 1 g protoplasts (Table 4). Cell wall analysis and enzyme capacity. Protoplasts exposed to simulated weightlessness (0 g) for 3 d started the incorporation of cellulosic wall compounds at the same time as the 1 g protoplasts, but the thickness of the wall in 6-d-old 0 g protoplasts was only half that of the 1 g protoplasts at the same age (Table 5). The cellulosic wall components were detected by the use of Calcofluor White (CW) staining. These results are consistent with the results obtained after 14 d under micro-g conditions on Biokosmos 9, suggesting that the reduced wall synthesis in the micro-g protoplasts was due to the loss of weight (no 1 g centrifuge was present on board). Peroxidase activity in protoplasts exposed to simulated weightlessness was shown to be similar to the activity in the 1 g control (Table 3). In contrast, the flight samples (0 g and 1 g on-board control) had a slightly, but not significantly, decreased activity compared to the parallel ground controls (1 g and 1.4 g). The finding that peroxidase activity was not affected by simulated

TABLE 3 GUAIACOL-DEPENDENT PEROXIDASE ACTIVITY IN CRUDE EXTRACTS FROM DEVELOPING BRASSICA NAPUS PROTOPLASTS AFTER EXPOSURE TO MICRO-g AND SIMULATED WEIGHTLESSNESS ON A 2-D CLINOSTAT IML-1 Biokosmos 9

Flight

Ground

Ground experiments

Peroxidase activity

Flight

Ground

0g

1g

1g

1.4 g

Clinostat 0 g

Control 1 g

U (ml PCV)21 U (mg protein)21

15 2.36

34 2.48

60 ±

58 ±

49 2.65

50 2.73

± 0.10

± 0.10

Flight durations were 14 and 8 d for Biokosmos 9 and IML-1, respectively, whereas the simulated 0 g treatment on the 2-D clinostat was 3 d. Values from the Biokosmos 9 and IML-1 flights are from representative samples.

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SKAGEN AND IVERSEN TABLE 5

EFFECT OF DIFFERENT SIMULATED WEIGHTLESSNESS REGIMES* ON THE PERCENTAGE OF PROTOPLASTS WITH CALCOFLUOR WHITESTAINED CELLULOSIC COMPOUNDS Wall thickness² (mm)

Protoplasts with CW staining (%) Age of protoplasts (d) 3 6

1g

0g

1g

0g

77 ^ 12 89 ^ 5

79 ^ 6 80 ^ 2

± 1:6 ^ 0:4

± 0:8 ^ 0:2

*Treatments: 3 d with 0 g treatment on a 2-D clinostat; and after an additional 3 d cultivation under 1 g conditions (6 d in total); compared to protoplasts exposed to 1 g during the whole test period. ² Wall thickness was measured in the 6-d-old protoplasts by estimation of the diameter of the CW fluorescence on the protoplast's perimeter. Mean ^ SD.

Fig. 3. Representative pictures at the same magnification of freshly isolated rapeseed protoplasts with randomly oriented CMTs (A) and CMTs organized in parallel pattern (B). Horizontal bar, 10 mm.

weightlessness supports the interpretation that other environmental factors had an influence on the samples in space. Peroxidases are enzymes that play a key role in cross-binding of microfibrils in the cell wall, and their activities are claimed to be a prerequisite for the reconstitution of the cell wall in protoplasts (de Marco and Roubelakis-Angelakis, 1996). Peroxidases are located in

Fig. 4. Quantity of CMTs in individual protoplasts after 0, 24 and 48 h exposure to simulated weightlessness (0 g) on a 2-D clinostat. Vertical bars ^ SD.

the cytosol or connected to the wall. Since the reconstitution of the cell wall in protoplasts cultivated under micro-g conditions and simulated weightlessness was retarded, whereas the peroxidase activity was not significantly affected, the use of guaiacoldependent peroxidase activity in crude extracts as an early biochemical marker of wall development was not successful, at least not after 8 d. Corticular microtubules and initial cell regeneration. In an attempt to explain the lack of regeneration capacity in protoplasts exposed to micro-g, extensive experiments on the importance of CMTs have been performed on the ground. The quantity and organization of CMTs (Fig. 3) were observed after the protoplasts had been exposed to short periods of real and simulated weightlessness. Figure 4 shows the effect of gravity on the quantity of CMTs in individual protoplasts. The quantity expressed as CMT (mm2) was significantly …P , 0:05† increased in the 1 g protoplasts after 24 h compared to the CMT quantity in protoplasts immediately after isolation, whereas the CMT quantity in 0 g protoplasts was stable. After 48 h of g-treatment the CMT quantity in the 1 g protoplasts decreased again to the same level as the 0 g protoplasts (Fig. 4). All these tests were performed with protoplasts in a hormone-free medium which, over a prolonged stay, may result in depolymerization of CMTs. The low amount of CMTs in 0 g protoplasts after 24 h indicates a low level of repolymerization. The synthesis of cellulose has been shown to occur only in areas of protoplasts with a high density of CMTs (Meijer and Simmonds, 1988). Based on this, the low CMT quantity in 0 g protoplasts after 24 h could be a possible explanation for the reduced amount of cellulosic compounds found in the 0 g protoplasts after 6 d (Fig. 4; Table 5) and in the protoplasts exposed to micro-g (Table 2). Protoplasts exposed to simulated weightlessness (on a 3-D clinostat) or real weightlessness (on the FFM; see Mesland et al., 1996) had randomly oriented CMTs (,50%) after 48 h g-treatment. The CMTs in the stationary 1 g protoplasts and those exposed to 1 g on the FFM centrifuge were, in contrast, organized in parallel arrays (Fig. 5). The removal of the cell wall during protoplast isolation from mature tissue results to some extent in protoplasts with randomly oriented CMTs, whereas the organization of CMTs in the tissue is highly ordered. During protoplast regeneration, the CMT pattern reorganizes the randomly oriented CMTs to a parallel alignment (Hasezawa et al., 1988; Melan, 1990; Kuss-Wymer and Cyr, 1992). The CMTs are believed to act as templates for cellulose microfibrils

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PROTOPLASTS UNDER WEIGHTLESSNESS

Fig. 5. (A) Organization of CMTs in protoplasts exposed to simulated weightlessness on 2-D and 3-D clinostats, and in protoplasts exposed to cycles of real weightlessness on a free-fall machine (FFM) compared to protoplasts exposed to 1 g (48 h exposure time). (B) CMT organization in protoplasts exposed to 1 g on a centrifuge in the FFM (FFM 1 g) and a stationary 1 g control for 24 h.

deposited in the regenerating cell wall (Giddings and Staehelin, 1991; Kropf et al., 1998). Dijak and Simmonds (1988) found that the division pattern in protoplasts with randomly organized CMTs 2 d after isolation was disturbed compared to the protoplasts which had retained a parallel CMT array. The findings described here, that protoplasts exposed to various forms of simulated and real weightlessness on the ground had randomized CMTs, could therefore indicate that the low division capacity in 0 g protoplasts was correlated with the randomized CMT in these protoplasts (Fig. 5; Table 4). Conclusions A clear effect of exposing rapeseed protoplasts to a micro-g environment has been increased cell size. A similar effect was observed when protoplasts were exposed to simulated weightlessness on a 2-D clinostat (Table 1). A reduced regeneration capacity, with calluses making only shoots or roots, was also a general characteristic of the micro-g-exposed protoplasts, resulting in a failure to regenerate an intact plant (Figs. 1 and 2). The

regeneration ability is correlated with cell wall formation, and the low content of structural wall components in the flight samples (Biokosmos 9; Table 2) was suggested to be a result of decreased cell wall formation in orbit. As a 1 g control centrifuge was lacking on board the Biokosmos satellite, it was not possible to determine whether the reduced cellulose and hemicellulose content is an effect of 0 g or of other micro-g factors such as cosmic radiation. However, the results obtained on the ground demonstrating a reduced production of cellulosic compounds in protoplasts exposed to simulated weightlessness on a 2-D clinostat suggest that retarded wall regeneration in flight samples is due to weightlessness in orbit. Peroxidase activity has been claimed to be a prerequisite for the reconstitution of the cell wall in regenerating protoplasts (de Marco and Roubelakis-Angelakis, 1996). In addition, the total activity of peroxidases has been demonstrated to be higher in differentiating protoplast cultures than in recalcitrant protoplasts (Siminis et al., 1993; de Marco and Roubelakis-Angelakis, 1996). The effect of micro-g on guaiacol-dependent peroxidase activity in crude extracts from the rapeseed protoplasts is discussed in the following with respect to specific activity (U mg21 protein), as the use of catabolic units per ml PCV to indicate peroxidase activity is considered to be less reproducible. There were no significant differences between the micro-g samples and ground controls, but both the 0 g and 1 g flight samples had a slightly decreased guaiacol-dependent peroxidase activity compared to the controls on the ground (Table 3). However, the fact that the 1 g flight control also had reduced peroxidase activity indicates that conditions other than 0 g gave the reduced activity observed. This suggestion is supported by the results obtained using the 2-D clinostat to gain simulated 0 g, where no differences between 0 g and 1 g were detected after 3 d of g-treatment. Differences in peroxidase activity after further gtreatment on the clinostat cannot be excluded. Attempts have been made to link the observed effects of micro-g conditions, simulated weightlessness and 1 g with the protoplast CMT characteristics. The results indicate that the quantity and quality of CMTs are clearly g-dependent, and may be correlated to a low level of repolymerization which may cause a low level of cellulose synthesis and a randomized CMT array which, in turn, could lead to abnormalities in the cell division. The present research group will, as a follow-up of these results, approach ESA with an experimental set-up for the ISS. The focus will be on cell wall biogenesis in protoplasts and intact roots of Arabidopsis thaliana exposed to hypo-g (real weightlessness; 3-D clinostats) and hyper-g (centrifuges). By observing mRNA and using microarrays, the expression of a substantial number of genes can be followed as a consequence of variable g-exposures. Of special interest will be the different touch genes (Braam et al., 1996) which have been found to be involved in cell wall biogenesis.

Acknowledgments We would like to thank Prof. Anders Johnsson and his group at the Department of Physics, Norwegian University of Science and Technology for the use of the hyper-g centrifuge, and Dr. Augusto Cogoli and the rest of the Space Biology Team at the ETH Technopark in Zurich, Switzerland, for use of the 3-D clinostat and the FFM.

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