Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.     

GraPhoBox: Gravitropism and phototropism inArabidopsis thaliana

The morphology of plants is directed by the directional growth of roots and shoots. Gravity and light direction are the two major environmental stimuli important for directional growth. The ’GraPhoBox’ experiment, flown on the Dutch DELTA mission to the ISS in April 2004, tries to elucidate the different effects of gravitropism and phototropism on plants, and their combined effects on plant morphology. Wild-type Arabidopsis thaliana (L.), phototropic-deficient mutants phot1 and gravitropic-deficient mutant pgm1 seeds were germinated in microgravity and in Earth gravity, in low light conditions and darkness. The angle of directional growth of roots and shoots was then assessed. Light is -even in the absense of gravity- the most important environmental cue for directional growth of shoots, while for roots gravity is by far the most important cue, and light is only a very minor factor due to their poor phototropic capacity. Compared to roots, shoots are deviated more than roots in microgravity and therefore less gravity-dependent. All results together suggests that environmental cues are differently percepted by roots and shoots which also adapt differently. Furthermore, environmental cues are probably transferred little or not to the opposite side of the plant.     

Germination of Arabidopsis Seed in Space and in Simulated Microgravity: Alterations in Root Cell Growth and Proliferation

Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions.     

The Impact of Simulated Microgravity on the Growth of Different Genotypes of the Model Legume Plant <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Simulated microgravity has been a useful tool to help understand plant development in altered gravity conditions. Thirty-one genotypes of the legume plant Medicago truncatula were grown in either simulated microgravity on a rotating clinostat, or in a static, vertical environment. Twenty morphological features were measured and compared between these two gravity treatments. Within-species genotypic variation was a significant predictor of the phenotypic response to gravity treatment in 100% of the measured morphological and growth features. In addition, there was a genotype–environment interaction (G × E) for 45% of the response variables, including shoot relative growth rate (p <?0.0005), median number of roots (p ～ 0.02), and root dry mass (p <?0.005). Our studies demonstrate that genotype does play a significant role in M. truncatula morphology and affects the response of plants to the gravity treatment, influencing both the magnitude and direction of the gravity response. These findings are discussed in the context of improving future studies in plant space biology by controlling for genotypic differences. Thus, manipulation of genotype effects, in combination with M. truncatula’s symbiotic relationships with bacteria and fungi, will be important for optimizing legumes for cultivation on long-term space missions.     

Flowering in space

The reproductive success of plants is often dependent on their flowering time being adapted to the terrestrial environment, in which gravity remain constant. Whether plants can follow the same rule to determine their flowering time under microgravity in space is unknown. Although numerous attempts have been made to grow a plant through a complete life cycle in space, apparently no published information exists concerning the flowering control of plants under microgravity in space. Here, we focused on two aspects. Firstly the environmental and intrinsic factors under microgravity related to flowering control. Secondly, the plant-derived regulators are involved in flowering control under microgravity condition. The potential environmental and intrinsic factors affect plant flowering under microgravity may include light, biological circadian clock as well as long-distance signaling, while the plant-derived flowering regulators in response to microgravity could include gibberellic acid, ethylene, microRNA and sugar. The results we have obtained from the space experiments on board the Chinese recoverable satellites (the SJ-8 and the SJ-10) and the experiment on the Chinese space lab TG-2 are also introduced. We conclude by suggesting that long-term space experiments from successive generations and a systematic analysis of regulatory networks at the molecular level is needed to understand the mechanism of plant flowering control under microgravity conditions in space.     

Facilities for Simulation of Microgravity in the ESA Ground-Based Facility Programme

Knowledge of the role of gravity in fundamental biological processes and, consequently, the impact of exposure to microgravity conditions provide insight into the basics of the development of life as well as enabling long-term space exploration missions. However, experimentation in real microgravity is expensive and scarcely available; thus, a variety of platforms have been developed to provide, on Earth, an experimental condition comparable to real microgravity. With the aim of simulating microgravity conditions, different ground-based facilities (GBF) have been constructed such as clinostats and random positioning machines as well as magnets for magnetic levitation. Here, we give an overview of ground-based facilities for the simulation of microgravity which were used in the frame of an ESA ground-based research programme dedicated to providing scientists access to these experimental capabilities in order to prepare their space experiments.     

The effect of simulated microgravity on bacteria from the mir space station

The effects of simulated microgravity on two bacterial isolates, Sphingobacterium thalpophilium and Ralstonia pickettii (formerly Burkholderia pickettii), originally recovered from water systems aboard the Mir space station were examined. These bacteria were inoculated into water, high and low concentrations of nutrient broth and subjected to simulated microgravity conditions. S. thalpophilium (which was motile and had flagella) showed no significant differences between simulated microgravity and the normal gravity control regardless of the method of enumeration and medium. In contrast, for R. pickettii (that was non-motile and lacked flagella), there were significantly higher numbers in high nutrient broth under simulated microgravity compared to normal gravity. Conversely, when R. pikkettii was inoculated into water (i.e., starvation conditions) significantly lower numbers were found under simulated microgravity compared to normal gravity. Responses to microgravity depended on the strain used (e.g., the motile strain exhibited no response to microgravity, while the non-motile strain did), the method of enumeration, and the nutrient concentration of the medium. Under oligotrophic conditions, non-motile cells may remain in geostationary orbit and deplete nutrients in their vicinity, while in high nutrient medium, resources surrounding the cell may be sufficient so that high growth is observed until nutrients becoming limiting.     

Live Imaging Technique for Studies of Growth and Development of Chinese Cabbage Under Microgravity in a Recoverable Satellite (SJ-8)

The live imaging techniques have been developed and applied to investigate for the first time the growth and development of Chinese cabbage for 18 days under microgravity conditions on board the Chinese SJ-8 recoverable satellite. These experiments offer insight into plant behaviors operating during plant development in space. Two automatic, preprogrammed CCD cameras were installed in the plant experimental chamber. The experimental objectives were: (1) seed germination; (2) seedling growth; (3) flower opening and pollination. The growth of seedlings and flowers were followed by time lapse photography at 2 h intervals. Serial real-time images of the Chinese cabbage plant growth under microgravity were successfully obtained through the remote operating system. The image data obtained from space experiment, in comparison with the results from ground control (1 g) and 3D clinostat stimulate experiments, showed that the height of plant and the number of leaves were significantly reduced under the microgravity conditions, but characters of leaf arrangement and leaf shape were not altered obviously. Flower opening and expansion were inhibited by exposed to space flight condition. The petals of flowers from both SJ-8 grown plants and clinostat rotated plants couldn’t fully expand before wilted. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Vegetative growth of higher plants on a three-dimensional clinostat

Seedlings of rice, maize, cress, pea, and azuki bean were grown on a three-dimensional clinostat and changes in their vegetative growth processes were analyzed. A balanced relationship among the length or the weight of each organ was observed in these species even on the clinostat. Growth of pea second internodes is supported by the transport of sugars from the cotyledons, which was not influenced by the clinostat rotation. Thus, growth correlation and the translocation of sugars normally occurred even under simulated microgravity conditions. In contrast, morphogenesis was clearly changed by the clinostat rotation. The axiality along the gravity vector disappeared and so seedlings formed themselves into a sphere-like shape on the clinostat. The dorsiventrality was indistinct in growth of maize coleoptiles on the surface of the earth, but the clinostat rotation induced a clear dorsinventral bending. These changes in morphogenesis may influence the long-term growth phenomena and modify the life cycle of higher plants under a microgravity environment.     

Seed Germination and Seedling Growth under Simulated Microgravity Causes Alterations in Plant Cell Proliferation and Ribosome Biogenesis

The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception.     

Photoperiod-controlling Guttation and Growth of Rice Seedlings Under Microgravity on Board Chinese Spacelab TG-2

Guttation has been shown to play a crucial role in controlling plant growth and development by involvement of the transport of water, but how does this water transport in plant from roots to leaves work against pull of gravity is still unknown. The aim of the present study was to evaluate the influence of microgravity on photoperiod-controlling guttation and growth of rice (Oryza sativa L.) seedlings on board the Chinese Spacelab TongGong-2(TG-2). The growth rate of rice seedlings was closely correlated with guttation under both the microgravity and the ground conditions, which was increased by microgravity under both a 16-h long-day (LD) and an 8-h short-day (SD) photoperiod conditions. In addition, guttation of the TG-2 grown rice under the LD condition was more significant in comparison with that under the SD condition. These results indicated that microgravity affected the photoperiod-controlling growth of rice seedlings could be related to the enhanced guttation in space.     

Effects of Simulated Microgravity on Otolith Growth of Larval Zebrafish using a Rotating-Wall Vessel: Appropriate Rotation Speed and Fish Developmental Stage

Stimulus dependence is a general feature of developing animal sensory systems. In this respect, it has extensively been shown earlier that fish inner ear otoliths can act as test masses as their growth is strongly affected by altered gravity such as hypergravity obtained using centrifuges, by (real) microgravity achieved during spaceflight or by simulated microgravity using a ground-based facility. Since flight opportunities are scarce, ground-based simulators of microgravity, using a wide variety of physical principles, have been developed to overcome this shortcoming. Not all of them, however, are equally well suited to provide functional weightlessness from the perspective of the biosystem under evaluation. Therefore, the range of applicability of a particular simulator has to be extensively tested. Earlier, we have shown that a Rotating-Wall Vessel (RWV) can be used to provide simulated microgravity for developing Zebrafish regarding the effect of rotation on otolith development. In the present study, we wanted to find the most effective speed of rotation and identify the appropriate developmental stage of Zebrafish, where effects are the largest, in order to provide a methodological basis for future in-depth analyses dedicated to the physiological processes underlying otolith growth at altered gravity. Last not least, we compared data on the effect of simulated microgravity on the size versus the weight of otoliths, since the size usually is measured in related studies due to convenience, but the weight more accurately approximates the physical capacity of an otolith. Maintaining embryos at 10 hours post fertilization for three days in the RWV, we found that 15 revolutions per minute (rpm) yielded the strongest effects on otolith growth. Maintenance of Zebrafish staged at 10 hpf, 1 day post fertilization (dpf), 4 dpf, 7 dpf and 14 dpf for three days at 15 rpm resulted in the most prominent effects in 7 dpf larvae. Weighing versus measuring the size of otoliths yielded basically similar results, but the data gained by weighing were more distinct. Overall, our results clearly support the concept that the environmental gravity vector regulates fish otolith growth in terms of the pendulum model of otolith test masses, and that wall vessel rotation is a valuable means to provide functional weightlessness from the perspective of developing Zebrafish. We recommend that Zebrafish embryos staged 7 dpf (or possibly slightly elder) are rotated at 15 rpm in a Rotating-Wall Vessel as used in the present study for further experiments designed to elucidate the mechanisms underlying (altered gravity affected) otolith growth.     

Actin dynamics in mouse fibroblasts in microgravity

After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.     

Effect of convective disturbances induced by g-jitter on the periodic precipitation of lysozyme

Numerical simulations are carried out to investigate the crystallization process of a protein macromolecular substance under two different conditions: pure diffusive regime and microgravity conditions present on space laboratories. The configuration under investigation consists of a protein reactor and a salt chamber separated by an “interface”. The interface is strictly related to the presence of agarose gel in one of the two chambers. Sedimentation and convection under normal gravity conditions are prevented by the use of gel in the protein chamber (pure diffusive regime). Under microgravity conditions periodic time-dependent accelerations (g-jitter) are taken into account. Novel mathematical models are introduced to simulate the complex phenomena related to protein nucleation and further precipitation (or resolution) according to the concentration distribution and in particular to simulate the motion of the crystals due to g-jitter in the microgravity environment. The numerical results show that gellified lysozyme (crystals “locked” on the matrix of agarose gel) precipitates to produce “spaced deposits”. The crystal formation results modulated in time and in space (Liesegang patterns), due to the non-linear interplay among transport, crystal nucleation and growth. The propagation of the nucleation front is characterized by a wavelike behaviour. In microgravity conditions (without gel), g-jitter effects act modifying the phenomena with respect to the on ground gellified configuration. The role played by the direction of the applied sinusoidal acceleration with respect to the imposed concentration gradient (parallel or perpendicular) is investigated. It has a strong influence on the dynamic behaviour of the depletion zones and on the spatial distribution of the crystals. Accordingly the possibility to obtain better crystals for diffraction analyses is discussed.     

Simulating Parabolic Flight like <Emphasis Type="Italic">g</Emphasis>-Profiles on Ground - A Combination of Centrifuge and Clinostat

Clinostats and centrifuges are widely used to create simulated microgravity or hypergravity, respectively, in order to study the impact of gravity on biosystems. Here, we used a clinostat and a centrifuge in alternating modes of operation in order to create a simulated parabolic flight like g-profile. To our knowledge, it is the first time that both devices were run in connection. In order to test the method, we investigated the production of reactive oxygen species of immune cells (macrophages) during oxidative burst in an on-line kinetic approach, which has been extensively studied under real (parabolic flight) and simulated microgravity (clinostat) as well as under hypergravity conditions (centrifuge). Our results indicate that clinostat and centrifuge can be operated in an alternating way to simulate the repetitive changes of gravity during parabolic flight. Although the switch from one gravity level to the other could not be carried out as quickly as it takes place during actual parabolic flight due to technical and operational reasons, it can be concluded that running experiments in a clinostat aboard a centrifuge on ground are suitable for studying gravity-related phenomena.     

Fish Inner Ear Otolith Growth Under Real Microgravity (Spaceflight) and Clinorotation

Using late larval stages of cichlid fish (Oreochromis mossambicus) we have shown earlier that the biomineralization of otoliths is adjusted towards gravity by means of a neurally guided feedback loop. Centrifuge experiments, e.g., revealed that increased gravity slows down otolith growth. Microgravity thus should yield an opposite effect, i.e., larger than normal otoliths. Consequently, late larval cichlids (stage 14, vestibular system operational) were subjected to real microgravity during the 12 days FOTON-M3 spaceflight mission (OMEGAHAB-hardware). Controls were kept at 1g on ground within an identical hardware. Animals of another batch were subsequently clinorotated within a submersed fast-rotating clinostat with one axis of rotation (2d-clinostat), a device regarded to simulate microgravity. Temperature and light conditions were provided in analogy to the spaceflight experiment. Controls were maintained at 1g within the same aquarium. After all experiments, animals had reached late stage 21 (fish can swim freely). Maintenance under real microgravity during spaceflight resulted in significantly larger than normal otoliths (both lapilli and sagittae, involved in sensing gravity and the hearing process, respectively). This result is fully in line with an earlier spaceflight study in the course of which otoliths from late-staged swordtails Xiphophorus helleri were analyzed. Clinorotation resulted in larger than 1g sagittae. However, no effect on lapilli was obtained. Possibly, an effect was present but too light to be measurable. Overall, spaceflight obviously induces an adaptation of otolith growth, whereas clinorotation does not fully mimic conditions of microgravity regarding late larval cichlids.     

Effects of Gravity on Ignition and Combustion Characteristics of Externally Heated Polyethylene Film

The objective of this research is to investigate the effects of gravity on the ignition and the combustion characteristics of the Polyethylene (PE) film by outer heating. Combustion experiments of PE film were carried out in a normal gravity field and the microgravity field. In the microgravity experiments, it was carried out in 50 m-class drop facility. Here it can be realized 10^??4G microgravity field in about 2.5-3.0 second. The PE film is heated by the inserted high-temperature chamber. In the experiments, the PE was used film type. The chamber temperature was fixed at 900 K and 1000 K. In the case of microgravity field, the ignition delay period has become about 50 percent shorter than that in the case of the normal gravitational field. In the normal gravity field, since the PE surface layer is cooled by natural convection, the ignition delay period is considered to be longer than that in the microgravity field. The combustion time in the normal gravity was about 0.8 sec. In the microgravity field, the combustion time was more than 2 sec, and it could not be measured during the free fall period.     

Embedding Arabidopsis Plant Cell Suspensions in Low-Melting Agarose Facilitates Altered Gravity Studies

Gravity plays a role in modulating plant growth and development and its alteration induces changes in these processes. Microgravity research has recently been extended to the use of in vitro plant cell cultures which are considered as an ideal model system to study cell proliferation and growth. In general, among the ground-based facilities available for microgravity simulation, the 2D pipette clinostat had been previously considered a suitable facility to be used for unicellular biological models although studies using single plant cell cultures raised some concerns. The incompatibility comes from the standard requirement of shaking a suspension culture for assuring its viability and active proliferation status in the control samples. Moreover, a related issue applies to the use of the random positioning machine (RPM) for cell suspension experiments. Here, we demonstrate an alternative culture method based on the immobilization of the culture before the altered gravity treatment occurs, such that it behaves as a solid object. Our immobilization procedure preserved plant cell culture viability without compromising basic cell properties as viability, morphology, cell cycle phases distribution, or chromatin organization, when compared with a standard cell suspension under shaking as a control. This approach should allow the space biology community to improve the quantity and quality of plant cell results in future simulated microgravity experiments or spaceflight opportunities.     

Thyroid Cells Exposed to Simulated Microgravity Conditions – Comparison of the Fast Rotating Clinostat and the Random Positioning Machine

The ground-based facilities 2D clinostat (CN) and Random Positioning Machine (RPM) were designed to simulate microgravity conditions on Earth. With support of the CORA-ESA-GBF program we could use both facilities to investigate the impact of simulated microgravity on normal and malignant thyroid cells. In this review we report about the current knowledge of thyroid cancer cells and normal thyrocytes grown under altered gravity conditions with a special focus on growth behaviour, changes in the gene expression pattern and protein content, as well as on altered secretion behaviour of the cells. We reviewed data obtained from normal thyrocytes and cell lines (two poorly differentiated follicular thyroid cancer cell lines FTC-133 and ML-1, as well as the normal thyroid cell lines Nthy-ori 3-1 and HTU-5). Thyroid cells cultured under conditions of simulated microgravity (RPM and CN) and in Space showed similar changes with respect to spheroid formation. In static 1g control cultures no spheroids were detectable. Changes in the regulation of cytokines are discussed to be involved in MCS (multicellular spheroids) formation. The ESA-GBF program helps the scientists to prepare future spaceflight experiments and furthermore, it might help to identify targets for drug therapy against thyroid cancer.     

不同重力环境对AlN-玻璃复相陶瓷显微结构的影响

采用SHS化学炉为热源,在抛物线飞行飞机上进行了一系列不同重力环境下的A1N－玻璃复相材料的液相烧结,研究了烧结过程中,不同重力水平对材料显微结构和扩散行为的影响。研究结果表明：超重力条件下物相会发生偏析,而策重力条件下,物质扩散均匀,可促进晶粒的正常发育,有利于材料显微结构的均匀性。