Possible actions of gravity on the cellular machinery.

Since the first flight of the ESA Biorack on the German Spacelab Mission D1 in 1985 evidence has been obtained that biological cells and small unicellular organisms function differently under conditions of microgravity. However, there is still lack of scientific proof that these effects are caused by a direct influence on the cells in the weightlessness condition. The question how normal gravity may play a role in cellular activity is being addressed and the results show that gravity may provide important signals during certain state transitions in the cell. These would be gravity-sensitive windows in the biological process. Also, by amplification mechanisms inside the cell, the cell may assume a state that is typical for normal gravity conditions and would change in microgravity. Experimental tools are discussed that would provide the conditions to obtain evidence for direct action of gravity and for the possible existence of gravity-sensitive windows.     

Gravitational force regulates elongation growth of Arabidopsis hypocotyls by modifying xyloglucan metabolism.

Growth of dark-grown Arabidopsis hypocotyls was suppressed under hypergravity conditions (300 g), or was stimulated under microgravity conditions in space (Space Shuttle STS-95). The mechanical extensibility of cell walls decreased and increased under hypergravity and microgravity conditions, respectively. The amounts of cell wall polysaccharides (pectin, hemicellulose-I, hemicellulose-II and cellulose) per unit length of hypocotyls increased under hypergravity conditions, and decreased under microgravity conditions. The amount and the molecular mass of xyloglucans also increased under the hypergravity conditions, while those decreased under microgravity conditions. The activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls decreased and increased under hypergravity and microgravity conditions, respectively. These results indicate that the amount and the molecular mass of xyloglucans are affected by the magnitude of gravity and that such changes are caused by changes in xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell walls by gravity stimulus may be the primary event determining the cell wall extensibility, thereby regulating the growth rate of Arabidopsis hypocotyls.     

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.     

Structural and functional organisation of regenerated plant protoplasts exposed to microgravity on Biokosmos 9.

Preparatory experiments for the IML-1 mission using plant protoplasts, were flown on a 14-day flight on Biokosmos 9 in September 1989. Thirty-six hours before launch of the biosatellite, protoplasts were isolated from hypocotyl cells of rapeseed (Brassica napus) and suspension cultures of carrot (Daucus carota). Ultrastructural and fluorescence analysis of cell aggregates from these protoplasts, cultured under microgravity conditions, have been performed. In the flight samples as well as in the ground controls, a portion of the total number of protoplasts regenerated cell walls. The processes of cell differentiation and proliferation under micro-g did not differ significantly from those under normal gravity conditions. However, in micro-g differences were observed in the ultrastructure of some organelles such as plastids and mitochondria. There was also an increase in the frequency of the occurrence of folds formed by the plasmalemma together with an increase in the degree of complexity of these folds. In cell cultures developed under micro-g conditions, the calcium content tends to decrease, compared to the ground control. Different aspects of using isolated protoplasts for clarifying the mechanisms of biological effects of microgravity are discussed.     

The role of gravity in the evolutionary emergence of multicellular complexity: Microgravity effects on arthropod development and aging

While experiments carried out in Space with isolated cells have shown that eucaryotic cells are able to sense and respond to the absence of gravity by modifying their reactions, experiments in which more complex processes have been investigated, such as Biological Systems undergoing development under Microgravity, have been surprisingly unaffected by the space environment. This can be considered a curious result since all organisms are evolutionarily adapted to the current level of the gravity force in our planet and should eventually change if this parameter will vary in a permanent manner. In fact, the small effects of the modifications in gravity on development in short term experiments may be equivalent to the difficulties in detecting the involvement of other basic physical processes such as diffusion-controled auto-organizative reactions in currently developing biological systems. An apparent exception to this lack of effect is experiments where brine shrimp dormant gastrulae directly exposed to the space environment accumulate developmental defects as a consequence of cosmic irradiation. In this article we discuss the idea that at a certain stage during the evolutionary emergence of multicellular organisms the cues laid by generic forces such as gravity were involved in the evolutionary organization of these primitive organisms. As evolution proceed, these early mechanisms may have been obscured and/or made redundant by the appearance of new internal, environment-independent biological regulatory mechanisms. On the other hand, behavioral responses that may be important, for example, in setting the life-spans of organisms may still be more readily susceptible to manipulation by external cues as experiments carried out by our group in Space and on the ground with Drosophila melanogaster indicate.     

Experiments with air-dried seeds of Arabidopsis thaliana (L) Heynh. and Crepis capillaris (L) Wallr., aboard Salyut 6.

Space flight factors resulted in the accumulation of genetic damage in embryonic meristem cells of seeds of Arabidopsis thaliana and Crepis capillaris in flights of different duration (49, 226, 408 and 827 days) aboard the orbital station Salyut 6. As a result, the viability of seeds and seedlings was reduced, and the sterility of plants grown from seeds exposed on Salyut 6 was increased. The effect depended upon the flight duration. The data obtained suggest an acceleration of seed aging under flight conditions.     

微重力下胶原蛋白纤维化及羟基磷灰石结晶的初步研究

在长期空间飞行过程中, 骨质丢失是一个严重问题. 羟基磷灰石(HAP)晶体是骨骼的主要成分, 骨骼中的胶原蛋白纤维在HAP生长结晶过程中起到关键作用. 研究了胶原蛋白纤维化过程在模拟微重力和常重力条件下的变化, 对以胶原 蛋白纤维作为模板生长出的HAP晶体形貌进行了观察. 结果表明, 不同浓度胶原蛋白溶液中形成的胶原蛋白纤维, 其内部孔隙数量和尺寸在模拟微重力条件下要明显大于常重力条件下, 胶原蛋白纤维内部孔隙的分布也不同于常重力条 件下的结果. 以模拟微重力条件下形成的胶原蛋白纤维为模板生长出的HAP 晶体主要为立方体状, 而以常重力条件下形成的胶原蛋白纤维为模板生长出的 HAP晶体形貌主要为板状. 该结果有助于未来进一步阐明空间骨质丢失的机理.      

Ontogeny of plants under various gravity condition.

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.     

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, --> ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.     

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.     

微重力对拟南芥长势影响的代谢网络流平衡分析

微重力作为典型的空间环境因素,对植物生长发育的影响机制是空间生命科学的研究热点。微重力环境直接或间接影响植物代谢,并引起许多生理适应。 随着系统生物学的发展,代谢网络模型使微重力环境下的植物代谢建模成为可能。采用流平衡分析方法对模式植物拟南芥不同组织的代谢网络进行分析,研究微重力对拟南芥生长发育的影响机制。通过比较空间与地面条件下拟南芥的生物质产量,发现空间条件下拟南芥黄化幼苗、幼苗、芽、根、下胚轴的生物量分别下降了33.00%,51.52%,6.89%,12.53%,11.70%,与空间环境下拟南芥的长势变化趋势一致。代谢通路富集分析发现,微重力使得拟南芥的碳固定等通路下调,而磷酸戊糖途径上调,初步解析了微重力对拟南芥生长发育的影响机制,也验证了流平衡方法用于微重力生物学效应研究中的可行性。      

Space flight, microgravity, stress, and immune responses.

Exposure of animals and humans to space flight conditions has resulted in numerous alterations in immunological parameters. Decreases in lymphocyte blastogenesis, cytokine production, and natural killer cell activity have all been reported after space flight. Alterations in leukocyte subset distribution have also been reported after flight of humans and animals in space. The relative contribution of microgravity conditions and stress to the observed results has not been established. Antiorthostatic, hypokinetic, hypodynamic, suspension of rodents and chronic head-down tilt bed-rest of humans have been used to model effects of microgravity on immune responses. After use of these models, some effects of space flight on immune responses, such as decreases in cytokine function, were observed, but others, such as alterations in leukocyte subset distribution, were not observed. These results suggest that stresses that occur during space flight could combine with microgravity conditions in inducing the changes seen in immune responses after space flight. The biological/biomedical significance of space flight induced changes in immune parameters remains to be established. Grant Numbers: NCC2-859, NAG2-933.     

Seed-to-seed growth of Arabidopsis thaliana on the International Space Station.

The assembly of the International Space Station (ISS) as a permanent experimental outpost has provided the opportunity for quality plant research in space. To take advantage of this orbital laboratory, engineers and scientists at the Wisconsin Center for Space Automation and Robotics (WCSAR), University of Wisconsin-Madison, developed a plant growth facility capable of supporting plant growth in the microgravity environment. Utilizing this Advanced Astroculture (ADVASC) plant growth facility, an experiment was conducted with the objective to grow Arabidopsis thaliana plants from seed-to-seed on the ISS. Dry Arabidopsis seeds were anchored in the root tray of the ADVASC growth chamber. These seeds were successfully germinated from May 10 until the end of June 2001. Arabidopsis plants grew and completed a full life cycle in microgravity. This experiment demonstrated that ADVASC is capable of providing environment conditions suitable for plant growth and development in microgravity. The normal progression through the life cycle, as well as the postflight morphometric analyses, demonstrate that Arabidopsis thaliana does not require the presence of gravity for growth and development.     

Radiosensibility of higher plant seeds after space flight.

The influence of long-term storage of higher plant seeds under space flight conditions (49 to 827 days) on their radiosensibility was studied in the experiments on the orbital stations Salyut 6 and 7. Short-term storage has been proved to have no effect on radiosensitivity of Crepis capillaris seeds. Only in the case of maximal exposure duration the frequency of chromosome aberrations in post-flight irradiated seeds significantly exceeded the chromosome aberration frequency in the ground-based irradiated control. A statistically significant increase in the number of cells with multiple chromosome aberrations was also observed in this experiment. After gamma-irradiation of Arabidopsis thaliana seeds the germinating ability and survival rate of plants decreased depending on the duration of seed storage. Flight-exposed seeds were more sensitive to irradiations with respect to these parameters. A statistically significant increase in the frequency of recessive lethal mutations was observed only in two experiments of long exposure duration.     

Microtubule self-organisation depends upon gravity.

The molecular processes by which gravity is transduced into biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical structures do not depend upon gravity. It has been proposed that biological systems might show a gravity dependence by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. We have found that in-vitro preparations of microtubules, an important element of the cellular cytoskeleton, show this type of behaviour. On earth, the solutions show macroscopic self-ordering, and the morphology of the structures that form depend upon the orientation of the sample with respect to gravity at a critical moment at an early stage in the development of the self-organised state. An experiment carried out in a sounding rocket, showed that as predicted by theories of this type, no self-organisation occurs when the microtubules are assembled under low gravity conditions. This is an experimental demonstration of how a very simple biochemical system, containing only two molecules, can be gravity sensitive. At a molecular level this behaviour results from an interaction of gravity with macroscopic concentration and density fluctuations that arise from the processes of microtubule contraction and elongation.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

Modification of plant growth and development by acceleration and vibration: concerns and opportunities for plant experimentation in orbiting spacecraft.

Growth, development, and orientation of higher plants is altered by physical disturbances such as shaking, touching, or vibration. Plant growth responses to thigmic (contact rubbing) forces are almost always negative, whereas growth responses to periodic seismic (shaking) or vibric (vibrational) disturbances may be positive or negative, depending on intensity and duration of force, and prevailing environmental conditions. Seedlings are most sensitive to mechanical stress when grown in darkness or under the low-light conditions typically available in plant flight hardware. Brief exposure to physical perturbation causes immediate growth inhibition of dark-grown seedlings followed by gradual recovery of growth rate beginning 10-12 minutes later. For mild vibration, growth rate may overshoot that of undisturbed control plants within an hour of a stress episode, whereas for thigmic stress recovery may remain incomplete for 24 hours or longer. Lack of physical stimulation by gravity should make plants even more responsive to random physical perturbation. Threshold growth response of seedlings to vibrational parameters needs to be determined under real spaceflight conditions.     

Thin film bioreactors in space.

Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization and cellular differentiation. Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers us an opportunity to learn more about basic biological systems with one important variable removed. The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions. Studies on cell cultures grown in microgravity would enable us to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.