The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9.

An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 ("Kosmos-2044") in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.     

Microgravity effects on neural retina regeneration in the newt.

Data on forelimb and eye lens regeneration in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mullerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates.     

Influence of clinorotation and fettering stress on tail regeneration of Triturus vulgaris (Urodela).

Tail-amputated adult Triturus vulgaris, fettered in cuvettes of a fast-rotating clinostat were exposed to simulated weightlessness (60 rpm; equiv. to 10(-3)-10(-4) g), during a 14-day period. To feed and clean the animals rotation was stopped once a day for approx. 10 min. To test the influence of the fettering stress, a second series of animals was kept separately under normal earth conditions without rotation. A further control series was kept in a dark container without any handicap. While tail regeneration of the rotated animals was markedly accelerated, the fettered-only animals showed a considerably less marked acceleration effect. At the end of the 14-day period, all regenerates were reamputated together with an additional 5 mm of the tail stump. Although this second level of amputation was distant from the first, the regenerative growth rate of the rotated series was accelerated 123% in contrast to both the control and the fettered-only series. Our results demonstrate that the growth acceleration is induced by clinorotation. Fettering stress has no comparable influence. The growth promoting effect is not limited to the regenerating area.     

Early development of Xenopus embryos is affected by simulated gravity.

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) "morphological patterning" such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) "the timing of embryonic events" such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.     

Histochemical investigations on the influence of long-term altered gravity on the CNS of developing cichlid fish: results from the 2nd German Spacelab Mission D-2.

The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brains as well as in individual brain nuclei of developing cichlid fish larvae had been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing accelerations from near weightlessness (spaceflight) via 1g controls to 3g hyper gravity (centrifuge) resulted in slightly increasing "all over the brain" (total brain) SDH reactivity. When focusing on distinct neuronal integration centers within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity only within vestibulum related brain parts were obtained.     

Gravitational neurobiology of fish.

In vertebrates (including man), altered gravitational environments such as weightlessness can induce malfunctions of the inner ears, based on irregular movements of the semicircular cristae or on dislocations of the inner ear otoliths from the corresponding sensory epithelia. This will lead to illusionary tilts, since the vestibular inputs are not confirmed by the other sensory organs, which results in an intersensory conflict. Vertebrates in orbit therefore face severe orientation problems. In humans, the intersensory conflict may additionally lead to a malaise, commonly referred to as space motion sickness (SMS), a kinetosis. During the first days at weightlessness, the orientation problems (and SMS) disappear, since the brain develops a new compensatory interpretation of the available sensory data. The present review reports on the neurobiological responses--particularly of fish--observed at altered gravitational states, concerning behaviour and neuroplastic reactivities. Recent investigations employing microgravity (spaceflight, parabolic aircraft flights, clinostat) and hyper-gravity (laboratory centrifuges as ground based research tools) yielded clues and insights into the understanding of the respective basic phenomena.     

Changes in symbiotic and associative interrelations in a higher plant-bacterial system during space flight.

The miniature cenosis consisting of the water fern Azolla with its associated symbiotic nitrogen-fixing cyanobacterium Anabaena and the concomitant bacteria was investigated. Ecological closure was shown to produce sharp quantitative and qualitative changes in the number and type of concomitant bacteria. Changes in the distribution of bacterial types grown on beef-extract broth after space flight were recorded. Anabaena azollae underwent the most significant changes under spaceflight conditions. Its cell number per Azolla biomass unit increased substantially. Thus closure of cenosis resulted in a weakening of control over microbial development by Azolla. This tendency was augmented by spaceflight factors. Reduction in control exerted by macro-organisms over development of associated micro-organisms must be taken into account in constructing closed ecological systems in the state of weightlessness.     

Circumnutation augmented in clinostatted plants by a tactile stimulus

Dark-grown, 4-day old, $\text{Helianthus annuus}$ seedlings were rotated for 20 hr on horizontal clinostats to minimize the amplitude of circumnutation. Then a Plexiglas sheet was placed gently against the tip of the cotyledons. By time-lapse video imaging (using intermittent IR illumination to which the plants were insensitive) movements of the clinostatted plants were observed before, during, and after the period of mechanical contact. Immediately after the Plexiglas sheet was removed residual nutation increased in amplitude almost three-fold, then declined over the next 7 hr to the prestimulation level. This demonstration of enhancement of circumnutation by mechanical contact is consistent with the model of an endogenous oscillator that can be stimulated by factors other than gravity.     

失重因素对航天员体温调节影响的分析

系统地总结了实际载人航天及地面模拟中失重因素引起的航天员生理性体温调节功能变化的种种现象和内在联系。指出在失重环境下 ,自然对流消失 ,血液重新分布 ,排尿性失水增加 ,血浆容积变小 ,心血管系统功能下降 ,最大氧摄入量降低 ,这些都可导致体温调节能力受损 ,最终使航天员的高温耐力明显下降。文章同时指出 ,在体温调节系统中 ,行为性调节是生理性调节的补充和延伸 ,前者只有通过后者才能发挥作用 ,因此 ,在航天器座舱温控系统及航天服通风 -液冷系统的设计中 ,应充分考虑失重因素的影响。     

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.     

Putative graviperception mechanisms of protists.

Many (if not all) free-living cells use the gravity vector for their spatial orientation (gravitaxis). Additional responses may include gravikinesis as well as changes in morphological and physiological parameters. Though using essentially different modes of locomotion, ameboid and ciliated cells seem to rely on common fundamental graviperception mechanisms. Uniquely in the ciliate family Loxodidae a specialized intracellular gravireceptor organelle has been developed, whereas in all other cells common cell structures seem to be responsible for gravisensing. Changes in direction or magnitude of acceleration (from 0 to 5 g) as well as experiments in density-adjusted media strongly indicate that either the whole cytoplasm or dense organelles like nuclei act as statoliths and open directly or via cytoskeletal elements mechano-sensitive ion channels in the cell membrane. A recent spaceflight experiment (S/MM-06) demonstrated that prolonged (9 d) actual weightlessness did not affect the ability of Loxodes to respond to acceleration stimuli. However, prolonged cooling (> or = l4 d, 4-10 degrees C) destroyed the ability for gravitactic orientation of Paramecium. This may reflect a profound effect either on the gravireceptor itself or on the gravity-signal processing. In gravity signalling the ubiquitous second messenger cAMP may be involved in acceleration-stimulus transduction.     

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Understanding the organization of the amphibian egg cytoplasm: gravitational force as a probe.

A combination of hypergravity (centrifugation) and hypogravity (clinostat) studies have been carried out on amphibian (frog, Xenopus) eggs. The results reveal that the twinning caused by centrifugation exhibits substantial spawning to spawning variation. That variation can be attributed to the apparent viscosity of the egg's internal cytoplasm. Simulated hypogravity results in a relocation of the egg's third (horizontal) cleavage furrow, towards the equator. Substantial egg-to-egg variation is also observed in this "cleavage effect". For interpreting spaceflight data and for using G-forces as probes for understanding the egg's architecture the egg variation documented herein should be considered.     

Polarity of root statocytes—Relevance for graviperception

During outgrowth of the radicle of cress ( L.) the statocytes of the root cap develop a structural polarity with the nucleus at the proximal cell pole and a complex of endoplasmic reticulum (ER) at the distal cell pole. Amyloplasts sediment upon this complex of ER. During all stages of development of the cytoskeleton (microtubules, microfilaments) is involved in positioning of the ER. The structural polarity of the statocytes develops independently of gravity, as indicated by corresponding results from fast and slow rotating clinostats and roots grown under microgravity in orbit. Disturbance of the structural polarity is possible by application of drugs, influencing microtubules and microfilaments. If, by rotation of roots on slow rotating clinostats or centrifugation, the structural polarity of the statocytes is changed, the ability of the roots to perceive gravity is affected also.     

Spaceflight hardware for conducting plant growth experiments in space: the early years 1960-2000.

The best strategy for supporting long-duration space missions is believed to be bioregenerative life support systems (BLSS). An integral part of a BLSS is a chamber supporting the growth of higher plants that would provide food, water, and atmosphere regeneration for the human crew. Such a chamber will have to be a complete plant growth system, capable of providing lighting, water, and nutrients to plants in microgravity. Other capabilities include temperature, humidity, and atmospheric gas composition controls. Many spaceflight experiments to date have utilized incomplete growth systems (typically having a hydration system but lacking lighting) to study tropic and metabolic changes in germinating seedlings and young plants. American, European, and Russian scientists have also developed a number of small complete plant growth systems for use in spaceflight research. Currently we are entering a new era of experimentation and hardware development as a result of long-term spaceflight opportunities available on the International Space Station. This is already impacting development of plant growth hardware. To take full advantage of these new opportunities and construct innovative systems, we must understand the results of past spaceflight experiments and the basic capabilities of the diverse plant growth systems that were used to conduct these experiments. The objective of this paper is to describe the most influential pieces of plant growth hardware that have been used for the purpose of conducting scientific experiments during the first 40 years of research.     

Plant growth, development and embryogenesis during Salyut-7 flight.

The growth and geotropic movements of roots and hypocotyls of lettuce have been studied on board the Salyut 7 station in a stationary position and on the centrifuge at 0.01, 0.1 and 1 g. On the centrifuge at 0.1 and 0.01 g as well as under weightlessness, the final length of hypocotyls was by 8-16% greater than in control plants on the centrifuge at 1 g. The length of roots, however, was reduced by 17% at 0.01 g and under weightlessness; at 0.1 g their growth is much the same as at 1 g. On the Earth, while growing in a vertical position, and in space at 0 < or = g, the roots and hypocotyls deviate from the longitudinal axis of the seed. Average values of deviation eagles on the Earth are always equal to zero, while this is not always the case in space, which indicates the biological effect of microgravity conditions on board a spacecraft. The threshold of geotropic sensitiveness of lettuce hypocotyls, calculated from the linear regression parameters of the dependence of the response geotropic reaction upon the value of the centrifugal force, comprised 2.9 x 10(-3) g. In the Fiton 3 micro-greenhouse under spaceflight conditions, the plants of Arabidopsis thaliana (L) Heynh have, for the first time, undergone a full cycle of individual development. The seeds sown during the flight germinated, performed growth processes, formed vegetative and generative organs and, judging by the final result, they succeeded in fecundation, embryogenesis and ripening. Despite the noted modification of growth and development of plants in space, 42% of formed seeds appeared to be valuable biologically.     

Clinostation influence on regeneration of cell wall in Solanum tuberosum L. protoplasts.

Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in a regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.     

The state of gravity sensors and peculiarities of plant growth during different gravitational loads.

In the primary roots of lettuce shoots grown under altered gravitational conditions--180 degrees inversion on the centrifuged clinostat, horizontal clinostat and in dynamic weightlessness--localization of the cellular organelles, cell morphology and peculiarities of growth have been studied. Significant changes took place in the localization of amyloplasts on the horizontal clinostat. The changes of amyloplast position in the cap cells on the horizontal clinostat and under weightlessness are similar. A change of the normal shoot position (180 degrees inversion and horizontal clinostat) causes an inhibition of growth. Weightlessness increases the length of axial organs and cells in the zone of elongation, but decreases the nitotic index in comparison to the centrifuged control. The anlysis of the formation of generative organs has been carried out for Arabidopsis plants grown on board the orbital station Salyut-6. The ability of plants to undergo vegetative growth and to pass through early phases of generative development under weightlessness was confirmed.     

Effects of microgravity on muscle and cerebral cortex: a suggested interaction.

The "slow" antigravity muscle adductor longus was studied in rats after 14 days of spaceflight (SF). The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light and electron microscopy revealed myofiber atrophy, segmental necrosis and regenerative myofibers. Regenerative myofibers were N-CAM immunoreactive (N-CAM-IR). The neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles, degenerative changes, vacant axonal spaces and changes suggestive of axonal sprouting. No alterations of muscle spindles was seen either by light or electron microscopy. These observations suggest that muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight. In a separate study, GABA immunoreactivity (GABA-IR) was evaluated at the level of the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension ("simulated" microgravity). A reduction in number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-IR terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system after spaceflight and hindlimb suspension it is suggested that after limb unloading there are alterations of afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the changes observed in GABA immunoreactivity of cells and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.     

Amphibian egg cytoplasm response to altered g-forces and gravity orientation

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) non-yolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.