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.     

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.     

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.     

First flight of the ASTROCULTURE (TM) experiment as a part of the U.S. Shuttle/MIR program.

A number of space-based experiments have been conducted to assess the impact of microgravity on plant growth and development. In general, these experiments did not identify any profound impact of microgravity on plant growth and development, though investigations to study seed development have indicated difficulty in plants completing their reproductive cycle. However, it was not clear whether the lack of seed production was due to gravity effects or some other environmental condition prevailing in the unit used for conducting the experiment. The ASTROCULTURE (TM) flight unit contains a totally enclosed plant chamber in which all the critically important environmental conditions are controlled. Normal wheat (Triticum aestivum L.) growth and development in the ASTROCULTURE (TM) flight unit was observed during a ground experiment conducted prior to the space experiment. Subsequent to the ground experiment, the flight unit was transported to MIR by STS-89, as part of the U.S. Shuttle/MIR program, in an attempt to determine if super dwarf wheat plants that were germinated in microgravity would grow normally and produce seeds. The experiment was initiated on-orbit after the flight unit was transferred from the Space Shuttle to MIR. The ASTROCULTURE (TM) flight unit performed nominally for the first 24 hours after the flight unit was activated, and then the unit stopped functioning abruptly. Since it was not possible to return the unit to nominal operation it was decided to terminate the experiment. On return of the flight unit, it was confirmed that the control computer of the ASTROCULTURE (TM) flight unit sustained a radiation hit that affected the control software embedded in the computer. This experience points out that at high orbital inclinations, such as that of MIR and that projected for the International Space Station, the danger of encountering harmful radiation effects are likely unless the electronic components of the flight hardware are resistant to such impacts.     

Effects of long duration space flight on rice seed (or embryo) radiation sensitivity and element microlocalizations.

In long duration space experiments Rice caryopses and embryos, which are able to remain alive 10 years (or more) and tolerate extreme physical conditions (temperature, few water content) during irradiation and post-irradiation storage, were used (8, 40, 201 and 457 days on board of Salyut 7, 2107 days on LDEF). In certain experiments (Salyut 7), samples were irradiated either before or after the flight. Effects of the flight and radiosensitivity were observed in Rice seedlings cultivated in in vitro conditions. Statistical results indicate an increase in radiosensitivity when irradiations occur before the flight. Microanalyses were made in different parts of one caryopsis and of one embryo, and the results compared with those of control samples. With caryopses and embryos of the same Rice varieties, but from LDEF, we made the same kinds of experiments to compare results.     

Morphological and molecular changes of maize plants after seeds been flown on recoverablf satellite

Dry seeds of Zea mays, heterozygous for Lw₁/lw₁ alleles, sandwiched between nuclear track detectors aboard Chinese satellite for 15 days, were recovered and mutations in morphological characters on plants developed from these seeds, as well as their selected progenies, were investigated. The dosimetric results indicated that 85% of the seeds received at least 1 hit with Z≥20. About 10% of plants developed from flown seeds and 40% of observed selfed lines from the first generation plants showed some morphological changes, such as yellow stripes displayed on leaves, dwarf, anomogensis of floral organs and yellow-green seedlings, when compared with those from ground control. Using yellow stripes on leaves as the main endpoint for evaluating mutation induced in space environment, the frequency of stripe occurrence was 4.6% in the first generation plants, comparable with the results obtained from Long Duration Exposure Facility (LDEF) mission (Mei et al., 1994), but much lower than those from ground based ⁶⁰ Co-gamma treatment at a dose of 100 Gy, which reached 35.5% in the selfed lines of the second generation. One hundred and ten random primers were screened in RAPD analysis to detect the variation on genomic DNA of plants with stripes on leaves. Of these primers, 10.9% were able to generate polymorphic bands between mutated plants and control, also, common band patterns in several progenies with the same mutation phenotype were observed. These results demonstrated that space radiation environment could induce inheritable mutagenic effects on plant seeds, and verified the change in genetic material in the mutants. Further study will be needed for a better understand of the nature and mechanism of this induction of mutation.     

Mutational effects of space flight on Zea mays seeds.

The growth and development of more than 500 Zea mays seeds flown on LDEF were studied. Somatic mutations, including white-yellow stripes on leaves, dwarfing, change of leaf sheath color or seedling color were observed in plants developed from these seeds. When the frequency of white-yellow formation was used as the endpoint and compared with data from ground based studies, the dose to which maize seeds might be exposed during the flight was estimated to be equivalent to 635 cGy of gamma rays. Seeds from one particular holder gave a high mutation frequency and a wide mutation spectrum. White-yellow stripes on leaves were also found in some of the inbred progenies from plants displayed somatic mutation. Electron microscopy studies showed that the damage of chloroplast development in the white-yellow stripe on leaves was similar between seeds flown on LDEF and that irradiated by accelerated heavy ions on ground.     

Seedling growth and development on space shuttle.

Young pine seedlings, and mung bean and oat seeds were flown on shuttle flights, STS-3 and STS-51F, in March, 1982 and July/August, 1985, respectively. The plant growth units built to support the two experiments functioned mechanically as anticipated and provided the necessary support data. Pine seedlings exposed to the microgravity environment of the space shuttle for 8 days continued to grow at a rate similar to ground controls. Pine stems in flight seedlings, however, averaged 10 to 12% less lignin than controls. Flight mung beans grew slower than control beans and their stems contained about 25% less lignin than control seedlings. Reduced mung bean growth in microgravity was partly due to slower germination rate. Lignin also was reduced in flight oats as compared to controls. Oats and mung beans exhibited upward growing roots which were not observed in control seedlings. Chlorophyll A/B ratios were lower in flight tissues than controls. The sealed PGCs exhibited large variations in atmospheric gas composition but the changes were similar between flight and ground controls. Ethylene was present in low concentrations in all chambers.     

The role of weightlessness in the genetic damage from preflight gamma-irradiation of organisms in experiments aboard the Salyut 6 orbital station

The effect of weightlessness on chromosomal aberration frequency in preflight irradiated $\text{Crepis capillaris}$ seeds, on the viability, fertility and mutation frequency in $\text{Arabidopsis thaliana}$, and on the frequency of nondisjunction and loss of X chromosomes in preflight irradiated $\text{Drosophila melanogaster}$ gametes was studied aboard the Salyut 6 orbital station. The following effects were observed: a flight-time dependent amplification of the effects of preflight

-irradiation in $\text{A}$. $\text{thaliana}$ with respect to all the parameters studied; unequal effects in seeds and seedlings of $\text{Crepis capillaris}$; and a significant increase in the frequency of nondisjunction and loss of chromosomes during meiosis in $\text{Drosophila}$ females. These observations are discussed in terms of the data of ground-based model experiments and flight experiments with a different time of exposure of objects to weightlessness. An attempt is made to elucidate the role of weightlessness in the modification of ionizing radiation effects.     

Inhibition of reproductive growth of Arabidopsis in airtight vessels

Airtight vessels have various advantages for space experiments. However, Arabidopsis thaliana plants scarcely produced seeds when grown in such vessels. The mechanism by which reproductive growth is inhibited in airtight vessels was studied. The length of the flower stalk was shorter when the plants were grown in airtight vessels. Thus, there was a possibility that the inhibition of reproductive growth was due to the inhibition of vegetative growth. However, even when the plants which has grown under non-airtight conditions and has reached to the flowering stage were transferred to airtight vessels, silique formation was inhibited, suggesting that the airtight environment directly influences reproductive growth. In airtight vessels, anther dehiscence was inhibited, which appears to be the cause of inhibition of silique formation and seed development. Reproductive growth recovered when silica gel was added to the vessels. These results suggest that in airtight vessels, high humidity causes a suppression of anther dehiscence, resulting in the inhibition of reproductive growth. Therefore, the control of humidity by ventilation should be taken into consideration in designing a growth chamber for space experiments.     

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.     

Viability of barley seeds after long-term exposure to outer side of international space station

Barley seeds were exposed to outer space for 13 months in a vented metal container without a climate control system to assess the risk of physiological and genetic mutation during long-term storage in space. The space-stored seeds (S0 generation), with an 82% germination rate in 50 seeds, lost about 20% of their weight after the exposure. The germinated seeds showed normal growth, heading, and ripening. The harvested seeds (S1 generation) also germinated and reproduced (S2 generation) as did the ground-stored seeds. The culm length, ear length, number of seed, grain weight, and fertility of the plants from the space-stored seeds were not significantly different from those of the ground-stored seeds in each of the S0 and S1 generation. Furthermore, the S1 and S2 space-stored seeds respectively showed similar β-glucan content to those of the ground-stored seeds. Amplified fragment length polymorphism analysis with 16 primer combinations showed no specific fragment that appears or disappears significantly in the DNA isolated from the barley grown from the space-stored seeds. Though these data are derived from nine S0 space-stored seeds in a single exposure experiment, the results demonstrate the preservation of barley seeds in outer space for 13 months without phenotypic or genotypic changes and with healthy and vigorous growth in space.     

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.     

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.     

Leaf proteomic analysis of three rice heritable mutants after seed space flight

To explore the proteomic changes of heritable variant rice plants induced by space environment, three mutants were selected after seed space flight by comparing the phenotypes with their on-ground controls. R955 grew more tillers and became dwarf, 971-5 acquired higher grain yield and better stress resistance, 974-5 matured earlier. Leaf proteins were extracted during the tiller development and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). More than 300 proteins were detected as reproducible Coomassie Brilliant Blue stained spots with pI values from around 4.0 to 7.0. Five proteins that changed significantly over the controls were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The main functions of these proteins were photosynthesis, stress defense and metabolism including RuBisCO activase, glycine rich RNA binding protein, peroxidase, triosephosphate isomerase and phosphoenolpyruvate carboxylase, which might be probably associated with the altered phenotypes. Quantitative analyses were also applied: less total protein spots and more down-regulated protein spots were detected in the mutants, indicating there might be a major loss of protein in heritable variant rice plants after seed space flight. These results may provide new insights to understand the biological effects of space environment to rice.     

Growth restoration in azuki bean and maize seedlings by removal of hypergravity stimuli.

Hypergravity stimuli, gravitational acceleration of more than 1 x g, decrease the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls by decreasing the cell wall extensibility via an increase in the molecular mass of matrix polysaccharides. An increase in the pH in the apoplastic fluid is hypothesized to be involved in the processes of the increase in the molecular mass of matrix polysaccharides due to hypergravity. However, whether such physiological changes by hypergravity are induced by normal physiological responses or caused by physiological damages have not been elucidated. In the present study, we examined the effects of the removal of hypergravity stimuli on growth and the cell wall properties of azuki bean and maize seedlings to clarify whether the effects of hypergravity stimuli on growth and the cell wall properties are reversible or irreversible. When the seedlings grown under hypergravity conditions at 300 x g for several hours were transferred to 1 x g conditions, the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls greatly increased within a few hours. The recovery of growth rate of these organs was accompanied by an immediate increase in the cell wall extensibility, a decrease in the molecular mass of matrix polysaccharides, and an increase in matrix polysaccharide-degrading activities. The apoplastic pH also decreased promptly upon the removal of hypergravity stimuli. These results suggest that plants regulate the growth rate of shoots reversibly in response to hypergravity stimuli by changing the cell wall properties, by which they adapt themselves to different gravity conditions. This study also revealed that changes in growth and the cell wall properties under hypergravity conditions could be recognized as normal physiological responses of plants. In addition, the results suggest that the effects of microgravity on plant growth and cell wall properties should be reversible and could disappear promptly when plants are transferred from microgravity to 1 x g. Therefore, plant materials should be fixed or frozen on orbit for detecting microgravity-induced changes in physiological parameters after recovering the materials to earth in space experiments.     

Auxin polar transport in Arabidopsis under simulated microgravity conditions — Relevance to growth and development

Activity of auxin polar transport in inflorescence axes of Arabidopsis thaliana grown under simulated microgravity conditions was studied in relation to the growth and development. Seeds were germinated and allowed to grow on an agar medium in test tubes on a horizontal clinostat. Horizontal clinostat rotation substantially reduced the growth of inflorescence axes and the productivity of seeds of Arabidopsis thaliana (ecotypes Landsberg erecta and Columbia), although it little affected seed germination, development of rosette leaves and flowering. The activity of auxin polar transport in inflorescence axes decreased when Arabidopsis plants were grown on a horizontal clinostat from germination stage, being ca. 60% of 1 g control. On the other hand, the auxin polar transport in inflorescence axes of Arabidopsis grown in 1 g conditions was not affected when the segments were exposed to various gravistimuli, including 3-dimensional clinorotation, during transport experiments. Pin-formed mutant of Arabidopsis, having a unique structure of the inflorescence axis with no flower and extremely low levels of the activity of auxin polar transport in inflorescence axes and endogenous auxin, did not continue its vegetative growth under clinostat rotation. These facts suggest that the development of the system of auxin polar transport in Arabidopsis is affected by microgravity, resulting in the inhibition of growth and development, especially during reproductive growth.     

Investigations on-board the biosatellite Cosmos-83.

The program of the 5 day flight of the biosatellite Cosmos-1514 (December 1983) envisaged experimental investigations the purpose of which was to ascertain the effect of short-term microgravity on the physiology, growth and development of various animal and plant species. The study of Rhesus-monkeys has shown in that they are an adequate model for exploring the mechanisms of physiological adaptation to weightlessness of the vestibular apparatus and the cardiovascular system. The rat experiment has demonstrated that mammalian embryos, at least during the last term of pregnancy, can develop in microgravity. This finding has been confirmed by fish studies. The experiment on germinating seeds and adult plants has given evidence that microgravity produces no effect on the metabolism of seedlings and on the flowering stage.     

"From seed-to-seed" experiment with wheat plants under space-flight conditions.

An important goal with plant experiments in microgravity is to achieve a complete life cycle, the "seed-to-seed experiment." Some Soviet attempts to reach this goal are described, notably an experiment with the tiny mustard, Arabidopsis thaliana, in the Phyton 3 device on Salyut 7. Normal seeds were produced although yields were reduced and development was delayed. Several other experiments have shown abnormalities in plants grown in space. In recent work, plants of wheat (Triticum aestivum) were studied on the ground and then in a preliminary experiment in space. Biometric indices of vegetative space plants were 2 to 2.5 times lower than those of controls, levels of chlorophyll a and b were reduced (no change in the ratio of the two pigments), carotenoids were reduced, there was a serious imbalance in major minerals, and membrane lipids were reduced (no obvious change in lipid patterns). Following the preliminary studies, an attempt was made with the Svetoblock-M growth unit to grow a super-dwarf wheat cultivar through a life cycle. The experiment lasted 167 d on Mir. Growth halted from about day 40 to day 100, when new shoots appeared. Three heads had appeared in the boot (surrounded by leaves) when plants were returned to earth. One head was sterile, but 28 seeds matured on earth, and most of these have since produced normal plants and seeds. In principle, a seed-to-seed experiment with wheat should be successful in microgravity.     

Effects of a hypogeomagnetic field on gravitropism and germination in soybean

Any plants grown during long-term space missions will inevitably experience an extremely low magnetic field (i.e. a hypogeomagnetic field, HGMF). It is possible that the innate adaptation of plants to the earth’s magnetic field (i.e. the geomagnetic field, GMF) would be disrupted. Effects of the HGMF on plant physiological and metabolic processes are unclear. In this study we established a hypogeomagnetic incubation system on the ground and investigated the effects of the HGMF on the gravitropism and germination of soybean seeds. The gravitropism angle, germination percentage, germination speed, water absorbance ratio, seed weight, radicle length, radicle weight, and radicle weight ratio of soybean seeds grown in the local field and the HGMF were compared. In general, the gravitropism angle in the HGMF was smaller than that in the local field when seeds were positioned before emergence in such a way that the direction of the radicle was opposite to that of gravity. The germination percentage, germination speed, and radicle weight ratio increased in the HGMF compared to the control. Our results indicate that the germination and gravitropism of soybean seeds are affected by elimination of the geomagnetic field.