Light microscopic analysis of the gravireceptor in Xenopus larvae developed in hypogravity.

The paper describes an investigation of the influence of gravity on the early differentiation of gravity receptors in Xenopus embryos and larvae. There is evidence that the expression of crystals in the saccus endolymphaticus was statistically greater when the embryos developed in near weightlessness (hypogravity) than on earth. The function of these crystals is unknown but they may contribute to the functioning of the vestibular apparatus.     

Automorphosis of higher plants on a 3-D clinostat.

On a three-dimensional (3-D) clinostat, various plant organs developed statocytes capable of responding to the gravity vector. The graviresponse of primary roots of garden cress and maize grown on the clinostat was the same as the control roots, whereas that of maize coleoptiles was reduced. When maize seedlings were grown in the presence of 10(-4) M gibberellic acid and kinetin, the graviresponse of both roots and shoots was suppressed. The corresponding suppression of amyloplast development was observed in the clinostatted and the hormone-treated seedlings. Maize roots and shoots showed spontaneous curvatures in different portions on the 3-D clinostat. The hormone treatment did not significantly influence such an automorphic curvature. When the root cap was removed, maize roots did not curve gravitropically. However, the removal suppressed the automorphic curvatures only slightly. On the other hand, the removal of coleoptile tip did not influence its graviresponse, whereas the spontaneous curvature of decapitated coleoptiles on the clinostat was strongly suppressed. Also, cytochalasin B differently affected the gravitropic and the automorphic curvatures of maize roots and shoots. From these results it is concluded that the graviperception and the early processes of signal transmission are unnecessary for automorphoses under simulated microgravity conditions. Moreover, the results support the view that the amyloplasts act as statoliths probably via an interaction with microfilaments.     

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.     

Interaction of growth-determining systems with gravity

The experiments have been carried out with lettuce shoots on board the Salyut-7 orbital station, the Kosmos-1667 biological satellite and under ground conditions at 180° plant inversion. By means of the centrifuge Biogravistat-1M the threshold value of gravitational sensitivity of lettuce shoots has been determined on board the Salyut-7 station. It was found to be equal to 2.9 × 10⁻³g for hypocotyls and 1.5 × 10⁻⁴g for roots. The following results have been received in the experiment performed on board the Kosmos-1667 satellite: a) under microgravity the proliferation of the meristem cells and the growth of roots did not differ from the control; b) the growth of hypocotyls in length was significantly enhanced in microgravity; c) under microgravity transverse growth of hypocotyls (increase in cross sectional area) was significantly increased due to enhancement of cortical parenchyma cell growth. At 180° inversion in Earth's gravity root extension growth and rate of cell division in the root apical meristem were decreased. The determination of DNA-fuchsin value in the nuclei of the cell root apexes showed that inversion affected processess of the cell cycle preceeding cytokinesis.     

Development of space-fertilized eggs and formation of primordial germ cells in the embryos of Medaka fish.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly 'space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.     

The role of calcium ions in cytological effects of hypogravity

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema $\text{Funaria}$$\text{hygrometrica}$ grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.     

Gravity effects on the [correction of thr] growth and development of moss secondary protonemata.

The superficial cells of dark-grown moss shoots give rise to negatively gravitropic protonemata, whatever the orientation of the shoot. Shoot orientation, however, does affect from which side of the shoot the protonemata form and the direction of their growth. Protonemata from horizontal shoots grow out at a near-right angle to their supporting axes and are initiated more or less evenly along the upper side of the stem. Protonemata arising from vertically-oriented shoots in either an upright or an inverted position grow straight at an acute angle to the stem axis. The difference in the growth direction of the protonemata seems to be conditioned by the different position of the growth zone of the protonemal outgrowths, and subsequently that of the apical protonemal cells, with respect to the gravity vector. Observations suggest that the shoot protonemata, in conditions of clinorotation, persist in their original growth direction. Results also indicate that, in darkness, gravity determines only the site of protonemata initiation, not the process of initiation itself. Light, by contrast, by acting through both phytochrome and high-energy reaction systems, triggers the initiation process and defines the location of protonemata.     

Possible use of a 3-D clinostat to analyze plant growth processes under microgravity conditions.

A three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles was constructed, and various growth processes of higher plants grown on this clinostat were compared with ground controls, with plants grown on the conventional horizontal clinostat, and with those under real microgravity in space. On the 3-D clinostat, cress roots developed a normal root cap and the statocytes showed the typical polar organization except a random distribution of statoliths. The structural features of clinostatted statocytes were fundamentally similar to those observed under real microgravity. The graviresponse of cress roots grown on the 3-D clinostat was the same as the control roots. On the 3-D clinostat, shoots and roots exhibited a spontaneous curvature as well as an altered growth direction. Such an automorphogenesis was sometimes exaggerated when plants were subjected to the horizontal rotation, whereas the curvature was suppressed on the vertical rotation. These discrepancies in curvature between the 3-D clinostat and the conventional ones appear to be brought about by the centrifugal force produced. Thus, the 3-D clinostat was proven as a useful device to simulate microgravity.     

Oscillations of axial plant organs.

The tips of roots and shoots commonly show lateral movements as they grow forwards. These occur as both circumnutations (with long periods and large amplitudes) and micronutations (with short periods and small amplitudes). Their properties are reviewed, with emphasis on roots, and possible ways in which they could be regulated are discussed. The mechanisms could include long-range controls (for circumnutations) that depend on transmissible signals using steps common to gravitropism, and short-range controls (for micronutations) that operate within the elongation zone. The former are a property of the apex as a whole, while the latter may be confined to localized groups of cells. Simulation of nutations is presented with a view to isolating key physiological processes. However, this approach is limited by the current inadequate understanding of the growth mechanisms involved.     

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.     

Operation of magnetically assisted fluidized beds in microgravity and variable gravity: experiment and theory.

The conversion of solid waste into useful resources in support of long duration manned missions in space presents serious technological challenges. Several technologies, including supercritical water oxidation, microwave powered combustion and fluidized bed incineration, have been tested for the conversion of solid waste. However, none of these technologies are compatible with microgravity or hypogravity operating conditions. In this paper, we present the gradient magnetically assisted fluidized bed (G-MAFB) as a promising operating platform for fluidized bed operations in the space environment. Our experimental and theoretical work has resulted in both the development of a theoretical model based on fundamental principles for the design of the G-MAFB, and also the practical implementation of the G-MAFB in the filtration and destruction of solid biomass waste particles from liquid streams.     

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.     

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。      

The promotive effect of latrunculin B on maize root gravitropism is concentration dependent.

The cytoskeleton has been proposed to be a key player in the gravitropic response of higher plants. A major approach to determine the role of the cytoskeleton in gravitropism has been to use inhibitors to disrupt the cytoskeleton and then to observe the effect that such disruption has on organ bending. Several investigators have reported that actin or microtubule inhibitors do not prevent root gravitropism, leading to the conclusion that the cytoskeleton is not involved in this process. However, there are recent reports showing that disruption of the actin cytoskeleton with the actin inhibitor, latrunculin B, promotes the gravitropic response of both roots and shoots. In roots, curvature is sustained during prolonged periods of clinorotation despite short periods of gravistimulation. These results indicate that an early gravity-induced signal continues to persist despite withdrawal of the constant gravity stimulus. To investigate further the mechanisms underlying the promotive effect of actin disruption on root gravitropism, we treated maize roots with varying concentrations of latrunculin B in order to determine the lowest concentration of latrunculin B that has an effect on root bending. After a 10-minute gravistimulus, treated roots were axially rotated on a one rpm clinostat and curvature was measured after 15 hours. Our results show that 100 nM latrunculin B induced the strongest promotive effect on the curvature of maize roots grown on a clinostat. Moreover, continuously gravistimulated roots treated with 100 nM latrunculin B exhibited stronger curvature responses while decapped roots treated with this concentration of latrunculin B did not bend during continuous gravistimulation. The stronger promotive effect of low concentrations of latrunculin B on the curvature of both clinorotated and continuously gravistimulated roots suggests that disruption of the finer, more dynamic component of the actin cytoskeleton could be the cause of the enhanced tropic responses of roots to gravity.     

Optimization of moisture content for wheat seedling germination in a cellulose acetate medium for a space flight experiment.

The Porous Tube Plant Nutrient Delivery System (PTPNDS), a hydrophilic, microporous ceramic tube hydroponic system designed for microgravity, will be tested in a middeck locker of the Space Shuttle. The flight experiment will focus on hardware operation and assess its ability to support seed germination and early seedling growth in microgravity. The water controlling system of the PTPNDS hardware has been successfully tested during the parabolic flight of the KC-135. One challenge to the development of the space flight experiment was to devise a method of holding seeds to the cylindrical porous tube. The seed-holder must provide water and air to the seed, absorb water from the porous tube, withstand sterilization, provide a clear path for shoots and roots to emerge, and be composed of flight qualified materials. In preparation for the flight experiment, a wheat seed-holder has been designed that utilizes a cellulose acetate plug to facilitate imbibition and to hold the wheat seeds in contact with the porous tube in the correct orientation during the vibration of launch and the microgravity environment of orbit. Germination and growth studies with wheat at a range of temperatures showed that optimal moisture was 78% (by weight) in the cellulose acetate seed holders. These and other design considerations are discussed.     

Differential flank growth.

With the mathematical basis for the precise analysis of developmental processes in plants, the patterns of growth in phototropic and gravitropic responses have become better understood. A detailed temporal and spatial quantification of a growth process is an important tool for evaluating hypotheses about the underlying physiological mechanisms. Studies of growth rates and curvature show that the original Cholodny-Went hypothesis cannot explain the complex growth patterns during tropic responses of shoots and roots. In addition, regulating factors other than the lateral redistribution of hormones must be taken into account. Electrophysiological studies on roots led to a modification of the Cholodny-Went hypothesis in that redistributions of bioelectrical activities are observed.     

The influence of gravity on the structure and functions of plant material

Growth process generate plant form and relate to most physiological functions. The Earth's gravity force affects plant growth in both obvious and subtle ways. It is a major environmental influence on morphology and physiology of plants. Gravity is less important as an agent for plant stress than as an environmental signal to guide growth. The plant's bioaccelerometers are remarkably sensitive, especially in hypogravity. Simulation (clinostat) studies and experiments in satellite laboratories are needed to understand the sensing, transduction, and response characteristics of g related mechanisms. By examining how plants alter growth processes to accomplish developmental or physiological “objectives” we may find it pragmatically desirable to ask ourselves how we might design a plant to achieve such responses to environmental influences. Examples of this design engineering approach for gravity related effects are described as an aid to experimentation.     

Genetic analysis of the gravitropic set-point angle in lateral roots of Arabidopsis.

Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their characteristically non-vertical growth orientation are critical for the determination of plant form. For example, in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth of new lateral roots appears to be determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation.     

Biological effects due to weak magnetic field on plants.

Throughout the evolution process, Earth's magnetic field (MF, about 50 microT) was a natural component of the environment for living organisms. Biological objects, flying on planned long-term interplanetary missions, would experience much weaker magnetic fields, since galactic MF is known to be 0.1-1 nT. However, the role of weak magnetic fields and their influence on functioning of biological organisms are still insufficiently understood, and is actively studied. Numerous experiments with seedlings of different plant species placed in weak magnetic field have shown that the growth of their primary roots is inhibited during early germination stages in comparison with control. The proliferative activity and cell reproduction in meristem of plant roots are reduced in weak magnetic field. Cell reproductive cycle slows down due to the expansion of G1 phase in many plant species (and of G2 phase in flax and lentil roots), while other phases of cell cycle remain relatively stable. In plant cells exposed to weak magnetic field, the functional activity of genome at early pre-replicate period is shown to decrease. Weak magnetic field causes intensification of protein synthesis and disintegration in plant roots. At ultrastructural level, changes in distribution of condensed chromatin and nucleolus compactization in nuclei, noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids in meristem cells were observed in pea roots exposed to weak magnetic field. Mitochondria were found to be very sensitive to weak magnetic field: their size and relative volume in cells increase, matrix becomes electron-transparent, and cristae reduce. Cytochemical studies indicate that cells of plant roots exposed to weak magnetic field show Ca2+ over-saturation in all organelles and in cytoplasm unlike the control ones. The data presented suggest that prolonged exposures of plants to weak magnetic field may cause different biological effects at the cellular, tissue and organ levels. They may be functionally related to systems that regulate plant metabolism including the intracellular Ca2+ homeostasis. However, our understanding of very complex fundamental mechanisms and sites of interactions between weak magnetic fields and biological systems is still incomplete and still deserve strong research efforts.     

Calcium balance in pea root statocytes under both clinorotation and Ca2+ channel blockers' influence.

We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca2+ level in pea root statocytes. A rise in [Ca2+]i may be a serious problem for plants in microgravity environment. It is hypothesized that involvement of Ca2+ channel blockers in the growth medium may rescue a plant from abundance of Ca2+ ions. Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca2+ channel blocker (1 micromole D600 or nicardipine, 12 hr) causes decreasing the Ca2+ concentration in pea root statocytes in comparison with clinorotation alone. Redistribution of Ca(2+)-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation. Our data support the hypothesis that Ca2+ channel blockers may act as protectors for plants against rise in [Ca2+]i. The role for Ca2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca2+ balance in plant cells in space flights are discussed.