The effects of extended clinorotation on potato minituber formation and structure.

Formation and structure of potato minitubers grown aseptically for 30 days on a horizontal clinostat and in stationary control have been studied by light and electron microscopy. It was demonstrated that the number of plants that formed minitubers, their size and fresh weight, was higher when clino-rotated than in the stationary control. It was revealed that the amount of amyloplasts in parenchyma cell sections was doubled in minitubers formed under clino-rotation. Other factors (shape of minitubers and size of reserve parenchyma cells) did not differ from the stationary control. The changes in amyloplast ultrastructure suggest accelerated cell maturity of potato reserve parenchyma in extended clino-rotation.     

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.     

Soybean cotyledon starch metabolism is sensitive to altered gravity conditions.

We have demonstrated that etiolated soybean seedlings grown under the altered gravity conditions of clinorotation (1 rpm) and centrifugation (5xg) exhibit changes in starch metabolism. Cotyledon starch concentration was lower (-28%) in clinorotated plants and higher (+24%) in centrifuged plants than in vertical control plants. The activity of ADP-glucose pyrophosphorylase in the cotyledons was affected in a similar way, i.e. lower (-37%) in the clinorotated plants and higher (+22%) in the centrifuged plants. Other starch metabolic enzyme activities, starch synthase, starch phosphorylase and total hydrolase were not affected by the altered gravity treatments. We conclude that the observed changes in starch concentrations were primarily due to gravity-mediated differences in ADP-glucose pyrophosphorylase activity.     

Growth of pea epicotyl in low magnetic field implication for space research

A magnetic field is an inescapable environmental factor for plants on the earth. However, its impact on plant growth is not well understood. In order to survey how magnetic fields affect plant, Alaska pea seedlings were incubated under low magnetic field (LMF) and also in the normal geo-magnetic environment. Two-day-old etiolated seedlings were incubated in a magnetic shield box and in a control box. Sedimentation of amyloplasts was examined in the epicotyls of seedlings grown under these two conditions. The elongation of epicotyls was promoted by LMF. Elongation was most prominent in the middle part of the epicotyls. Cell elongation and increased osmotic pressure of cell sap were found in the epidermal cells exposed to LMF. When the gravitational environment was 1G, the epicotyls incubated under both LMF and normal geomagnetic field grew straight upward and amyloplasts sedimented similarly. However, under simulated microgravity (clinostat), epicotyl and cell elongation was promoted. Furthermore, the epicotyls bent and amyloplasts were dispersed in the cells in simulated microgravity. The dispersion of amyloplasts may relate to the posture control in epicotyl growth under simulated microgravity generated by 3D clinorotation, since it was not observed under LMF in 1G. Since enhanced elongation of cells was commonly seen both at LMF and in simulated microgravity, all elongation on the 3D-clinostat could result from pseudo-low magnetic field, as a by-product of clinorotation. (i.e., clinostat results could be based on randomization of magnetic field together with randomization of gravity vector.) Our results point to the possible use of space for studies in magnetic biology. With space experiments, the effects of dominant environmental factors, such as gravity on plants, could be neutralized or controlled for to reveal magnetic effects more clearly.     

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.     

Effects of clinorotation on the polysaccharide content of resynthesized walls of protoplasts.

Changes in cellulose and callose content during cell wall regeneration in Brassica oleracea protoplasts have been examined by cytofluorimetry following their exposure to the conditions of the horizontal clinostat (2 r.p.m.) for 10 days. In comparison with controls, cellulose content decreased 4-fold and 28% of the protoplasts failed to resynthesize a wall in the clinorotated sample. The callose content was almost doubled in clinostated cells. Callose synthesis fluctuated in both control and clinorotated protoplasts. The results support the idea that inhibition of cellulose synthesis in protoplasts grown on the clinostat is caused by a change of plasmalemma fluidity and functioning, and also by a disturbance to the state of cytoplasmic calcium under conditions of simulated microgravity.     

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.     

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.     

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.     

Comparative characteristic of mitochondria ultrastructural organization in Chlorella cells under altered gravity conditions.

Results from experiments that used cells from the unicellular alga Chlorella vulgaris (strain Larg-1) grown on a clinostat, demonstrated the occurrence of rearrangements in cellular organelles, including changes in the mitochondrial ultrastructure compared to controls. Changes in mitochondrial structure were observed in auto- and heterotrophic regimes of cells grown in altered gravity conditions, especially in long-term experiments. The mitochondrial rearrangements become apparent during cell proliferation, which resulted in an increase in the relative volume of mitochondria per cell: up to 2.7 +/- 0.3% in short-term clino-rotation (2.2 +/- 0.1% in the control) and up to 5.3 +/- 0.4% and 5.1 +/- 0.4% in long-term clinorotation (2.3 +/- 0.2% in the control). The size of the mitochondria and their cristae increased in cells grown under long-time clinorotation. In addition, hypertrophied organelles, not typical for this strain, were observed. These changes in the cells were accompanied by increased electron density of the matrix and a well-ordered topography of the cristae. To examine the separation of oxidative phosphorylation and respiration, an inhibitory agent 2,4-dinitrophenol (2,4-DNP) was applied to cells which resulted in insignificant volume changes of the mitochondria (2.5 +/- 0.4% versus 2.1 +/- 0.2% in the control). The increase of mitochondrial size with regularly arranged cristae, with more condensed matrix and extension of cristae areas of clino-rotated cells, may demonstrate higher functional activity of the mitochondria under altered gravity conditions. Changes observed early in clinorotated cells, in particular the increased level of respiration, adenylate content (especially ATP) and more intensive electron-cytochemical reactions of Mg2(+)-ATPase and succinate [correction of succinat] dehydrogenase (SDH) in mitochondria (including hypertrophic organelles), also suggest increased activity of mitochondria from cells grown under altered gravity conditions compared to controls.     

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.     

The role of cellulases in the mechanism of changes of cell walls of Funaria hygrometrica moss protonema at clinostating.

Using electroncytochemical and biochemical methods, differences between the cytochemical reaction intensity and activity of the cellulosolytic enzymes in Funaria hygrometrica moss cells grown for 30 days in the horizontal clinostat (2 rev/min) and in control have been studied. It has been shown that on clinostating the precipitate amount and size increases with the cellulase activity enhancement in the periplasmic space and protonema cell walls, when compared to control. Using biochemical methods it has been found that the activity of both endo-1,4-beta-glucanase and exo-1,4-beta-glucanase was higher under these conditions. A decrease of cellulose total content, its crystalline form, and pectic substances as well as an increase of hemicellulose content have been revealed in the clinostated material compared to control. Data obtained are discussed regarding the possible mechanism of cellulase activation and synthesis inhibition and cellulose crystallization in plant cell walls at clinostating.     

Crystallisation of alpha-crustacyanin, the lobster carapace astaxanthin-protein: results from EURECA.

Crystallisation of alpha-crustacyanin, the lobster carapace astaxanthin-protein was attempted under microgravity conditions in EURECA satellite using liquid-liquid diffusion with polyethyleneglycol (PEG) as precipitant; in a second reaction chamber phenol and dioxan were used as additives to prevent composite crystal growth. Crystals of alpha-crustacyanin grown under microgravity from PEG were larger than those grown terrestrially in the same apparatus under otherwise identical conditions. On retrieval, the crystals from PEG were shown to be composite and gave a powder diffraction pattern. The second reaction chamber showed leakage on retrieval and had also been subjected to rapid temperature variation during flight. Crystal fragments were nevertheless recovered but showed a powder diffraction pattern. It is concluded, certainly for liquid-liquid diffusion using PEG alone, that, for crustacyanin, although microgravity conditions resulted in an increase in dimensions of crystals, a measurable improvement in molecular ordering was not achieved.     

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.     

Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

Energetic metabolism response in algae and higher plant species from simulation experiments with the clinostat.

Adenylate state is acknowledged to be among the most convenient approaches in the study of physiological changes in plant cells under simulation of altered gravity condition with the clinostat. Adenylate levels and the ATP/ADP ratio in cytoplasmic and mitochondrial extracts of cultivated cells of Haplopappus gracilis and algae cells of Chlorella vulgaris under initial stages of the fast-rotating and slow-rotating clinorotation, as well as the long-term clinorotation, have been investigated. For analysis of ATP and ADP levels in the plant cells under the clinorotation, we applied a high-sensitive bioluminescence method using the luciferase and piruvate kinase enzyme systems. It has been shown that the adenylate ratio is already increased during at the start of clinorotation with the different speed of rotation in the biological material tested. The considerable changes in mitochondrial ultrastructure of Chlorella cells, as well as the rising ATP level and dropping of the ATP/ADP ratio appear after long-duration clinorotation if compared to control material. It is probably connected with the distinctions in ATP-synthetase functioning in mitochondria of the cells under the clinorotation conditions.     

The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.     

Patterns of cortical microtubules formed in epidermis of Beta vulgaris L. roots under clinorotation.

Changes of cortical microtubules (MTs) from the normal transverse arrangement were observed in epidermal cells of Beta vulgaris roots under clinorotation. We hypothesize that the epidermis is sensitive to clinorotation and that the microtubular cytoskeleton plays a key role in the ensuing growth response.     

In vitro plant cell growth in microgravity and on clinostat.

For the study of gravity's role in the processes of plant cell differentiation in-vitro, a model "seed-seedling-callus" has been used. Experiments were carried out on board the orbital stations Salyut-7 and Mir as well as on clinostat. They lasted from 18 to 72 days. It was determined that the exclusion of a one-sided action of gravity vector by means of clinostat and spaceflight conditions does not impede the formation and growth of callus tissue; however, at cell and subcellular levels structural and functional changes do take place. No significant changes were observed either on clinostat or in space concerning the accumulation of fresh biomass, while the percentage of dry material in space is lower than in control. Both in microgravity (MG) and in control, even after 72 days of growth, cells with a normally developed ultrastructure are present. In space, however, callus tissue more often contains cells in which the cross-section area of a cell, a nuclei and of mitochondria are smaller and the vacuole area--bigger than in controls. In microgravity a considerable decrease in the number of starch-containing cells and a reduction in the mean area of starch grains in amyloplasts is observed. In space the amount of soluble proteins in callus tissue is 1.5 times greater than in control. However, no differences were observed in fractions when separated by the SDS-PAGE method. In microgravity the changes in cell wall material components was noted. In the space-formed callus changes in the concentration of ions K, Na, Mg, Ca and P were observed. However, the direction of these changes depends on the age of callus. Discussed are the possible reasons for modification of morphological and metabolic parameters of callus cells when grown under changed gravity conditions.     

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.