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.     

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.     

Structure of cress root statocytes in microgravity (Bion-10 mission).

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.     

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.     

Differenciation et proliferation cellulaires dans des racines de mais cultive en microgravite (Biocosmos 1985)

A cytological study was performed on Maize roots ( s) which were grown in space (flight Biocosmos 1985) and on control roots on earth. Two criteria were selected : cell elongation in the cortical zone in the four mm of the extremity of the root and mitotic activity of the meristem. The results show that in microgravity the length of the meristem is reduced of 1/3 and that its mitotic activity increases of about twofold comparatively to the synchronous control. In parallel the cell differentiation begin closer to the root cap junction. These results are discussed relative to the influence of gravistimulation on cell proliferation and cell differentiation in roots.     

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.     

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.     

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.     

Agravitropic mutant for the study of hydrotropism in seedling roots

Roots have been shown to respond to a moisture gradient by positive hydrotropism. Agravitropic mutant plants are useful for the study of the hydrotropism in roots because on Earth hydrotropism is obviously altered by the gravity response in the roots of normally gravitropic plants. The roots are able to sense water potential gradient as small as 0.5 MPa mm⁻¹. The root cap includes the sensing apparatus that causes a differential growth at the elongation region of roots. A gradient in apoplastic calcium and calcium influx through plasmamembrane in the root cap is somehow involved in the signal transduction mechanism in hydrotropism, which may cause a differential change in cell wall extensibility at the elongation region. We have isolated an endoxy loglucan transferase (EXGT) gene that is strongly expressed in pea roots and appears to be involved in the differential growth in hydrotropically responding roots. Thus, it is now possible to study hydrotropism in roots by comparing with or separate from gravitropism. These results also imply that microgravity conditions in space are useful for the study of hydrotropism and its interaction with gravitropism.     

Force sensitivity of plant gravisensing.

Rotation at 4, 10, 50 and 100 rpm on a horizontal clinostat and in microgravity exerts limited effects on the morphogenesis of lettuce and cress root statocytes and statoliths if compared with the vertical control or 1 g spaceflight reference centrifuge. However, the average distance of statoliths from the distal wall increases. The pattern of plastid location of microgravity-grown and that of clino-rotated samples has been determined at 10, 50, and 100 rpm. Experiments on the centrifuge-clinostat and spaceflight centrifuge (acceleration forces of 0.005 to 1 g) revealed that the average statolith location depends on the amplitude of acropetally or basipetally directed mass acceleration. Decreasing the acropetally directed force from 1 g to 0.4 g dislocates statoliths towards the cell center possibly mediated by the elastic forces of the cytoskeleton. In statocytes formed on the clinostat or in microgravity, the majority of statoliths are located at the center of the cell. To force the statoliths from the center of the statocyte towards one of its poles, a threshold mass acceleration of 0.01 g is required. Statocytes with centrally-located statoliths are considerably more effective in transducing a gravistimulus than those with distally-located plastids. The latent time of the graviresponse is shorter and the response itself is enhanced in roots grown on the clinostat compared to vertically grown samples. The early phases of graviperception are independent of root growth conditions since presentation time and g-threshold are similar for roots grown stationary and those on a clinostat. We propose a sequence of events in gravitropic stimulation that considers not only the lateral displacement of statoliths, as predicted by the starch-statolith hypothesis, but also its longitudinal motion, together with differential gravisensitivity of mechanotransducing structures along the lower-most longitudinal cell wall.     

Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10⁵ cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.     

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

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

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.     

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.     

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.     

Development of plant protoplasts during the IML-1 mission.

During the 8 day IML-1 mission, regeneration of cell walls and cell divisions in rapeseed protoplasts were studied using the Biorack microscope onboard the Space Shuttle "Discovery". Samples from microgravity and 1g protoplast cultures were loaded on microscope slides. Visual microscopic observations were reported by the payload specialist Roberta Bondar, by down-link video transmission and by use of a microscope camera. Protoplasts grown under microgravity conditions do regenerate cell walls but to a lesser extent than under 1g. Cell divisions are delayed under microgravity. Few cell aggregates with maximum 4-6 cells per aggregate are formed under microgravity conditions, indicating that microgravity may have a profound influence on plant cell differentiation.     

Modelling the effects of microgravity on the permeability of air interface respiratory epithelial cell layers

Although it has been suggested that microgravity might affect drug absorption in vivo, drug permeability across epithelial barriers has not yet been investigated in vitro during modelled microgravity. Therefore, a cell culture/diffusion chamber was designed specifically to accommodate epithelial cell layers in a 3D-clinostat and allow epithelial permeability to be measured under microgravity conditions in vitro with minimum alteration to established cell culture techniques. Human respiratory epithelial Calu-3 cell layers were used to model the airway epithelium. Cells grown at an air interface in the diffusion chamber from day 1 or day 5 after seeding on 24-well polyester Transwell cell culture inserts developed a similar transepithelial electrical resistance (TER) to cells cultured in conventional cell culture plates. Confluent Calu-3 layers exposed to modelled microgravity in the 3D-clinostat for up to 48 h maintained their high TER. The permeability of the paracellular marker ¹⁴C-mannitol was unaffected after a 24 h rotation of the cell layers in the 3D-clinostat, but was increased 2-fold after 48 h of modelled microgravity. It was demonstrated that the culture/diffusion chamber developed is suitable for culturing epithelial cell layers and, when subjected to rotation in the 3D-clinostat, will be a valuable in vitro system in which to study the influence of microgravity on epithelial permeability and drug transport.     

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.     

Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.     

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.