Growth and development in higher plants under simulated microgravity conditions on a 3-dimensional clinostat.

Growth and development of etiolated pea (Pisum sativum L. cv. Alaska) and maize (Zea mays L. cv. Golden Cross Bantam) seedlings grown under simulated microgravity conditions were intensively studied using a 3-dimensional clinostat as a simulator of weightlessness. Epicotyls of etiolated pea seedlings grown on the clinostat were the most oriented toward the direction far from cotyledons. Mesocotyls of etiolated maize seedlings grew at random and coleoptiles curved slightly during clinostat rotation. Clinostat rotation promoted the emergence of the 3rd internodes in etiolated pea seedlings, while it significantly inhibited the growth of the 1st internodes. In maize seedlings, the growth of coleoptiles was little affected by clinostat rotation, but that of mesocotyls was suppressed, and therefore, the emergence of the leaf out of coleoptile was promoted. Clinostat rotation reduced the osmotic concentration in the 1st internodes of pea seedlings, although it has little effect on the 2nd and the 3rd internodes. Clinostat rotation also reduced the osmotic concentrations in both coleoptiles and mesocotyls of maize seedlings. Cell-wall extensibilities of the 1st and the 3rd internodes of pea seedlings grown on the clinostat were significantly lower and higher as compared with those on 1 g conditions, respectively. Cell-wall extensibility of mesocotyls in seedlings grown on the clinostat also decreased. Changes in cell wall properties seem to be well correlated to the growth of each organ in pea and maize seedlings. These results suggest that the growth and development of plants is controlled under gravity on earth, and that the growth responses of higher plants to microgravity conditions are regulated by both cell-wall mechanical properties and osmotic properties of stem cells.     

Effects of gravistimuli on osmoregulation in azuki bean epicotyls

The effects of hypergravity on growth and osmoregulation were examined in dark-grown azuki bean epicotyls. Elongation growth of epicotyls was promptly suppressed by hypergravity at 300g. On the contrary, the increase in fresh weight of epicotyls during incubation was not suppressed by hypergravity at 300g at least up to 6 h. Also, the level of total osmotic solutes increased during epicotyl growth for 6 h, which was not affected by hypergravity. These results suggest that azuki bean epicotyls are capable of maintaining osmoregulation even under 300g conditions for a short period. On the other hand, the increase in fresh weight of epicotyls was suppressed, in addition to suppression of elongation growth, when seedlings were treated with 300g for 24 h. The increase in level of total osmotic solutes was also inhibited by 24 h hypergravity treatment, which was accounted by the reduced levels of organic solutes, such as sugars and amino acids. Furthermore, the dry weight of seeds decreased during incubation for 24 h, but the decrease was inhibited by hypergravity at 300g. Hypergravity treatment at 300g for 24 h also increased the pH value of apoplastic solution in epicotyls. Taken together, these results suggest that the translocation of organic solutes from the seed to epicotyls is inhibited by prolonged hypergravity treatment, which may underlie the suppression of epicotyl growth, and that the breakdown of H⁺ gradient across the plasma membrane in epicotyl cells may be at least partly involved in the reduction of organic solute accumulation under hypergravity conditions.     

Gravitational force regulates elongation growth of Arabidopsis hypocotyls by modifying xyloglucan metabolism.

Growth of dark-grown Arabidopsis hypocotyls was suppressed under hypergravity conditions (300 g), or was stimulated under microgravity conditions in space (Space Shuttle STS-95). The mechanical extensibility of cell walls decreased and increased under hypergravity and microgravity conditions, respectively. The amounts of cell wall polysaccharides (pectin, hemicellulose-I, hemicellulose-II and cellulose) per unit length of hypocotyls increased under hypergravity conditions, and decreased under microgravity conditions. The amount and the molecular mass of xyloglucans also increased under the hypergravity conditions, while those decreased under microgravity conditions. The activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls decreased and increased under hypergravity and microgravity conditions, respectively. These results indicate that the amount and the molecular mass of xyloglucans are affected by the magnitude of gravity and that such changes are caused by changes in xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell walls by gravity stimulus may be the primary event determining the cell wall extensibility, thereby regulating the growth rate of Arabidopsis hypocotyls.     

Changes in membrane lipid composition in azuki bean epicotyls under hypergravity conditions: Possible role of membrane sterols in gravity resistance

Seedlings of azuki bean (Vigna angularis Ohwi et Ohashi) were cultivated under hypergravity conditions, and changes in membrane lipid composition in their epicotyls were analyzed. Under hypergravity conditions at 300g, the levels of total sterols, phospholipids, and fatty acids per fresh weight were kept higher, as compared with 1g controls. In particular, sterol levels were prominently increased by hypergravity. On the other hand, hypergravity did not clearly influence the levels of each phospholipid and glycolipid class, or their fatty acid compositions. Thus, the effect of hypergravity on membrane lipid metabolism was specific for sterol biosynthesis. In various regions of azuki epicotyls, high growth rate was associated with high sterol levels. Hypergravity suppressed elongation growth and stimulated lateral expansion of azuki epicotyls. In the presence of lovastatin, an inhibitor of sterol biosynthesis, at 30 μM, such changes in growth parameters occurred even under 1g conditions, suggesting that lovastatin made epicotyls hypersensitive to the gravitational force. These results support the hypothesis that membrane sterols are involved in maintenance of normal growth capacity of plant organs against gravity.     

Automorphogenesis and gravitropism of plant seedlings grown under microgravity conditions.

Plant seedlings exhibit automorphogenesis on clinostats. The occurrence of automorphogenesis was confirmed under microgravity in Space Shuttle STS-95 flight. Rice coleoptiles showed an inclination toward the caryopsis in the basal region and a spontaneous curvature in the same adaxial direction in the elongating region both on a three-dimensional (3-D) clinostat and in space. Both rice roots and Arabidopsis hypocotyls also showed a similar morphology in space and on the 3-D clinostat. In rice coleoptiles, the mechanisms inducing such an automorphic curvature were studied. The faster-expanding convex side of rice coleoptiles showed a higher extensibility of the cell wall than the opposite side. Also, in the convex side, the cell wall thickness was smaller, the turnover of the matrix polysaccharides was more active, and the microtubules oriented more transversely than the concave side, and these differences appear to be causes of the curvature. When rice coleoptiles grown on the 3-D clinostat were placed horizontally, the gravitropic curvature was delayed as compared with control coleoptiles. In clinostatted coleoptiles, the corresponding suppression of the amyloplast development was also observed. Similar results were obtained in Arabidopsis hypocotyls. Thus, the induction of automorphogenesis and a concomitant decrease in graviresponsiveness occurred in plant shoots grown under microgravity conditions.     

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.     

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.     

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.     

Possible mechanisms of plant cell wall changes at microgravity.

Space and clinostatic experiments revealed that changes of plant cell wall structure and its function depend on type of tissue and duration of influence. It was shown that clinostat conditions reproduce the part of weightlessness biological effects. It is established that various responses of wall structural-metabolic organization occur at microgravity: changes of cell walls ultrastructure and organelles structure; decrease of synthesis of primary plant cell wall; rearrangements of polysaccharides content. It is shown that mechanisms of plant cell wall changes at microgravity are connected with decrease of cellulose crystallization, activation of pectolytic enzymes and rearrangement of calcium balance of apoplast and cytoplasm.     

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.     

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.     

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, --> ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.     

Effects of altered gravity on plant cell processes: results of recent space and clinostatic experiments.

Space and clinostatic experiments revealed that plant cell structure and metabolism rearrangements depend on taxonomical position and physiological state of objects, growth phase and real or simulated microgravity influence duration. It was shown that clinostat conditions reproduce only a part of microgravity biological effects. It is established that various responses occur in microgravity: 1) rearrangements of cytoplasmic organelles ultrastructure and calcium balance; 2) physical-chemical properties of the plasmalemma are changed; 3) enzymes activity is often enhanced. These events provoke the acceleration of growth and differentiation of cells and their aging as a result; at the same time some responses can be considered as cell adaptation to microgravity.     

Leaf senescence under various gravity conditions: relevance to the dynamics of plant hormones.

Effects of simulated microgravity and hypergravity on the senescence of oat leaf segments excised from the primary leaves of 8-d-old green seedlings were studied using a 3-dimensional (D) clinostat as a simulator of weightlessness and a centrifuge, respectively. During the incubation with water under 1-g conditions at 25 degrees C in the dark, the loss of chlorophyll of the segments was found dramatically immediately after leaf excision, and leaf color completely turned to yellow after 3-d to 4-d incubation. In this case kinetin (10 micromolar) was effective in retarding senescence. The application of simulated microgravity conditions on a 3-D clinostat enhanced chlorophyll loss in the presence or absence of kinetin. The loss of chlorophyll was also enhanced by hypergravity conditions (ca. 8 to 16 g), but the effect was smaller than that of simulated microgravity conditions on the clinostat. Jasmonates (JAs) and abscisic acid (ABA) promoted senescence under simulated microgravity conditions on the clinostat as well as under 1-g conditions. After 2-d incubation with water or 5-d incubation with kinetin, the endogenous levels of JAs and ABA of the segments kept under simulated microgravity conditions on the clinostat remained higher than those kept under 1-g conditions. These findings suggest that physiological processes of leaf senescence and the dynamics of endogenous plant hormone levels are substantially affected by gravity.     

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.     

Behavioural changes in Paramecium and Didinium exposed to short-term microgravity and hypergravity.

The swimming behaviour of two ciliate species, Paramecium caudatum and Didinium nasutum was analyzed under microgravity and hypergravity. In Paramecium the differences between former upward and downward swimming rates disappeared under weightlessness. At microgravity the swimming rates equalled those of horizontally swimming cells at 1g. In contrast, the swimming rates of Didinium increased under microgravity conditions, being larger than horizontal swimming rates at 1g. These findings are in accordance with a hypothesis of gravireception in ciliates based on electrophysiological data, which considers the different topology of mechanoreceptor channels in theses species. The hypothesis received further support by data recorded under hypergravity conditions.     

Molecular developmental neurobiology of formation, guidance and survival of primary vestibular neurons.

Untangling the molecular and physiological interactions that generate the proper connections of the primary vestibular neurons in normal gravity requires two parallel approaches. One approach needs to use mutant mice to delineate the molecular basis of developmental mechanisms that govern ear development, including formation and differentiation of neurons and establishment of their peripheral and central connections. Beyond that and in addition to it, we need physiological investigations using microgravity and/or hypergravity, as well as absence of otoconia, to understand the role played by vestibular stimuli to fine tune connections of primary and secondary vestibular neurons. This paper provides an overview of some of the molecular mechanisms uncovered over the last few years that guide development, differentiation and survival of primary vestibular neurons of the mammalian ear. Briefly, several genes that are essential for primary neuron formation have been identified, all genes that govern embryonic survival are known and the first genes and mechanisms that guide formation of proper connections are being revealed. While still incomplete, the progress has been astounding and the completion of the mouse genome project will further accelerate the pace. Such data pave the way to put the research on the influence of altered gravity stimulation within a molecular framework.     

Effects of gravity and cosmic rays on cell proliferation kinetics in Paramecium tetraurelia.

Space flights resulted in a stimulating effect on kinetics of proliferation in Paramecium tetraurelia. Additional experiments were performed in order to determine the origin of this phenomena. Paramecia were cultivated in balloon flights or in a slow clinostat, or were exposed to different levels of hypergravity. The results suggest that changes in cell proliferation rate are related to cosmic rays and to a direct effect of microgravity.     

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity.

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion molecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.     

The biological clock of Neurospora in a microgravity environment.

The circadian rhythm of conidiation in Neurospora crassa is thought to be an endogenously derived circadian oscillation; however, several investigators have suggested that circadian rhythms may, instead, be driven by some geophysical time cue(s). An experiment was conducted on space shuttle flight STS-9 in order to test this hypothesis; during the first 7-8 cycles in space, there were several minor alterations observed in the conidiation rhythm, including an increase in the period of the oscillation, an increase in the variability of the growth rate and a diminished rhythm amplitude, which eventually damped out in 25% of the flight tubes. On day seven of flight, the tubes were exposed to light while their growth fronts were marked. Some aspect of the marking process reinstated a robust rhythm in all the tubes which continued throughout the remainder of the flight. These results from the last 86 hours of flight demonstrated that the rhythm can persist in space. Since the aberrant rhythmicity occurred prior to the marking procedure, but not after, it was hypothesized that the damping on STS-9 may have resulted from the hypergravity pulse of launch. To test this hypothesis, we conducted investigations into the effects of altered gravitational forces on conidiation. Exposure to hypergravity (via centrifugation), simulated microgravity (via the use of a clinostat) and altered orientations (via alterations in the vector of a 1 g force) were used to examine the effects of gravity upon the circadian rhythm of conidiation.