Clinorotation Effect on Response of Cress Leaves to Red and Far-Red Light

Responses of cress (Lepidium sativum L.) seedling leaves to separate and simultaneous illumination with red (660 nm) and far-red (735 nm) light were studied under fast clinorotation (50 rpm) and usual gravity (1g) conditions. The monochromatic light emitting diodes (LEDs) have been used for illumination of seedlings from above. The growth and spatial orientation of leaves and the location of presumable gravisensors in petioles were analysed. Clinorotation in the dark promoted the radial expansion of leaf lamina and unfolding of leaves. It was shown that clinorotation in red light inhibited significantly the elongation of petioles as compared with that under the action of gravity force. Simultaneous red and far-red illumination promoted the growth of petioles under clinorotation, but did not affect the orientation of laminas, which remained the same as of the 1-g control ones. Red light, applied separately and simultaneously with far-red light, guided the bending of laminas as well as the unfolding of leaf petioles in both usual and clinorotation conditions. Histological and cytological analyses of petioles revealed the presence of movable amyloplasts in endodermic cells in proximal region of petioles. Comparison of intracellular positioning of amyloplasts in petioles of leaves grown under clinorotation and the action of gravity allows a presumption that these plastids may be identified as gravisensors of garden cress leaves. The obtained data imply that clinorotation and exposition to monochromatic red light or simultaneous illumination by red and far-red light affect the elongation of petioles of cress seedling leaves. Spectral components guide the unfolding of laminas in a gravity-independent manner.     

Chlorophyll,Carotenoid and Anthocyanin Accumulation in Mung Bean Seedling Under Clinorotation

The accumulation of plant pigments in mung bean (Vigna radiata L.) seedlings was measured after clinorotation (2 rpm for 2-4 days), and compared to a stationary control. The pigments measured included chlorophyll and carotenoid in primary leaves, and the anthocyanin in seedlings. While significant changes in chlorophyll and carotenoid accumulation were not observed during the initial 2 to 4 days of cultivation, by day 4 the seedlings grown on the clinostat had lower levels of anthocyanin, compared to those in the control seedlings. To further detail the cause for the observed reduction in anthocyanin accumulation under altered gravity conditions, seedlings were grown in the presence of silver nitrate, a known ethylene inhibitor, for 4 days, since it is known ethylene has a negative impact on anthocyanin accumulation. Silver nitrate promoted anthocyanin accumulation in the clinostat seedlings, and as a result there was no significant difference between the control and clinostat seedlings in anthocyanin accumulation. The results suggest that slow clinorotation negatively impacts anthocyanin pigmentation in mung bean seedlings, with endogenous ethylene suspected to be involved in this.     

Analysis of Statoliths Displacement in <Emphasis Type="Italic">Chara</Emphasis> Rhizoids for Validating the Microgravity-Simulation Quality of Clinorotation Modes

In single-celled rhizoids of the green algae Chara, positively gravitropic growth is governed by statoliths kept in a dynamically stable position 10–25 μ m above the cell tip by a complex interaction of gravity and actomyosin forces. Any deviation of the tube-like cells from the tip-downward orientation causes statoliths to sediment onto the gravisensitive subapical cell flank which initiates a gravitropic curvature response. Microgravity experiments have shown that abolishing the net tip-directed gravity force results in an actomyosin-mediated axial displacement of statoliths away from the cell tip. The present study was performed to critically assess the quality of microgravity simulation provided by different operational modes of a Random Positioning Machine (RPM) running with one axis (2D mode) or two axes (3D mode) and different rotational speeds (2D), speed ranges and directions (3D). The effects of 2D and 3D rotation were compared with data from experiments in real microgravity conditions (MAXUS sounding rocket missions). Rotational speeds in the range of 60–85 rpm in 2D and 3D modes resulted in a similar kinetics of statolith displacement as compared to real microgravity data, while slower clinorotation (2–11 rpm) caused a reduced axial displacement and a more dispersed arrangement of statoliths closer to the cell tip. Increasing the complexity of rotation by adding a second rotation axis in case of 3D clinorotation did not increase the quality of microgravity simulation, however, increased side effects such as the level of vibrations resulting in a more dispersed arrangement of statoliths. In conclusion, fast 2D clinorotation provides the most appropriate microgravity simulation for investigating the graviperception mechanism in Chara rhizoids, whereas slower clinorotation speeds and rotating samples around two axes do not improve the quality of microgravity simulation.     

Cytosolic Calcium Concentration Changes in Neuronal Cells Under Clinorotation and in Parabolic Flight Missions

All life on earth has been established under conditions of stable gravity of 1g. Nevertheless, in numerous experiments the direct gravity dependence of biological processes has been shown on all levels of organization, from single molecules to humans. To study the effects especially of microgravity on biological systems, a variety of platforms are available, from drop towers to the ISS. Due to the costs of these platforms and their limited availability, as an alternative, numerous simulators have been developed for so called “simulated” microgravity. A classical systems is a clinostat, basically rotating a sample around one axis, and by integration of the gravity vector for 360° arguing that thus the effects of gravity are depleted. Indeed, a variety of studies has shown that taking out the direction of gravity from a biological system often results in consequences similar to the exposure of the system to real microgravity. Nevertheless, the opposite has been shown, too, and as a consequence the relevance of clinostats in microgravity research is still under discussion. To get some more insight into this problem we have constructed a small fluorescence clinostat and have studied the effects of clinorotation on the cytosolic calcium concentration of neuroglioma cells. The results have been compared to experiments with identical cells in real microgravity, utilizing parabolic flight missions. Our results show that in case of a cell suspension used in a small florescence clinostat within a tube diameter of 2mm, the effects of clinorotation are comparable to those under real microgravity, both showing a significant increase in intracellular calcium concentration.     

The stress protein level under clinorotation in context of the seedling developmental program and the stress response

Heat-shock proteins (HSP70 and HSP90) are present in plant cells under the normal growth conditions. At the same time, a variety of environmental disruptions results in their rapid synthesis as a substantial part of adaptation. HSP amounts can be indicative of a cellular stress level. Altered gravity (clinorotation) is unnatural for plants, so it may be a kind of stress. The aim of this study was to analyze the influence of horizontal clinorotation on the HSP70 and HSP90 level during seedling development. Pea (Pisum sativum L.) seedlings grown for 3 days from seed imbibitions in stationary control and under slow clinorotation (2 rpm) are used for this investigation. Western blot analysis indicated that HSP70 and HSP90 were abundant in the embryos of dry seeds and their amount decreased significantly during seed germination. But under horizontal clinorotation, their level in seedlings remained higher compared to the control. Furthermore, a comparison of the influence of horizontal and vertical clinorotation on the HSP level was carried out. On the ELISA data, HSP70 and HSP90 amounts in the 3-day old seedlings were higher after horizontal clinorotation than after vertical. The obtained data show an increased HSP70 and HSP90 level in pea seedlings under clinorotation. Both, rotation and change in the cell position relatively to a gravity vector affect the HSP level.     

Comparison Study of Gravity-Dependent Displacement of Amyloplasts in Statocytes of Cress Roots and Hypocotyls

In this study, the kinetics of gravity-dependent movement of amyloplasts (statoliths) along root statocytes and hypocotyls (endodermis cells) has been analyzed and compared in order to testify cytoskeleton involvement in the displacement of statoliths in cress (Lepidium sativum L.) seedling statocytes. After 32 h of growth at 1 g or under a fast clinorotation (50 rpm), the seedlings were treated for 24 min as follows: exposition to clinorotation or 180° inversion and the action of gravitational force in root tip or hypocotyl tip direction. Statolith displacement was studied by light microscopy on semi-thin longitudinal sections of hypocotyls and root caps, measuring the distance between the centre of plastids and morphological cell bottom. Considerable temporal differences have been determined between the kinetics of the longitudinal motion of amyloplasts in root and hypocotyl statocytes of 1-g seedlings upon exposition to fast clinorotation and inversion. In statocytes of both organs of seedlings grown under fast clinorotation, the gravity provoked displacement of statoliths in the direction of its action; however, the displacement was much faster in hypocotyl than in root statocytes. It has been assumed that the gravity-determined longitudinal transport of amyloplasts, both in hypocotyl endodermic cells and root statocytes of cress seedlings, is modulated by the cytoskeleton.     

Tubulin Cytoskeleton in Arabidopsis thaliana Root Cells Under Clinorotation

The effect of horizontal clinorotation (2 rpm) on the spatial organization of cortical microtubules (CMTs) in the living Arabidopsis root cells has been investigated. MTs were visualized by using a stably transformed line of transgenic Arabidopsis thaliana expressing GFP-MAP4 fusion protein. To monitor total changes in MT dynamics, the sensitivity to MT-inhibitor 5 μM/l oryzalin was used as an indicator. It was shown that CMTs in the distal elongation zone (DEZ) of clinorotated 5 day old seedlings exhibited a higher degree of disorder and also they were more sensitive to oryzalin. We suggest that rearrangement of CMTs in DEZ cells under clinorotation is caused by fast rate of MT dynamic turnover.     

Physiological Responses of Synechocystis sp. PCC 6803 under Clinorotation

Photosystem efficiency and the characteristic on oxidative stress were examined to elucidate the metabolic responses of Synechocystis sp. PCC 6803 to short-term clinorotation. Results compiled when using clinostat to simulate microgravity for 60?h, showed that clinorotation clearly prohibited the photochemical quantum yield, but promoted the synthesis of chlorophyll and total protein. This may be a compensatory mechanism for the algal cell to maintain its normal metabolism. An increased malondialdehyde (MDA) content of algal cell upon clinorotation, together with an enhanced catalase (CAT) activity was observed during the whole period of clinorotation. One conclusion is that short-term clinorotation acts as a kind of stress, and that these physiological responses may be a special way for an algal cell to adapt itself to a different environment other than earth gravity.     

Vibration spectroscopy study of hydrolyzed precursors for sintering calcium phosphate bio-ceramics

Diffuse reflectance infrared Fourier transform and Raman spectroscopy is applied to investigate the structure of hydrolyzed CaHPO₄ · 2H₂O (DCPD) processed to non-stoichiometric apatite to be used as starting material for preparing sintered calcium phosphate materials. The spectra of hydrolyzed DCPD samples, obtained under differing experimental conditions, are analyzed to check how the sintered phase composition correlates with the structure of the hydrolyzed precursor. It is shown that the addition of extra Ca²⁺ ions in the alkaline solution during the process of hydrolyzing affects strongly the degree of disorder in the structure of the hydrolyzed materials, thus increasing the relative amount of hydroxyapatite in the final sintered product. The addition of F^– ions to the synthesis mixture strengthens slightly the P-O bonds in the precursors and increases the content of hydroxyapatite phase in the sintered material. As a result bi-phase ceramics of highest content of hydroxyapatite can be produced using both fluorine additives and calcium compensators in the synthesis suspension.     

Seed Germination and Seedling Growth under Simulated Microgravity Causes Alterations in Plant Cell Proliferation and Ribosome Biogenesis

The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception.     

Clinorotation Affects the Ultrastructure of Pea Root Mitochondria

Effects of clinorotation on the mitochondrial ultrastructure in cells of meristem, distal and central elongation zones of 3- and 5-day-old etiolated roots of pea seedling roots were studied. It was shown that mitochondria in cells of examined root growth zones revealed a different sensitivity to clinorotation. The ultrastructure of mitochondria in the meristem and central elongation zone cells did not substantially change in comparison with stationary control. At the same time, changes in the mitochondrial ultrastructure in cells of the distal elongation zone under clinorotation were observed, namely: decrease in the size of mitochondria, as well as increase in both matrix electron density and crista volume. Such changes in the mitochondrial ultrastructure under clinorotation are supposed to display the rearrangements of energy metabolism in cells of the distal elongation zone in these conditions.     

Rocket seedling production on the international space station: Growth and nutritional properties

Producing sprouts directly during space missions may represent an interesting opportunity to offer high-quality fresh ready to eat food to the astronauts. The goal of this work was to compare, in terms of growth and nutritional quality, rocket (Eruca sativa Mill.) seedlings grown in the International Space Station during the ENEIDE mission with those grown in a ground-based experiment (in presence and absence of clinorotation). The rocket seedlings obtained from the space-experiment were thinner and more elongated than those obtained in the ground-based experiment. Cotyledons were often closed in the seedlings grown in the space experiment. Quantitative (germination, fresh and dry weight) and qualitative (glucose, fructose, sucrose and starch) traits of rocket seedling were negatively affected by micrograv-ity, especially those recorded on seedlings grown under real microgravity conditions The total chlorophyll, and carotenoids of seedlings obtained in the space experiment were strongly reduced in comparison to those obtained in the ground-based experiment (presence and absence of clinorotation). The results showed that it is possible to produce rocket seedlings in the ISS; however, further studies are needed to define the optimal environmental conditions for producing rocket seedlings with high nutritional value     

The Impact of Simulated Microgravity on the Growth of Different Genotypes of the Model Legume Plant <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Simulated microgravity has been a useful tool to help understand plant development in altered gravity conditions. Thirty-one genotypes of the legume plant Medicago truncatula were grown in either simulated microgravity on a rotating clinostat, or in a static, vertical environment. Twenty morphological features were measured and compared between these two gravity treatments. Within-species genotypic variation was a significant predictor of the phenotypic response to gravity treatment in 100% of the measured morphological and growth features. In addition, there was a genotype–environment interaction (G × E) for 45% of the response variables, including shoot relative growth rate (p <?0.0005), median number of roots (p ～ 0.02), and root dry mass (p <?0.005). Our studies demonstrate that genotype does play a significant role in M. truncatula morphology and affects the response of plants to the gravity treatment, influencing both the magnitude and direction of the gravity response. These findings are discussed in the context of improving future studies in plant space biology by controlling for genotypic differences. Thus, manipulation of genotype effects, in combination with M. truncatula’s symbiotic relationships with bacteria and fungi, will be important for optimizing legumes for cultivation on long-term space missions.     

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.     

Anti-tumor activity of self-charged (Eu,Ca):WO3 and Eu:CaWO4 nanoparticles

Non-stoichiometric (Eu,Ca):WO₃ and Eu:CaWO₄ nanoparticles with anti-tumor activity are synthesized in a sol?Cgel method by adding excessive Eu^3?+? and Ca^2?+? ions to tungsten oxide crystal structure. Colorimetric assay shows that 10?nm (Eu,Ca):WO₃ and Eu:CaWO₄ nanoparticles can effectively inhibit growth of mammary cancer cells without any harm to normal cells. Nanoparticles are characterized by X-ray diffraction, high resolution transmission electron microscopy and fluorescence optical spectrometry. Nanomaterials, insoluble in synthesized water, have complicated self-charging surfaces that trap mammary cancer cells. Surface self-charging effect is suggested as the inhibition mechanism.     

Effects of Clinorotation on Growth and Chlorophyll Content of Rice Seeds

Microgravity, as a different environment, has been shown to affect plant growth and development (Sievers et al. 1996; Sack 1997). In the present study, effects of slow clinorotation (2 rpm) on growth and chlorophyll content in rice (variety: PRH-10) seedlings were investigated. Rice seeds were clinorotated continuously for 3, 5 and 7 days under ambient conditions. Root and shoot lengths and weights of rice seedlings were measured on the third, fifth and seventh day. Chlorophyll was extracted using N, N-Dimethylformamide (DMF). Absorption and fluorescence spectra of chlorophyll were recorded. Chlorophyll a, chlorophyll b and total chlorophyll contents were calculated from absorption spectra using Arnon’s method. Results showed an increase in root and shoot lengths in clinorotated samples. Similar results were obtained for root and shoot weights. Absorption spectra of chlorophyll showed no shift in the absorption peaks. Chlorophyll content was increased in clinorotated samples as compared to the controls. Interestingly, the difference between chlorophyll content in control and clinorotated samples decreased as the number of days of clinorotation increased. Chlorophyll a/b ratio was lowered in clinorotated samples as compared to the controls. These results suggest that slow clinorotation (2 rpm) affects plant growth and chlorophyll content in rice seedlings.     

Photoperiod-controlling Guttation and Growth of Rice Seedlings Under Microgravity on Board Chinese Spacelab TG-2

Guttation has been shown to play a crucial role in controlling plant growth and development by involvement of the transport of water, but how does this water transport in plant from roots to leaves work against pull of gravity is still unknown. The aim of the present study was to evaluate the influence of microgravity on photoperiod-controlling guttation and growth of rice (Oryza sativa L.) seedlings on board the Chinese Spacelab TongGong-2(TG-2). The growth rate of rice seedlings was closely correlated with guttation under both the microgravity and the ground conditions, which was increased by microgravity under both a 16-h long-day (LD) and an 8-h short-day (SD) photoperiod conditions. In addition, guttation of the TG-2 grown rice under the LD condition was more significant in comparison with that under the SD condition. These results indicated that microgravity affected the photoperiod-controlling growth of rice seedlings could be related to the enhanced guttation in space.     

Thyroid Cells Exposed to Simulated Microgravity Conditions – Comparison of the Fast Rotating Clinostat and the Random Positioning Machine

The ground-based facilities 2D clinostat (CN) and Random Positioning Machine (RPM) were designed to simulate microgravity conditions on Earth. With support of the CORA-ESA-GBF program we could use both facilities to investigate the impact of simulated microgravity on normal and malignant thyroid cells. In this review we report about the current knowledge of thyroid cancer cells and normal thyrocytes grown under altered gravity conditions with a special focus on growth behaviour, changes in the gene expression pattern and protein content, as well as on altered secretion behaviour of the cells. We reviewed data obtained from normal thyrocytes and cell lines (two poorly differentiated follicular thyroid cancer cell lines FTC-133 and ML-1, as well as the normal thyroid cell lines Nthy-ori 3-1 and HTU-5). Thyroid cells cultured under conditions of simulated microgravity (RPM and CN) and in Space showed similar changes with respect to spheroid formation. In static 1g control cultures no spheroids were detectable. Changes in the regulation of cytokines are discussed to be involved in MCS (multicellular spheroids) formation. The ESA-GBF program helps the scientists to prepare future spaceflight experiments and furthermore, it might help to identify targets for drug therapy against thyroid cancer.     

Calcium release activated membrane channels of osteoblast-like cells: targets and entrance gates for metal cations?

Enossal metal implants release ions which alter e. g. the Ca²⁺-based signaling of bone cells. One crucial element of this Ca²⁺ signaling is the calcium release activated calcium flux (CRAC) which is involved in the refilling of the cell's intracellular Ca²⁺ stores (ICS). Properties of this CRAC were studied in cultured osteoblast-like (OBL) cells using the Ca²⁺ sensitive fluorescent dye fura-2. CRAC channels were opened by depleting ICS in the absence of extracellular calcium ([Ca²⁺]_e) by either thapsigargin (5 μM) or 4-bromo-A23187 (2 μM). Elevation of [Ca²⁺]_e to 1.8 mM then increased the free intracellular Ca²⁺ ([Ca²⁺]_i). This Ca²⁺ influx was a typical CRAC and could be blocked by flufenamic acid (100 μM) which is a characteristic inhibitor of cation channels with low selectivity. Induction of CRAC enhanced the influx of extracellular Mn²⁺ (2 mM) 4.3fold, as measured by quenching of flura-2 fluorescence. Ni²⁺, on the one hand, potentiated the immediate CRAC while, on the other hand, it reversibly inhibited Ca²⁺ influx at a later stage. Similarly, Pb²⁺ (> 5 μM) dose dependently inhibited the immediate CRAC. Using the high affinity of fura-2 to Pb²⁺ and the resulting Ca²⁺ binding-like change of the optical signal, the permeation of Pb²⁺ into OBL cells could be detected. This binding could be reversed by the cell permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). At the single cell level, the permeation of Pb²⁺ correlated with increasing amounts of CRAC. Even low concentrations of Pb²⁺ (1 μM), which have been found in the blood of human individuals, clearly increased the fura-2 signal. These results highlight CRAC channels of OBL cells as a possible entrance gate and / or a target for metal ions which may be released from metal implant materials.     

Ions and water transmembrane transport in nervous and testicular cultured cells in low gravity conditions

Aim of the present study was to investigate on the possible alterations induced by on ground modeled microgravity on ion-water transport proteins at cellular level. For the purpose we used astrocytes, C₆ line, neurons (NT₂ line from human teratocarcinoma) and testicular cells (germ cells, Sertoli cells, and Leydig cells; primary cultures from trypsinised prepuberal pig testes). Modeled microgravity was achieved by a desktop 3D Random Positioning Machine, cultures were kept rotating for 30′, 1h and 24h. After 30′, immunopositivity for the antibodies to Na⁺/K⁺ATPase and Na⁺/K⁺/Cl^? co-transporters was greatly diminished, the plasma membrane appeared to be altered, and the mitochondria inner cristae were disrupted. Immunostaining to the antibody to the water channel aquaporin 4 was very bright. After 1h at random rotation immunostaining for the heat shock protein Hsp27 was visible, After 24h, immunostaining for the ion transport proteins was again like that of the controls, plasma membrane and the mitochondria were again normal. Immunostaining for aquaporin 4 become again similar to that of the controls. We conclude that low gravity induces only transient alterations in the cell’s transmembrane ion-water transport: the cells are able to adapt to the gravity vector changes in few hours.