Clinostating effects on biochemical characteristics and productivity of healthy and virus-infected wheat plants of dwarf Apogee variety.

The effects of clinostating on physiological processes and biochemical characteristics of wheat plants (Triticum aestivum L.) both healthy and infected by the wheat streak mosaic virus (WSMV) were studied. In six experiments, each lasting over 30 days, healthy and infected plants of the dwarf Apogee variety were grown under conditions of continuous horizontal and vertical clinostating with 2 rpm at 21 +/- 2 degrees C and 6000 1x (the optimal moisture of a substrate being maintained). The control variants (healthy and infected) were simultaneously grown under the same conditions of temperature and illumination in stationary containers and in open pots. During the experiment, visual observations were carried out over the state of tested plants. After completing the experiment, biometric indices, pigment, carbohydrate and dry matter contents were determined in all the plants. It was shown that clinostating sharply reduced the reproductive function of healthy plants and considerably affected their biomass (productivity) and concentration of chlorophylls and sugars. The viral infection resulted in further reduction of these characteristics. In control variants the viral effect was more significant. We speculate that clinostating reduced the rate of reproduction and spread of the virus.     

Clinostation influence on regeneration of cell wall in Solanum tuberosum L. protoplasts.

Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in a regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.     

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.     

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.     

Lipid peroxidation of plants under microgravity and its simulation.

In series of space experiments aboard the biosatellites "Cosmos 1887", "Bion 9", the orbital stations "Salut", "Mir" and under clinostating, changes of lipid peroxidation (LPO) and antioxidation activity (AOA) of Chlorella, Haplopappus tissue culture, wheat and pea roots were determined. The changes had a complex fluctuation character; three steps of response were established: LPO decreasing accompanied by AOA increase; stabilization LPO <==> AOA balance; secondary LPO activation. Most early and highly amplitude decreasing of LPO were fixed in mitochondria. The rate of response have been increased on multicellular level of plants organization.     

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.     

Effect of simulated microgravitation on phytohormones and cell structure of tropical orchids.

When studying the effect of two month clinostating on the phytohormonal system of orchids with different types of shoot system branching and different shoot morphology, it was determined that, as a result of simulated microgravitation, endogenous growth regulators changed less in the species with sympodial branching than in species with monopodial branching and without pseudobulbs. Stimulators prevail in the balance of growth regulators in species of the first type and inhibitors in species of the second type. Besides this, comparative analysis of structural organization of juvenile leaf surface tissue of tested orchids was carried out. Variability of size, number and structure of stomatal organization were found according to species belonging to each branching type after clinostating. Electronic microscope studies show some structural peculiarities of epidermal and mesophilous cells.     

The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9.

An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 ("Kosmos-2044") in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.     

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.     

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.     

Ultrastructural analysis of organization of roots obtained from cell cultures at clinostating and under microgravity.

Data are presented of a comparative analysis on rhizogenesis in the Arabidopsis thaliana tissue culture growing in a solid nutrient medium under stationary conditions, clinostatic conditions and microgravity. Tissue samples weighing 100 mg. were set in the Petri dishes and placed in a horizontal slow clinostat /2 revs/min/. After 14 days of growth they were analyzed. On clinostating the number of roots formed from the callus cells was approximately one half the control. The formed root cap manifested no essential differences, in comparison with the stationary control, in the number of layers and cell sizes in its layers. In callusogenic roots, formed from clinostated cells, differentiation including root cap cells, proceeds without noticeable deviations from the norm. At the same time, gravireceptor cells do not function under these conditions. This is clearly displayed at a structural level in the location of amyloplasts-statoliths throughout the cytoplasm. The callus cell cultures experienced microgravity for 8 days. The number of formed roots under the influence of this factor was 36% relative to the stationary control. Root cap formation was abnormal. Gravireceptor cells did not formed under microgravity.     

Seasonal variation of total electron content at crest of equatorial anomaly station during low solar activity conditions

The variability of total electron content (TEC) over the crest of equatorial anomaly station Bhopal has been studied during the low solar activity period (2005–2006) using global positioning system (GPS) data. Diurnal variation of TEC is studied for different seasons. Interesting features like the winter anomaly, semiannual anomaly and noon bite out in TEC have been reported. GPS derived TEC is then compared with International Reference Ionosphere (IRI) 2001 model and the difference between predictions and observation is being studied. Using the variability index we have also studied the TEC variability for different seasons and also during quiet and disturbed conditions. A higher variability is observed on quiet days as compared to disturbed days during daytime and nighttime hours.     

On different sensitivities of microorganisms to lowered gravitation

The influence of lowered gravitation on biomass and CO₂ production in $\text{B.megaterium}$, a xerophyte, and $\text{Spirillum azotocolligens}$, an aqueous spirillum, in liquid nutrient medium on a horizontal clinostat at 0.1 $\text{g}$ has been studied. As controls we considered: 1) growth under stationary conditions of cultivation with test tubes oriented horizontally; 2) growth on a synchronously revolving centrifuge; and 3) growth on a swing with stirring. A horizontal clinostat at 0.1 g stimulates biomass production and CO₂ release in $\text{B.megaterium}$ as compared with the controls. $\text{Spirillum azotocolligens}$ growth is reduced as a result of clinostating. The best development and CO₂ production are observed under stationary conditions. The results do not support the assumption that microorganisms living in water are more resistant to lowered gravitation than those living in soil.     

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.     

The role of calcium ions in cytological effects of hypogravity

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

High phosphorylase activity is correlated with increased potato minituber formation and starch content during extended clinorotation.

The major purpose of these experiments were to investigate growth of potato storage organs and starch synthesis in minitubers at slow horizontal clinorotation (2 rpm), which partly mimics microgravity, and a secondary goal was to study the activity and localization of phosphorylase (EC 2.4.1.1) in storage parenchyma under these conditions. Miniplants of Solanum tuberosum L. (cv Adreta) were grown in culture for 30 days for both the vertical control and the horizontal clinorotation. During long-term clinorotation, an acceleration of minituber formation, and an increase of amyloplast number and size in storage parenchyma cells, as well as increased starch content, was observed in the minitubers. The differences among cytochemical reaction intensity, activity of phosphorylase, and carbohydrate content in storage parenchyma cells of minitubers grown in a horizontal clinostat were established by electron-cytochemical and biochemical methods. It is shown that high phosphorylase activity is correlated with increased starch content during extended clinorotation. The results demonstrate the increase in carbohydrate metabolism and possible accelerated growth of storage organs under the influence of microgravity, as mimicked by clinorotation; therefore, clinorotation can be used as a basis for future studies on mechanisms of starch synthesis under microgravity.     

Statistical analyses of influence of solar and geomagnetic activities on car accident events.

Statistical analyses of the influence of Solar and geomagnetic activity, sector structure of the interplanetary magnetic field and galactic cosmic ray Forbush effects on car accident events in Poland for the period of 1990-1999 have been carried out. Using auto-correlation, cross-correlation, spectral analyses and superposition epochs methods it has been shown that there are separate periods when car accident events have direct correlation with Ap index of the geomagnetic activity, sector structure of the interplanetary magnetic field and Forbush decreases of galactic cosmic rays. Nevertheless, the single-valued direct correlation is not possible to reveal for the whole period of 1990-1999. Periodicity of 7 days and its second harmonic (3.5 days) has been reliably revealed in the car accident events data in Poland for the each year of the period 1990-1999. It is shown that the maximum car accident events take place in Poland on Friday and practically does not depend on the level of solar and geomagnetic activities.     

ASTROCULTURE (TM) root metabolism and cytochemical analysis.

Physiology of the root system is dependent upon oxygen availability and tissue respiration. During hypoxia nutrient and water acquisition may be inhibited, thus affecting the overall biochemical and physiological status of the plant. For the Astroculture (TM) plant growth hardware, the availability of oxygen in the root zone was measured by examining the changes in alcohol dehydrogenase (ADH) activity within the root tissue. ADH activity is a sensitive biochemical indicator of hypoxic conditions in plants and was measured in both spaceflight and control roots. In addition to the biochemical enzyme assays, localization of ADH in the root tissue was examined cytochemically. The results of these analyses showed that ADH activity increased significantly as a result of spaceflight exposure. Enzyme activity increased 248% to 304% in dwarf wheat when compared with the ground controls and Brassica showed increases between 334% and 579% when compared with day zero controls. Cytochemical staining revealed no differences in ADH tissue localization in any of the dwarf wheat treatments. These results show the importance of considering root system oxygenation in designing and building nutrient delivery hardware for spaceflight plant cultivation and confirm previous reports of an ADH response associated with spaceflight exposure.     

Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells.

The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw. cultivated on a clinostat (2rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive peculiarity of the cells cultivated on clinostat. It testifies to the fact that increasing of free calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weightlessness.