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

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

Long clinostation influence on the ultrastructure of Funaria hygrometrica moss protonema cells

Changes in the ultrastructure of protonema cells of Funaria hygrometrica, cultivated during 20 days on a horizontal clinostat (2 rev/min), were determined by the electron microscopy method. About 20% of the cells were almost identical to those in the control, 20% were destructive cells, and in 60% ultrastructure changes were observed. The heterogeneity of the reaction demonstrated the evidence of sensitive cells on the clinostation process. Changes affected the ultrastructure of plastids, wall of the cell, and the form of the nucleus as well. Starch disappeared from chloroplasts practically completely, thylakoids swelled, granas frequently disappeared from plastids. Peroxisomes number in cells increased appreciably, width of cell walls decreased by almost half their size. Ca⁺⁺-binding sites were revealed in cytoplasma of cells. Electronocytochemical exposure of ATPases activity with the presence of Mg⁺⁺ and Ca⁺⁺ ions showed that Mg²⁺-ATPase activity localization in clinosted cells was not too different from the control, while Ca²⁺-ATPase location presented differences in plasmalemma and Ca-sites. These changes are perhaps connected with the membranes permeability breaking and affect the plant cells adaptation to the influence of hypogravitation.     

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.     

Short-term and long-term clinostat and vibration-induced biochemical changes in dwarf marigold stems.

Stems of 21-day dwarf Marigold plants cultivated on the clinostat were compared with plants cultivated on vertical axis rotators ("vibrational controls") and stationary controls for long-term changes in cell wall composition. Stems of 21-day plants grown under stationary conditions and subsequently exposed to the clinostat for 24 hours were also analyzed. Among the long-tern markers, calciun, lignin, and protein-bound hemicellulose (possibly cell wall glycoprotein) clearly differentiated the effects of vibration from those of the clinostat. Short-term differential responses included rate of ethylene production, nastic movement and peroxidase activity of the cell wall, but not of the protoplast.     

The state of gravity sensors and peculiarities of plant growth during different gravitational loads.

In the primary roots of lettuce shoots grown under altered gravitational conditions--180 degrees inversion on the centrifuged clinostat, horizontal clinostat and in dynamic weightlessness--localization of the cellular organelles, cell morphology and peculiarities of growth have been studied. Significant changes took place in the localization of amyloplasts on the horizontal clinostat. The changes of amyloplast position in the cap cells on the horizontal clinostat and under weightlessness are similar. A change of the normal shoot position (180 degrees inversion and horizontal clinostat) causes an inhibition of growth. Weightlessness increases the length of axial organs and cells in the zone of elongation, but decreases the nitotic index in comparison to the centrifuged control. The anlysis of the formation of generative organs has been carried out for Arabidopsis plants grown on board the orbital station Salyut-6. The ability of plants to undergo vegetative growth and to pass through early phases of generative development under weightlessness was confirmed.     

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.     

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.     

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.     

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

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

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

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

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.     

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.     

Cytochemical localization of calcium in soybean root cap cells in microgravity.

The antimonate precipitation technique was used to evaluate the effects of microgravity and ethylene on the cellular and subcellular distribution of free calcium ions in soybean root apices. Soybean (Glycine max L. [Merr.]) dry seeds were launched, activated by hydration, and germinated in the presence of KMnO4 (to remove ethylene) and in its absence onboard the space shuttle Columbia during the STS-87 mission. Primary root apices of 6-day old seedlings were fixed for electron microscopy after landing. Ultrastructural studies indicated that antimonate precipitation appeared as individual electron-dense particles which were more or less round in shape and varied in diameter from 10 nm (minimum size beginning from which the particles were well identified) to 90 nm. It was revealed that analyzed root cap cells varied in both the precipitate particle sizes and the amount particles per unit of the cellular area. In both flight and ground control treatments, antimonate precipitation level increases from apical meristem cells to peripheral (secretory) cells of root apices. In root cap statocytes, subcellular localization of precipitate particles was revealed in the cytoplasm, nucleus and small vacuoles. The quantitative analysis showed a reduction of precipitate density in the cytoplasm and the nucleus, and an increase in precipitate density in the vacuoles from statocytes of both spaceflight treatments in comparison with ground controls.     

Calcium movements and the cellular basis of gravitropism.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.     

The theoretical consideration of microgravity effects on a cell.

Mechanical processes and factors involved in gravireception of a plant cell qualitatively considered and their changes caused by microgravity and clinostat modeling conditions are discussed. It is supposed that the most of the cell microgravity effects as well as clinostat modeling effects on a cell may be attributed to the generalized unspecific reaction of a cell to external influence.     

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.     

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.     

Ultrastructural and growth indices of Chlorella culture in multicomponent aquatic systems under space flight conditions.

Submicroscopic organization of Chlorella cells cultivated under space flight conditions in three-component aquatic system has been studied. Comparison of the experimental cells with that of the controls revealed certain rearrangements of cell organelles particularly, a reduction in the amount of reserve polysaccharides in chloroplasts, increase of cell vacuolization and mitochondrion volume, complication configuration of plasmalemma evaginations and invaginations, and also disturbances in the process of cytokinesis. More over an increase in the number of Chlorella cells infected by bacteria was shown in the experimental variant. No considerable differences were established in the growth characteristics of the experimental and control populations. A comparative cytological analysis revealed general regularities of organelles in Chlorella cells cultivated under space flight condition in the uni- and multicomponent systems.     

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.     

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.