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.     

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.     

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.     

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.     

The effects of extended clinorotation on potato minituber formation and structure.

Formation and structure of potato minitubers grown aseptically for 30 days on a horizontal clinostat and in stationary control have been studied by light and electron microscopy. It was demonstrated that the number of plants that formed minitubers, their size and fresh weight, was higher when clino-rotated than in the stationary control. It was revealed that the amount of amyloplasts in parenchyma cell sections was doubled in minitubers formed under clino-rotation. Other factors (shape of minitubers and size of reserve parenchyma cells) did not differ from the stationary control. The changes in amyloplast ultrastructure suggest accelerated cell maturity of potato reserve parenchyma in extended clino-rotation.     

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.     

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.     

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.     

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.     

Prospects of autotrophic link functioning in biological life-support systems based on cell biology studies

On the basis of using of modern methods of cytological analysis the cell growth peculiarities, reproduction and structure of plant cells of different level organisation (unicellular algae, mosses, angiosperms, cell cultures of higher factors--weightlessness, hypogravity, magnetic fields of various intensity, vibration and acceleration were cleared. It is shown that many discovered biological effects of space flight are connected with the degree of complexity of the object organisation and intensified with the increasing duration of influence.