Growth of Prunus tree stems under simulated microgravity conditions.

Stem growth of Prunus trees under simulated microgravity conditions was examined using a three-dimensional clinostat. The stems elongated with bending under such conditions. Stem elongation and leaf expansion were both promoted, whereas the formation of xylem in the secondary thickening growth was inhibited under the simulated microgravity condition. In secondary xylem, sedimentable amyloplasts were observed in the 1g control. The present results suggest that stem elongation and leaf expansion may be inhibited at 1g, while growth direction and secondary xylem formation depend on a gravity stimulus. A space experiment is expected to advance research on thickening growth in trees.     

Growth of Prunus tree stems under simulated microgravity conditions

Stem growth of Prunus trees under simulated microgravity conditions was examined using a three-dimensional clinostat. The stems elongated with bending under such conditions. Stem elongation and leaf expansion were both promoted, whereas the formation of xylem in the secondary thickening growth was inhibited under the simulated microgravity condition. In secondary xylem, sedimentable amyloplasts were observed in the 1g control. The present results suggest that stem elongation and leaf expansion may be inhibited at 1g, while growth direction and secondary xylem formation depend on a gravity stimulus. A space experiment is expected to advance research on thickening growth in trees.     

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.     

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.     

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.     

Microgravity effects on neural retina regeneration in the newt.

Data on forelimb and eye lens regeneration in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mullerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates.     

Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

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.     

Sedimentable amyloplasts in starch sheath cells of woody stems of Japanese cherry.

We examined whether sedimentable amyloplasts act as statolith in the perception of gravity in woody stems using the elongated internodes of Japanese cherry (Prunus jamasakura Sieb. ex Koidz.). In the internode of the seedlings grown on earth, amyloplasts were found sedimented at the distal end of each cell of the endodermal starch sheath tissue. In the internode grown on three-dimensional (3-D) clinostat, amyloplasts were dispersed throughout the cell matrix in the endodermal starch sheath tissue. After changing the positions of the internode from vertical to horizontal, re-sedimentation of amyloplasts toward the direction of gravity was completed in 1h, whereas the bending of the internode was observed after 12 days. We propose that sedimentable amyloplasts in the endodermal starch sheath cells may play a role in gravity perception leading to secondary xylem formation in the secondary thickening growth and eccentric growth in gravi-bending of tree stems.     

Auxin polar transport in Arabidopsis under simulated microgravity conditions — Relevance to growth and development

Activity of auxin polar transport in inflorescence axes of Arabidopsis thaliana grown under simulated microgravity conditions was studied in relation to the growth and development. Seeds were germinated and allowed to grow on an agar medium in test tubes on a horizontal clinostat. Horizontal clinostat rotation substantially reduced the growth of inflorescence axes and the productivity of seeds of Arabidopsis thaliana (ecotypes Landsberg erecta and Columbia), although it little affected seed germination, development of rosette leaves and flowering. The activity of auxin polar transport in inflorescence axes decreased when Arabidopsis plants were grown on a horizontal clinostat from germination stage, being ca. 60% of 1 g control. On the other hand, the auxin polar transport in inflorescence axes of Arabidopsis grown in 1 g conditions was not affected when the segments were exposed to various gravistimuli, including 3-dimensional clinorotation, during transport experiments. Pin-formed mutant of Arabidopsis, having a unique structure of the inflorescence axis with no flower and extremely low levels of the activity of auxin polar transport in inflorescence axes and endogenous auxin, did not continue its vegetative growth under clinostat rotation. These facts suggest that the development of the system of auxin polar transport in Arabidopsis is affected by microgravity, resulting in the inhibition of growth and development, especially during reproductive growth.     

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.     

空间飞行与回转器回旋条件下青菜开花与花粉发育的研究

比较研究了SJ-8返回式卫星留轨舱微重力条件与地面三维回转模拟微重力条件下青菜生长与发育情况.研究发现空间微重力条件下青菜开花过程需要大约18 h,明显长于地面对照5 h左右.回转器模拟实验结果表明,改变重力影响了花瓣的伸展与发育及花粉的产量,回转条件下花粉细胞中的微管排列明显不同于静止对照.细胞骨架受到干扰可能是改变重力条件下花粉产量降低的原因之一.本研究首次报道了在空间飞行试验中成功地采用了显微实时图像技术观察植物的开花过程,并获得了从花蕾到开花结束各阶段清晰的图像.      

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.     

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.     

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.     

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.     

Clinorotation reduces number, but not size, of cartilaginous nodules formed in micromass cultures of mouse limbbud cells.

In previous studies we used a ground based model to investigate the cellular responses to microgravity by exposing micromass cultures of embryonic limb cells to simulated weightlessness on a clinostat. Cultures set up in T-flasks and rotated at 30 rpm showed that clinostatted cultures had less chondrocyte differentiation than stationary or rotation controls, as assessed by number of nodules/culture stained with cartilage specific Alcian blue. In the current study, nodule size and shape of these nodules was assessed by interactive measurement of area, perimeter, circularity, and equivalent diameters, using the Optimas imaging software. Results show no significant difference in any of the measurements, indicating that clinorotation has no effect on expansion of the nodules either by differentiation of cells within the nodule, or by recruitment of cells into the nodule. The reduction in number of nodules without an alteration in size and shape indicates that the effect of simulated microgravity is to reduce the cell interactions required for the initial condensation of cells into a nodule, probably by interference with cell adhesion molecules.     

Cortical Microtubule Reorientation and Its Relation to Cell Surface Texture of Epidermal Cells of Arabidopsis Thaliana Hypocotyls under Simulated Microgravity Conditions

Gravitropic curvature growth of Arabidopsis hypocotyls mainly occurred in the rapid growing Elongation Zone (EZI), not in the slow-growing Elongation Zone (EZⅡ). By examining reorientation of Microtubules (MT) and phenotype of the cell wall in the EZI and the EZⅡ of Arabidopsis hypocotyls under normal gravitational condition, it is found that MTs in the rapid growing epidermal cells were mainly in the transverse direction, while those in the non-growing epidermal cells were in the longitudinal directions. However, this difference in cortical MT arrays between the EZI and EZⅡ cells disappeared when the seedlings were exposed to the simulated microgravity condition on a horizontal clinostat. Field emission scanning electron microscopy revealed that the surface texture of epidermal cells, like the direction of the MT, in the EZI and the EZⅡ also became similar when exposed to the simulated microgravity condition. This result indicated that simulate microgravity could modify the potential differentiation between the EZI and the EZⅡ by affecting the orientation of cortical MT in the epidermal cells.      

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.     

三维回转器回旋条件下拟南芥种子发育分析

向重力性反应是植物适应地球重力环境的一个重要生理过程, 是植物正常生长发育不可缺少的反应机制, 但是, 微重力是否影响植物种子发育至今尚无一致性结论. 本文研究了三维回转器回旋模拟微重力对拟南芥种子发育(胚胎发育与代谢活动)的影响. 研究结果表明, 三维回转条件下, 拟南芥果荚出现不规则弯曲或扭曲形态, 形成的种子中可溶性糖和淀粉含量明显增加, 盐溶性贮藏蛋白质含量显著降低而碱溶性蛋白质含量显著升高, 球形胚时期的种子对三维回转处理最为敏感. 对球形胚时期的植株进行短期的三维回转处理可抑制胚柄细胞的分裂和伸长, 而胚柄伸长受阻可能会影响到营养物质向胚胎中输送, 进而导致部分胚胎败育. 对种子干燥脱水阶段的植株进行三维回转处理不影响胚胎的发育, 但会导致种子贮藏蛋白质含量下降.